research title about breast cancer

Advances in Breast Cancer Research

A polyploid giant cancer cell (PGCC) from triple-negative breast cancer.

NCI-funded researchers are working to advance our understanding of how to prevent, detect, and treat breast cancer. They are also looking at how to address disparities and improve quality of life for survivors of the disease.

This page highlights some of what's new in the latest research for breast cancer, including new clinical advances that may soon translate into improved care, NCI-supported programs that are fueling progress, and research findings from recent studies.

Early Detection of Breast Cancer

Breast cancer is one of a few cancers for which an effective screening  test, mammography , is available. MRI ( magnetic resonance imaging ) and  ultrasound  are also used to detect breast cancer, but not as routine screening tools for people with average risk.

Ongoing studies are looking at ways to enhance current breast cancer screening options. Technological advances in imaging are creating new opportunities for improvements in both screening and early detection.

One technology advance is 3-D mammography , also called breast tomosynthesis . This procedure takes images from different angles around the breast and builds them into a 3-D-like image. Although this technology is increasingly available in the clinic, it isn’t known whether it is better than standard 2-D mammography , for detecting cancer at a less advanced stage.

NCI is funding a large-scale randomized breast screening trial, the Tomosynthesis Mammographic Imaging Screening Trial (TMIST) , to compare the number of advanced cancers detected in women screened for 5 years with 3-D mammography with the number detected in women screened with 2-D mammography. 

Two concerns in breast cancer screening, as in all cancer screening, are:

• the potential for diagnosing tumors that would not have become life-threatening ( overdiagnosis )
• the possibility of receiving false-positive test results, and the anxiety that comes with follow-up tests or procedures

As cancer treatment is becoming more individualized, researchers are looking at ways to personalize breast cancer screening. They are studying screening methods that are appropriate for each woman’s level of risk and limit the possibility of overdiagnosis.

For example, the Women Informed to Screen Depending on Measures of Risk (WISDOM) study aims to determine if risk-based screening—that is, screening at intervals that are based on each woman’s risk as determined by her genetic makeup, family history , and other risk factors—is as safe, effective, and accepted as standard annual screening mammography.

WISDOM is also making a focused effort to enroll Black women in the trial. Past studies  tended to contain a majority of White women and therefore, there is less data on how screening can benefit Black women. Researchers are taking a number of steps to include as many Black women as possible in the study while also increasing the diversity of all women enrolled.

Breast Cancer Treatment

The mainstays of breast cancer treatment are surgery , radiation , chemotherapy , hormone therapy , and targeted therapy . But scientists continue to study novel treatments and drugs, along with new combinations of existing treatments.

It is now known that breast cancer can be divided into subtypes based on whether they:

• are hormone receptor (HR) positive which means they express  estrogen and/or progesterone receptors  ( ER , PR )

Shortening Radiation Therapy for Some with Early Breast Cancer

A condensed course was as effective and safe as the standard course for women with higher-risk early-stage breast cancer who had a lumpectomy.

As we learn more about the subtypes of breast cancer and their behavior, we can use this information to guide treatment decisions. For example:

• The NCI-sponsored TAILORx clinical trial. The study, which included patients with ER-positive, lymph node-negative breast cancer, found that a test that looks at the expression of certain genes can predict which women can safely avoid chemotherapy.
• The RxPONDER trial found that the same gene expression test can also be used to determine treatment options in women with more advanced breast cancer. The study found that some postmenopausal women with HR positive, HER-2 negative breast cancer that has spread to several lymph nodes and has a low risk of recurrence do not benefit from chemotherapy when added to their hormone therapy. 
• The OFSET trial is comparing the addition of chemotherapy to usual treatment ( ovarian function suppression plus hormone therapy) to usual treatment alone in treating premenopausal estrogen receptor (ER)-positive/HER2-negative breast cancer patients who are at high risk of their cancer returning. This will help determine whether or not adding chemotherapy helps prevent the cancer from returning.  

Genomic analyses, such as those carried out through  The Cancer Genome Atlas (TCGA) , have provided more insights into the molecular diversity of breast cancer and eventually could help identify even more breast cancer subtypes. That knowledge, in turn, may lead to the development of therapies that target the genetic alterations that drive those cancer subtypes.

HR-Positive Breast Cancer Treatment 

Hormone therapies have been a mainstay of treatment for HR-positive cancer. However, there is a new focus on adding targeted therapies to hormone therapy for advanced or metastatic HR-positive cancers. These treatments could prolong the time until chemotherapy is needed and ideally, extend survival. Approved drugs include:

Drug Combo Effective for Metastatic Breast Cancer in Younger Women

Ribociclib plus hormone therapy were superior to standard chemotherapy combos in a recent trial.

• Palbociclib (Ibrance) ,  ribociclib (Kisqali) , and  everolimus (Afinitor) have all been approved by the FDA for use with hormone therapy for treatment of advanced or metastatic breast cancer. Ribociclib has been shown to increase the survival of patients with metastatic breast cancer . It has also shown to slow the growth of metastatic cancer in younger women when combined with hormone therapy.
• Elacestrant (Orserdu) is approved for HR-positive and HER2-negative breast cancer that has a mutation in the ESR1 gene, and has spread. It is used in postmenopausal women and in men whose cancer has gotten worse after at least one type of hormone therapy.
• Abemaciclib (Verzenio) can be used with or after hormone therapy to treat advanced or metastatic HR-positive, HER2-negative breast cancer. In October 2021, the Food and Drug Administration ( FDA ) approved abemaciclib in combination with hormone therapy to treat some people who have had surgery for early-stage HR-positive, HER2-negative breast cancer.
• Alpelisib (Piqray)  is approved to be used in combination with hormone therapy to treat advanced or metastatic HR-positive, HER2-negative breast cancers that have a mutation in the PIK3CA gene .
• Sacituzumab govitecan-hziy (Trodelvy) is used for HR-positive and HER2-negative breast cancer that has spread or can't be removed with surgery. It is used in those who have received hormone therapy and at least two previous treatments. It has shown to extend the amount of time that the disease doesn't get worse ( progression-free survival ) and also shown to improve overall survival .

HER2-Positive Breast Cancer Treatment 

The FDA has approved a number of targeted therapies to treat HER2-positive breast cancer , including:

• Trastuzumab (Herceptin) has been approved to be used to prevent a relapse in patients with early-stage HER2-positive breast cancer. 
• Pertuzumab (Perjeta) is used to treat metastatic HER2-positive breast cancer, and also both before surgery ( neoadjuvant ) and after surgery ( adjuvant therapy ). 
• Trastuzumab and pertuzumab together can be used in combination with chemotherapy to prevent relapse in people with early-stage HER2-positive breast cancer.  Both are also used together in metastatic disease, where they delay progression and improve overall survival. 
• Trastuzumab deruxtecan (Enhertu) is approved for patients with advanced or metastatic HER2-positive breast cancer who have previously received a HER2-targeted treatment. A 2021 clinical trial showed that the drug lengthened the time that people with metastatic HER2-positive breast cancer lived without their cancer progressing. The trial also showed that it was better at shrinking tumors than another targeted drug, trastuzumab emtansine (Kadcyla).
• Tucatinib (Tukysa) is approved to be used in combination with trastuzumab and capecitabine (Xeloda) for HER2-positive breast cancer that cannot be removed with surgery or is metastatic. Tucatinib is able to cross the blood–brain barrier, which makes it especially useful for HER2-positive metastatic breast cancer, which tends to spread to the brain. 
• Lapatinib (Tykerb)  has been approved for treatment of some patients with HER2-positive advanced or metastatic breast cancer, together with capecitabine or letrozole.
• Neratinib Maleate (Nerlynx) can be used in patients with early-stage HER2-positive breast cancer and can also be used together with capecitabine (Xeloda) in some patients with advanced or metastatic disease.
• Ado-trastuzumab emtansine (Kadcyla) is approved to treat patients with metastatic HER2-positive breast cancer who have previously received trastuzumab and a taxane . It's also used in some patients with early-stage HER2-positive breast cancer who have completed therapy before surgery ( neoadjuvant ) and have residual disease at the time of surgery.

HER2-Low Breast Cancer

 A newly defined subtype, HER2-low, accounts for more than half of all metastatic breast cancers. HER2-low tumors are defined as those whose cells contain lower levels of the HER2 protein on their surface. Such tumors have traditionally been classified as HER2-negative because they did not respond to drugs that target HER2. 

However, in a clinical trial, trastuzumab deruxtecan (Enhertu) improved the survival of patients with HER2-low breast cancer compared with chemotherapy , and the drug is approved for use in such patients. 

Immunotherapy Improves Survival in Triple-Negative Breast Cancer

For patients whose tumors had high PD-L1 levels, pembrolizumab with chemo helped them live longer.

Triple-Negative Breast Cancer Treatment 

Triple-negative breast cancers (TNBC) are the hardest to treat because they lack both hormone receptors and HER2 overexpression , so they do not respond to therapies directed at these targets. Therefore, chemotherapy is the mainstay for treatment of TNBC. However, new treatments are starting to become available. These include:

• Sacituzumab govitecan-hziy (Trodelvy)  is approved to treat patients with TNBC that has spread to other parts of the body . Patients must have received at least two prior therapies before receiving the drug.
• Pembrolizumab (Keytruda)  is an immunotherapy drug that is approved to be used in combination with chemotherapy for patients with locally advanced or metastatic TNBC that has the PD-L1 protein. It may also be used before surgery (called neoadjuvant ) for patients with early-stage TNBC, regardless of their PD-L1 status.
• PARP inhibitors, which include olaparib (Lynparza) and talazoparib (Talzenna) , are approved to treat metastatic HER2-negative or triple-negative breast cancers in patients who have inherited a harmful BRCA gene mutation. Olaparib is also approved for use in certain patients with early-stage HER2-negative or triple-negative breast cancer. 
• Drugs that block the androgen receptors  or prevent androgen production are being tested in a subset of TNBC that express the androgen receptor.

For a complete list of drugs for breast cancer, see Drugs Approved for Breast Cancer .

NCI-Supported Breast Cancer Research Programs

Many NCI-funded researchers working at the NIH campus, as well as across the United States and world, are seeking ways to address breast cancer more effectively. Some research is basic, exploring questions as diverse as the biological underpinnings of cancer and the social factors that affect cancer risk. And some are more clinical, seeking to translate this basic information into improving patient outcomes. The programs listed below are a small sampling of NCI’s research efforts in breast cancer.

TMIST is a randomized breast screening trial that compares two Food and Drug Administration (FDA)-approved types of digital mammography, standard digital mammography (2-D) with a newer technology called tomosynthesis mammography (3-D).

The  Breast Specialized Programs of Research Excellence (Breast SPOREs)  are designed to quickly move basic scientific findings into clinical settings. The Breast SPOREs support the development of new therapies and technologies, and studies to better understand tumor resistance, diagnosis, prognosis, screening, prevention, and treatment of breast cancer.

The NCI Cancer Intervention and Surveillance Modeling Network (CISNET) focuses on using modeling to improve our understanding of how prevention, early detection, screening, and treatment affect breast cancer outcomes.

The Confluence Project , from NCI's Division of Cancer Epidemiology and Genetics (DCEG) , is developing a research resource that includes data from thousands of breast cancer patients and controls of different races and ethnicities. This resource will be used to identify genes that are associated with breast cancer risk, prognosis, subtypes, response to treatment, and second breast cancers. (DCEG conducts other breast cancer research as well.)

The Black Women’s Health Study (BWHS) Breast Cancer Risk Calculator allows health professionals to estimate a woman’s risk of developing invasive breast cancer over the next 5 years. With the NCI-funded effort, researchers developed a tool to estimate the risk of breast cancer in US Black women. The team that developed the tool hopes it will help guide more personalized decisions on when Black women—especially younger women—should begin breast cancer screening. 

The goal of the Breast Cancer Surveillance Consortium (BCSC) , an NCI-funded program launched in 1994, is to enhance the understanding of breast cancer screening practices in the United States and their impact on the breast cancer's stage at diagnosis, survival rates, and mortality.

There are ongoing programs at NCI that support prevention and early detection research in different cancers, including breast cancer. Examples include:

• The  Cancer Biomarkers Research Group , which promotes research in cancer biomarkers and manages the Early Detection Research Network (EDRN) . EDRN is a network of NCI-funded institutions that are collaborating to discover and validate early detection biomarkers. Within the EDRN, the Breast and Gynecologic Cancers Collaborative Group conducts research on breast and ovarian cancers.
• NCI's Division of Cancer Prevention  houses the Breast and Gynecologic Cancer Research Group which conducts and fosters the development of research on the prevention and early detection of  breast and gynecologic cancers.

Breast Cancer Survivorship Research

NCI’s Office of Cancer Survivorship, part of the Division of Cancer Control and Population Sciences (DCCPS), supports research projects throughout the country that study many issues related to breast cancer survivorship. Examples of studies funded include the impact of cancer and its treatment on physical functioning, emotional well-being, cognitive impairment , sleep disturbances, and cardiovascular health. Other studies focus on financial impacts, the effects on caregivers, models of care for survivors, and issues such as racial disparities and communication.

Breast Cancer Clinical Trials

NCI funds and oversees both early- and late-phase clinical trials to develop new treatments and improve patient care. Trials are available for breast cancer prevention , screening , and treatment . 

Breast Cancer Research Results

The following are some of our latest news articles on breast cancer research and study updates:

• Can Some People with Breast Cancer Safely Skip Lymph Node Radiation?
• Study Adds to Debate about Mammography in Older Women
• Pausing Long-Term Breast Cancer Therapy to Become Pregnant Appears to Be Safe
• A Safer, Better Treatment Option for Some Younger Women with Breast Cancer
• Shorter Course of Radiation Is Effective, Safe for Some with Early-Stage Breast Cancer
• Pembrolizumab Improves Survival in Advanced Triple-Negative Breast Cancer

View the full list of Breast Cancer Research Results and Study Updates .

Cochrane Breast Cancer

Top 10 breast cancer topics needing a cochrane systematic review.

Deciding which research topics to focus on in medicine and health depends on many factors. These factors can include the currency of a topic, feedback from people providing or receiving care, and the priorities of funders.

In late 2019, the Cochrane Breast Cancer Group (part of Cochrane’s Cancer Network) conducted a formal priority-setting exercise to help decide which review topics were most needed in the Cochrane Library. The Group did this by circulating a survey listing 25 new or existing review topics to a diverse group of individuals who are part of the international breast cancer community. The survey asked individuals to rank their top 10 topics from the list. Read details about the aims and methods used for this priority-setting exercise, which adhered to the standards outlined in Cochrane’s priority setting guidance note .

What were the top 10 review topics?

Read about the ranking of the 25 new or existing review topics .

What is next?

Support to author teams For the top 10 topics, the Cochrane Breast Cancer Group will prioritise these topics during the editorial and peer-review process.

For all breast cancer review topics registered with Cochrane, the Cochrane Breast Cancer Group continues to work on these topics with author teams as these remain important topics. There will be no noticeable change in the support provided to author teams.

Future topics The Cochrane Breast Cancer Group is open to receiving new topic ideas. If you have suggestions for new topics that are not currently covered in the Cochrane Library, please send your idea to [email protected] .

Repeating this priority-setting exercise The priority-setting exercise may be repeated every 3 years, depending on resources.

Who responded to the survey?

The survey was circulated to over 800 individuals. Of the 199 people who responded, 90 people (45%) provided complete responses. The respondents were doctors (59%), researchers (18%) and people who had received treatment or currently receiving treatment for breast cancer (14%). Most respondents were from the UK, followed by the USA, Argentina, and India.

How did we calculate the ranking for each review topic?

The average ranking was calculated for each topic. This method is commonly used to determine ranking scores from surveys. This approach considers the number of counts for each ranking on a topic, the weighting of each rank (where a ranking of 1 gets the most weight) and the total number of counts. 

[Cover image: foliage of the Yew tree. Taxanes, a class of chemotherapy drugs, were originally derived from the Yew tree]

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• Open access
• Published: 18 May 2024

Disparities in quality of life among patients with breast cancer based on surgical methods: a cross-sectional prospective study

• Yi Wang 1 ,
• Yibo He 1 ,
• Shiyan Wu 1 &
• Shangnao Xie 1  

Scientific Reports volume  14 , Article number:  11364 ( 2024 ) Cite this article

Metrics details

• Breast cancer
• Quality of life

To determine the impact of breast conservation on quality of life and identify treatment-related and other demographic factors associated with post-breast cancer treatment quality of life. A prospective study was conducted on 392 women who underwent breast cancer surgery at Hangzhou Cancer Hospital from January 1, 2013, to December 31, 2022. Operable breast cancer patients who had completed all treatments except endocrine therapy were included. Patients with tumor recurrence/metastasis, bilateral or male breast cancer, and other primary malignancies were excluded. After enrollment, patients were asked to complete the BREAST-Q scale, and their pathological and medical records were reviewed. Analysis of variance was used to compare the quality of life scores among the groups. Univariate and multivariate linear regression analyses were performed to identify independent factors associated with quality of life scores in different domains. Participants completed the BREAST-Q scale at a median of 4.6 years after surgery. Quality of life scores varied based on the therapeutic strategy. Breast conservation has significant advantages over mastectomy in terms of breast satisfaction, psychosocial, and sexual well-being. Compared to oncoplastic breast-conserving surgery, mastectomy was independently associated with decreased breast satisfaction, psychosocial, and sexual well-being, while conventional breast-conserving surgery showed comparable outcomes to oncoplastic breast-conserving surgery in terms of these factors. Breast conservation leads to an improvement in quality of life compared to mastectomy. Oncoplastic breast-conserving surgery does not lead to a decrease in quality of life compared to conventional breast-conserving surgery and offers better outcomes compared to mastectomy.

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Introduction.

Breast cancer is a prevalent global malignancy 1 , and breast-conserving surgery (BCS) with adjuvant radiotherapy (RT) is a well-established treatment for early-stage breast cancer 2 , 3 . However, up to 30% of BCS recipients express dissatisfaction with their postoperative appearance, necessitating corrective interventions 4 . In the 1980s, European surgeons introduced "oncoplastic breast-conserving surgery" (OBCS), which incorporates plastic surgery techniques for post-BCS breast defect reconstruction 5 .

While OBCS offers satisfactory long-term oncological results and broadens treatment possibilities for patients who would typically undergo mastectomies 6 , it involves more extensive incisions, additional tissue manipulation, and potential flap reconstruction in comparison to conventional breast-conserving surgery (cBCS) 7 , 8 . The procedures involved in OBCS are more complex, time-consuming, and costly. Given these complexities, is it still worthwhile to pursue breast conservation by OBCS? Some researchers have proposed whether the use of OBCS should be reduced 9 .

Understanding the impact on the quality of life of breast cancer survivors is crucial given its significant influence on medical decision-making 10 , 11 . Despite the widespread utilization of OBCS to conserve the breast and enhance its aesthetics, research on its impact on quality of life is limited and complicated due to the variability of surgical approaches. Consequently, this study aimed to assess the effect of breast conservation by OBCS on the quality of life of patients with operable breast cancer treated at Hangzhou Cancer Hospital from January 1, 2013, to December 31, 2022, and to elucidate the treatment and demographic factors associated with postoperative quality of life.

Materials and methods

This prospective, cross-sectional, case–control study was conducted at a single center. The inclusion criteria were operable breast cancer patients treated at Hangzhou Cancer Hospital between January 1, 2013, and December 31, 2022, who had completed all treatments except endocrine therapy and provided participation consent. The exclusion criteria were patients with tumor recurrence/metastasis, bilateral or male breast cancer, or other primary malignancies. Participants were categorized into two groups: BCS group (cBCS with RT subgroup and OBCS with RT subgroup), and unilateral MAST group (MAST with RT subgroup and MAST without RT subgroup). This study utilized the BREAST-Q scale 12 , which includes separate modules for BCS and MAST without reconstruction. The BCS module was used for the OBCS with RT subgroup because OBCS in this study predominantly referred to oncoplastic lumpectomy/glandular remodeling. BREAST-Q assesses six distinct domains: satisfaction with breasts, psychosocial well-being, physical well-being, sexual well-being, satisfaction with overall outcome, and satisfaction with care. Due to the elapsed time between surgery and questionnaire completion in this study, the domains of satisfaction with the overall outcome and satisfaction with care were excluded. Each domain was scored on a scale from 0 to 100, with higher scores indicating an enhanced quality of life. Differences in BREAST-Q scores were categorized as small (2–3 points), moderate (4–7 points), and large (8–10 points) 13 . Patient characteristics, collected using the questionnaire, included employment status, educational level, marital status, and economic status. Patients’ medical and pathological records were reviewed to determine the disease tumor, node, and metastasis (TNM) staging 14 , erythroblastic oncogene B (ERBB2; formerly HER2/neu or HER2) status, hormone receptor status, and body mass index (BMI). Information on surgery, chemotherapy (yes/no), RT, and endocrine therapy (yes/no) was obtained using a questionnaire in conjunction with medical records. The lymphedema status (yes/no) was assessed using the questionnaire's question regarding arm swelling. This study was approved by the Ethics Committee of Hangzhou Cancer Hospital, and all participants provided written informed consent. The study was performed in accordance with the Declaration of Helsinki and followed the guidelines of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) reporting guidelines.

Statistical analysis

The experimental data were statistically analyzed using SPSS (version 29.0) software, and categorical covariates were expressed as numbers (percentages). Analysis of variance (ANOVA) was used to compare quality of life scores among the different groups. Univariate and multivariate linear regression analyses were used to determine the independent factors associated with the quality of life scores in each domain. Variables with two-tailed P  ≤ 0.15 in the univariate analysis were included in the multivariate analysis model using a stepwise method to establish the final multivariate model. Differences with P  < 0.05 were considered statistically significant.

Ethics approval and consent to participate

This study was reviewed and approved by the ethics committee of Hangzhou Cancer Hospital (approval number: [hzch-2023] HS no.007). Written informed consent was obtained from every patient.

Patient enrollment

After screening, 623 eligible patients were invited, 456 provided written informed consent and completed the survey, but three were found to not meet the inclusion criteria after enrollment. After excluding 61 participants who only completed a brief questionnaire, a total of 392 patients’ data were included in the statistical analysis.

Patient, disease, and treatment characteristics

The interval between surgery and scale completion averaged 4.6 years (range: 0.33 to 9.83 years). Patient characteristics are detailed in Table 1 . Majority were married, employed, had moderate economic status (income ¥30,000–200,000 per year), and high school or higher education. At surgery, 324 (82.7%) patients had a body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) within the normal range (18.5 to 23.9 kg/m 2 ), and 56 (14.3%) patients had a BMI of 24 kg/m 2 or above. Among the patients, 39 (9.9%) had stage 0 breast cancer, 154 (39.3%) had stage I breast cancer, 158 (40.3%) had stage II breast cancer, and 41 (10.5%) had stage III breast cancer. The lesions on imaging before surgery of 253 (64.5%) patients measured two centimeters or less, 134 (34.2%) two to five centimeters, and 5 (1.3%) more than five centimeters. Chemotherapy was administered to 293 (74.7%) patients, with 121(30.9%) receiving neoadjuvant chemotherapy, and 273 (69.6%) patients received hormone therapy.

Treatment details including surgery, RT, and lymphedema are presented in Table 1 . Among the patients, 88 (22.4%) underwent OBCS, 51 (13.0%) underwent cBCS, and 253 (64.5%) underwent unilateral MAST, among which 100 (25.5%) patients who underwent unilateral MAST received postoperative RT. All patients underwent axillary surgery, with 255 (65.1%) patients undergoing sentinel lymph node biopsy only and 137 (34.9%) patients undergoing axillary lymph node dissection. 61 (15.6%) patients reported having lymphedema.

BREAST-Q results by breast surgery strategy

Figure  1 illustrates unadjusted mean BREAST-Q scores by breast surgery strategy. Satisfaction with breasts, psychosocial well-being and sexual well-being were significantly different among the groups ( P  < 0.001). BCS group showed higher scores in satisfaction with breasts (61.70), psychosocial well-being (76.01), physical well-being (83.52) and sexual well-being (55.06), while the scores for MAST group is lower (satisfaction with breasts: 57.30, psychosocial well-being: 70.83, physical well-being: 82.40 and sexual well-being: 49.21).

Unadjusted BREAST-Q mean scores by breast surgery strategy. BCS: breast-conserving surgery; MAST: mastectomy.

Satisfaction with breasts

Higher scores in satisfaction with breasts correlated independently with age ≥ 60 (β = 4.662; 95% CI = 2.345 to 6.979; P  < 0.001) and patient-reported income ≥ 200,000 (β = 5.068; 95% CI = 2.781 to 7.356; P  < 0.001). Lower scores were associated with BMI ≥ 24 (β = − 2.528; 95% CI = − 4.977 to − 0.079; P  = 0.043), axillary dissection (β = − 4.875; 95% CI = − 6.704 to − 3.046; P  < 0.001) and MAST (β = − 3.927; 95% CI = − 5.741 to − 2.113; P  < 0.001) (Fig.  2 A). Patient-reported income < 30,000 and lymphedema showed significance only in univariate analysis. Other factors exhibited no significant association.

Patient and treatment factors associated with breast satisfaction ( A ), psychosocial well-being ( B ), physical well-being ( C ) and sexual well-being ( D ) scores by breast surgery strategy. MAST: mastectomy; BCS: breast-conserving surgery; BMI: body mass index; CI: confidence interval.

Psychosocial well-being

Better psychosocial well-being correlated with age ≥ 60 (β = 2.564; 95% CI = 0.163 to 4.965; P  = 0.036), patient-reported income ≥ 200,000 (β = 4.820; 95% CI = 2.496 to 7.144; P  < 0.001), and ≥ 5y from surgery (β = 2.419; 95% CI = 0.523 to 4.315; P  = 0.013). Poor psychosocial well-being was linked to age < 35 (β = − 3.892; 95% CI = − 7.715 to − 0.069; P  = 0.046), BMI ≥ 24 (β = − 3.352; 95% CI = − 5.845 to − 0.859; P  = 0.009), patient-reported income < 30,000 (β = − 4.489; 95% CI = − 7.317 to − 1.660; P  = 0.002), axillary dissection (β = − 5.898; 95% CI = − 7.739 to − 4.058; P  < 0.001) and MAST (β = − 5.157; 95% CI = − 7.032 to − 3.283; P  < 0.001) (Fig.  2 B). Chemotherapy was only significant in univariate analysis. Other variables showed no significant association.

Physical well-being

Factors associated with better physical well-being were age ≥ 60 (β = 3.594; 95% CI = 1.554 to 5.634; P  = 0.001), patient-reported income ≥ 200,000 (β = 4.541; 95% CI = 2.559 to 6.524; P  < 0.001), and ≥ 5y from surgery (β = 2.311; 95% CI = 0.714 to 3.907; P  = 0.005). Conversely, patient-reported income < 30,000 (β = − 5.924; 95% CI = − 8.351 to − 3.497; P  < 0.001), axillary dissection (β = − 2.486; 95% CI = − 4.057 to − 0.914; P  = 0.002) and lymphedema (β = − 2.185; 95% CI = − 4.275 to − 0.094; P  = 0.041) were associated with poorer physical well-being (Fig.  2 C). < 1y from surgery was only significant in univariate analysis. Other factors lacked significant association.

Sexual well-being

Multivariate analysis indicated lower sexual well-being scores with BMI ≥ 24 (β = − 2.887; 95% CI = − 4.831 to − 0.943; P  = 0.004), < 1y from surgery (β = − 3.482; 95% CI = − 5.887 to − 1.077; P  = 0.005), axillary dissection (β = − 3.002; 95% CI = − 4.437 to − 1.567; P  < 0.001), and MAST (β = − 5.650; 95% CI = − 7.114 to − 4.187; P  < 0.001). Patient-reported income ≥ 200,000 (β = 2.272; 95% CI = 0.441 to 4.104; P  = 0.015) correlated with elevated sexual well-being (Fig.  2 D). Lymphedema was significant in univariate analysis. Other variables exhibited no significant correlation.

BREAST-Q results by local therapy strategy

To assess if there were enhancements in quality of life among women who underwent OBCS, we performed similar analyses among the subgroups. Figure  3 illustrates unadjusted mean BREAST-Q scores by local therapy strategy. All four domains were significantly different ( P  < 0.05). OBCS with RT group showed highest scores in satisfaction with breasts (61.99), psychosocial well-being (76.27) and sexual well-being (55.53). cBCS with RT group yielded the highest physical well-being score (84.10). The lowest domain scores were in MAST with RT group (satisfaction with breasts: 53.11, psychosocial well-being: 65.49, physical well-being: 79.89 and sexual well-being: 46.24).

Unadjusted BREAST-Q mean scores by local therapy strategy. RT: radiotherapy; cBCS: conventional breast-conserving surgery; OBCS: oncoplastic breast-conserving surgery; MAST: mastectomy.

Multivariate analysis indicated that MAST with RT was associated with poor breast satisfaction (β = − 8.381; 95% CI = − 10.858 to − 5.905; P  < 0.001), psychosocial well-being (β = − 11.491; 95% CI = − 14.039 to − 8.943; P  < 0.001), physical well-being (β = − 3.607; 95% CI = − 5.782 to − 1.432; P  = 0.001) and sexual well-being (β = − 9.493; 95% CI = − 11.454 to − 7.533; P  < 0.001). MAST without RT was associated with decreased breast satisfaction (β = − 2.536; 95% CI = − 4.817 to − 0.255; P  = 0.029), psychosocial well-being (β = − 3.171; 95% CI = − 5.487 to − 0.855; P  = 0.007) and sexual well-being (β = − 4.739; 95% CI = − 6.530 to − 2.947; P  < 0.001). cBCS with RT was not associated with BREAST-Q scores on univariate or multivariate analysis. The statistically significant factors correlated with BREAST-Q scores were mostly consistent with the outcomes of the breast surgery models (Fig.  4 ).

Patient and treatment factors associated with breast satisfaction ( A ), psychosocial well-being ( B ), physical well-being ( C ) and sexual well-being ( D ) scores by local therapy strategy. cBCS: conventional breast-conserving surgery; OBCS: oncoplastic breast-conserving surgery; MAST: mastectomy; RT: radiotherapy; BMI: body mass index; CI: confidence interval.

The rates of BCS and breast reconstruction after mastectomy are significantly lower in China than in Western countries 15 . One contributing factor is that Chinese women typically have smaller breast sizes than women in Western countries, while presenting with larger breast tumor volumes at the time of initial diagnosis, making BCS challenging. Additionally, some Chinese patients adhere to outdated beliefs and have concerns about potential impacts on treatment outcomes or cancer recurrence associated with BCS. OBCS provides acceptable long-term oncological outcomes and has extended treatment options for patients who would traditionally be candidates for mastectomies 6 . In recent years, there has been a clear change in the emphasis of surgical oncology in China, with a growing emphasis on utilizing modern oncoplastic surgical techniques to perform more breast conserving surgeries. Given the increasing prevalence of OBCS, it is essential to examine its impact on quality of life.

In this single-center prospective study, discernible disparities in quality of life surfaced among patients with breast cancer undergoing various local treatment strategies within ten years of surgery. Patients opting for more extensive surgery, particularly when combined with RT, experienced diminished quality of life; satisfaction with breasts; and psychosocial, physical, and sexual well-being. This aligns with findings from prior studies. Engel et al.’s study 16 has shown that patients undergoing BCS reports a higher quality of life compared to those opting for mastectomy. This improvement is often linked to the conservation of the breast and the associated psychological advantages. BCS enables breast conservation, leading to enhanced body image and self-esteem. Patients undergoing BCS may experience less psychological distress and enjoy better psychosocial well-being due to breast conservation. Additionally, BCS has a lesser impact on sexual well-being in comparison to mastectomy, as it retains natural breast tissue.

This study’s findings concur with those of Otsuka et al.’s study 17 in that oncoplastic surgery improved satisfaction with breasts. However, in Otsuka et al.’s study, the quality of life score was not elevated by OBCS (major breast surgery: 154.5 ± 24.6; minor breast surgery: 159.0 ± 20.8; OBCS: 158.7 ± 14.0). Although differences exist between major breast surgery and OBCS, the difference is not pronounced. In the present study, psychosocial and sexual well-being scores were elevated compared to MAST. Additionally, patients who underwent OBCS had better physical well-being scores than those who underwent MAST with RT and equal physical well-being scores than those who underwent MAST without RT. This may be attributable to the omission of RT, reduced chemotherapy and lymphedema in the MAST without RT group. Previous studies 18 , 19 have highlighted RT, chemotherapy, and lymphedema as adverse determinants of quality of life.

Rose et al. 20 suggested that patients who underwent OBCS showed significant improvement in the “psychosocial well-being” module compared to cBCS, while no significant differences were observed between the two groups in the “physical health,” “breast satisfaction,” and “sexual health” modules. Furthermore, a meta-analysis 21 indicated improved quality of life with OBCS compared with cBCS in patients with early-stage breast cancer, with better physical and psychological well-being, higher self-esteem, and a more stable body image, leading to improved social and emotional functioning. However, the clinical studies included in the meta-analysis were predominantly small- sample studies from single centers, and the surgical approaches varied. This study identified no significant differences in any of the quality of life modules between the patients who underwent OBCS and those who underwent cBCS, which is consistent with the findings of de Oliveira-Junior et al 22 . This may be because the present study’s follow-up time was longer, and several aspects of OBCS will decline over time 23 . In our study, the tumor lesion on imaging before surgery averaged 2.11 ± 0.67 cm in OBCS subgroup, and 1.62 ± 0.52 cm in cBCS subgroup. Smaller lesions are more likely to undergo cBCS, resulting in comparable cosmetic outcomes between the two surgical groups. Moreover, the limited number of BCS patients in our study is a significant factor that limits the ability to detect differences in quality of life between OBCS and cBCS subgroups.

In addition to the type of surgery, other clinical factors such as BMI (≥ 24), income (< 30,000), < 1y from surgery, axillary dissection, and lymphedema were negatively correlated with quality of life. Identifying these risk factors can facilitate early postoperative intervention and ultimately improve the postoperative quality of life of patients with breast cancer. Age (≥ 60) and ≥ 5y from surgery were associated with enhanced quality of life. Breast cancer patients can experience significant effects from the disease itself and the ongoing adjuvant therapies, both after diagnosis and during the treatment process 24 . These are all factors that lead to decreased quality of life within 5 years, especially within 1 year, rather than ≥ 5y after surgery. Moreover, good economic status was associated with better satisfaction with breasts, and psychosocial, physical, and sexual well-being. Patients with improved financial circumstances can access higher-quality healthcare services, opt for more expensive treatment options that may improve aesthetic outcomes. The financial advantage also affords patients more opportunities for supportive care, counseling, and resources to manage the challenges of breast cancer treatment and recovery, resulting in a decrease in stress, anxiety, and depression. These enhancements can have a positive impact on patients’ self-perception, confidence, and overall satisfaction with their breast appearance, all of which are closely connected to sexual health and intimacy. Notably, other studies 25 , 26 found an association between economic status and quality of life.

This study has some limitations. It was a cross-sectional, single-time, survey-based prospective study; therefore, the baseline quality of life of patients before surgery was not recorded, which may have influenced their choice of surgical approach and postoperative quality of life. Additionally, this study did not identify patients who chose MAST due to refusal of BCS; patients who selected MAST based on personal preferences may have different quality-of-life scores. Furthermore, this study did not include patients with postmastectomy breast reconstructions, which may improve quality of life of postmastectomy patients. Finally, given that this was a single-center small-sample study, studies with larger sample sizes are required to further confirm the findings of this study. Nevertheless, patient-reported questionnaires can provide basic information on quality of life and assist in identifying potential areas requiring intervention during the patient’s survival period.

OBCS is an acceptable option for patients with larger tumors who are not suitable for cBCS because it allows them to conserve their breasts 6 . This study demonstrated that patients who had their breast conserved reported a higher quality of life compared to mastectomy patients. Despite extensive incisions, additional tissue manipulation, and potential flap reconstruction, patients who underwent OBCS did not report a lower quality of life than those who underwent cBCS. Furthermore, they experienced significantly enhanced quality of life compared with patients who underwent MAST, particularly in the domains of satisfaction with breasts, psychosocial well-being, and sexual well-being. Quality of life data should be incorporated into decision support tools to assist patients with breast cancer in selecting the surgical approach, and discussions with patients should include information regarding quality of life to ensure that they understand the long-term impacts of different surgical approaches. This is particularly crucial because most patients with breast cancer have an extended postoperative survival period. Our data can support further improvements in Chinese breast surgical care for better survival and quality of life.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to Chinese law but are available from the corresponding author on reasonable request.

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The interpretation and reporting of these data are the sole responsibility of the authors, and no endorsement by the Hangzhou Cancer Hospital is intended nor should be inferred.

This research was financed by the Medical and Health Research Project of Zhejiang Province, China (No. 2023KY964).

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Wang, Y., He, Y., Wu, S. et al. Disparities in quality of life among patients with breast cancer based on surgical methods: a cross-sectional prospective study. Sci Rep 14 , 11364 (2024). https://doi.org/10.1038/s41598-024-62105-z

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Breast Cancer and the Environment: A Life Course Approach (2012)

Chapter: 7 recommendations for future research.

7 Recommendations for Future Research

A lthough much has been learned about breast cancer and its relation to environmental exposures, much remains unclear. As the preceding chapters have illustrated, this reflects a mixture of circumstances. First, the scientific community is faced with conflicting and inconclusive results from past studies of some risk factors. Second, growing knowledge of the complex biology of breast cancer suggests a need to reframe hypotheses by focusing more on exposures in early life, examining associations with tumors of specific types, and considering mechanistically driven gene–environment interactions. Third, for a wide array of exposures, data are simply inadequate because exposure assessment methodologies have not been developed, informative studies may be nearly impossible to conduct in humans, and/or the existing tools and resources to conduct relevant research in animals or in vitro systems are limited.

With the complexity of breast cancer as a disease and of the combinations of biological and environmental factors that are potential contributors to it, the committee is persuaded that no one perspective will be sufficient to guide the future research that is needed to reduce the toll of this disease. Bringing together the perspectives of many disciplines into a transdisciplinary approach will be needed to generate innovative and cost-effective approaches to framing research questions, designing and conducting studies, developing new tools for data collection and analysis, and translating the results of research on risk factors into interventions that can reduce the risk of breast cancer.

Drawing on the insights developed in the previous chapters, the committee presents in this final chapter recommendations for research that

range from further examination of elements of the biology of breast development and carcinogenesis to tests of potential interventions to reduce risk. Important components of the work recommended here provide support for the research necessary to develop better tools for assessing the carcinogenicity of chemicals and pharmaceuticals as well as tools needed to strengthen epidemiologic research. The importance of a life course perspective runs throughout these recommendations.

Many of these recommendations are directed to both researchers and research funders. Researchers will have to conduct the work described here, but they will need the resources that come from a variety of sources. The National Institutes of Health and other federal agencies are major funders of research on breast cancer or they have unique authority or responsibility in certain areas. But the nation’s portfolio of research on breast cancer is also shaped in important ways by funders and other organizations in the private sector, such as Susan G. Komen for the Cure, that have the flexibility to pursue research topics and approaches that federal agencies may not. The committee urges effective and innovative collaborations to answer the many unresolved questions about the causes of breast cancer.

APPLYING A LIFE COURSE PERSPECTIVE TO RESEARCH ON BREAST CANCER

Progress has been made in understanding the biology of breast development, molecular mechanisms of carcinogenesis, the influence of the tissue microenvironment on breast cancer development, and some aspects of risk and prevention. But gaps remain in understanding of the etiology of breast cancer and the extent of environmental influences on breast cancer development.

Most epidemiologic studies have been obliged to focus on events in the few years or perhaps one to two decades before a breast cancer diagnosis. As described in Chapter 5 , however, growing evidence suggests that events associated with breast carcinogenesis may occur much earlier—in young adulthood, puberty, childhood, and in utero. The effect of radiation, for instance, is greater when exposure occurs around the time of puberty or earlier. Although information about some early life events, such as age when first giving birth or age at menarche, can be reliably retrieved, few studies have collected information on nonreproductive environmental exposures that may influence the occurrence of clinically detectable breast cancer many decades later.

To address gaps in knowledge about the origins of breast cancer, the committee determined that research should increasingly focus on the influence of environmental factors during potential windows of susceptibility over the life course. It is possible that some exposures later in life, after

childbearing is complete, have little effect on breast cancer risk whereas similar exposures, if incurred early in life, before completion of breast development, may increase risk for breast cancer. On the other hand, exposures later in life may increase the growth of cancerous cells that have lain dormant for years and that would, without the exposure, have continued to be dormant. Thus the committee recommends that future research address the timing of exposures in relation to a woman’s life course and explore vulnerable windows for specific exposures of concern.

Recommendation 1: Breast cancer researchers and research funders should pursue integrated and transdisciplinary studies that provide evidence on etiologic factors and the determinants of breast cancer across the life course, with the goal of developing innovative prevention strategies that can be applied at various times in life.

• Such studies should seek to integrate animal models that capture the whole life course and human epidemiologic cohort studies that follow individuals over long periods of time and allow for investigation of so-called “windows of susceptibility” wherein breast tissue may be especially sensitive to environmental influences (e.g., prenatal, childhood, and adolescent, and childbearing periods). Long-term follow-up of cohorts is critical because new, unexpected evidence frequently arises with extended follow-up.

• Topics warranting attention include (but are not limited to) the biology of breast development; the mechanisms of carcinogenesis early in life, including the role of the tissue microenvironment in tumor suppression and development, and differences that may be related to tumor type; differences in risk by tumor type; the potential contribution of timing of exposure to variation in risk; and analytical tools for investigating the potential for interactions among exposures and the impact of mixtures of environmental agents on biologic processes.

Other work to aid investigation of environmental influences on breast cancer risk includes

• identifying cellular, biochemical, or molecular biomarkers of early events leading to breast cancer and validating their predictive value for future risk for breast cancer;

• determining whether intermediate endpoints, such as indicators of breast development or peak height growth velocity, are valid and predictive biomarkers of risk for breast cancer so that research can

effectively identify predictors of change in risk earlier in life or with shorter study periods;

• investigating the role that environmental factors may have in the origins of breast cancers of different types (e.g., estrogen or progestin receptor positive [ER+, PR+] or receptor negative [ER–, PR–]; HER2/neu positive or negative; or triple negative, meaning being negative for all three types of receptors) to better understand the potential contribution of these factors to disparities in the incidence of types of breast cancers among racial and ethnic groups;

• exploring the value of linking information across cohort studies focused on different stages of life as a way to overcome the challenges of mounting single long-term follow-up studies; and

• ensuring that cohorts established primarily to study genetic determinants of cancer and other diseases improve their capacity to capture information about environmental exposures over the life course.

TARGETING SPECIFIC CONCERNS

Rationale: From its examination of evidence on a selection of environmental factors, the committee sees particular benefit in further research to clarify the mechanisms underlying breast cancer.

Recommendation 2: Breast cancer researchers and research funders should pursue research to increase knowledge of mechanisms of action of environmental factors for which there is provocative, but as yet inconclusive, mechanistic, animal, life course, or human health evidence of a possible association with breast cancer risk.

High-priority topics include the following:

• Shift work : There is growing evidence that shift work resulting in the disruption of circadian rhythm is probably associated with increased risk for breast cancer. Currently, there are no known effective interventions other than avoidance of shift work, which will not be an option for many workers. The biological mechanisms and the potential contribution of light exposure during normal sleep periods are poorly understood. More needs to be learned about the biological processes and pathways through which shift work and circadian rhythm disruption, or other factors arising from shift work, relate to breast cancer. This includes investigation of hormonal effects of circadian disruption, the role of “clock genes” and signaling pathways in breast tissue development, how

disruption of those signaling pathways may contribute to initiation or progression of breast tumors, developing more detailed and standardized approaches to exposure assessment for use in epidemiologic research, and developing and testing the effectiveness of interventions that could mitigate the carcinogenic effects that may be associated with shift work.

• Endocrine activity : Exposure to chemicals with estrogenic or other properties relevant to sex steroid activity, such as bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), zearalenone, and certain dioxins and dioxin-like compounds, may influence breast cancer risk, especially if those exposures occur at certain life stages or in combination with exposure to other similar chemicals, certain dietary components, or other factors. Although the evidence on the association between breast cancer risk and individual chemicals in this category is not conclusive, current mechanistic hypotheses warrant further research to examine their activity, to investigate additive or greater potency across multiple chemicals, to explore the effects of timing of exposure, and to evaluate interactions with diet, body mass index, and other factors that may influence the relationship of these types of compounds to breast cancer risk.

• Genotoxicity : Animal studies have demonstrated that some mutagenic chemicals are capable of inducing malignant mammary tumors, and numerous animal models of breast carcinogenesis routinely use the potent mutagens 7,12-dimethylbenz[a]anthracene (DMBA) and N-methyl-N-nitrosourea (MNU) as reproducible initiators of those tumors. But these studies have shown that the effect is highly sensitive to the timing of the exposures and can be influenced by other factors. More research is needed to understand the degree to which mutagenic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), benzene, and ethylene oxide, acting alone or in combination with other exposures at specific life stages, may contribute to breast cancer risk at current levels of exposure.

• Epigenetic activity: Recent studies have demonstrated that some chemicals, including BPA, while not genotoxic per se, can have important influences on gene expression that may be relevant to breast cancer risk. Relatively little is known about the importance for breast cancer risk of such epigenetic modifications by environmental chemicals. More fundamental research on the role of epigenetic modifications in breast cancer risk is needed.

• Gene–environment interactions : Although few such interactions have been identified, to some extent this may reflect the small number of discrete exposures for which relevant genes are currently identifiable. Limited evidence indicates, for example, that

genes governing acetylation efficiency may describe a susceptible subset of the population for which exposure to tobacco smoke has substantial influence on breast cancer risk. Likewise, isozymes of different enzymes involved in alcohol metabolism may affect breast cancer risks, particularly among those with high alcohol intake.

EPIDEMIOLOGIC RESEARCH

Studies of Occupational Cohorts and Other Highly Exposed Populations

Rationale: Many known human carcinogens were first identified as a result of studies carried out in occupational settings where workers were subject to chemical and physical exposures that were typically higher than those experienced by the general population. When many of the early occupational studies were carried out, relatively few women were in the workforce. Changes in the typical workplace and the presence of more women in the workforce, both in the United States and internationally, make it appropriate to revisit occupational studies as a possible means to identify some exposures that increase risk for breast cancer. These studies should account for not only comparisons of breast cancer incidence associated with various work assignments or job titles, but also the distribution of known breast cancer risk factors among workers to ensure that the analyses of exposure-related risk are not confounded by differences among types of workers in the prevalence of these other known risk factors.

Outside the workplace, other events such as industrial accidents or contamination episodes can lead to high exposures for specific population groups. Sometimes these events provide opportunities to investigate the impact of specific timing of exposures, as in the case of the survivors of the atomic bombs in Hiroshima and Nagasaki, or the population living in the vicinity of the industrial accident in Seveso, Italy, and exposed to high levels of dioxin. High-dose or long-term medical exposures have also lent themselves to study through the assembly of cohorts from records of patients treated for specific diseases or conditions.

Recommendation 3: Breast cancer researchers and research funders should pursue studies of populations with higher exposures, such as occupational cohorts, persons with event-related high exposures, or patient groups given high-dose or long-term medical treatments. These studies should include collection of information on the prevalence of known breast cancer risk factors among the study population. Support for these studies should include resources for the development of improved exposure assessment methods to quantify chemical and other

environmental exposures potentially associated with the development of breast cancer.

New Exposure Assessment Tools

Rationale: A life course perspective on breast cancer suggests that critical periods of vulnerability may exist during in utero development, in childhood, adolescence, and early adulthood, and at older ages. Exposure assessment becomes particularly challenging if the interval between critical exposure events and the point at which breast cancer can be diagnosed extends over decades.

If evidence of exposure is retained in either environmental media or the human body, measurements made long after exposure may provide an adequate basis for estimating an earlier exposure. To be able to do so requires sufficient knowledge of the patterns of persistence of chemical compounds and their metabolites, the determinants of variability in retention, and the variation in exposure levels over time. If evidence of exposure is not retained, one-time measurements are unlikely to be an adequate basis for assessing true exposure unless it is known that an individual’s exposure is consistent over long periods.

To effectively study exposures over long time periods, research protocols may need to obtain measurements of exposure at multiple time points. However, because repeated measurements can be prohibitively burdensome, it may be necessary to develop alternative strategies that rely on external indicators of exposure. For instance, if, hypothetically, 50 percent of the body burden of a chemical exposure is from consumption of liquids from plastic bottles, then questionnaires about such behavior patterns may be a more reliable basis for assessing exposure than measurements of urinary metabolites. If, additionally, persons who consume fluids from plastic bottles do so consistently over years or decades, then this approach may be reasonable for establishing past exposures as well.

Recommendation 4: Breast cancer and exposure assessment researchers and research funders should pursue research to improve methodologies for measuring, across the life course, personal exposure to and biologically effective doses of environmental factors that may alter risk for or susceptibility to breast cancer.

Such research should encompass

• improving measurements in the environment and assessing variation over time and space;

• determining routes of exposures and how they vary over time and over the life course;

• evaluating how products are used and the extent to which actual usage deviates from label instructions (e.g., home pesticide applications) as a critical component of exposure assessment, and focusing on the impact on personal exposures;

• incorporating use of advanced environmental dispersion modeling techniques with accurate emissions and air monitoring data to characterize specific population exposures;

• measuring compounds and their metabolites in biospecimens, including specimens obtained by noninvasive means;

• understanding pharmacodynamics and pharmacokinetics and how they vary by lifestage, body weight, nutrition, comorbidity, or other factors;

• developing other biomarkers of exposure through early biologic effects (DNA adducts, methylation, tissue changes, gene expression, etc.);

• using existing and yet-to-be-established human exposure biomonitoring programs (e.g., breast milk repositories) by geographic areas; and

• validating exposure questionnaires through various strategies.

RESEARCH TO ADVANCE PREVENTIVE ACTIONS

Minimizing Exposure to Ionizing Radiation

Rationale: As discussed in Chapters 3 and 6 and Appendix F , some of the strongest evidence reviewed by the committee indicated a strong causal association between breast cancer and ionizing radiation. However, population exposures to ionizing radiation in medical imaging are increasing. Chapter 6 sets forth a series of steps that can be taken by various groups and in various settings to reduce exposures to ionizing radiation and therefore reduce risks for breast and other cancers. However, many unknowns remain about the best ways to achieve these reductions. This work might include investigation of the feasibility of developing cost-effective forms of imaging that do not rely on ionizing radiation. Further research is warranted to clarify the extent of population risks, unnecessary uses of medical radiography, and the best means to maximize its benefits and minimize its harms.

Recommendation 5: The National Institutes of Health, the Food and Drug Administration, and the Agency for Healthcare Research and Quality should support comparative effectiveness research to assess

the relative benefits and harms of imaging procedures and diagnostic/follow-up algorithms in common practice. This research effort should also assess the most effective ways to fill knowledge gaps among patients, health care providers, hospitals and medical practices, industry, and regulatory authorities regarding practices to minimize exposure to ionizing radiation incurred through medical diagnostic procedures.

Developing and Validating Preventive Measures

Rationale: Some breast cancer risk factors appear to be modifiable, but it is important to determine what modifications of these environmental exposures can be most effective in reducing risk and when during the life course these changes need to occur. For example, overweight and obesity are recognized as increasing risk for postmenopausal breast cancer, but the contribution of weight loss to reducing risk is much less clear.

Recommendation 6: Breast cancer researchers and research funders should pursue prevention research in humans and animal models to develop strategies to alter modifiable risk factors, and to test the effectiveness of these strategies in reducing breast cancer risk, including timing considerations and population subgroups likely to benefit most.

Particular aspects of prevention that require attention include

• when weight loss is most likely to be beneficial in reducing risk for postmenopausal breast cancer;

• effective strategies for achieving and maintaining weight loss in different risk groups;

• effective and sustainable methods to prevent obesity;

• the feasibility of interventions in early life and development that may influence breast cancer risk in adult life such as preventing childhood obesity, increasing physical activity, and minimizing exposures to potentially harmful environmental carcinogens;

• approaches to prevention that respond to the differing breast cancer experience of various racial and ethnic groups; and

• dissemination and adoption of effective prevention strategies.

Chemoprevention—Research on Medications to Reduce Breast Cancer Risk

Rationale: Breast cancer is likely to remain a major source of morbidity for many decades to come. However, if early life events are critical in breast cancer carcinogenesis, then most women may have already had some

critical exposures by mid-life, when the incidence of breast cancer increases. Avoiding other exposures later in life, such as hormone therapy, may delay or even prevent breast cancer in some women, but it may be that further reductions in risk later in life are most efficiently achieved through pharmaceutical interventions.

Research has demonstrated that drugs that alter responses to estrogen (e.g., tamoxifen, raloxifene) or production of estrogen (e.g., aromatase inhibitors) can substantially reduce risk of ER+ breast cancer (Cummings et al., 2009; Nelson et al., 2009; Goss et al., 2011). The Food and Drug Administration (FDA) has approved use of tamoxifen and raloxifene for this purpose by women who are considered at increased risk of breast cancer and are not at increased risk for cerebrovascular disease. Other medications, such as bisphosphonates and metformin, are under study to assess their potential role in reducing the risk of either ER+ or ER– breast cancer (Cuzick et al., 2011). But relatively few eligible women have chosen to use tamoxifen and raloxifene, at least in part because they are associated with increased risk for serious adverse health effects, including endometrial cancer and stroke (Fisher et al., 2005; Vogel et al., 2010).

The desirability of drugs that can reduce breast cancer risk must be balanced against any potential dangers associated with the use of those drugs. These dangers are of particular concern for the large numbers of women who would not have developed breast cancer even without medication, as well as for the smaller numbers of women who develop breast cancer despite using them.

Additional research into medications that can reduce risk for breast cancer with minimal added risk of other serious adverse health effects should be fostered and accelerated. Studies should include sufficient follow-up, both during the study when the medications are being used and after what is anticipated to be the typical period of use, to provide an adequate basis for determining the benefits and risks that may be associated with the medication. Furthermore, because the approved drugs only reduce the risk of ER+ breast cancer, research is critically needed to find effective ways to reduce the risk of other forms of breast cancer, including triple negative breast cancer and other hard-to-treat forms of breast cancer that may have a disproportionate impact at younger ages or among African American, Asian, or Hispanic women.

Recommendation 7: Breast cancer researchers and research funders should pursue continued research into new breast cancer chemoprevention agents that have minimal risk for other adverse health effects. This work should include efforts to identify chemopreventive approaches for hormone receptor negative breast cancer.

Adequately sized primary prevention studies will be needed to allow for estimation of both benefits and risks. Research plans should also include long-term follow-up to identify any changes in risk patterns for types of breast cancer or other effects that only become evident beyond the time frame of the initial study and analyses.

TESTING TO IDENTIFY POTENTIAL BREAST CARCINOGENS

In Vivo Testing for Carcinogenicity

Rationale: Testing in animals is currently an established component of the evaluation of the carcinogenicity of chemicals in industry and commerce, but it is unclear which whole-animal test protocols are best suited for screening for possible human breast carcinogens. Human sensitivity to breast cancer has been demonstrated for exposures in utero (e.g., diethylstilbestrol [DES]), before and during puberty (e.g., radiation), and postmenopausally (e.g., combination hormone therapy). Studies in animals have also demonstrated that some exposures early in life that are not themselves carcinogenic may alter susceptibility to carcinogens encountered later in life.

But these age windows are typically not included in standard cancer bioassays such as those used in conjunction with the registration of pesticides and pharmaceuticals. The standard protocols commonly begin exposures when animals are 7 to 8 weeks of age. Thus they miss the rapid mammary ductal growth and branching during pubertal development, a period of heightened sensitivity in the rat to adverse effects from chemical exposures. These protocols also miss gestational exposures and terminate the experiments at 2 years, which omits the older age period, a time of increasing incidence of breast cancer in humans.

Interpretation of rodent bioassays for mammary carcinogenicity is complicated by certain characteristics of the animals typically used for these studies. The mouse strains appear generally insensitive to hormonally induced mammary tumors. Conversely, a commonly used rat strain is overly sensitive to the occurrence of constant estrus and early reproductive senescence. Constant estrus and early reproductive senescence can tend to increase the incidence of mammary tumors, but this phenomenon may not be relevant for humans. Thus results of bioassays of hormonally active agents are confounded when mammary tumors are increased concomitantly with constant estrus in the treated rats. With the insensitivity of mice, negative results from tests in mice are not necessarily a reliable indicator of lack of mammary carcinogenicity.

To increase the ability to detect statistically significant increases in cancer rates in the limited number of animals that can be used in toxicity and carcinogenicity testing, chemicals are typically administered at dose

equivalents that are far higher than the exposures humans would normally have. Pharmacokinetic and metabolic differences between high- and low-dose chemical exposures complicate the prediction of risks at lower doses that would be more comparable to human experience.

Finally, standard bioassay protocols for regulatory testing generally test individual chemicals. However, humans are generally exposed throughout life to a myriad of hormonally active and genotoxic chemicals. Some experimental protocols used in cancer research employ mixed exposures (e.g., in utero exposure to one agent and subsequent high-dose exposure to a genotoxic chemical during a period of rapid ductal growth). Other tests look for abnormal development of the mammary gland following in utero or early in life exposure, to identify early predisposing events. In reports from some research studies, it is difficult to assess the level of attention devoted to important design issues such as randomization, blinded assessment of endpoints, and standardization of endpoints.

Recommendation 8: The research and testing communities should pursue a concerted and collaborative effort across a range of relevant disciplines to determine optimal whole-animal bioassay protocols for detection and evaluation of chemicals that potentially increase the risk of human breast cancer.

The development of these protocols should address several issues, including the following:

• potential differences in sensitivity to carcinogenic effects and during different life stages;

• the appropriateness and limitations of the rodent strains and species used for testing, and potential alternatives;

• the frequency, magnitude, and route of dosing, and the possible need for alternative protocols that provide improved relevance for predicting human risk;

• the utility of genetically engineered mouse models, which show promise for studying breast tumor formation and progression and the effectiveness of treatments; and

• standard practices for conducting and reporting results of animal studies.

This work will probably also require targeted mechanistic and pharmacokinetic studies to assess appropriate dosing levels in test protocols to better address human exposure circumstances, including the influence of life stage, genetic variability, and multiple chemical exposures.

New Approaches to Toxicity Testing

Rationale: Most of the thousands of chemicals used in industry and commerce have not been tested for their potential to contribute to breast and other cancers. Screening all chemicals with the standardized approaches used for pharmaceuticals and pesticides is impracticable because of the time and resources (including large numbers of test animals) that would be required (NRC, 2006, 2007). Furthermore, the tests are done chemical-by-chemical, which does not address the potential consequences of exposures to mixtures of chemicals or interactions with other ongoing exposures (e.g., dietary components). The high doses used in testing also introduce uncertainty and limitations for predicting risks at lower doses that are relevant to human exposures.

Under the broad umbrella of the Tox21 (EPA, 2011) and National Toxicology Program initiatives, new toxicity testing approaches are being developed to more rapidly and accurately screen and identify the toxicity of chemicals encountered in human environmental, occupational, and product exposures. This effort relies on the elucidation of key toxicity pathways involved in human disease, and on the development of sensitive, rapid testing approaches to determine a chemical’s potential to perturb such pathways and at what concentrations. A variety of tests are being developed and considered: high-throughput in vitro screens that use cell components and engineered cells; toxicogenomic responses following cellular, tissue, and organism exposures; novel animal systems (e.g., the roundworm, Caenorhabditis elegans ); and limited, targeted testing in laboratory animals to anchor test results and understand mechanisms, new chemistries, and pharmacokinetics (Dix et al., 2007; NRC, 2007).

The new approach also calls for the use of pharmacokinetic evaluations, human biomonitoring data, and epidemiologic results to establish the predictive ability of the tests. Pharmacokinetics will be an important consideration in understanding test results, in studying uptake and distribution to target cells, and in examining the biochemical transformations that make the chemical biologically active or inactive. This aspect of the effort is currently a significant challenge in the development of high-throughput and other in vitro tests.

Because breast cancer is a major contributor to morbidity among women, these tests should address pathways that underlie the basic mechanisms of breast cancer—mutagenesis, estrogen receptor signaling, epigenetic programming, growth promotion via mitogenic cell signaling, and modulation of immune functioning—with particular attention to cell types and environments relevant to breast cancer. They should also take into account alterations at the whole-organ level, and they should be relevant to typical human exposures, which often occur at low doses and as mixtures.

Recommendation 9:

a. The research and testing communities should ensure that new testing approaches developed to serve as alternatives to long-term rodent carcinogenicity studies include components that are relevant for breast cancer.

To be relevant for breast cancer, it will be necessary to be able to assess changes in susceptibility through the life course and mechanisms characteristic of hormonally active agents. The test development should also include exploring the predictive value of in vitro and in vivo experimental testing for site-specific cancer risks for humans.

b. A research initiative should assess the persistence and consequences for mammary carcinogenicity of abnormal mammary development and related intermediate outcomes observed in some toxicological testing.

As useful predictors of increased mammary cancer risk become available, intermediate outcomes may aid in identifying chemicals that may pose increased risk of human breast cancer when exposures occur early in life.

c. Research should be conducted to improve understanding of the potential cumulative effects of multiple, small environmental exposures on risk for breast cancer and the interaction of these exposures with other factors that influence risk for breast cancer.

Improved understanding of both mixed and serial low-dose exposures is critical for the interpretation of in vivo results and is of heightened importance for understanding the results of the emerging in vitro tests. Relevant exposures may come from sources that include food, pharmaceuticals, and the general environment. It is also critical for the understanding of epidemiologic and in vivo and in vitro experimental research results on the health effects of chemical mixtures that are characteristic of human environmental exposures.

Identifying Breast Cancer Risks Associated with Hormonally Active Pharmaceutical Products

The committee sees a need to ensure that mechanisms for detection and assessment of breast cancer risks associated with use of drugs regulated by FDA are adequate. It also recognizes that enhanced methods to detect breast cancer risks represent only one specific dimension of a more general interest in strengthening FDA’s ability to ensure the safety and timely avail-

ability of prescription and over-the-counter drugs (IOM, 2007a,b) and in strengthening the science to support FDA’s regulatory work (e.g., IOM, 2011).

Menopausal hormone therapy was originally developed to control menopausal symptoms. Some health professionals advocated long-term and substantially expanded use in anticipation that it would reduce age-related health problems, including cardiovascular disease and memory disorders, even before clear evidence was in hand. Although these products are effective in reducing menopausal symptoms and osteoporotic fractures while women are taking them, evidence from the Women’s Health Initiative examining multiple health outcomes in a randomized trial design showed that use of a combination of estrogen and progestin in postmenopausal hormone preparations increases risk of breast cancer and stroke and does not provide overall benefits for cardiovascular risk or memory disorders (Writing Group for the Women’s Health Initiative Investigators, 2002). This experience is an illustration of the dangers of exposing millions of healthy women to pharmacological doses of exogenous hormones without sufficient evidence of net benefit. Decades of study have also confirmed a small excess risk of breast cancer among current users of oral contraceptives (Collaborative Group on Hormonal Factors in Breast Cancer, 1996; Marchbanks et al., 2002; Strom et al., 2004; IARC, 2011). Although the increased risk of breast cancer that is associated with use of combination hormone therapies, including oral contraceptives, declines after treatment stops, women should be aware of the full range of potential harms as well as the benefits when they decide whether to use any form of hormone therapy, including those touted as safe because they are “bioidentical” or “natural.”

New Approaches to Testing Hormonally Active Candidate Pharmaceuticals

Rationale: Given the evidence for hormonal influences on the development of breast cancer, the committee is concerned that testing required to gain marketing approval for various hormonally active pharmaceuticals that are already on the market or that are being developed does not adequately address the potential impact on the risk for breast cancer. For example, the 2-year rodent carcinogenicity studies done for Prempro, the combined estrogen–progestin product used in the Women’s Health Initiative, showed a reduction in mammary tumors in rats (Ayerst Laboratories, 2003), and premarketing human safety and efficacy studies are generally too small and too brief to detect an effect on the incidence of breast cancer. Given that some hormonal products have been found to increase the risk of breast cancer, it is important that new postmenopausal hormone preparations, including those advertised as bioidentical or natural hormones, have

an adequate evidence base to support any claims that they do not cause breast cancer. It is also important to have an adequate understanding of the implications for breast cancer risk of the hormone composition and dosing schedules of new oral contraceptives (e.g., a preparation that causes a woman to have only four menstrual periods per year).

Identifying hormonally active substances is complex, in that various models are used to measure hormonal activity and the activity levels detected for a substance may differ depending on the model and dose used. It is important to assess the effectiveness of current testing protocols for hormonally active products in providing indicators of the potential for increased risk of breast cancer, and to develop and validate new testing practices where needed.

Recommendation 10: The pharmaceutical industry and other sponsors of research on new hormonally active pharmaceutical products should support the development and validation of better preclinical screening tests that can be used before such products are brought to market to help evaluate their potential for increasing the risk of breast cancer.

A suite of in vitro and in vivo tests will likely be needed to address the different mechanisms of action that may be relevant over the life course (in utero, early infancy, pre- and postpuberty, pregnancy, and pre- and postmenopause). If such tests can be developed and validated, FDA should require submission of the results as part of the process for approving the introduction of new hormonal preparations for prescription or over-the-counter use. These tests may also prove useful in testing environmental chemicals.

Postmarketing Studies of Hormonally Active Products

Rationale: With the demonstration that use of certain hormonally active prescription drugs is associated with an increased risk of breast cancer and other adverse health effects, it is important to investigate whether use of other hormonally active drugs is also associated with increased risk. The Food and Drug Administration Amendments Act of 2007 gave FDA the authority to require postmarketing studies or clinical trials for approved drugs when adverse event reporting would not be sufficient to assess a known or suspected serious risk (FDA, 2011). Because adverse event reporting systems are generally better suited to the detection of adverse events that occur soon after use of a drug than to events such as breast cancer that take years to develop, formally conducted studies appear necessary to assess the potential breast cancer risk.

Recommendation 11: FDA should use its authority under the Food and Drug Adminisration Amendments Act of 2007 to engage the pharmaceutical industry and scientific community in postmarketing studies or clinical trials for hormonally active prescription drugs for which the potential impact on breast cancer risk has not been well characterized.

Study oversight should be designed to mitigate against bias and conflict of interest of study sponsors. Special attention should be accorded to those products that represent a substantial change in pharmacologic composition or dosage schedule from products currently on the market. The studies should be adequately powered to quantitatively explore the possible contribution of the products to breast cancer risk, as well as other risks that have been associated with these classes of drugs (e.g., cardiovascular effects).

UNDERSTANDING BREAST CANCER RISKS

Researchers, health care providers, and the public all have an incomplete picture of the components of breast cancer risk. Further work is needed to clarify the contribution of recognized risk factors to differences and changes in the incidence of breast cancer and to determine the most effective ways to convey information about breast cancer risk.

Risk Modeling

Rationale: Public health messages about ways to reduce risk should rest on strong science on the attribution of risks to various causal factors. Systematic modeling approaches are needed to refine the estimates of the proportion of breast cancer in the United States and other countries that can be attributed to known factors, especially modifiable factors. Substantial proportions of the increase in breast cancer incidence rates in the United States over the past century, and of the differences in rates of breast cancer between less developed countries and more affluent countries, are probably due in large part to differences over time and between countries in the prevalence of established breast cancer risk factors (e.g., age at menarche, age at first birth, parity, use of menopausal hormone therapy, physical activity, weight and weight change). Few reliable estimates of these temporal and international differences in risk factor prevalence exist.

Developing data on changes in the prevalence of known risk factors, along with changes in breast cancer incidence, should permit statistical modeling of the size of these proportions associated with individual risk factors and combinations of these risk factors. This information will also help in determining the magnitude of risk associated with other unidentified

factors, which may include other environmental exposures. Of particular interest are the modifiable risk factors.

Risk modeling on both the individual and population levels will benefit greatly from improved understanding of the etiology of breast cancer. As the science improves, risk models can also help guide future research investments and policy decisions for population-level interventions. A collaborative approach, such as that used by the Cancer Intervention and Surveillance Modeling Network (CISNET) consortium, may be a cost-effective way to pursue some of this work.

Recommendation 12: Breast cancer researchers and research funders should support efforts to (1) develop statistical methodology for the estimation of risk of breast cancer for given sets of risk factors and that takes the life course perspective into account, (2) determine the proportion of the total temporal and geographic differences in breast cancer rates that can be plausibly attributed to established risk factors, and (3) develop modeling tools that allow for calculation of breast cancer risk, in both absolute and relative terms, with the goal of assessing potential risk reduction strategies, at both personal and public health levels.

Communicating About Breast Cancer Risks

Rationale: Accurate and effective communication of breast cancer risks is important for individuals, the public at large, and policy makers and public health officials. Individuals need to be able to make informed choices regarding risk factors, prevention opportunities, and health care appropriate to their risk circumstances. Research indicates that women may have a poor understanding of their risk of breast cancer, with both over- and underestimates of risk observed (Lipkus et al., 2001; Apicella et al., 2009; Waters et al., 2011). A systematic review under the auspices of the Cochrane Collaborative found that both health care providers and consumers understood risks of health outcomes better when those risks were presented as frequencies rather than as probabilities (Akl et al., 2011). Both thought the risks were lower when presented as absolute risk reduction than as relative risk reduction, and both were more persuaded by relative than absolute risks in terms of potential behavioral change. To allow a fair comparison of risks and benefits, supplementing presentation of relative measures with absolute ones is useful because other disease endpoints may be more or less common than breast cancer.

From a public health policy and practice perspective, it is important to determine where risks lie and the potential for benefit and risk at a population level. Uncertainty is inherent in risk prediction, and it can be difficult or impossible to establish that an exposure is not associated with cancer

risk. However, moderate or large risks can be ruled out with reasonable confidence when studies with robust and appropriate research designs and analyses have been conducted in populations with relevant exposures. Meaningful differences in risk need to be effectively communicated to the public, health care providers, and policy makers so that limited funds can be invested in the most promising research and intervention strategies.

Recommendation 13: Breast cancer researchers and research funders should pursue research to identify the most effective ways of communicating accurate breast cancer risk information and statistics to the general public, health care professionals, and policy makers.

Because people differ in their health literacy, their numeracy (ability to understand numerical information), and in their preferred modes of learning, multiple communication strategies, modes, and messaging tactics will be needed to reach diverse communities and stakeholders. Among the topics that should receive attention in this research are

• perception and comprehension of different ways to present messages (numbers, graphs, text), modalities of communication (audio, video, print, face-to-face, and multiple modalities, etc.), as well as the content of the messages themselves;

• ways that personal experiences (e.g., family history) affect the ability to absorb messages;

• determination of the similarities and differences in how individuals from diverse racial, ethnic, educational, and occupational groups understand and respond to breast cancer risk information that is presented various ways;

• comprehension of terms such as relative risks, absolute risks, and hazards;

• ways to improve translation of research results into messages that can effectively convey the implications of the results for women in different risk categories, women from diverse racial and ethnic groups, health care providers, and public health decision makers; and

• ways to convey information about chemicals for which there is suggestive evidence of risk from experimental studies.

CONCLUDING OBSERVATIONS

Breast cancer is a leading cause of cancer morbidity among women in the United States and many other countries. Major advances have been made in understanding its biology and diversity, but more needs to be

learned about the causes of breast cancer and how to prevent it. Familiar advice about healthful lifestyles appears relevant, but it remains difficult to discern what contribution a diverse array of other environmental factors may be making. Important targets for research are the biologic significance of life stages at which environmental risk factors are encountered, what steps may counter their effects, when preventive actions can be most effective, and whether opportunities for prevention can be found for the variety of forms of breast cancer.

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IOM (Institute of Medicine). 2007a. Challenges for the FDA: The future of drug safety, workshop summary. Washington, DC: The National Academies Press.

IOM. 2007b. The future of drug safety: Promoting and protecting the health of the public. Washington, DC: The National Academies Press.

IOM. 2011. Building a national framework for the establishment of regulatory science for drug development: Workshop summary . Washington, DC: The National Academies Press.

Lipkus, I. M., W. M. Klein, and B. K. Rimer. 2001. Communicating breast cancer risks to women using different formats. Cancer Epidemiol Biomarkers Prev 10(8):895–898.

Marchbanks, P. A., J. A. McDonald, H. G. Wilson, S. G. Folger, M. G. Mandel, J. R. Daling, L. Bernstein, K. E. Malone, et al. 2002. Oral contraceptives and the risk of breast cancer. N Engl J Med 346(26):2025–2032.

Nelson, H. D., R. Fu, J. C. Griffin, P. Nygren, M. E. Smith, and L. Humphrey. 2009. Systematic review: Comparative effectiveness of medications to reduce risk for primary breast cancer. Ann Intern Med 151(10):703–715, W-226–W-735.

NRC (National Research Council). 2006. Toxicity testing for assessment of environmental agents: Interim report . Washington, DC: The National Academies Press.

NRC. 2007. Toxicity testing in the 21st century: A vision and a strategy. Washington, DC: The National Academies Press.

Strom, B. L., J. A. Berlin, A. L. Weber, S. A. Norman, L. Bernstein, R. T. Burkman, J. R. Daling, D. Deapen, et al. 2004. Absence of an effect of injectable and implantable progestin-only contraceptives on subsequent risk of breast cancer. Contraception 69(5):353–360.

Vogel, V. G., J. P. Costantino, D. L. Wickerham, W. M. Cronin, R. S. Cecchini, J. N. Atkins, T. B. Bevers, L. Fehrenbacher, et al. 2010. Update of the National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: Preventing breast cancer. Cancer Prev Res (Phila) 3(6):696–706.

Waters, E. A., W. M. Klein, R. P. Moser, M. Yu, W. R. Waldron, T. S. McNeel, and A. N. Freedman. 2011. Correlates of unrealistic risk beliefs in a nationally representative sample. J Behav Med 34(3):225–235.

Writing Group for the Women’s Health Initiative Investigators. 2002. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: Principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288(3):321–333.

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Breast cancer remains the most common invasive cancer among women. The primary patients of breast cancer are adult women who are approaching or have reached menopause; 90 percent of new cases in U.S. women in 2009 were diagnosed at age 45 or older. Growing knowledge of the complexity of breast cancer stimulated a transition in breast cancer research toward elucidating how external factors may influence the etiology of breast cancer.

Breast Cancer and the Environment reviews the current evidence on a selection of environmental risk factors for breast cancer, considers gene-environment interactions in breast cancer, and explores evidence-based actions that might reduce the risk of breast cancer. The book also recommends further integrative research into the elements of the biology of breast development and carcinogenesis, including the influence of exposure to a variety of environmental factors during potential windows of susceptibility during the full life course, potential interventions to reduce risk, and better tools for assessing the carcinogenicity of environmental factors. For a limited set of risk factors, evidence suggests that action can be taken in ways that may reduce risk for breast cancer for many women: avoiding unnecessary medical radiation throughout life, avoiding the use of some forms of postmenopausal hormone therapy, avoiding smoking, limiting alcohol consumption, increasing physical activity, and minimizing weight gain.

Breast Cancer and the Environment sets a direction and a focus for future research efforts. The book will be of special interest to medical researchers, patient advocacy groups, and public health professionals.

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Scientists create tailored drug for aggressive breast cancer

Scientists have used breast cancer cells' weakness against themselves by linking a tumour-selective antibody with a cell-killing drug to destroy hard-to-treat tumours.

The research, published today in Clinical Cancer Research by a team from King's College London and funded by Breast Cancer Now, marks a new method in cancer treatment.

The discovery is particular to triple negative breast cancer, which makes up 15% of all diagnosed breast cancer. This type of breast cancer is typically aggressive, resistant to chemotherapy, has a lower survival rate and is more common in women under 40.

Usual treatment involves surgery, chemotherapy and radiotherapy, however this type of cancer can evade the drugs and return to spread again.

The scientists conducted data analysis using over 6000 breast cancer samples to investigate the properties of breast cancer cells that are associated with aggressive and chemotherapy-resistant cancers.

They studied the cancer's biology, what is expressed in the tumour and the cell surface, and the cell's insides to understand how the cancer cells escape from cancer drugs. They established the presence of the cancer cell surface marker EGFR along with oncogenic molecules cyclin-dependent kinases (CDK), which are responsible for cell division and proliferation.

They used this knowledge against the cancer cells to link cetuximab, a tumour-selective antibody that targets the EGFR protein expressed in this type of cancer, with a CDK-blocking drug to create a tailored drug for breast cancer. Because the antibody drug conjugate specifically targets the cancer cell, it may be possible to administer a lower inhibitor dose than usual which means it's less toxic for the patient.

Lead author Professor Sophia Karagiannis, from King's College London, said: "We were on the hunt for cancer's vulnerabilities and now we've found out how we can guide our therapies to one of these. We combined these two drugs to create a tailored antibody drug conjugate for patients with this aggressive cancer. The antibody guides the toxic drug directly to the cancer cell which offers the possibility for a lower dose and less adverse side effects to be experienced.

"More work needs to be done before this therapy can reach the clinic, but we expect that this can offer new treatment options for cancers with unfavourable prognosis. Beyond this antibody drug conjugate, we hope that our concept will lead the way for new antibody drug conjugates of this type to be tailored to patient groups likely to benefit."

Lead research scientist Dr Anthony Cheung from King's College London said: ''Triple negative breast cancer represents a molecularly and clinically diverse disease. By exploiting EGFR overexpression and dysregulated cell cycle molecules in selected patient groups, the antibody drug conjugate, but not the antibody alone, could stop the cancer cell from dividing and engender cytotoxic functions specifically against the cancer cells.''

Dr Simon Vincent, director of services, support and influencing at Breast Cancer Now, which funded this research, said: "Each year, around 8,000 women in the UK are diagnosed with triple negative breast cancer, which is typically more aggressive than other breast cancers and more likely to return or spread following treatment.

"This exciting research has not only improved our understanding of the properties of aggressive breast cancer cells that are resistant to chemotherapy but has also brought us closer to developing a targeted therapy that destroys these cancer cells while minimising side effects for patients.

"While further research is needed before this treatment can be used in people, this is an exciting step forward in developing targeted therapies for triple negative breast cancer, and we look forward to seeing how these findings could lead to new and effective ways of tackling this devastating disease."

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Materials provided by King's College London . Note: Content may be edited for style and length.

Journal Reference :

• Anthony Cheung, Alicia M. Chenoweth, Annelie Johansson, Roman Laddach, Naomi Guppy, Jennifer Trendell, Benjamina Esapa, Antranik Mavousian, Blanca Navarro-Llinas, Syed Haider, Pablo Romero-Clavijo, Ricarda M. Hoffmann, Paolo Andriollo, Khondaker Miraz Rahman, Paul Jackson, Sophia Tsoka, Sheeba Irshad, Ioannis Roxanis, Anita Grigoriadis, David E. Thurston, Christopher J. Lord, Andrew N.J. Tutt, Sophia N. Karagiannis. Anti-EGFR antibody-drug conjugate carrying an inhibitor targeting CDK restricts triple-negative breast cancer growth . Clinical Cancer Research , 2024; DOI: 10.1158/1078-0432.CCR-23-3110

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Study examines metabolic reprogramming of breast cancer tumors during neoadjuvant chemotherapy

by Karolinska Institutet

In a study published in Nature Communications , scientists have made significant strides in understanding the complex interplay between the immune system and cancer metabolism in breast cancer treatment. The research offers new insights into how the immune state and cancer metabolism evolve during neoadjuvant chemotherapy (NAC).

The study utilized proteomics, genomics, transcriptomics and histopathology to analyze tumor tissue samples from breast cancer patients before, during, and after NAC.

"We used multiple 'omic' analyses to examine metabolic reprogramming within the tumor microenvironment (TME) in breast cancer and studied whether and how it evolves during neoadjuvant chemotherapy.

"We identified targetable vulnerabilities of immunometabolism with the potential to be exploited in immunotherapy combination strategies," says Theodoros Foukakis, researcher at the Department of Oncology-Pathology, Karolinska Institutet, who led the study.

The researchers discovered that changes in immune state, tumor metabolic proteins, and tumor cell gene expression are linked to treatment response. Potential drivers of immunometabolism were identified and validated in vitro, suggesting that targeting tumor metabolism could be used for immunomodulation.

Implications for future therapies

The findings underscore the potential of targeting tumor metabolism to enhance the effectiveness of immunotherapies. By understanding the dynamic relationship between tumor-intrinsic metabolic states and the tumor microenvironment , new prognostic biomarkers and therapeutic targets may emerge, paving the way for precision medicine in breast cancer treatment .

This study not only sheds light on the intricate ecosystem of breast cancer but also opens the door to novel treatment strategies that could significantly improve patient outcomes. This approach to characterizing the genomic and proteomic landscape of breast cancer represents a promising direction for future cancer research and therapy.

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Breast cancer research and treatment, breast cancer research and treatment onlinefirst articles, clinical breast exam contribution to breast cancer diagnosis in brca mutation carriers vs. average to intermediate risk women, the ki67 dilemma: investigating prognostic cut-offs and reproducibility for automated ki67 scoring in breast cancer, clinical benefit of post-trastuzumab deruxtecan treatment in patients with her 2-positive unresectable or metastatic breast cancer: a single-institution retrospective observational study, pathomic model based on histopathological features and machine learning to predict ido1 status and its association with breast cancer prognosis, measuring serum oestrogen levels in breast cancer survivors using vaginal oestrogens: a systematic review, changes in expression of breast cancer tumor biomarkers between primary tumors and corresponding metastatic sites: common patterns and relationships with survival, prognostic significance and value of further classification of lymphovascular invasion in invasive breast cancer: a retrospective observational study, assessment of efficacy and safety of dose-dense doxorubicin and cyclophosphamide (ddac) in combination with immunotherapy in early-stage triple-negative breast cancer, intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery, long-term outcomes of neoadjuvant trastuzumab emtansine + pertuzumab (t-dm1 + p) and docetaxel + carboplatin + trastuzumab + pertuzumab (tcbhp) for her2-positive primary breast cancer: results of the randomized phase 2 jbcrg20 study (neo-peaks), latest issues, breast cancer research and treatment 3/2024, breast cancer research and treatment 2/2024, breast cancer research and treatment 1/2024, breast cancer research and treatment 3/2023.

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About this journal

Breast Cancer Research and Treatment provides the surgeon, radiotherapist, medical oncologist, endocrinologist, epidemiologist, immunologist or cell biologist investigating problems in breast cancer a single forum for communication. The journal creates a "market place" for breast cancer topics which cuts across all the usual lines of disciplines, providing a site for presenting pertinent investigations, and for discussing critical questions relevant to the entire field. It seeks to develop a new focus and new perspectives for all those concerned with breast cancer. Oncology is undoubtedly the most rapidly growing subspecialty in the field of medicine, and breast cancer is one of the most serious problems of oncology. It is the leading cause of death of women in many countries, and is truly a multidisciplinary problem without geographic restrictions. Yet this very multidisciplinary aspect accounts for breast cancer literature appearing in any of the dozens of existing medical journals. None of these journals provides a focus on the unique problems of breast cancer. There has been no convenient arena for the discussion and resolution of ongoing controversies in breast cancer treatment, or for the consideration of thoughtful speculation and comments on current work. Breast Cancer Research and Treatment aims to fill this need. Each issue contains several articles dealing with original laboratory investigations and articles dealing with clinical studies. There are sections devoted to review articles, pro and con discussions of controversial subjects, meeting reports, and editorials. The panel discussions encourage experts to consider important topics.There is a section for letters to the editor, which provides for a lively exchange of opinions on previously published articles or other topics of interest. There is also an opportunity to publish the proceedings of special workshops, symposia, etc., devoted to breast cancer. All man uscripts are peer reviewed by a distinguished group of advisory editors from many countries covering all of the various disciplines of breast cancer.

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Can diet help with advanced breast cancer? All indications are positive, researchers say

University of Rochester Medical Center

Women with breast cancer who exclusively ate a whole-foods, plant-based diet lost weight, improved cholesterol levels and other key metabolic factors, had less fatigue, and perceived that they felt sharper mentally and generally more well.

The outcomes are from a small study by researchers at the University of Rochester Medical Center and Wilmot Cancer Institute . Study participants were individuals with stage 4 breast cancer, who will be on lifelong treatment.

These patients are typically excluded from dietary studies, but with their survivorship numbers growing, it presented an opportunity to make an impact both short- and long-term, said research leader Thomas M. Campbell, MD , an assistant professor of Family Medicine at URMC and an expert on using plant-based diets to improve health.

What Did the Clinical Trial Require?

The study included 30 patients who were on stable treatment and could tolerate food.

Researchers randomly divided participants into two groups: One received standard care, and the intervention group ate meals provided by the research team for eight weeks. The diet consisted solely of fruits, vegetables, whole grains (including whole grain pasta), legumes (beans), potatoes, and nuts and seeds. Participants agreed to avoid animal-based foods (meat, eggs, and dairy), and all oils and added solid fats. They also took a daily multivitamin.

Weekly assessments occurred, and the study reported 95 percent compliance.

“It’s exciting to see that these major dietary changes were feasible, well-tolerated, and acceptable to the clinical trial participants,” Campbell said.

No calorie restriction was involved. Individuals were encouraged to eat as often as they wanted of food that was “on plan.”

How A Clean, Plant-Based Diet Makes a Difference

The women started with an average BMI of 29.7, which is borderline obese. The patients in the whole-foods plant-based group lost one-two pounds per week for eight weeks, without mandated exercise.

This is significant because individuals with breast cancer often gain weight during treatment, which is risky. Why? Too much body weight increases insulin levels and hormones (estrogen and testosterone) in the blood, which can fuel cancer.

Another encouraging study result: researchers saw a reduction in blood samples of IGF-1, a growth factor that has been associated with many common cancers, as well as less inflammation.

“Although we cannot say anything yet about whether the diet can stop cancer progression from this small study, we saw preliminary results that suggest favorable changes within the body, which is very positive,” Campbell said.

To better understand the implications for cancer growth, the team is collaborating with Isaac Harris, PhD , at Wilmot, in a bench-to-clinic investigation recently funded by the American Cancer Society.

Scientists know that cancer cells rely on amino acids to survive, and the patients who followed the plant-based diet had changes in their blood levels of amino acids. Harris is studying the effect of amino acid composition on cancer cell survival, and the effect of the amino acids on various cancer drugs.

The journal Breast Cancer Research and Treatment published the primary study , which is believed to be the first of its kind. The breast cancer trial had enough significant results that two additional papers were also published from the dietary intervention: a second study in the same journal, and a third study in Frontiers in Nutrition, all in March 2024.

How to Start Making Healthy Changes

Patients should first consult with their oncologists or healthcare providers before making major dietary changes, Campbell said. This is especially important for people who take blood thinners or insulin medications.

Examples of food provided in the breast cancer clinical trial included peanut soba noodles, steel cut oatmeal, banana flax muffins, sweet potato enchiladas, and Mediterranean white bean soup.

To get started with plant-based recipes and meal ideas that are simple and affordable, Campbell recommends these websites: plantyou.com , shaneandsimple.com , and monkeyandmekitchenadventures.com . 

Several factors influence a person’s motivation to eat healthier, Campbell added, including family support, taste preferences, and cooking ability.

Whether a person makes dramatic changes overnight, or simply decides to swap out an occasional meal in favor of a plant-based recipe can be a good choice: “You only need five-10 plant-based recipes that are easy, tasty, and convenient enough that you will make them regularly to have a substantial overhaul in your diet,” he said. 

Higher food costs are often cited as a reason to shirk a plant-based diet, but in 2023 co-author Erin Campbell, MD, published a separate study showing that the diets leading to the biggest health improvements — including Dietary Approaches to Stop Hypertension or the DASH diet, which is also plant-based — were the same or cheaper in terms of food costs compared to standard American diets with ultra-processed foods and restaurant take-out.

Enhancing health with a plant-based diet is the cornerstone of Thomas Campbell’s professional career. He is founder and co-director of the UR Medicine/Highland Hospital Nutrition in Medicine Research Center , and an obesity medicine specialist. With his father, T. Colin Campbell, a renowned biochemist and professor emeritus at Cornell University, Campbell wrote The China Study , a best-selling book about nutrition and health. It is partly based on a long-term study of people in 65 counties in China that investigated rates of cancer and other serious diseases. Later, the younger Campbell wrote a companion volume, the China Study Solution , touted as a simple way to lose weight and reverse illness.

Breast Cancer Research and Treatment

10.1007/s10549-024-07266-1

Method of Research

Randomized controlled/clinical trial

Subject of Research

Article title.

A whole-food, plant-based randomized controlled trial in metastatic breast cancer: weight, cardiometabolic, and hormonal outcomes

Article Publication Date

Coi statement.

TMC: Royalties from general interest books about plant-based nutrition (Benbella Books, Penguin Random House) and income from a lifestyle medicine practice, Thomas M. Campbell, MD PLLC; EKC: Conflicts of spouse (TMC); AH: MJH Healthcare Holdings (OncLive), Mediflix (Skipta/Informa); RGM: Consultant for Fujirebio Diagnostics. Research funding from Angle plc. The rest of the authors declare no competing interests.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

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Recent Advances in Breast Cancer Research

Daniela grimm.

1 Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Aarhus C, 8000 Aarhus, Denmark; kd.ua.demoib@ggd ; Tel.: +45-21379702; Fax: +45-8612-8804

2 Department of Microgravity and Translational Regenerative Medicine, University Clinic for Plastic, Aesthetic and Hand Surgery, Otto von Guericke University, Pfälzer Str. 2, 39106 Magdeburg, Germany

3 Research Group “Magdeburger Arbeitsgemeinschaft für Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen” (MARS), Otto von Guericke University, 39106 Magdeburg, Germany

This Special Issue (SI), titled “Recent Advances in Breast Cancer Research”, covers 12 research articles and 1 communication in the field of breast cancer (BC) research. It includes publications reporting the results of cell biological, animal, and human studies.

BC is the second most common cancer in females, with skin cancer being the most prevalent cancer. BC accounts for about 30% (or 1 in 3) of new female cancers each year [ 1 ]. In recent years, BC incidence rates have increased by 0.5% per year [ 1 ]. The American Cancer Society provided estimates of BC in the United States for 2023, which are detailed in the following data: new cases of invasive BC will affect 297,790 female patients, and about 43,700 female deaths will occur as a result of BC. An estimated 2.3 million new cases of BC are diagnosed globally each year [ 2 , 3 ], and 684,996 BC-related deaths occur worldwide per year [ 3 ].

BC exhibits seven molecular BC subtypes with varying characteristic morphologies, treatment strategies, and future outcomes [ 4 , 5 ]. Patient survival depends on the tumor’s size, the specific hormone receptor profile, and tumor progression at the time of diagnosis.

The main BC treatment strategies are surgery and radiation-based treatment. Typical BC therapies include chemotherapy, immunotherapy, hormone therapy, and drug-targeted treatment strategies [ 6 ]. However, several available drugs can cause adverse effects. Another problem is the development of drug resistance in BC patients. Therefore, intensive efforts to advance cancer research, together with unified interdisciplinary attempts to identify novel strategies and drug targets, are necessary [ 6 ].

This SI covers several human studies (patients and specimens derived from human tissues) that used modern molecular biological technologies in BC research [ 7 , 8 , 9 , 10 , 11 , 12 ]. One study investigated the samples of male BC patients, focusing on the tumor microenvironment and infiltration of immune cells [ 9 ].

In addition, this SI includes seven cell culture studies [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Moreover, several papers of this SI focused on potential biomarkers [ 7 , 11 , 17 ] that predict either disease progression or a therapeutic response.

In total, this SI comprises 12 research articles [ 7 , 8 , 9 , 10 , 11 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] and one communication [ 12 ]. These 13 excellent papers were published as detailed in Table 1 .

Contributions to the Special Issue “Recent Advances in Breast Cancer Research”.

Fischer et al. [ 7 ] investigated 47 patients with metastatic BC and reported that the expression levels of circulating miR-200 family members were significantly increased during disease progression, which was predictive of circulating tumor cell (CTC) status. Both elevated CTCs and increased circulation of miR-200 content in blood plasma were associated with reduced OS and PFS. These factors are promising biomarkers for optimizing the clinical management of metastatic BC [ 7 ]. Another study investigated tumor specimens from 10 triple-negative breast cancer (TNBC) patients [ 8 ]. This BC type exhibited profound intratumoral heterogeneity. Therefore, single biopsy specimens may show only a portion of genetic aberrations of the entire tumor [ 8 ]. Genetic aberrations are involved in various cancer-specific biological processes, like tumorigenicity, induction of cell signaling, senescence, angiogenesis, migration, and response to treatment [ 8 ]. The authors concluded that medications used on the basis of the molecular profile of diagnostic biopsies may fail to remove the tumor, thus resulting in tumor recurrence. Knowledge of the molecular mechanisms that drive intratumoral heterogeneity in TNBC supports the development of new therapeutic targets.

A further article reports the results of a retrospective histological analysis of 113 cases of male BC, focusing on sTILs and programmed cell death ligand-1 (PD-L1) expression [ 9 ]. The authors demonstrated that a subset of male BC patients harbors an immunological environment characterized by an increase in sTILs with PD-L1 expression. Male BC are not only ER related and endocrine dependent, but also frequently HER2 low. These patients may benefit from immune checkpoint inhibitor therapy. In addition, frequent HER2-low status provides new options for anti-HER2 therapy in male BC patients [ 9 ].

Sokolenko et al. [ 10 ] investigated 229 BC patients. Among these patients treated via neoadjuvant chemotherapy (NACT) were 25 BRCA1 carriers and 204 women without recurrent BRCA1 alterations. NACT often results in a pathologic complete response (pCR). The authors found a lack of visible tumor cells in the post-NACT tumor bed to be a reliable indicator of the complete elimination of transformed clones [ 10 ].

Zaib et al. [ 11 ] investigated histological specimens of 97 patients with TNBC, showing that CD22 is highly expressed in this tumor type. The authors suggest that CD22 is a suitable prognostic biomarker in TNBC patients [ 11 ].

A further human study published as a communication focused on gene expression profiling of fibro-epithelial lesions (FELs) in the breast [ 12 ]. The authors studied the expression of 750 tumor-related genes in 34 FELs (5 fibroadenomas (FAs), 9 cellular FAs, 9 benign phyllodes tumors (PTs), 7 borderline PTs, and 4 malignant PTs). The overall gene expression profiles of benign PTs, cellular FAs, and FAs were similar. Borderline and benign PTs only slightly differed, whereas greater difference was detected between borderline and malignant PTs. This gene-expression-profiling-based approach supports further stratification of FELs, thus improving understanding of pathogenesis and diagnosis of BC [ 12 ].

Moreover, this SI covers several in vitro studies that investigated different human cell types [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Two such publications used data sourced from animal experiments [ 15 , 19 ].

Two studies focused on BC cells exposed to simulated microgravity conditions using an RPM, which is a device designed to create conditions of weightlessness on Earth [ 13 , 14 ]. Known microgravity-induced changes in human cancer cells include alterations in the cytoskeleton and changes in the ECM, adhesion, migration, differentiation, proliferation, survival, and apoptosis [ 20 ]. Differential expression of various genes and protein production and secretion were reported in benign and malignant cells [ 21 , 22 , 23 ].

Wise et al. [ 13 ] analyzed the supernatants of MCF-7 BC cells in order to determine extracellular vesicles (EVs). The cells had been exposed to an RPM for 5 or 10 days. A clear rise in released vesicles following RPM exposure was measured at both time points. Moreover, changes in the distribution of subpopulations related to surface protein expression were reported. Studying BC cells under microgravity led to an improved in vitro model that focused on changes in small EVs. Cancer research in space will extend our knowledge of cell communication in the tumor microenvironment and contribute to finding new therapies for BC [ 13 ].

Another microgravity experiment studied MCF-7 and MDA-MB-231 BC cells for 14 days using an RPM [ 14 ]. Both cell types grew in one of two phenotype forms: (1) adherent two-dimensional monolayers or (2) three-dimensional multicellular spheroids (MCSs). ERK1 , AKT1 , MAPK14 , EGFR , CTNNA1 , CTNNB1 , ITGB1 , COL4A5 , ACTB , and TUBB genes of MCSs were differentially regulated in both cell lines. Bioinformatic analyses revealed a positive association between the real metastatic microtumor environment and MCSs regarding EGF/MAP signaling, focal adhesion, cytoskeleton, and the ECM, depending on the BC type. This long-term investigation improved pre-existing knowledge of tumor spheroid formation and growth [ 14 ].

The third cell culture study applied different types of BC cells and reported distinct roles for the Grainyhead-like 2 ( GRHL2 ) gene in luminal and basal BC [ 15 ]. GRHL2 gene silencing performed via a mouse model revealed a decrease in primary tumor growth and reduced the number and size of lung colonies. Altogether, GRHL2 influences growth and motility. It is negatively associated with patient survival and growth suppression.

Meligova et al. [ 16 ] conducted a pharmacological study that investigated MCF-7 BC cells and their responses to antiestrogens and retinoids [ 16 ]. The authors showed that ERβ1 is a marker of responsiveness; in contrast, ERβ2 was shown to be an indicator of MCF7 cells’ resistance to antiestrogens alone and in combination with all-trans retinoic acid (ATRA) [ 16 ]. The authors concluded that the five unique hub genes ( PPARG , HIPK2 , ZFP36L1 , HMGB2 , and ALDH1A3 ) create a gene expression signature that specified the therapeutic response of ERβ1-expressing and ERα-positive BC cells to 4-hydroxytamoxifen and ATRA therapy [ 16 ].

Moreover, a BC cell study showed that NUF2 promotes BC development and, thus, serves as a new tumor stem cell indicator [ 17 ]. The findings of this study were as follows: the overexpressed NUF2 upregulated the proliferation and tumor stemness ability of BC cell lines MCF-7 and Hs-578T [ 17 ].

Piasna-Słupecka et al. [ 18 ] found that the young shoots of red beet are a richer source of total polyphenols that have anti-carcinogenic properties and exhibit higher antioxidant activity. The polyphenolic profile of the juice from young shoots of beetroot and the apoptosis mechanisms induced by subjecting the juices to in vitro gastrointestinal digestion and absorption were studied [ 18 ]. The authors demonstrated the antiproliferative and apoptotic effects of the evaluated types of beetroot juice, in particular those made of young shoots or roots that were subjected to the process of digestion and absorption in an in vitro gastrointestinal tract model, against BC cells [ 18 ].

A combined cell culture and animal study (mice) investigated CCL2 ’s role in mediating stromal interactions [ 19 ]. THP-1-derived macrophages and mammary fibroblasts were co-cultured for 72 h, which induced an M2 phenotype and a rise in CCL2 gene expression. Mice that overexpressed CCL2 in the mammary glands were analyzed for global gene expression via RNAseq, showing upregulation of cancer-associated gene pathways. The CCL2 -overexpressing mice showed enhanced macrophage infiltration and tumorigenesis [ 19 ].

Taken together, these 13 publications demonstrate novel findings in the field of BC research. The authors investigated several genes and molecular pathways, increasing our understanding of BC to aid improved diagnosis and the development of novel therapies. Studies that applied modern molecular biological approaches and bioinformatic analyses were published in this SI.

I wish to thank all of the authors who contributed to this SI. I am convinced that application of modern molecular biological technologies, together with a personalized medicine, will enable prevention and diagnosis of and new therapies for BC. Currently, many investigations are applying OMICS technologies and bioinformatics to identify new proteins that may serve as new mortality-decreasing drug targets or detect novel biomarkers that aid improved diagnosis of BC.

Acknowledgments

I would like to thank Herbert Schulz, Otto von Guericke University Magdeburg, Germany, for his help with EndNote and making important suggestions.

Abbreviations

Funding statement.

D.G. was funded by Deutsches Zentrum für Luft-und Raumfahrt (DLR), BMWK, projects 50WB2219 and 50WK2270G.

Conflicts of Interest

The author declares no conflict of interest.

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• Open access
• Published: 21 May 2024

All-trans- retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells

• Rita El Habre 1 ,
• Rita Aoun 1 ,
• Roula Tahtouh 1 &
• George Hilal 1  

BMC Cancer volume  24 , Article number:  615 ( 2024 ) Cite this article

Metrics details

Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans -Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.

MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student’s t-test or one-way ANOVA was used to analyze data from replicates.

Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.

Conclusions

To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Peer Review reports

Cancer is one of the most prominent causes of mortality. Among females, breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death [ 1 ]. BC can be categorized into the following groups: cells expressing estrogen receptor (ER+) or progesterone receptor (PR+), cells expressing human epidermal receptor 2 (HER2+), and triple-negative BC cells (TNBC) (ER−, PR−, HER2−). The treatment approaches of cells should be based on these molecular characteristics [ 2 ]. Estrogen receptor (ER) expression is the main indicator of potential responses to hormonal therapy, and approximately 70% of human BCs are hormone-dependent cells [ 3 ]. ER is produced by BC cells as two isoforms, the estrogen receptors alpha ER(α) and beta ER(β), which are the products of separate genes [ 4 ]. In fact, ER(α) overexpression is related to increased proliferation and metastasis [ 5 ], in addition to inhibited apoptosis of BC cells [ 6 ]. However, the role of ER(β) in BC remains elusive, as ER(β) may have a bi-faceted role in BC; it has both antiproliferative and pro-apoptotic activities, while a smaller number of studies suggest that ER(β) promotes the invasion and metastasis of BC [ 7 ]. ER is therefore a valuable target for BC therapy [ 8 ]. Fulvestrant is a pure antiestrogen with no known agonistic activity, contrasting tamoxifen. The steroidal agent fulvestrant prevents estradiol binding to ER(α) to a stronger extent than tamoxifen. It also has a distinct mode of action that causes severe receptor conformational changes, promoting receptor degradation and downregulation of ER protein level and depletion of ER transcriptional activation [ 9 ]. ERs provide a potential role in the regulation of long non-coding RNAs (lncRNAs), including H19 [ 10 ], and in the transcriptional regulation of human telomerase reverse transcriptase (hTERT) [ 11 ]. Telomerase is a nuclear reverse transcriptase enzyme that increases the length of telomeres; afterwards, it has recently emerged as an attractive target for cancer, as it is a crucial factor required for the tumor immortalization of cells [ 12 ]. In BC, the expression of hTERT is regulated by epigenetic, transcriptional, post-translational modification mechanisms and DNA variation [ 13 ]. Given the overexpression of hTERT in most BC cells, the detection of hTERT and its associated molecules are potential for enhancing early screening and prognostic evaluation of BC. Although still in its early stages, therapeutic approaches targeting hTERT and its regulatory molecules show promise as viable strategies for BC treatment [ 14 ]. Increased telomerase activity is observed in most malignant tumors [ 15 ]; therefore, different therapeutic approaches for telomerase, mainly specific inhibitors, have been developed to reduce tumorigenicity in BC [ 16 ]. Additionally, lncRNAs are involved in transcription, translational regulation, and cell development. They participate in the regulation of a variety of cell activities, such as cell differentiation, proliferation, invasion, and apoptosis, which may also occur through interacting with microRNAs (miRNAs) [ 17 ]. One of the lncRNAs with a crucial function in both embryonic development and tumorigenesis is the oncofetal lncRNA H19 [ 18 ]. . H19 lncRNA is highly expressed in a variety of human cancers and overexpressed in approximately 70% of BC [ 19 ]. H19 can play differential roles depending on the tissue type and developmental stage; it is an oncogene in BC and is highly expressed in cancer tissues compared with normal tissues [ 20 ]. In fact, the expression of H19 is higher in ER(α) positive cells than in ER(α) negative MDA-MB-231 cells, where overexpression of H19 is associated with increased proliferation, indicating that H19 favors BC development via different mechanisms [ 21 ]. H19 can regulate gene expression in BC at multiple levels, including epigenetic, transcriptional and posttranscriptional. The abnormal expression of H19 is closely associated with the tumorigenesis and progression of BC via different underlying molecular mechanisms. Indeed, a large number of clinical studies have suggested that H19 can serve as a potential biomarker for the diagnosis of BC [ 22 ]. Interestingly, H19 may interfere with the activity of the telomerase complex in cancer cells [ 23 ]. The impact of H19 on the metastatic abilities of human BC cells could be due to the sponging of miRNAs, such as regulating members of the let-7 miRNA family, which all play important roles in development, glucose metabolism, and cancer [ 24 ]. In addition, the overexpression of hTERT might enhance the invasiveness and metastatic ability of cancer cells through an interaction with miRNAs [ 25 ]. MiRNAs might play an important role in oncogenesis; therefore, abnormal miRNA expression can affect cell survival, tumor cell proliferation, apoptosis, metastasis, and invasion [ 26 ]. H19 interacts with miRNA pathways to regulate the expression of their targets. MicroRNA let-7 (MiR-let-7) is one of the earliest discovered miRNAs and has been reported to regulate self-renewal and tumorigenicity of BC cells; microRNA let-7a (miR-let-7a) is a new identified miRNA; it has been featured as a tumor suppressor in different human tumors by targeting genes implicated in tumors signaling pathways [ 27 ], which may open novel perspectives for clinical treatments against BC [ 28 ]. Decreased expression of miR-let-7a or impaired function of miR-let-7a could be associated with increased tumor metastasis [ 29 ]. Glycolysis is one of the signaling pathways regulated by miRNAs by targeting major transcription factors, enzymes, and oncogenes [ 30 ]. Therefore, the Warburg effect is a metabolic phenotype observed in tumor cells, in which the deregulation of miRNAs contributes to high glycolysis [ 31 ]. Pyruvate kinase (PK) and lactate dehydrogenase A (LDHA) are two crucial glycolytic enzymes that facilitate this process, conferring a growth advantage for tumor cells [ 32 ]. First, PK catalyzes the last step of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate with concomitant ATP production [ 33 ]. Among the four isoforms of pyruvate kinase (PK) in mammals, L, R, M1, and M2, tumor cells predominantly express the M2 isoform PKM2 [ 34 ]. Second, LDHA is another crucial glycolytic enzyme that converts pyruvate to lactate and oxidizes the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD + to sustain glycolysis [ 35 ]. Upregulation of pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) has been associated with tumorigenesis and reported in several malignancies, including BC cells; subsequently, these glycolytic enzymes pathways could be regulated by miRNAs, such as miR-let-7a [ 36 , 37 ]. Retinoids are a family of signaling molecules that are natural and synthetic vitamin A derivatives [ 38 ], and they are known to inhibit the growth of hormone-dependent but not hormone-independent BC cells [ 39 ]. All − trans −  Retinoic acid (ATRA), the prototype of retinoids, is involved in the regulation of multiple biological processes by activating specific genomic pathways or by influencing key signaling proteins [ 40 ]. ATRA has been widely investigated in preclinical and clinical trials to be used in the treatment of BC. It inhibits BC cell growth and prevents mammary carcinogenesis in animal models with the induction of cell apoptosis and cell-cycle arrest [ 41 ]. In addition, ATRA shows greater growth inhibition of BC cell for ER-positive than ER-negative cells, while triple negative BC cell such as MDA-MB-231 cell is poorly responsive to ATRA treatment [ 42 ]. Thereafter, ATRA could be considered a promising agent in the management of certain hematologic malignancies and solid tumors, including BC.

Subsequently, targeting H19, telomerase, and specific miRNAs, such as miR-let-7a, offers promising avenues for the treatment of BC by disrupting key processes involved in tumor progression and metastasis, which can enhance therapeutic efficacy, overcome resistance, and improve patient outcomes in a personalized manner.

The main objective of our study is to investigate a possible relationship among ATRA, H19, telomerase, and glucose metabolism in BC cells. This study focuses particularly on the modulation of the expression and activity of the enzyme PKM2 and the expression of LDHA in the glycolysis pathway, as well as the variation in the expression of miR-let-7a between MCF-7 (ER+) and MDA-MB-231 (ER-) BC cell lines.

Cell culture and treatment with reagents

The present study was performed on two BC cell lines, namely, MCF-7 (ATCC ®HTB-22™) and MDA-MB-231 (ATCC ®HTB-26™). The two cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Cells were cultured in 4.5 g/L Dulbecco’s modified Eagle’s medium (DMEM) (Sigma‒Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma‒Aldrich) and 1% penicillin/streptomycin (PS; Sigma‑Aldrich) according to the manufacturer’s protocol. All cells were cultured in a humidified atmosphere at 37 °C with 5% CO 2 .

Cells were seeded in 6‑well plates (2 × 10 5 cells/well) or in 100 mm petri dishes (1.5 × 10 6 cells/dish) for cell culture. At 80% confluence, cells were treated for 48 h with the following inhibitors: 5 µM all − trans −  Retinoic acid (ATRA) inhibitor (Sigma‒Aldrich) and/or 100 nM fulvestrant (Sigma‒Aldrich). The negative control corresponded to non‑treated cells maintained in the same conditions as treated cells.

Cytotoxicity assay

The cytotoxicity of ATRA and/or fulvestrant was evaluated using a WST-8 cell counting kit according to the manufacturer’s instructions (Sigma‒Aldrich, Germany). Briefly, 10 4 cells per well were seeded in 96-well plates and incubated for 48 h in DMEM, 4.5 g/L (10% FBS, 1% PS). The medium was then removed and replaced with ATRA (1, 5, 10, 20 µM) and/or fulvestrant (0.1, 0.5, 1, 2, 5, 10, 20 µM). After 48 h, 10 µl of tetrazolium salt was added to each well. This assay uses tetrazolium salt, which is converted to the fluorescent product formazan by metabolically active cells. Fluorescence was monitored at 450 nm by a Multiskan Go ELISA reader.

RNA extraction and quantitative reverse transcription polymerase chain reaction (RT‒qPCR)

Total cellular RNA from three independent experiments (biological replicates) was extracted using NucleoZol (MACHEREY–NAGEL; Bethlehem, PA, USA) reagent according to the manufacturer’s instructions. The RNA concentration and A260/A280 ratio were determined using a NanoDropTM 1000 spectrophotometer (Thermo Scientific). A total of 1000 ng of total RNA was reverse transcribed in a 20 µL total volume using the iScript cDNA Synthesis Kit (Bio-Rad, USA). The relative expression of the genes mentioned below was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Complementary DNA (cDNA) was amplified using a SYBR Green PCR Kit with a CFX Connect Real-Time PCR Detection System (Bio-Rad). The RNA levels were quantified using the 2 ‑ΔΔCq method, and the treated samples were compared with their control. Primer sequences for GAPDH, H19, hTERT, PKM2, LDHA, ER (α) and ER (β) amplification are shown in Table  1 below.

Quantitative RT‒PCR for detection of miRNAs

Total RNA was extracted as previously described. The expression of miR-let-7a was quantified by RT‒qPCR. Single strand RNA was first polyadenylated by poly(A) polymerase before reverse transcription into cDNA using qScript RT with a proprietary adapter oligo(dT) primer using the “miRCUY® LNA® RT Kit” (Qiagen) following the manufacturer’s protocol. The amplification step was carried out using the CFX Connect Real-Time PCR Detection System (Bio-Rad). U6 served as an internal control. The miRNA-specific primers are listed in Table  2 below.

ER(α) and ER(β) expression constructs

The ER(α) expression plasmid pEGFP-C1-ER alpha, ER(β) expression plasmid pCDNA3.1-nv5-ER beta and scramble vector Pbabe-neo were purchased from Addgene (Addgene plasmids #28,230, #22,770, and #1767, respectively). After being transformed using the heat shock technique, the Escherichia coli DH5α strain was spread using a sterile loop onto a prepared lysogeny broth (LB) agar plate containing kanamycin or ampicillin respectively, to isolate individual colonies of bacteria carrying the plasmids cited above and incubated overnight at 37 \(^ \circ {\rm{C}}\) C. After 24 h, one colony was transferred into LB media with the corresponding antibiotic and incubated at 37 \(^ \circ {\rm{C}}\) C for while shaking. After incubation, bacterial growth was characterized by a cloudy haze in the media. The plasmids were extracted and purified from the transformed and proliferated Escherichia coli DH5α using the GenElute HP Plasmid Maxiprep kit (Sigma‒Aldrich). MDA-MB-231 cells were then transfected using the traditional protocol with Attractene Transfection Reagent (Qiagen) following the manufacturer’s instructions. After 48 h of transfection, RNA and proteins were extracted as previously described.

Western blot analysis

MCF-7 and MDA-MB 231 cells treated and/or transfected were harvested in PBS and lysed in 1% Triton lysis buffer supplemented in the presence of sodium orthovanadate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride (PMSF), all purchased from Sigma‒Aldrich, USA. The supernatant containing the protein was collected and concentrated by ultracentrifugation, and the protein concentration was measured using the BCA protein assay kit (Bio-Rad, USA). To evaluate the expression of PKM2 (58 kDa), ER(α) (59 kDa), ER(β) (59 kDa), and LDHA (38 kDa), proteins were separated on Tris-Glycine gradient polyacrylamide gels and transferred onto Immuno-Blot PVDF membranes (Bio-Rad). Membranes were incubated in blocking buffer, probed with antibodies specific for PKM2 (Cell Signaling, 1:1000 dilution), β-actin (Cell Signaling, 1:1000 dilution), LDHA (Cell Signaling, 1:1000 dilution), ER(α) (Cell Signaling, 1:1000 dilution), and ER(β) (Sigma, 1:500 dilution) at 4 °C overnight, washed, and then incubated with the appropriate peroxidase-conjugated secondary antibodies (Cell Signaling, 1:2000 dilution) at room temperature. Antibody binding was detected by incubation with enhanced chemiluminescence (ECL) reagents (Abcam) and exposure of the membrane in an ECL machine. The expression of the desired protein was compared to that of β-actin, which served as an internal control.

SiRNA analysis

Small interfering RNAs (siRNAs) against H19 and hTERT, a positive control (all star cell death), and a negative control siRNA (all stars negative control) were purchased from Qiagen (Qiagen Inc., Valencia, CA, USA); transfection was performed according to the manufacturer’s recommendations. Briefly, 20 nM siRNA diluted with serum-free medium and Hi-perfect transfection reagent (Qiagen Inc.) were added to the wells (24-well plates and 6-well plates were used), with or without ATRA, and incubated at room temperature. After 15 min, MCF-7 cells were seeded and incubated for 72 h. The knockdown efficacy of H19 and hTERT, in addition to studying the effect of gene expression inhibition on ER(α), ER(β), H19, telomerase, miR-let-7a, PKM2, and LDHA in combination or not with ATRA treatment, was confirmed by RT‑qPCR.

Assay of telomerase activity

Samples for telomerase activity assays were extracted from cells for use following standard methods. First, the cells were trypsinized, washed with PBS, centrifuged and resuspended in lysis buffer 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate, 10 mM Tris pH 8.0 (Sigma‒Aldrich, USA). Second, the lysate was incubated on ice for 30 min and centrifuged at 13000 rpm at 4°C for 20 min. A BCA protein assay (Bio-Rad) was used to determine the protein concentration in the extracts. Heat-inactivated samples were used as negative controls. A real-time quantitative telomeric repeat amplification protocol (qTRAP) assay was performed. Briefly, reactions were carried out using a SYBR Green PCR Kit (Bio-Rad, USA). 1 µL cell lysate, telomerase primer TS (5’-AATCCGTCGAGCAGAGTT-3’) and reverse primer ACX.

(5ʹ-GCGCGGCTTACCCTTACCCTTACCCTAACC-3ʹ) were used. Samples were amplified for 40 cycles. Data analysis was performed with a CFX Connect Real-Time PCR Detection System (Bio-Rad) that incorporates the real-time PCR effectiveness that was calculated by successive dilutions of the most active sample.

PKM2 activity assay

For activity, cells were lysed in buffer as described previously. Activity was measured using a NADH/lactate dehydrogenase (LDH) coupled assay. The decrease in OD at 340 nm due to the oxidation of NADH was monitored using a spectrophotometer. The reaction was started by adding 50 µg cell lysate to a mixture containing 50 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 1.25 mM ADP, 0.5 mM PEP, 0.28 mM NADH and 8 units of LDH. Specific PKM2 activity was calculated per mg of cell lysate.

Statistical analysis

One-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons test or an unpaired, two-tailed Student’s t-test was performed to analyze data from biological replicates using GraphPad Prism software. All experiments were repeated independently at least three times. A p value of ˂0.05 was considered statistically significant.

ATRA and fulvestrant cytotoxicity on MCF-7 and MDA-MB-231 cells

To indicate the concentrations that should be used for each inhibitor, a cytotoxicity test was performed, and the concentrations were chosen according to the highest concentration that has no toxic effect and therefore no effect on the viability of each cell line. After choosing and settling the appropriate concentration of ATRA (5 µM), the cytotoxicity of the ATRA and fulvestrant (various concentrations) combination toward the cells was evaluated as well. The cytotoxicity assay demonstrated that ATRA (Fig.  1 . a-b) or fulvestrant (Fig.  1 . c-d), as well as the combination of the two, was not cytotoxic toward MCF-7 (Fig.  1 . e) and MDA-MB-231 (Fig.  1 . f) cell lines at any concentration tested compared to the control after 48 h of treatment.

Effect of ATRA and/or fulvestrant on cell viability. MCF-7 and MDA-MB-231 cells were seeded at a density of 10 4 cells per well in 96-well plates. Cell viability was calculated after 48 h of incubation and treatment and expressed as a percentage of control cells. This assay uses tetrazolium salt, which is converted to the fluorescent product formazan by metabolically active cells. Fluorescence was monitored at 450 nm by a Multiskan Go ELISA reader. The ATRA cytotoxicity effect on the cell lines was determined using different concentrations (1, 5, 10, and 20 µM) ( a, b ), and the fulvestrant cytotoxicity effect on the cell lines was determined using different concentrations (0.1, 0.5, 1, 2, 5, 10, and 20 µM) ( c, d ). After choosing and settling the appropriate concentration of ATRA (5 µM), the cytotoxicity of the ATRA and fulvestrant (various concentrations) combination toward the cells was evaluated as well (e, f) . Five replicates ( n  = 5) of each experimental condition were performed. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, as indicated

Effect of ATRA treatment on ER isoforms, H19 and hTERT RNA expression in MCF-7 and MDA-MB-231 cells

Considering ATRA as a promising agent for BC cell treatment that could be involved in the regulation of multiple biological processes by influencing specific genomic pathways and with the aim of differentiating the effects of ATRA on MCF-7 and MDA-MB-231 cells, both cell lines were treated with 5 µM ATRA for 48 h in 4.5 g/L high glucose DMEM. RNA expression of ER(α) and ER(β) was evaluated in ER-positive cells; however, H19 and hTERT were evaluated in both cell lines. RNA expression was quantified using the primer sequences mentioned in Table  1 . As shown in Fig.  2 . We found that in MCF-7 cells, ATRA significantly decreased ER(α) (38.3%) ( p  = 0.0088) (Fig.  2 . a), H19 (17%) ( p  = 0.0022) (Fig.  2 . c), and hTERT (43%) ( p  < 0.0001) (Fig.  2 . d), whereas ATRA significantly increased ER(β) (94.7%) ( p  = 0.0017) (Fig.  2 . b). However, in MDA-MB-231 cells, ATRA significantly increased H19 (19.5%) ( p  = 0.03) (Fig.  2 . c) and hTERT (37.7%) ( p  < 0.0001) expression (Fig.  2 . d).

Effect of ATRA on the estrogen receptor isoforms H19 and hTERT in MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were seeded at a density of 1.5 × 10 6 cells per dish and treated with 5 µM ATRA for 48 h. RNA was extracted from both cell lines, and the quantification of the expression of ER(α) ( a ), ER(β) ( b ), H19 ( c ), and hTERT ( d ) was performed by qPCR using SYBR green mix. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Effect of ATRA and/or fulvestrant on ER(α), ER(β), H19, and telomerase in MCF-7 cells, as well as on H19 and telomerase in MDA-MB-231 cells

Knowing that ER is a valuable target for BC therapy, to evaluate the effect of ATRA on MCF-7 ER-positive cell lines, which act as hormone-dependent cells, and to highlight the importance of ER isoforms in modulating H19 and hTERT expression, cells were treated with 5 µM ATRA inhibitor and/or with 100 nM fulvestrant for 48 h. RNA expression of ER(α), ER(β), H19, and hTERT was evaluated by Q-PCR. ER(α) and ER(β) proteins were quantified using Western blot analysis, and telomerase activity was evaluated with a qTRAP assay. The results shown in Fig.  3 reveal the variation in MCF-7 cells treated. ER(α) mRNA (Fig.  3 . a) and protein expression (Fig.  3 . c-d) decreased significantly after all the treatments; mainly, the inhibitor combination showed a very strong significant decrease in ER(α) (84.75%) ( p  < 0.0001). Our results showed a significant increase in ER(β) mRNA (Fig.  3 . b) and protein (Fig.  3 . e-f) expression; note that the treatment combination significantly increased ER(β) mRNA (160%) ( p  = 0.0001) and protein expression (164.4%) ( p  < 0.05). hTERT, which acts as a crucial factor required for the tumor immortalization of cells, was subjected to treatment combination and showed a significant decrease in mRNA and activity, with expression decreasing significantly at the mRNA level (Fig.  3 . g) by 81.43% ( p  < 0.0001) and activity (Fig.  3 . h) by 58.83% ( p  = 0.002). H19, an oncogene in BC development, showed a highly significant decrease after treatment combination (Fig.  3 . i) (70.83%) ( p  < 0.0001). However, to differentiate the variations mentioned above in TNBC cells, MDA-MB-231 cells were subjected to the same treatment conditions as MCF-7 cells. In MDA-MB-231 treated cells, we found that hTERT mRNA expression increased significantly, mostly after inhibitor combination (Fig.  4 . a) (61.3%) ( p  = 0.001), while Fig.  4 . b shows no significant variation in telomerase activity after treatment. Although, H19 RNA expression increased significantly mainly after the combination treatment (Fig.  4 . c) (53.75%) ( p  < 0.0001).

ATRA and/or fulvestrant modulates ER(α), ER(β), H19, and telomerase in MCF-7 cells. MCF-7 cells were seeded at a density of 1.5 × 10 6 cells per dish and treated with 5 µM ATRA and/or 100 nM fulvestrant for 48 h. RNA was extracted from cells, and the quantification of the expression of ER(α) ( a ), ER(β) ( b ), hTERT ( g ), and H19 (i) was performed by RT‒qPCR using SYBR green mix. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. After treatment, the cells were lysed, and 50 µg of extracted proteins was analyzed using Western blotting with β-actin as an internal control for MCF-7 cells. Representative Western blot showing the change in protein levels of ER(α) ( c ) and ER(β)( e ) compared to β-actin. The bar graph shows the quantified protein levels ( d-f ). Three independent experiments were carried out, and a representative image is shown. Full-length blots are presented in Supplementary Figs.  1 and 2 . After treatment, telomerase activity was detected using a qTRAP assay ( h , i ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Effect of ATRA and/or fulvestrant on H19 and telomerase in MDA-MB-231 cells

MDA-MB-231 cells were seeded at a density of 1.5 × 10 6 cells per dish and treated with 5 µM ATRA and/or 100 nM fulvestrant for 48 h. RNA was extracted from cells, and the quantification of the expression of hTERT (a) and H19 (c) was performed by RT‒qPCR using SYBR green mix. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. After treatment, telomerase activity was detected using a qTRAP assay (b) . Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated.

Glycolysis modulation after ATRA and/or fulvestrant treatment of MCF-7 and MDA-MB-231 cells

Considering that upregulated PKM2 and LDHA, which are two crucial glycolytic enzymes, facilitate the growth advantage of tumor cells, and to determine the effect of ATRA on glycolysis in ER-positive and triple-negative cells, we examined the variation in the mRNA and protein expression of LDHA and PKM2, as well as PKM2 activity, in treated MCF-7 and MDA-MB-231 cells. In MCF-7 cells, we found that LDHA mRNA expression (Fig.  5 . a) decreased significantly after treatment, mostly after the combination treatment (56.37%) ( p  < 0.0001); moreover, LDHA protein expression (Fig.  5 . c-d) decreased significantly upon ATRA treatment without or with fulvestrant, by 63.53% ( p  < 0.01) and 40.63% ( p  < 0.05), respectively. Then, PKM2 variation after treatments was evaluated, and mRNA, protein, and activity expression decreased significantly. Hence, after inhibitor combination, mRNA expression (Fig.  5 . b) decreased by 48.5% ( p  = 0.0002), protein expression (Fig.  5 . e-f) decreased by 23% ( p  < 0.05), and PKM2 activity (Fig.  5 . g) decreased by 32.81% ( p  = 0.003). However, MDA-MB-231 treated cells showed a significant increase in LDHA mRNA expression (Fig.  6 . a), mostly after inhibitor combination (57.67%) ( p  = 0.0018), while there was no significant variation in LDHA protein expression (Fig.  6 . c-d) after treatment. In addition, PKM2 mRNA expression (Fig.  6 . b) increased significantly with ATRA or fulvestrant treatment, with no significant increase after the combination treatment. Furthermore, following the same conditions, PKM2 protein expression (Fig.  6 . e-f) and activity (Fig.  6 . g) indicated no significant modulation after treatments.

ATRA and/or fulvestrant modulates glycolytic enzymes in MCF-7 cells. MCF-7 cells were seeded at a density of 1.5 × 10 6 cells per dish and treated with 5 µM ATRA and/or 100 nM fulvestrant for 48 h. Effectively, mRNA was extracted from cells, and the quantification of the expression of LDHA ( a ) and PKM2 ( b ) was performed by RT‒qPCR using SYBR green mix. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. After treatment, cells were lysed, and extracted proteins were analyzed using Western blotting with β-actin as an internal control for MCF-7 cells. Representative Western blot showing the change in protein levels of LDHA ( c ) and PKM2 ( e ) compared to the loading control. Full-length blots are presented in Supplementary Figs.  3 and 4 . The quantitative analysis of the intensity of the bands is shown in the bar graph ( d-f ). After treatment, PKM2 activity was examined using an NADH/lactate dehydrogenase (LDH) coupled assay ( g ), where a decrease in optical density (OD) at 340 nm indicates a decrease in cell PKM2 activity. Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Effect of ATRA and/or fulvestrant on glycolytic enzymes in MDA-MB-231 cells. MDA-MB-231 cells were seeded at a density of 1.5 × 10 6 cells per dish and treated with 5 µM ATRA and/or 100 nM fulvestrant for 48 h. First, mRNA was quantified, and the expression of LDHA ( a ) and PKM2 ( b ) was detected by RT‒qPCR. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. After treatment, cells were lysed, and extracted proteins were analyzed using Western blotting with β-actin as an internal control for MDA-MB-231 cells. Then, a representative Western blot shows the change in protein levels of LDHA ( c ) and PKM2 ( e ) compared to the loading control. Full-length blots are presented in Supplementary Figs.  5 and 6 . The bar graph shows quantified protein levels ( d-f ). Thus, PKM2 activity was examined using an NADH/lactate dehydrogenase (LDH) coupled assay ( g ), in which the variation in OD at 340 nm indicates a variation in cell PKM2 activity. Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

ATRA combined with downregulated H19 or hTERT regulates H19 and telomerase expression in MCF-7 cells

For a better understanding of ATRA importance and mechanism of action on BC cell development and to investigate the involvement of H19 and hTERT in these processes in MCF-7 cells, mainly after showing reduced H19 and hTERT expression caused by ATRA treatment, MCF-7 cells were treated with ATRA alone or coupled with H19 siRNA transfection first or with hTERT siRNA transfection second. Our data indicate a highly significant decrease in hTERT mRNA expression (Fig.  7 . a), activity (Fig.  7 . b), and H19 expression (Fig.  7 . c) after cell transfection and treatment under all conditions, mainly after ATRA combination with siH19 or sihTERT. ATRA treatment combined with downregulated H19 significantly decreased hTERT mRNA expression by 64.8% ( p  < 0.0001), telomerase activity by 66% ( p  < 0.001), and H19 by 81.3% ( p  < 0.0001). Moreover, ATRA treatment combined with downregulated hTERT significantly decreased hTERT mRNA expression by 76.3% ( p  < 0.0001), telomerase activity by 71.6% ( p  < 0.0001), and H19 by 53.3% ( p  < 0.0001). In summary, the RNA expression pattern and telomerase activity obtained by gene silencing were similar to those obtained after treatment with the inhibitors.

ATRA combined with downregulated H19 or hTERT regulates telomerase and H19 expression in MCF-7 cells. MCF-7 cells were seeded at a density of 2 × 10 6 cells per dish. At 80% confluence, cells were treated with 5 µM of ATRA and/or transfected with H19 siRNA or hTERT siRNA using Hi-perfect transfection reagent. Then, cells were harvested for RNA extraction, followed by quantification performed by RT‒qPCR of the expression of hTERT ( a ) and H19 ( c ). Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. After treatment, telomerase activity was compared to that of the control and detected using a qTRAP assay ( b ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

The implication of H19 and hTERT, in addition to ATRA, in the regulation of glycolytic enzymes in MCF-7 cells

As mentioned above, ATRA downregulates glycolytic enzymes levels in MCF-7 cells. In the interest to determine whether H19 and hTERT are implicated in LDHA and PKM2 direct regulation, MCF-7 cells were treated with ATRA alone or coupled with H19 siRNA transfection first or with hTERT siRNA transfection second to determine whether the targeted inhibition of these genes could modulate glycolysis. Our results show a significant decrease in the mRNA and protein expression of LDHA and PKM2, as well as in PKM2 activity. In fact, as provoked by ATRA, LDHA mRNA expression (Fig.  8 . a) decreased significantly after siH19 (29%) ( p  < 0.05) or sihTERT (31.8%) ( p  < 0.01) transfection; likewise, for ATRA combined with downregulated H19 or hTERT. A similar expression pattern was observed at the protein LDHA levels (Fig.  8 . c-d). Similar to LDHA, PKM2 mRNA (Fig.  8 . b) and protein (Fig.  8 . e-f) expression, as well as PKM2 activity (Fig.  8 . g), showed a highly significant decrease, nearly 35%, after treatment. Based on these results, a correlation among H19, hTERT, and glycolytic enzymes could be assessed.

ATRA reduces glycolytic enzymes expression through H19 and hTERT in MCF-7 cells. After showing reduced H19 and hTERT expression caused by ATRA treatment in MCF-7 cells, 2 × 10 6 cells were seeded and treated with 5 µM ATRA alone or coupled with siRNA transfection of H19 (20 nM) first or with hTERT siRNA (20 nM) second. Hence, cells were harvested for RNA extraction, followed by RT‒qPCR quantification of LDHA ( a ) and PKM2 ( b ) expression. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. Next, extracted proteins from transfected and/or treated cells were analyzed using Western blotting with β-actin as an internal control for MCF-7 cells. Representative Western blot showing the change in protein levels of LDHA ( c ) and PKM2 ( e ) compared to the loading control. Full-length blots are presented in Supplementary Figs.  7 and 8 . The quantitative analysis of the intensity of the bands is shown in the bar graph ( d-f ). Furthermore, PKM2 activity was examined using an NADH/lactate dehydrogenase (LDH) coupled assay ( g ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Overexpression of ER(α) or ER(β) modulates H19 and telomerase in MDA-MB-231 cells

Considering that ER isoforms alpha and beta are involved in BC progression and glycolysis, to examine whether this biological process could occur through H19 and hTERT and with the aim of exploring whether ATRA could regulate this signaling pathway, we transfected treated or untreated MDA-MB-231 cells with ER(α) or ER(β) expression plasmids for 48 h. The outcome of ATRA treatment, ER(α) plasmid DNA transfection (ER(α)/pcDNA), ER(β) plasmid DNA transfection (ER(β)/pcDNA), and the combination of ATRA and each plasmid DNA transfection induced a significant increase in hTERT mRNA expression (Fig.  9 . a), almost 45% ( p  < 0.05). Hence, telomerase activity (Fig.  9 . b) increased significantly after ER(α) plasmid transfection (47%) ( p  < 0.05) or ER(β) plasmid transfection (41%) ( p  < 0.05) and after ATRA combination with ER(β) plasmid transfection (43%) ( p  < 0.05). However, telomerase activity showed no significant variation following ATRA treatment and after ATRA combined with ER(α) plasmid transfection. In addition, H19 (Fig.  9 . c) was upregulated significantly by almost 55% following the aforementioned treatments, except for ER(β) plasmid transfection, in which H19 expression variation was nonsignificant.

Estrogen receptor alpha or beta overexpression modulates H19 and telomerase in MDA-MB-231 cells. To clarify ER(α) and ER(β) function in regulating H19 and hTERT, MDA-MB-231 cells were transfected with ER(α) or ER(β) expression plasmids. MDA-MB-231 cells were seeded at a density of 1.5 × 10 6 cells per dish, treated with 5 µM ATRA, and/or transfected with plasmid coding estrogen receptor alpha (ER(α)/pcDNA) or beta (ER(β)/pcDNA) expression for 48 h using the Attractene Transfection Reagent protocol. Then, RNA was extracted from cells and quantified for the expression of hTERT ( a ) and H19 ( c ). Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. Following transfection and treatments, telomerase activity was detected using a qTRAP assay ( b ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Effect of upregulated ER(α) or ER(β) in modulating LDHA and PKM2 enzymes in MDA-MB-231 cells

By taking into account that ER(α) overexpression is related to increased proliferation and metastasis in BC, while ER(β) function remains elusive, it may have a bi-faceted role in BC, based on our results showing that H19 and telomerase levels increase after ER(α) or ER(β) overexpression and considering that the latter can be involved in glycolysis modulation, ATRA treated or untreated MDA-MB-231 cells, triple-negative cells, were transfected with ER(α) or ER(β) expression plasmids for 48 h. Subsequently, LDHA and PKM2 regulation after transfection and treatment was detected. Similar to the effect caused by ATRA, LDHA mRNA expression (Fig.  10 . a) increased significantly following ER(α) plasmid transfection (53%) ( p  < 0.05), ER(β) plasmid transfection (55%) ( p  < 0.05), and after ATRA combined with ER(β) plasmid transfection (69%) ( p  < 0.01); however, no significant variation was observed after ATRA combination with ER(α) plasmid transfection. Thus, LDHA protein (Fig.  10 . c-d) expression increased significantly only after ER(α) or ER(β) transfection by almost 108% ( p  < 0.05). Interestingly, as presented for ATRA, PKM2 mRNA (Fig.  10 . b) presented a highly significant increase after ER(α) or ER(β) transfection by 49% and 53%, respectively ( p  < 0.01). Only after ER(α) or ER(β) transfection, PKM2 protein (Fig.  10 . e-g) expression and activity (Fig.  10 . f), increased significantly, while nonsignificant variation was indicated after the other treatments and combinations.

Upregulated estrogen receptor alpha or beta modulates LDHA and PKM2 in MDA-MB-231 cells. To find a direct relationship between estrogen receptors and glycolytic enzymes regulation, we transfected MDA-MB-231 cells with ER(α)/pcDNA or ER(β)/pcDNA plasmid and then evaluated LDHA and PKM2 expression variation. MDA-MB-231 cells were seeded, treated with 5 µM ATRA, and/or transfected for 48 h using the Attractene Transfection Reagent protocol. Thus, mRNA was quantified, and the expression of LDHA ( a ) and PKM2 ( b ) was detected by qPCR. Data are the mean ± SD from three independent experiments with differences calculated using the delta-delta Ct method relative to the expression of the reference gene GAPDH. Extracted proteins were analyzed using Western blotting with β-actin as an internal control for MDA-MB-231 cells. Representative Western blot showing the change in protein levels of LDHA ( c ) and PKM2 ( e ) compared to the loading control. Full-length blots are presented in Supplementary Figs.  9 and 10 . The quantitative analysis of the intensity of the bands is shown in the bar graph ( d-g. ) Afterwards, PKM2 activity was examined using an NADH/lactate dehydrogenase (LDH) coupled assay ( f ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

ATRA regulates miR-let-7a through H19 and hTERT in MCF-7 and MDA-MB-231 cells

Given that miR-let-7a acts as a tumor suppressor by targeting some genes to affect signaling pathways by binding to the mRNA sequences, resulting in translational repression and mRNA degradation, we investigated miR-let-7a modulation in MCF-7 and MDA-MB-231 following ATRA treatment and transfections aforementioned for the two cell lines. MiR-let-7a RNA expression was quantified using the primer sequences mentioned in Table  2 . Interestingly, in MCF-7 cells, miR-let-7a indicated a highly significant increase after ATRA and/or fulvestrant (Fig.  11 . a); however, miR-let-7a decreased strongly and significantly following the same treatments in MDA-MB-231 cells (Fig.  11 . b). Moreover, ATRA-treated or untreated MCF-7 cells transfected with siH19 or sihTERT showed a highly significant increase in miR-let-7a, mostly after the ATRA and siH19 combination (375%) ( p  < 0.001), as well as after the ATRA and sihTERT combination (584%) ( p  < 0.0001) (Fig.  11 . c). However, ATRA-treated or untreated MDA-MB-231 cells transfected with ER(α) or ER(β) plasmid showed a highly significant decrease in miR-let-7a, almost 50%, under all conditions executed (Fig.  11 . d).

ATRA regulates miR-let-7a via H19 and hTERT in MCF-7 and MDA-MB-231 cells. To examine miR-let-7a implication in glycolytic enzymes regulation through H19 and hTERT, MCF-7 and MDA-MB-231 cells were seeded and subjected to the same conditions of treatments and transfections aforementioned for the two cell lines. Thus, miR-let-7a was quantified by Q-PCR, and its modulation was evaluated in MCF-7 cells treated with ATRA and/or fulvestrant ( a ) and in MDA-MB-231 cells treated with ATRA and/or fulvestrant ( b ). Next, miR-let-7a regulation was evaluated after ATRA-treated or untreated MCF-7 cells were transfected with siH19 or sihTERT ( c ) and after ATRA-treated or untreated MDA-MB-231 cells were transfected with ER(α) or ER(β) plasmid expression ( d ). Each value represents the mean of three assays. Data are expressed as the mean ± SD of triplicates. ns; p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 as indicated

Having a better characterization of the known and newly discovered potential markers would be of importance for the care and treatment of breast cancer [ 43 ]. LncRNAs particularly H19 [ 44 , 45 ] and hTERT [ 16 ] are important biomarkers in breast cancer based on their main roles in glycolysis [ 46 , 47 ]. Indeed, retinoic acid has inhibitory effects on proliferation and cancer cell migration by targeting cell proliferation proteins, such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) [ 41 ]. Consequently, the ultimate aim of our study is to investigate a possible relationship between H19, hTERT, and glycolytic metabolism that could be modulated by ATRA in breast cancer. In particular, we focused on the modulation of the expression and activity of PKM2 and the expression of LDHA in the glycolysis pathway, as well as, on the expression of miR-let-7a in MCF-7 and MDA-MB-231 breast cancer cell lines. As reported by Prat et al. [ 48 ], the effect of ATRA on breast cancer may be linked to the heterogeneity of this tumor; thus, the identification of specific markers defining breast cancer subtypes with particular sensitivity to ATRA represents a priority in our study. We first assessed the effect of ATRA on ER(α) and ER(β) mRNA expression in MCF-7 cells; however, H19 and hTERT were evaluated in both cell lines. Our experiments demonstrated that ER(α), H19, and hTERT RNA expression was reduced in MCF-7 cells compared to control cells, whereas ER(β) expression was increased, while in MDA-MB-231 cells, H19 and hTERT expression was increased. These differences confirm the fact that retinoids are known to affect hormone-dependent breast cancer cells only [ 49 ]. Recent studies have revealed that ATRA in combination with anti-tumor agents holds promise to enhance and improve anti-carcinogenic therapies [ 50 ]. In fact, combining ATRA with ER inhibitors such as tamoxifen inhibits growth and induces apoptosis of breast cancer cells [ 51 ]. Using the previous conditions, the combinatory effect of ATRA and fulvestrant on MCF-7 and MDA-MB-231 cells was evaluated. Regarding the expression of genes, the results were discordant between the two cell types. We found that in MCF-7 cells, following ATRA and/or fulvestrant treatment, the expression of ER(α), H19, hTERT, PKM2, and LDHA was reduced, as well as PKM2 activity and telomerase activity. However, ER(β) and miR-let-7a expression was increased. ER(α) is well known to be upregulated in the majority of breast cancers; it stimulates cancer cell proliferation, and its expression is a hallmark of hormone-dependent tumor growth [ 52 ]. Over the years, much evidence has shown the vital effect of ER(β) in breast cancer. Although there is controversy among scientists. ER(β) is generally thought to have antiproliferative effects in disease progression. In fact, the structure of ER(β) is homologous to that of ER(α), suggesting that while ER(β) could bind the same target genes as ER(α), it might have different specific ligands [ 53 ]. The role of ER(β) in BC initiation and proliferation has not yet been clearly established. In fact, several studies have suggested and demonstrated that ER(β) inhibits the proliferation, migration, and invasion of BC cells [ 54 ]; thus, ER(β) exhibits an inhibitory action on ER(α) mediated gene expression and, in many instances, opposes the actions of ER(α) [ 55 ]. Moreover, the expression of ER(β) may be regulated by DNA methylation, a reaction that is catalyzed by DNA methyltransferase (DNMT). Inhibition of DNA methyltransferase (DNMT) by fulvestrant increased the levels of ER(β), which exerted similar potency on DNMT activity as made by DNMT inhibitor [ 56 ]. This finding is in line with our study, where ATRA or/and fulvestrant reduced ER(α) and increased ER(β) expression. Thereafter, in this regard, it can be expected that these disorders caused by ATRA are accompanied by a change in upregulated oncogenes, tumor biomarkers, and glycolysis enzymes. As proven by SUN et al., H19 knockdown in MCF-7 cells resulted in a decrease in viable cell number and a blockade of estrogen-induced cell proliferation, indicating that H19 plays a significant role in cell survival and estrogen-induced cell proliferation in MCF-7 cells [ 10 ]. Second, increased telomerase activity and hTERT expression are reported in almost all human malignancies [ 57 ]. Recent studies have shown that certain miRNA expression correlate with tumor aggressiveness, and treatment responses suggesting that miRNAs can be used as diagnostic or prognostic markers [ 58 ]. Thereafter, dysregulated miRNA expression is frequently associated with the development of many types of human tumors, of which reduced expression of let-7 miRNA has been reported in breast cancer [ 59 ]. As discussed by Howard et al., let-7 miRNA is considered to be regulated by estrogen via ER(α), and estrogen signaling has been shown to regulate let-7 miRNA through direct ER(α) binding site interactions in estrogen receptor-positive breast cancer cells [ 60 ]. In this context, an increase in miR-let-7a expression upon ATRA treatment in MCF-7 cells could suppress the expression of several cancer-related genes in breast cancer, subsequently affecting biological processes such as glycolysis [ 61 ]. PKM2 is upregulated in breast cancer and can regulate tumor progression by promoting tumor cell viability, indicating thereafter that PKM2 is a potentially therapeutic target in breast cancer. YAO et al. have shown that miRNA let-7a can induce breast cancer cell apoptosis and inhibit cell proliferation, migration, and invasion; therefore, miR-let-7a inhibits aerobic glycolysis and proliferation of breast cancer cells by inhibiting PKM2 expression [ 36 ]. Together with our results, CHU et al. reported that the knockdown of PKM2 decreases the activity of pyruvate kinase in adenocarcinoma cells, and Shikonin which represents a novel PKM2 inhibitor, reduced PKM2 activity, which decreases cancer cell proliferation and survival [ 62 ]. Furthermore, Shikonin inhibits the rates of cellular lactate production and glucose consumption, in which LDHA plays a crucial role. Similar to PKM2, the regulation of LDHA is critical in cancer cells. One study showed that targeting LDHA with siRNA or small molecule inhibitors increased oxygen consumption and reactive oxygen species production, reduced glucose uptake and lactate production, and decreased tumor cell growth [ 32 ]. Afterward, miR-let-7a could be considered a microRNA that acts as a potent regulator due to its known role in regulating glycolysis in cancer cells. Taken together, our experiments demonstrated that ATRA regulates PKM2 and LDHA via miR-let-7a by inhibiting H19 and hTERT expression through estrogen receptors in MCF-7 cells. Besides, for MDA-MB-231 cells, upon ATRA and/or fulvestrant treatment, H19, hTERT, LDHA, and PKM2 RNA expression increased, while miR-let-7a expression decreased. The same treatment did not show a significant variation either on LDHA and PKM2 protein expression, or on PKM2 and telomerase activity. Increased expression of previously described RNAs after treatment is reported by the MDA-MB-231 cell line response caused by resistance to ATRA treatment. Liu et al. demonstrated that multidrug resistance is a major problem in successful cancer chemotherapy, leading to gene and enzymes overexpression [ 63 ]. As previously mentioned, the expression of miR-let-7a was significantly lower in breast cancer cells than in corresponding adjacent normal tissues, which suggested that miR-let-7a downregulation was associated with the development of breast cancer. Based on our results, ATRA and/or fulvestrant decreased miR-let-7a expression, thereby activating glycolysis, which induced an increase in PKM2 and LDHA mRNA expression. Nonsignificant protein expression of PKM2 and LDHA, neither on PKM2 activity was shown. However, the increase of these genes at the mRNA level could be due to post-transcriptional regulation that regulate cancer progression [ 64 ]. Interestingly, despite the ATRA effect on RNA level gene variation, no significant effect was observed on protein levels in MDA-MB-231 cells. To further investigate the direct implication of H19 and hTERT in miR-let-7a and glycolysis regulation in MCF-7 cells, mainly after their inhibition upon ATRA treatment, siRNA knockdown of each of the previous molecules alone or coupled with ATRA treatment was performed. After MCF-7 cells were treated with ATRA alone or coupled with H19 siRNA transfection or with hTERT siRNA transfection, a highly significant decrease in H19, hTERT, PKM2, and LDHA expression, as well as in PKM2 and telomerase activities were detected. However, miR-let-7a expression was increased. Interestingly, regulated expression patterns obtained by gene silencing were similar to those obtained after treatment with ATRA and/or fulvestrant, indicating that ATRA induces an inhibitory effect on PKM2 and LDHA, with an increase in miR-let-7a expression, via H19 and hTERT. Our results are compatible with Kallen et al. who confirmed that H19 antagonizes let-7 microRNAs, in which it modulates let-7 availability by acting as a molecular sponge, affecting, thereafter, the expression of endogenous let-7 targets [ 65 ]. In addition, Hrdlicˇkova´ et al. reported that hTERT is regulated by multiple miRNAs, such as let-7 g, that regulates hTERT expression and decreases telomerase activity [ 66 ]. These results are in line with our findings in which hTERT knockdown increased miR-let-7a expression. Moreover, telomerase mRNA and activity decrease after H19 inhibition and vice versa, indicating the presence of interconnection between these aforementioned tumor biomarkers. This interconnection was demonstrated by El Hajj et al., where telomerase was regulated by H19 in human acute promyelocytic leukemia cells [ 23 ]. As previously described, the implication of ER(α) and ER(β) in the modulation of the expression of tumor biomarkers and glycolysis has been demonstrated. To further evaluate their function in MDA-MB-231 cells, treated or untreated cells were transfected with ER(α) or ER(β) expression plasmids. Overexpression of ER(α) or ER(β) is directly related to an increase in PKM2 and LDHA expression, while ATRA combined with ER(α) or ER(β) overexpression restored PKM2 and LDHA expression. Indeed, JavanMoghadam et al. demonstrated that ER(α) modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle [ 67 ]. These findings are consistent with our results showing that ER(α) promotes glycolysis enzymes expression in MDA-MB-231 cells, thus, enhancing other biological processes, implicated in cancer cell progression. Contrary to the results observed in MCF-7 cells concerning ER(β) function, in MDA-MB-231 cells, ER(β) promotes glycolysis. This goes in Iine with our previous interpretation that ER(β) may have a bi-faceted role in breast cancer. Mishra et al. reported that the alteration in the expression of ER(α)/ER(β) balance is a critical step in breast cancer development and progression; the role of ER(β) in breast cancers expressing ER(β) alone, without ER(α), is less clear to date [ 56 ].

The present study investigated the effect of ATRA on H19, telomerase, miR-let-7a, PKM2, and LDHA in MCF-7 and MDA-MB-231 cells, which are ER-positive and triple-negative cells, respectively. Our study elucidates a signaling pathway regulated by ATRA in breast cancer cells. Indeed, we confirmed that MCF-7 cell treatment with ATRA alone or coupled with fulvestrant inhibited PKM2 and LDHA, increased miR-let-7a, and inhibited H19 and hTERT expression by modulating estrogen receptors alpha and beta, with an interconnection between H19 and hTERT. However, no significant regulation of glycolytic enzymes or telomerase activity was detected in MDA-MB-231 cells upon the same treatment (Fig.  12 ). These results highlight that ATRA acts as a tumor suppressor, demonstrating therapeutic potential with its combination with fulvestrant in ER-positive cells. Further investigations are required to clarify the effect of ATRA on ER(β) isoforms, to assess direct binding sites of miR-let-7a on the other tumor biomarkers and to evaluate metastasis and invasion of cancer cells after treatment. Finally, these in vitro observations should be validated using an in vivo model with ATRA and fulvestrant combination for the treatment of breast cancer and presenting afterwards an advantage in BC patient’s response to fulvestrant.

Summary diagram showing the variation in results after ATRA and/or fulvestrant treatment of MCF-7 and MDA-MB-231 cells. MCF-7 cell treatment with ATRA alone or coupled with fulvestrant inhibited PKM2 and LDHA, increased miR-let-7a, and inhibited H19 and hTERT expression by modulating ER(α) and ER(β). In addition, an interconnection between H19 and hTERT is noted, with a direct regulation carried out by each of them on miR-let-7a, PKM2, and LDHA. Effectively, increased expression of miR-let-7a and reduced expression of PKM2 and LDHA obtained by gene silencing were similar to those obtained after treatment with ATRA and/or fulvestrant. However, no significant variation in glycolytic enzymes expression or telomerase activity was detected in MDA-MB-231 cells upon the same treatment

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

American Type Culture Collection

Adenosine triphosphate

All-trans -Retinoic acid

• Breast cancer

Bicinchoninic acid

Complementary DNA

Carbon dioxide

Cycle threshold

Dulbecco’s modified Eagle’s medium

Deoxyribonucleic acid

Enhanced chemiluminescence

Estrogen Receptor

Estrogen receptor alpha

Estrogen receptor beta

Estrogen receptor alpha plasmid coding DNA

Estrogen receptor beta plasmid coding DNA

Fetal Bovine Serum

• Fulvestrant

Glyceraldehyde 3-phosphate dehydrogenase

Human epidermal receptor 2

Human telomerase reverse transcriptase

Insulin-like growth factor 2

Lysogeny broth

• Lactate dehydrogenase A

long non-coding RNA

MicroRNA let-7a

Messenger RNA

Nicotinamide adenine dinucleotide

Nonsignificant

Optical density

Phosphoenolpyruvate

Pruvate kinase

• Pyruvate kinase M2

Phenylmethylsulfonyl fluoride

Progesterone receptor

Penicillin/Streptomycin

Ribonucleic acid

Reverse-transcription polymerase chain reaction

Small interfering RNA of H19

Small interfering RNA of hTERT

Small interfering RNA

• Triple-negative breast cancer

Quantitative polymerase chain reaction

Quantitative telomeric repeat amplification protocol

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Acknowledgements

We would like to express our special thanks to Pathogen Laboratory, Faculty of Pharmacy at Saint-Joseph University, Beirut, Lebanon, for access to the diagnostic and microbiological research platform.

This work was supported by the Research Council of Saint-Joseph University and by the Lebanese National Council for Scientific Research (CNRS). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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R.E.H. performed the experiments, designed the project, analyzed the data, and wrote the manuscript. R.A. participated in data analysis and in manuscript revision. R.T. made substantial contributions to the manuscript’s section editing. G.H. supervised the study, provided guidance and edited the manuscript. All the authors have read and approved the final manuscript.

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El Habre, R., Aoun, R., Tahtouh, R. et al. All-trans- retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells. BMC Cancer 24 , 615 (2024). https://doi.org/10.1186/s12885-024-12379-3

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AI Might Help Spot Breast Cancer's Spread Without Biopsy

AI Might Help Spot Breast Cancer's Spread Without Biopsy

By Dennis Thompson HealthDay Reporter

FRIDAY, May 24, 2024 (HealthDay News) -- New AI can help detect breast cancer that is spreading to other parts of the body, without the need for biopsies, a new study finds.

The AI analyzes MRI scans to detect the presence of cancer cells in the lymph nodes under the arms, researchers said.

In clinical practice, the AI could help avoid 51% of unnecessary surgical biopsies to test lymph nodes for cancer, while correctly identifying 95% of patients whose breast cancer had spread, results showed.

Most breast cancer deaths are due to cancer that’s spread elsewhere, and the cancer typically first spreads to an armpit lymph node, explained lead researcher Dr. Basak Dogan , director of breast imaging research at UT Southwestern Medical Center.

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Finding cancer that’s spread to a lymph node “is critical in guiding treatment decisions, but traditional imaging techniques alone do not have enough sensitivity” to effectively detect it, Dogan said in a medical center news release.

Patients with benign findings from MRI exams or needle biopsies often must undergo surgical lymph node biopsy anyway, because those tests can miss a good number of cancer cells that have spread past the breast, Dogan said.

Researchers trained the AI by feeding the program MRI scans from 350 newly diagnosed breast cancer patients known to have cancer in their lymph nodes.

Testing showed that the newly developed AI was significantly better at identifying these patients than human doctors using MRI or ultrasound, researchers reported recently in the journal Radiology: Imaging Cancer .

“That’s an important advancement because surgical biopsies have side effects and risks, despite having a low probability of a positive result confirming the presence of cancer cells,” Dogan explained. “Improving our ability to rule out [cancer cells in lymph nodes] during a routine MRI -- using this model -- can reduce that risk while enhancing clinical outcomes.”

More information

The American Cancer Society has more about breast cancer .

SOURCE: UT Southwestern, news release, May 21, 2024

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• Risk Factors
• Breast Cancer Resources to Share
• What CDC Is Doing About Breast Cancer
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Screening for Breast Cancer

• Breast cancer screening can help find breast cancer early, when it is easier to treat.
• The US Preventive Services Task Force recommends that women who are 40 to 74 years old and are at average risk for breast cancer get a mammogram every 2 years.

Breast cancer screening means checking a woman's breasts for cancer before there are signs or symptoms of the disease. All women need to be informed by their health care provider about the best screening options for them. When you are told about the benefits and risks of screening and decide with your health care provider whether screening is right for you—and if so, when to have it—this is called informed and shared decision-making.

Although breast cancer screening cannot prevent breast cancer, it can help find breast cancer early, when it is easier to treat. Talk to your doctor about which breast cancer screening tests are right for you, and when you should have them.

Screening recommendations

The US Preventive Services Task Force is an organization made up of doctors and disease experts who look at research on the best way to prevent diseases and make recommendations on how doctors can help patients avoid diseases or find them early.

The Task Force recommends that women who are 40 to 74 years old and are at average risk for breast cancer get a mammogram every 2 years. Women should weigh the benefits and risks of screening tests (see below).

When Should I Start Getting Mammograms?

CDC's Dr. Lisa Richardson talks about the best time for women to start getting mammograms in this video.

Types of tests

A mammogram is an x-ray of the breast. For many women, mammograms are the best way to find breast cancer early, when it is easier to treat and before it is big enough to feel or cause symptoms. Having regular mammograms can lower the risk of dying from breast cancer. At this time, a mammogram is the best way to find breast cancer for most women of screening age.

Breast magnetic resonance imaging (MRI)

A breast MRI uses magnets and radio waves to take pictures of the breast. Breast MRI is used along with mammograms to screen women who are at high risk for getting breast cancer. Because breast MRIs may appear abnormal even when there is no cancer, they are not used for women at average risk.

Other exams

• Clinical breast exam: A clinical breast exam is an examination by a doctor or nurse, who uses his or her hands to feel for lumps or other changes.
• Breast self-awareness: Being familiar with how your breasts look and feel can help you notice symptoms such as lumps, pain, or changes in size that may be of concern. These could include changes found during a breast self-exam. You should report any changes that you notice to your doctor or health care provider.

Having a clinical breast exam or doing a breast self-exam has not been found to lower the risk of dying from breast cancer.

Benefits and risks of screening

Every screening test has benefits and risks, which is why it's important to talk to your doctor before getting any screening test, like a mammogram.

Benefit of screening

The benefit of screening is finding cancer early, when it's easier to treat.

Risks of screening

Harms can include false positive test results, when a doctor sees something that looks like cancer but is not. This can lead to more tests, which can be expensive, invasive, and time-consuming, and may cause anxiety.

Tests also can lead to overdiagnosis, when doctors find a cancer that would not have gone on to cause symptoms or problems, or even may go away on its own. Treatment of these cancers is called overtreatment. Overtreatment can include treatments recommended for breast cancer, such as surgery or radiation therapy. These can cause unnecessary and unwanted side effects. Other potential harms from breast cancer screening include pain during procedures and radiation exposure from the mammogram test itself. While the amount of radiation in a mammogram is small, there may be risks with having repeated x-rays.

Mammograms may also miss some cancers, called false negative test results, which may delay finding a cancer and getting treatment.

Where can I go to get screened?

You can get screened for breast cancer at a clinic, hospital, or doctor's office. If you want to be screened for breast cancer, call your doctor's office. They can help you schedule an appointment.

Most health insurance plans are required to cover screening mammograms every 1 to 2 years for women beginning at age 40 with no out-of-pocket cost (like a co-pay, deductible, or co-insurance).

Find a mammography facility near you.

Are you worried about the cost?‎

Breast cancer.

Talk to your doctor about when to start and how often to get a mammogram.

For Everyone

Public health.

Submission guidelines

Our 3-step submission process, before you submit.

Now you’ve identified a journal to submit to, there are a few things you should be familiar with before you submit.

• Make sure you are submitting to the most suitable journal - Aims and scope
• Understand the costs and funding options - Fees and funding
• Make sure your manuscript is accurate and readable - Language editing services
• Understand the copyright agreement - Copyright

Ready to submit

To give your manuscript the best chance of publication, follow these policies and formatting guidelines.

• Check the quality of your writing - Free Language check
• General formatting rules for all article types - Preparing your manuscript
• Make sure your submission is complete - Prepare supporting information
• Copyright and license agreement - Conditions of publication
• Read and agree to our Editorial Policies - Editorial policies

Submit and promote

After acceptance, we provide support so your article gains maximum impact in the scientific community and beyond.

Please note that manuscript can only be submitted by an author of the manuscript and may not be submitted by a third party. 

• Who decides whether my work will be accepted? - Peer-review policy
• Want to submit to a different journal? - Manuscript transfers
• Spreading the word - Promoting your publication

Submit manuscript

• Editorial Board
• Manuscript editing services
• Instructions for Editors
• Sign up for article alerts and news from this journal
• Collections
• Follow us on Twitter

Annual Journal Metrics

2022 Citation Impact 7.4 - 2-year Impact Factor 7.4 - 5-year Impact Factor 1.764 - SNIP (Source Normalized Impact per Paper) 2.408 - SJR (SCImago Journal Rank)

2023 Speed 20 days submission to first editorial decision for all manuscripts (Median) 129 days submission to accept (Median)

2023 Usage  2,432,781 downloads 1,561 Altmetric mentions

• More about our metrics

Breast Cancer Research

ISSN: 1465-542X

• Submission enquiries: [email protected]