Obesity, defined as a body mass index (BMI) ≥ 30, is an established risk factor for breast cancer among women in the general population after menopause. Whether elevated BMI is a risk factor for women with a germline mutation in BRCA1 or BRCA2 is less clear because of inconsistent findings from epidemiological studies and a lack of mechanistic studies in this population. Here, we show that DNA damage in normal breast epithelia of women carrying a BRCA mutation is positively correlated with BMI and with biomarkers of metabolic dysfunction. In addition, RNA sequencing showed obesity-associated alterations to the breast adipose microenvironment of BRCA mutation carriers, including activation of estrogen biosynthesis, which affected neighboring breast epithelial cells. In breast tissue explants cultured from women carrying a BRCA mutation, we found that blockade of estrogen biosynthesis or estrogen receptor activity decreased DNA damage. Additional obesity-associated factors, including leptin and insulin, increased DNA damage in human BRCA heterozygous epithelial cells, and inhibiting the signaling of these factors with a leptin-neutralizing antibody or PI3K inhibitor, respectively, decreased DNA damage. Furthermore, we show that increased adiposity was associated with mammary gland DNA damage and increased penetrance of mammary tumors in Brca1 +/− mice. Overall, our results provide mechanistic evidence in support of a link between elevated BMI and breast cancer development in BRCA mutation carriers. This suggests that maintaining a lower body weight or pharmacologically targeting estrogen or metabolic dysfunction may reduce the risk of breast cancer in this population.

Background: Elevated bodyweight is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 and BRCA2. However, the mechanistic basis for this association is unknown. Breast adipose tissue undergoes significant changes in the setting of weight gain and obesity, including elevation in aromatase expression which leads to the increased biosynthesis of estrogens. Given that estrogens and estrogen metabolites have known pro-proliferative and genotoxic effects, we hypothesized that in BRCA1/2 mutation carriers, obesity may be positively associated with breast epithelial cell DNA damage, thereby increasing the risk of tumorigenesis. Furthermore, we examined the impact of inhibiting estrogen signaling or production on breast epithelium DNA damage in BRCA1/2 mutation carriers. Methods: Tissue microarrays were generated from non-cancerous breast tissue derived from 72 women carrying a mutation in BRCA1 or BRCA2 with known body mass index (BMI, kg/m2). Breast epithelium DNA damage was quantified by immunofluorescence (IF) staining of the DNA damage marker γH2AX. RNA-Seq was performed on breast organoids to assess differences in gene expression in relation to BMI. Associations between DNA damage and biomarkers of estrogen biosynthesis and bioavailability, including aromatase expression in the breast and circulating steroid hormone binding globulin (SHBG), were also evaluated. To explore the effect of blocking estrogen signaling or production on DNA damage, non-tumorous breast tissue explants from BRCA1/2 mutation carriers were cultured with fulvestrant, an estrogen receptor degrader, or metformin, an anti-diabetic drug that also reduces aromatase expression in the breast. Breast epithelial cell DNA damage was measured in control vs treated explants by γH2AX IF staining after 24 hours of treatment. Results: BMI was positively correlated with DNA damage in the breast epithelium of BRCA1/2 mutation carriers. Upstream analysis of gene expression in organoids derived from women with a BMI ≥ 30 compared to <25, revealed activation of estrogen signaling. Further supporting a contribution of locally-derived and circulating estrogens to obesity-related DNA damage, breast aromatase expression was found to be positively correlated with DNA damage while circulating SHBG levels showed a negative correlation. Targeting estrogen signaling with fulvestrant significantly reduced breast epithelium DNA damage in breast explants from women carrying a mutation in either BRCA1 or BRCA2. Interestingly, metformin, also caused a significant reduction in DNA damage in breast explants. Conclusion: These data provide mechanistic evidence for the link between obesity and breast cancer in BRCA1 and BRCA2 mutation carriers through identification of a positive association between BMI and breast epithelial cell DNA damage. Importantly, these studies demonstrate that fulvestrant and metformin, drugs already approved for clinical use, decrease breast epithelial cell DNA damage. Further studies are warranted to determine whether targeting estrogens or use of metformin may be effective risk reduction strategies in BRCA1/2 mutation carriers with excess bodyweight who are at high risk for breast cancer development and currently have limited options for prevention beyond surgical intervention. Support: NIH R01CA215797, NIH F31CA236306, Anne Moore Breast Cancer Research Fund Citation Format: Priya Bhardwaj, Neil M. Iyengar, Sofya Oshchepkova, Phoebe Piloco, Rohan Bareja, Olivier Elemento, Dilip D. Giri, Michael Pollak, Monica Morrow, Jason A. Spector, Kristy A. Brown. Obesity is associated with DNA damage in the breast epithelium of BRCA1 and BRCA2 mutation carriers: A role for estrogens & strategies for prevention [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-03.

Obesity is an established risk factor for breast cancer among women in the general population after menopause. Whether elevated bodyweight is a risk factor for women with a germline mutation in BRCA1 or BRCA2 is less clear due to inconsistent findings from epidemiological studies and lack of mechanistic studies in this population. Here, we show that DNA damage in normal breast epithelium of BRCA mutation carriers is positively correlated with body mass index and with biomarkers of metabolic dysfunction. Additionally, RNA-sequencing reveals significant obesity-associated alterations to the breast adipose microenvironment of BRCA mutation carriers, including activation of estrogen biosynthesis, which impacts neighboring breast epithelial cells. We found that blockade of estrogen biosynthesis or estrogen receptor activity decreases DNA damage, whereas treatment with leptin or insulin increases DNA damage in BRCA heterozygous epithelial cells. Furthermore, we show that increased adiposity is associated with mammary gland DNA damage and increased penetrance of mammary tumors in Brca1 +/- mice. Overall, our results provide mechanistic evidence in support of a link between bodyweight and breast cancer development in BRCA mutation carriers and suggests that maintaining a healthy bodyweight or pharmacologically targeting estrogen or metabolic dysfunction may reduce the risk of breast cancer in this population. One Sentence Summary Elevated bodyweight is positively associated with DNA damage in breast epithelium of BRCA mutation carriers

Background: Obesity is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 or BRCA2. Previously, we found that obesity was positively associated with DNA damage in breast epithelium from BRCA mutation carriers. Furthermore, factors secreted by obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells, suggesting a cross-talk between breast epithelial cells and the adipose tissue that surrounds them. We hypothesized that leptin, a hormone secreted in abundance by obese adipose tissue, may be a driver of DNA damage and/or decrease capacity for DNA repair in breast epithelial cells. If true, this would provide a molecular target for intervention to reduce the risk of tumor formation in this high-risk population of women. Methods: RNA-seq followed by Ingenuity Pathway Analysis (IPA) was conducted on primary breast epithelial organoids isolated from lean and obese BRCA mutation carriers. Breast adipose tissue obtained from lean and obese women were cultured as explants for 24 hours to produce lean and obese conditioned media (CM). The effect of leptin on DNA damage was assessed in a non-cancerous breast epithelial cell line (MCF10A) carrying a heterozygous BRCA1 mutation. Immunofluorescence staining of the DNA damage marker ƴH2AX was carried out after treatment with leptin (100ng-800ng), CM, or CM+leptin antibody, used to neutralize leptin. To test whether leptin affects DNA repair capacity, BRCA1+/- MCF10A cells were treated with leptin or vehicle and then irradiated (1Gy) to induce DNA damage. Resolution of damage was quantified at 0, 0.5, 4, 12, and 24 hrs post-irradiation. Results: IPA analysis identified leptin signaling as significantly upregulated in breast epithelial organoids from obese women compared with lean women. Both obese CM and leptin treatment induced DNA damage in BRCA1+/- MCF10A cells while lean CM did not have this effect. Neutralizing leptin in obese CM was sufficient to inhibit obese CM-mediated induction of DNA damage. No significant difference was observed between leptin or vehicle treatments on DNA repair capacity after irradiation of BRCA+/- MCF10A cells. Conclusions: These data identify leptin, an adipose-derived hormone, as a novel driver of DNA damage in breast epithelial cells. To date, no studies have elucidated the molecular mechanisms that explain the increased penetrance of breast cancer in obese BRCA mutation carriers compared to lean BRCA mutation carriers. This work suggests that leptin may be a mediator of the link between obesity and breast cancer development in this population. Further studies are warranted to determine if targeting the leptin signaling axis will be an effective risk reduction strategy in BRCA mutation carriers who have excess adiposity.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, it remains the second leading cause of cancer-related death in the US. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. One major area of research relates to its reported effects to increase insulin sensitivity in diabetics, this being the basis for the development of unacylated ghrelin analog, AZP531 or livoletide, now in clinical trials for the treatment of Prader-Willi Syndrome. The mechanism of action of unacylated ghrelin is largely uncharacterized, in any system, because it does not bind to or activate the cognate ghrelin receptor, GHSR1a. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP531. We report potent effects of unacylated ghrelin, at picomolar doses, on the growth of breast cancer cells, dependent on 3D culture and activation of Gαi. Suppression of MAPK signaling by unacylated ghrelin leads to cell cycle arrest and apoptosis. AZP531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Obesity is a risk factor for estrogen receptor-positive (ER+) breast cancer after menopause. The pro-proliferative effects of estrogens are well characterized and there is a growing body of evidence to also suggest an important role in tumorigenesis. Importantly, obesity not only increases the risk of breast cancer, but it also increases the risk of recurrence and cancer-associated death. Aromatase is the rate-limiting enzyme in estrogen biosynthesis and its expression in breast adipose stromal cells is hypothesized to drive the growth of breast tumors and confer resistance to endocrine therapy in obese postmenopausal women. The molecular regulation of aromatase has been characterized in response to many obesity-related molecules, including inflammatory mediators and adipokines. This review is aimed at providing an overview of our current knowledge in relation to the regulation of estrogens in adipose tissue and their role in driving breast tumor development, growth and progression.

INTRODUCTION: Breast cancer (BC) research encompasses not only the study of tumor behavior, but also requires understanding the innumerable interactions that occur between breast cellular components and the extracellular matrix (ECM) in the development and progression of the disease. Despite extensive research, BC continues to be a leading cause of morbidity and mortality for women across the globe. The lack of three-dimensional models that are able to accurately replicate the tumor microenvironment and tumor-associated stroma has resulted in the unsuccessful translation of most preclinical studies into the clinic. It has been suggested that adipose tissue is a drug-metabolizing organ that protects tumor cells from chemotherapeutic agents. We have developed a tissue engineered, three-dimensional, high-throughput, patient specific, biomimetic model of BC that incorporates patient-derived adipocytes, adipose stromal cells, breast-duct organoids, and BC cells, and studied the effect of treatment with the chemotherapeutic agent doxorubicin on BC cell survival. METHODS: Under an approved IRB, breast tissue was acquired from patients and differentially processed to isolate mature adipocytes, stromal cells, and breast organoids which were subsequently co-cultured with cancer cells in a 0.6% type I collagen matrix. Mixtures containing fluorescently tagged MDA-MB-231 or MDA-MB-468 triple negative BC cells at a concentration of 200,000 cells/mL were plated onto 96 well plates. BC cells in plain collagen and biomimetic hydrogels without BC served as controls. Groups composed of three replicates of both Biomimetic and collagen only controls were treated with one time with serially diluted doses of doxorubicin at 0, 0.001, 0.01, 0.1, 1, and 10 uM, in cell culture media and fixed after 3 days in culture, stained, and fluorescently imaged using confocal microscopy. Tissues from three patients were used to test each cell line. Analysis was performed using Imaris™ software. RESULTS: After 3 days of doxorubicin treatment, MDA-MB-231 and MDA-MB-468 cell lines showed increased survival at 10mM doxorubicin (p<0.05) in the biomimetic platform derived from patient tissue, when compared to BC cells cultured in the collagen only controls. Moreover, fluorescent confocal microscopy demonstrated doxorubicin uptake by adipocytes that was directly proportional to increasing doxorubicin concentrations. No increased doxorubicin fluorescence was detected within the collagen-only controls. CONCLUSION: When cultured within a tissue engineered biomimetic platform comprised of patient tissue components, we observed significantly increased survival of two different triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) when compared to cultures in collagen alone. These data clearly demonstrate that the tumor microenvironment and neighboring tissue components modulates the effect of doxorubicin therapy on two different BC cell lines at least partially through adipocyte sequestration of doxorubicin. The ability of our platform to be personalized for individual patients may allow us to not only elucidate how the microenvironment affects BC cells, but also how patient specific tissue impacts the effects of various chemotherapeutic agents.

INTRODUCTION: Breast cancer (BC) is the most common non-skin cancer in females, affecting 12.5% of women throughout their lifetime. Partly due to a lack of models that accurately mimic the tumor microenvironment of individual patients, many preclinical successes fail translation into the clinic. Current two-dimensional models fail to replicate the cellular behaviors and interactions that occur in vivo, and newer three-dimensional systems, though promising, all lack vasculature which is crucial to understanding the mechanisms of tumor cell invasion and metastasis. We have engineered an advanced three-dimensional biomimetic platform derived from patient specific tissues that contains all components of the breast tumor microenvironment (glandular organoids, adipocytes, stromal vascular fraction (SVF)) surrounding engineered vascular structures that can be precisely positioned at predetermined distances from BC spheroids (BCS) allowing for highly novel ex-vivo investigations of the interactions between human tumor cells and blood vessels. METHODS: Polydimethylsiloxane (PDMS) wells were created using 3D-printed molds that include stages for localization of BCS, and putative vascular channels (VC). Type I collagen was neutralized at 0.3% and 0.6% w/v. Red-fluorescent MDA-MB-231 cells were mixed with 0.6% collagen at a density of 40,000 cells/uL; 1uL of the collagen/cancer cell mix was plated on stages of the PDMS molds and allowed to nucleate. A biomimetic platform made with BODIPY stained adipocytes and all other patient-derived tissue components mixed within both concentrations of collagen was plated in the pre-designed well, surrounding the BCS and VC. Twenty-four hours after plating, fluorescently labeled endothelial cells (EC) and smooth muscle cells (SMC) were seeded within the channel at a concentration of 5 million cells/mL. Control constructs were made by generating vascular structures and BCS within a collagen-only matrix, and by creating full biomimetic platforms with vascular channels in the absence of cancer cells. Constructs were cultured for 7 days, formalin-fixed, counterstained with DAPI, and analyzed with confocal microscopy. RESULTS: After 7 days in culture, confocal microscopy revealed successful fabrication of biomimetic platforms with a type I collagen (different concentrations) extracellular matrix containing patient-derived adipocytes, SVF and breast duct organoids. Patent VC lined with fluorescently labeled SMC and EC were visualized within the platform, with vascular walls located within 1mm of the red fluorescent, triple-negative MDA-MB-231 cancer foci. Invasion of BC cells into the surrounding tissue was identified by the presence of red fluorescent cells within the biomimetic platform in constructs containing type I collagen at both 0.3% and 0.6% w/v. Decreased vascular integrity was observed in constructs containing BCS when compared to those without BC cells. CONCLUSION: With the aid of three-dimensional printing technology, we have successfully engineered an advanced, patient-specific, biomimetic platform of the breast cancer microenvironment that not only replicates patient tissue characteristics, but also includes vascular structures and cancer foci that closely resemble early tumors. Observed BC invasion into the surrounding microenvironment, and the platform’s ability to mimic patient specific tissue with extremely high fidelity, make this platform a highly versatile and powerful tool that holds significant promise for diagnostic and therapeutic applications in the study of breast cancer.

Background: Obese women who carry a mutation in the DNA repair enzymes BRCA1 and/or BRCA2 are at a greater risk of developing breast cancer compared with lean BRCA1/2 mutation carriers. Causative factors that drive the increased penetrance of breast cancer in obese women are poorly characterized. Previously, we showed that BMI was positively correlated with DNA damage in breast epithelium of BRCA mutation carriers. We hypothesized that obesity-associated dysfunctional adipose tissue may cause DNA damage in neighboring breast epithelial cells. If true, this would provide evidence for the initial insults associated with increased risk of tumor formation in obese women. Methods: To test the effect of locally secreted factors on DNA damage induction, conditioned media was collected over 24 hours from primary breast adipose explants obtained from lean and obese women. Conditioned media was used to treat normal breast epithelial cells (MCF10A) harboring a heterozygous BRCA1 mutation or wildtype for BRCA. Immunofluorescence (IF) staining of the DNA damage marker ƴH2AX was carried out after treatment. Cell line findings were validated in primary breast epithelial organoids isolated by collagenase digestion of breast tissue obtained from the clinic. Additionally, RNA-seq was conducted on primary breast epithelial organoids isolated from lean and obese women to identify potential drivers of gene expression differences associated with obesity and DNA damage. Results: Conditioned media from obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells while lean conditioned media did not have this effect. Similar findings were made in primary breast epithelial organoids, suggesting a role for locally secreted factors in mediating DNA damage. Ingenuity Pathway Analysis of RNA-seq data from lean and obese primary breast organoids identified several upstream regulators of gene expression differences, including signaling by estradiol and pro-inflammatory cytokines, both of which are elevated in obese breast tissue and are known inducers of DNA damage. Conclusions: These data show for the first time that local factors produced by obese breast adipose tissue induce breast epithelial cell DNA damage. Our results suggest that therapies targeting weight or dysfunctional breast adipose tissue may reduce the increased DNA damage observed in obese BRCA mutation carriers and thereby decrease tumor formation in this high-risk population of women.