Triple-negative Breast Cancer In Women Research Articles

Abstract IA017: Targeting the mTOR pathway for the prevention of triple-negative breast cancer

Abstract Phase III cancer prevention clinical trials have shown that breast cancer prevention is feasible using anti-estrogen drugs. However, these drugs have not been widely accepted because of concerns about toxicity. In addition, anti-estrogen drugs do not prevent estrogen-negative breast cancers, including the most aggressive form of breast cancer, triple-negative breast cancer (TNBC), that does not express the estrogen receptor (ER), progesterone receptor, or the human epidermal growth factor receptor 2 (HER2). Our laboratory is focused on identifying growth-regulatory molecules that are essential for the growth of TNBCs. For this study, we identified mTOR as an essential growth-regulatory molecule that is highly expressed in TNBCs. We then investigated whether the mTOR inhibitor everolimus can prevent mammary tumors in transgenic mouse models including 4 models of TNBC and one model of ER-negative/HER2-positive breast cancer. Everolimus treatment significantly delayed mammary tumor formation but with a varying degree in all five mouse models. Everolimus treatment for up to 1 year was well tolerated with no observable toxicity. These results suggest that mTOR inhibitors may be promising drugs for the prevention of ER-negative and triple-negative breast cancers in women at risk of these aggressive breast cancers. Grant support: This research was supported by an NCI-PREVENT contract to P. Brown and A. Mazumdar (HHSN261201500018I/HHSN26100006). Citation Format: Powel H. Brown, Abhijit Mazumdar, William Tahaney, Jamal Hill, Yun Zhang, Sumankalai Ramachandran, Jitesh Kawedia, Jing Qian, Alejandra Contreras, Michelle Savage, Lana Vornik. Targeting the mTOR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA017.

Abstract 954: Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT)

Abstract Metastatic breast cancer is a leading cause of death of women in the United States. The metastatic process involves epithelial cells losing their cellular polarity, adhesion to the basement membrane, and acquiring migratory and invasive properties. Metastatic epithelial cells take on a mesenchymal phenotype, hence this process is termed epithelial-to-mesenchymal transition (EMT). EMT is characterized by a reduction in epithelial cell marker expression (E-cadherin, cytokeratin 8, and cytokeratin 18), and an increased expression of mesenchymal cell markers (N-cadherin, vimentin, and fibronectin). Previous studies have demonstrated that dietary intake of γ-tocotrienol and/or vitamin D3 may provide benefit in the preventing the growth and development of metastatic breast cancer. γ-Tocotrienol is a natural isoform of vitamin E that shows potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 has also been shown to display potent antiproliferative and antiangiogenic activity against human breast cancer cells, particularly triple negative breast cancers. Studies were conducted here to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, and epithelial to mesenchymal transition. Treatment with 10-16 µM γ-tocotrienol or 20-40 nM vitamin D3 induced a significant dose-responsive decrease in MDA-MB-231 growth after a 4-day culture period. Combined treatment with sub-effective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) induced a synergistically inhibition of MDA-MB-231 cell growth, as determined isobologram analysis. Studies using the matrigel invasion assay showed that similar combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 motility. These effects appear to result from a reversal in EMT. Western blot analysis shows that combined treatment resulted in an increased expression of the epithelial cell markers E-cadherin, cytokeratins 8 and cytokeratins 18 and a corresponding reduction in mesenchymal cell markers N-cadherin and vimentin. Taken together, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 synergistically inhibit TNBC MDA-MB-231 breast cancer cell growth, viability, motility, and induced a reversal of EMT. These findings strongly suggest that combined γ-tocotrienol and vitamin D3 treatment may provide significant benefits in the treatment of metastatic TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. Rafi Anwar, Taylor A. Hurst, Paul W. Sylvester. Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 954.

Scoping Review: Relationship between Overweight and Obesity with Triple Negative Breast Cancer in Woman

Breast cancer is still one of the main problems for global public health. Breast cancer has several pathological characteristics, including triple negative breast cancer (TNBC) characterized by negative ER, PR, HER2 results and has a poor prognosis. Risk factors for TNBC are closely related to Body Mass Index (BMI) as an individual description of obesity. It is known in women, overweight and obesity can trigger breast cancer. This study aims to analyze the relationship between overweight and obesity based on BMI with the incidence of TNBC in women. This study used a scoping review to analyze scientific writings from three data sources, they are PubMed, ScienceDirect and SpringerLink. In the initial search, 797 articles were generated, then adjusted according to the inclusion, exclusion and eligibility criteria to produce 5 articles summarized in the PRISMA diagram. Of the five articles that were predominantly conducted in Asia, four articles stated that women with a BMI ≥25 kg/m2 had a higher risk of TNBC. One article stated that the risk factors for TNBC can be assessed from waist circumference other than BMI. In addition, there is one article that mentioned the opposite, there is no relationship between BMI at the age of 25-54 years and TNBC. The conclusion of this study is that obesity have a relationship with the incidence of TNBC in women.

Abstract 2573: The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro

Abstract Epidemiological studies suggest that dietary intake of some natural products may be effective in reducing the risk of breast cancer. Furthermore, various phytochemicals have been shown to interfere with cell signaling pathways involved in regulating breast cancer cell proliferation and viability. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 is an abundant phytochemical which also displays potent antiproliferative and antiangiogenic activity against human breast cancer cells. Studies were conducted to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, colony formation, invasion, cell cycle progression, and apoptosis. Results showed that combined treatment with subeffective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) synergistically inhibited the growth of MDA-MB-231 cells, as determined by the MTT assay and isobologram analysis. Additional studies indicated that this combination treatment resulted in a significant inhibition in cancer cell migration and a significant reduction in cancer cell colony formation. Studies using the Matrigel invasion assay showed that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 invasiveness. Flow cytometry analysis showed that similar combined treatment of γ-tocotrienol and vitamin D3 significantly increased the percentage of cells found in G0/G1 phase, as compared to cells in the vehicle-treated control group. After 4 days of combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, the percentage of cells in the G0/G1 phase of the cell cycle increased to 55% (p<0.05, all p values compared to untreated cells), as compared to 37.48% for cells in the vehicle-treated control group. In addition, combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly increased the number of apoptotic and necrotic cells, as comparted to cells in the vehicle-treated control group. In summary, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 act synergistically to inhibit MDA-MB-231 human breast cancer cell growth and viability, and strongly suggest that combined treatment with these agents may provide some benefits in the treatment of TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. R. Anwar, A.K.M.N. Hossian, Georgios Matthaiolampakis, Paul W. Sylvester. The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2573.

Multiethnic PDX models predict a possible immune signature associated with TNBC of African ancestry.

Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors. We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment. In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets. Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.

Abstract P1-05-05: The effect of microRNA dysregulation on health disparities observed in African American women in triple negative breast cancer

Abstract BACKGROUND: Triple-negative breast cancers (TNBC) represent an important subtype characterized by an absence of ER, PR, and HER2, and therefore are not responsive to current targeted therapies. TNBC is considered a clinically aggressive subtype of breast cancer with lower survival rates and a higher risk of metastasis development. There is a higher prevalence of TNBC in women of African descent. Furthermore, there is evidence that African American (AA) women have worse clinical outcomes compared with Caucasian American (CA) women with TNBC. Several studies have begun to evaluate the major drivers of this health disparity including socio-economic status, cultural influences, and biological factors; however, to date, researchers have not been able to elucidate the mechanisms behind the aggressive biology seen in AA women with TNBC. Recently miRNAs were identified to play a role in breast cancer through the regulation of cellular processes associated with aggressive tumor phenotypes, such as TNBC. MiRNAs are a likely therapeutic target because they are involved in the regulation of several essential functions in cancer cells such as proliferation, migration, invasion, and apoptosis. For miRNAs to reliably be used as cancer therapeutic agents in clinical settings, miRNA signatures of specific cancer subtypes based on ethnicity must be identified. Here, we hypothesize that differential expression of miRNAs contributes to the more aggressive phenotype observed in AA patients compared to CA patients with TNBC. In this study, our primary goal is to characterize and compare the miRNA expression profiles of breast tumor tissue from AA and CA patients. METHODS: miRNA expression data of TNBC patients, contained in the TCGA database, were analyzed to identify a set of miRNAs with prognostics significance to be used as potential biomarkers. From the acquired miRNA-seq data we determined a total of 175 differentially expressed miRNAs between AAW and CA triple negative breast cancer tissue and decided to characterize 8 miRNAs that have been previously determined to have a role in breast cancer. RESULTS: In AA tumor tissue there was the marked loss of the following tumor suppressors: let-7b, miR-200b,-130b, and -375 and the overexpression of the following oncogenic miRNAs: miR-181c, 17, 21, 29c. In TNBC, miR-200 and 130b are known to sensitize cells to apoptosis, inhibit metastasis formation, and are associated with patient survival. Additionally, the expression of oncogenic miRNAs; miR-181c, 17, 21, 29c are associated with worse prognosis in TNBC. Target gene prediction using miRBase revealed that several of the dysregulated miRNAs were involved in the epithelial-mesenchymal transition, a key step in metastasis formation. Next, we evaluated how miRNA dysregulation could potentially affect patient survival. Using data from the TCGA we were able to extrapolate survival probability data (combined CAW and AAW n=203). We determined that 5-year survival was significantly decreased in patients that had high expression of miR-181c and miR-17 and let-7b and miR-200b had protective effects. CONCLUSIONS: These results support that there is a distinct miRNA expression signature in AAW and CAW with TNBC which may correlate to prognosis. Our results will contribute to the application of miRNAs as novel therapeutic tools against TNBC in AA patients and represents an innovative approach to address the health disparities seen in the AA population. Citation Format: Ches'que M. Phillips, Yaguang Xi. The effect of microRNA dysregulation on health disparities observed in African American women in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-05.

Molecular subtypes of triple-negative breast cancer in women of different race and ethnicity.

Molecular subtypes of triple negative breast cancer (TNBC) are associated with variation in survival and may assist in treatment selection. However, the association of patient race or ethnicity with subtypes of TNBC and clinical outcome has not been addressed. Using nCounter Gene Expression Codesets, we classified TNBCs into subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES) in 48 Hispanic, 12 African-American, 21 Asian, and 34 White patients. Mean age at diagnosis was significantly associated with subtype, with the youngest mean age (50 years) in MES and the oldest mean age (64 years) in LAR (p < 0.0005). Subtype was significantly associated with tumor grade (p = 0.0012) and positive lymph nodes (p = 0.021), with a marginally significant association of tumor stage (p = 0.076). In multivariate Cox-proportional hazards modeling, BLIS was associated with worst survival and LAR with best survival. Hispanics had a significantly higher proportion of BLIS (53%, p = 0.03), whereas Asians had a lower proportion of BLIS (19%, p = 0.05) and a higher proportion of LAR (38%, p = 0.06) compared to the average proportion across all groups. These differences in proportions of subtype across racial and ethnic groups may explain differences in their outcomes. Determining subtypes of TNBC facilitates understanding of the heterogeneity of the TNBCs and provides a foundation for developing subtype-specific therapies and better predictors of TNBC prognosis for all races and ethnicities.

A Provocative Molecular Link between Mammographic Density and BRCA1-loss associated TNBC.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that has a high mortality rate and disproportionately affects young African American (AA) women who carry mutations in the BRCA1 gene. Approximately 80% of breast cancers which develop in BRCA1-mutant carriers will have TNBC and the molecular mechanism facilitating tumor development is unclear. Our earlier work suggested Ubc9 to play a critical role in BRCA1 loss mediated TNBC cell migration and metastasis. Collagen is one of the major components of the stromal extracellular matrix (ECM) network that influences tissue density. Its re-organization act as a scaffold aiding cancer cells to migrate causing metastasis. Ubc9 is known to increase the production of collagen, a key component of fibroglandular breast tissue, as well as tumorigenesis. Our work is based on the hypothesis that loss of BRCA1 in women with high breast density causes abnormal Ubc9 levels which upregulates collagen, fibronectin and inhibits SIRT1, β-catenin expression facilitating TNBC. We tested this hypothesis by studying the expression of total collagen, fibronectin, Ubc9, SIRT1, β-catenin in BRCA1 mutant TNBC cells and tumor sample derived from patient with dense breasts using immunofluorescence, immunohistochemistry, and collagen assay. Our results suggest for the first time that mutation or loss of BRCA1 function in women with fibrocystic breasts can lead to over expression of Ubc9, induction of collagen and; fibronectin, inhibition of SIRT1 and nuclear accumulation of β-catenin which could contribute to TNBC development. This network will aid not only in the identification of potential mechanism-based biomarkers that could detect disease early, but also enforce preventive measures that could reduce the risk for TNBC in women with high MD thus reducing the mortality associated with these cancers to achieve health equity.

Racial Disparity and Triple-Negative Breast Cancer in African-American Women: A Multifaceted Affair between Obesity, Biology, and Socioeconomic Determinants.

Triple negative breast cancer (TNBC) is a molecularly heterogeneous disease whose incidence is disproportionately higher in African American (AA) women compared to European American (EA) women. Earlier onset, more advanced stage at diagnosis, and aggressive tumor phenotype are some of the characteristic features of TNBC in women with African ethnicity in comparison to EA women, denoting one of the most significant examples of racial disparity in oncology. It is still contentious whether health disparities result in aggressive behavior of TNBC in AA women or it is indeed a molecularly distinct disease. Given the “gaps-in-knowledge” surrounding racial disparity in TNBC, this review discusses various socioeconomic factors and the genetic predispositions contributing to poor prognosis of TNBC in AA women. While socioeconomic factors may contribute to poorer survival, multiple preclinical and clinical studies suggest inherent genetic risk factors and aberrant activation of oncogenic pathways in AA TNBC. Additionally, AA women are more likely to be obese and obesity is known to drive a molecular circuitry resulting in aggressive tumor progression indicating a potential obesity-TNBC axis at work in AA women. Given the multifactorial nature of AA TNBC, a transdisciplinary approach may help bridge the disparity that exists between AA and EA TNBC.

Abstract C57: Knowledge of breastfeeding to reduce breast cancer risk

Abstract Background: Increasing evidence suggests that higher parity and lack of breastfeeding associates strongly with the risk of developing the aggressive triple-negative breast cancer (TNBC). There is a 25-50% reduction in risk of developing TNBC in women who have breastfed &amp;gt;4-6 months when compared to parous women who have never breastfed. Higher rates of breastfeeding and lower incidence of BC observed in native African women support this postulate. Observational studies and recent national statistics indicate that the prevalence of breastfeeding is markedly low among African-American women (AAW) (58.9%) compared to Caucasian women (CW) (75.2%). Due to the remarkable health and economic benefit of breastfeeding, we investigated whether patients received information about breast cancer risk reduction as part of breastfeeding counseling and whether this knowledge affected participants' decision to initiate and sustain breastfeeding. Methods: A cross-sectional survey study was conducted from September 2016 to June 2017 at The Ohio State University Comprehensive Cancer Center (OSUCCC) on women aged 18-50 who had at least one live-born child. Participants completed an electronic survey that included demographic information and details of breastfeeding history. Recruitment occurred via primary care secure patient portal electronic messaging (MyChart) and an online national clinical research registry (Research Match). Summary statistics were used to describe responses. Wilcoxon Rank Sum test was used to compare length of breastfeeding. Results: A total of 724 women (496 CW and 152 AAW and 76 unknown) completed the online survey; 503 responses through Research Match, 197 through MyChart, and 24 unknown. 92% of those who responded had breastfed. 56% (60.7% Caucasians and 46.1% AA) of women said they were aware breastfeeding reduced the risk of cancer. Of those who were aware of the risk, 36.4% said that this knowledge affected their decision to breastfeed. Of the 39 who did not breastfeed, 23 (59.0%) said being aware of this risk would have influenced their decision. Only 16.6% of women reported they received this information from health care providers; others were informed through magazines and Internet. The mean length of breastfeeding among women who were aware of this benefit was significantly longer than the women who were not aware (13.2 months vs 9.3 months, respectively, p&amp;lt;0.001). 39.5% of AAW breastfed any one child for less than 6 months compared to 28% of CW. A greater percentage of women with a high school degree or below breastfed any one child for less than 6 months (59.0%) compared to women with some college or higher (30.1%). Most women (66.3%) went back to the workforce within the first year of their most recent birth, and 39.2% of women marked returning to work as a barrier to breastfeeding. Conclusions: Our study showed that there are disparities between AAW and CW in the knowledge that breastfeeding reduces the risk of breast cancer and in the duration of breast feeding. In addition, we showed that increased knowledge impacts decision-making regarding breastfeeding. Hence, development of culturally relevant communication strategies, especially in AAW, can impact initiation and maintenance of breastfeeding, and ultimately result in aggressive breast cancer reduction. Citation Format: Akaansha Ganju, Anupama Suresh, Julie Stephens, Marilly Palettas, Diana Burke, Laura Miles, Maryam Lustberg, Seuli Brill, Bhuvaneswari Ramaswamy. Knowledge of breastfeeding to reduce breast cancer risk [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C57.

Abstract C23: High Kaiso expression correlates with increased TGFβ signaling and aggressive TNBC in women of African Ancestry (WAA)

Abstract Despite increased awareness, early detection and improved therapies in the past 20 years, breast cancer (BC) remains the most frequent female cancer and a leading cause of female deaths worldwide. One of the most clinically challenging BC is the Triple Negative Breast Cancer (TNBC) subtype that is estrogen receptor (ER), progesterone receptor (PR) &amp; human epidermal growth factor receptor-2 (HER2) negative. TNBC tumors are aggressive and metastatic, and have a poor prognosis since they cannot be treated with standard therapies (Tamoxifen or Herceptin). Intriguingly TNBC is most prevalent in young women of African Ancestry (WAA) who, despite having lower BC incidence rates than Caucasian women, have higher mortality rates. The propensity of TNBC to metastasize suggests a deregulation of the epithelial-to-mesenchymal transition (EMT) pathway that endows cells with increased motility, and is implicated in tumor progression. Our lab and others recently found a significant correlation between high expression of the POZ-ZF transcription factor Kaiso, EMT, estrogen receptor (ER) negativity in WAA, and shorter metastasis-free survival. Notably when Kaiso is depleted in TNBC cell lines, the cells exhibited decreased TGFβ signaling (a known promoter of EMT), become less motile and invasive in vitro, and did not metastasize to lungs or liver in mouse xenograft models. These findings led us to hypothesize that Kaiso plays a role in the spread of aggressive BC possibly via up-regulation of TGFβ signaling. To gain insight into Kaiso's role in aggressive breast cancers and the racial disparity associated with BC outcomes, we generated a tissue microarray (TMA) comprised of TNBC tissues from Barbadian and Nigerian WAA, and performed expression profiling of Kaiso and various TGFβ signaling proteins (TGFβR1, TGFβR2 and p-Smad2) on the WAA-TMA. We found that high Kaiso expression correlated positively with increased TGFβ signalling in WAA TNBC tissues, thus supporting our hypothesis that Kaiso plays a role in TGFβ signaling and TNBC aggressiveness. Notably, we also observed that Kaiso depletion resulted in delayed onset of mouse xenograft tumors derived from MDA-231 TNBC cells. Mechanistic in vitro studies further revealed that Kaiso depletion reduced cell proliferation and increased apoptosis of TNBC cells, especially in response to the DNA-damaging drug Etoposide. Our findings linking high Kaiso expression to TNBC cell survival and active TGFβ signalling in aggressive TNBCs from WAA, raises the exciting possibility that Kaiso may be developed as a diagnostic and prognostic marker for TNBC patients regardless of race. Ongoing experiments are focused on elucidating Kaiso's exact role in aggressive breast tumors and the racial disparities in breast cancer outcomes. Citation Format: Blessing I. Bassey-Archibong, Lyndsay GA Rayner, Shawn Hercules, Sinthu Pathmanapan, Juliet M. Daniel. High Kaiso expression correlates with increased TGFβ signaling and aggressive TNBC in women of African Ancestry (WAA). [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C23.

Abstract B48: Clues to the causes of the abundance of triple-negative breast cancer in women of African descent

Abstract Background: Triple negative breast cancer (TNBC) is over-represented in indigenous African women and in women of African descent. The majority of women diagnosed with breast cancer in Nigeria, Uganda and Kenya have TNBC. Studies of TNBC in the US consistently report the percentage of TNBC to be 10-24% greater in African-Americans (AA) than in Caucasians (C). The abundance of TNBCs in both African and African-American women has yet to be explained and the incidence rates suggest that there may be a genetic predisposition to this particularly aggressive form of breast cancer. Methods: Epithelial cell lines were established from normal, healthy breast tissue donated to the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. Six cell lines from AA donors were matched on the basis of age, menopausal status and Gail-risk score to six cell lines from C donors. RNA was extracted from the cells and gene expression assayed using the Illumina HumanWG6 v3 chip. Significant differences in gene expression were identified using PADE (PAirwise Differential Expression; Expression Analysis, Durham, NC). Results: 1552 genes were differentially expressed (p≤ 0.05). The largest number of genes over-expressed in AAs mapped to the long arm of chromosome 1. Copy number gains have been observed in this region in multiple TNBC data sets. Many of these genes are in the 1q21 region, a region frequently rearranged in epithelial tumors as well as soft tissue and bone. Analysis of differentially expressed genes using MetaCore from Thomson Reuters revealed that the over-expressed genes are responsible for skin development, cell adhesion/cell junctions, and functions of the innate immune system. Pattern recognition is in the vanguard of the innate immune system. The pattern recognition pathways mediated by RAGE (receptor for advanced glycation end products) and the Toll-like receptors are activated in the normal AA epithelium. The expression of genes in these pathways, specifically S100A12, S100A8, S110A9, and TLR5, is known to increase in the presence of Salmonella and Shigella. MDA5 recognizes rotavirus infection. LYST and DUSP4 are active in the control of Leishmania infection. LCN2 disrupts iron acquisition, which protects against a number of pathogens including Salmonella, Klebsiella and mycobacteria. Conclusions: The results of the above gene expression analysis are best understood in light of our recently published findings using these same cells (Sauder et al, BMC Cell Biology, 2014). Epithelial cells grown from mammary explant culture are basal-like and phenotypically plastic. They can assume a wide range of cell types depending on the cues provided by the microenvironment. When grown in basement membrane matrix or on human dermal fibroblasts, these cells produce a stratified squamous epithelium. Although the cells used for the gene expression analysis were grown in plastic tissue culture flasks and grew as a monotonous monolayer, the data suggest that they are, to some extent, transcriptionally “squamous”. For example, they express involucrin and the genes that code for the corneodesmosome proteins: corneodesmin, desmoglein and desmocollin. The skin of humans living in sub-Saharan Africa has evolved to provide optimal barrier protection. It specifically protects against water loss and, pathogenic microorganisms and parasites. Mammary glands are ectodermal appendages of the epithelium and they may share similar immune defense mechanisms. The increased expression of genes involved in innate immunity in AA breast epithelial cells is of significant interest. We hypothesize that a subclinical, chronic inflammatory state is incited within basal breast cells in response to a pathogen that is endemic to Africa and overrepresented in low income communities in the US. This persistent and unresolved innate immunity predates the development of TNBC and can thus participate in its initiation and promotion. Citation Format: Candice A.M. Sauder, Jillian E. Koziel, MiRan Choi, Brittney-Shea Herbert, Susan E. Clare. Clues to the causes of the abundance of triple-negative breast cancer in women of African descent. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B48.

Abstract IA19: Regulation of 53BP1 in BRCA1-deficient cells: Why vitamin D matters and how disparities in vitamin D levels could promote aggressive TNBC in women of color

Abstract Breast cancer is the leading cause of cancer death in women worldwide. BRCA1-deficient and triple-negative breast cancers (TNBC) are particularly aggressive, strike younger, Hispanic and African American women, and are most difficult to treat. These tumors exhibit DNA repair defects, making them vulnerable to DNA damaging therapies. Currently, the most promising therapeutic agents are poly(ADP-ribose) polymerase inhibitors (PARPi), which render cells already deficient in DNA repair, with an unbearable load of DNA damage. Excitement about PARPi has been fueled by clinical trials showing that these compounds, either as single agents or in combination with chemotherapy, have very encouraging outcomes in BRCA1-related and TNBC and with minimal toxicity. However, a significant fraction of these cancers acquire resistance to PARPi. Understanding the molecular mechanisms that confer PARPi resistance will have a big therapeutic impact. Elegant studies demonstrated that loss of the DNA repair factor 53BP1 contributes to progression of BRCA1-deficient and TNBC and to their resistance to PARPi. Thus, raising the levels of 53BP1 in these tumors could represent a new therapeutic strategy. Our team identified a new pathway modulating the levels of 53BP1, which could be exploited to stabilize protein levels. We found that BRCA1-deficient cells activate the degradation of 53BP1 by the protease cathepsin-L (CTSL), and that CTSL inhibition by vitamin D stabilizes 53BP1, reduces proliferation, and increases PARPi and radiation sensitivity. Our studies in human breast tumors also suggest that deficiencies in vitamin D/vitamin D receptor (VDR) axis, more prevalent in women of color, could underlie the activation of CTSL-mediated degradation of 53BP1 in BRCA1-deficient and TNBC. Our current studies are testing the ability of vitamin D treatment to reduce the growth of BRCA1-deficient and TNBC cells, as well as their resistance to PARPi, providing a novel modality of treatment for these cancer patients. Citation Format: Susana Gonzalo. Regulation of 53BP1 in BRCA1-deficient cells: Why vitamin D matters and how disparities in vitamin D levels could promote aggressive TNBC in women of color. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr IA19.

Triple-negative vimentin-positive heterogeneous feline mammary carcinomas as a potential comparative model for breast cancer.

BackgroundHuman breast cancer is a heterogeneous disease classified by molecular subtyping into luminal A, luminal B, HER2-overexpressing, basal-like, claudin-low and normal-breast like. The routinely applied and standardized immunohistochemical-based surrogates of this classification group together the last three entities as triple-negative breast cancer (TNBCs) that show the most diverse and complex heterogeneity and represent a therapeutic challenge.In the present work 156 feline mammary lesions consisting of feline mammary carcinomas (FMCs), benign neoplasms, and hyperplastic/dysplastic tissues were evaluated histologically and by immunohistochemistry for expression of basal and luminal cytokeratins (CK), vimentin, alpha-smooth muscle actin, calponin, estrogen receptor (ER) alpha (a), and progesterone receptor (PR). Thirty-seven FMCs with 27 matched non-neoplastic controls were also investigated for gene expression of ERa, ER beta, PR, and HER2.ResultsA large group of hormone receptors (HRs)-negative aggressive carcinomas - that did not overexpress HER2 - could be distinguished from the less aggressive (10.8%) and benign (8%) HRs + tumors, that showed bilineage (luminal and myoepithelial) differentiation. Immunohistochemical evaluations of cytoplasmic filaments indicated that HRs- FMCs are vimentin+, CK14+, and CK5_6+ carcinomas that may resemble the TNBCs (basal like/claudin low) described in women. The identification of luminal and myoepithelial progenitors within the mammary ductal system suggested potential cells/sites of origin of these tumors. A diffuse and never previously described CKs/vimentin luminal cell co-expression was detected in the non-neoplastic ducts, indicating a potential bilineage progenitor.ConclusionsThese results indicate and potentially explain the high incidence of triple-negative, vimentin + aggressive tumors in cats that may used to elucidate some of the challenging features of TNBCs in women.

Prevalence of BRCA2 mutations and other clinical characteristics in women with triple-negative breast cancer.

160 Background: Triple-negative breast cancers (TNBC) represent 10%–20% of invasive breast cancers. Current guidelines recommend genetic testing for women who are diagnosed with TNBC. Studies have shown that BRCA1 mutations are associated with TNBC, but there is little information on the relationship of BRCA2 mutations and TNBC. The purpose of this study was to look at the clinical characteristics of TNBC compared to non-TNBC in a cohort of women with newly diagnosed breast cancer. Methods: The Breast Cancer Database at our institution was queried for patients with invasive breast cancer. We included the following variables: age, race, BRCA1,2, tumor characteristics, and personal history of breast cancer (PHBC). Statistical analyses included Pearson’s Chi-Square and Fisher’s Exact Tests. Results: Out of a total of 1,332 women, 125 (9%) had TNBC. The median age for both TNBC and non-TNBC was 59 years. Majority of women had early stage breast cancer (92%) with ductal carcinoma (80%). There was a significantly higher proportion of Blacks and Asians with TNBC (p &lt; 0.0001). Women with TNBC had higher Ki-67 (p &lt; 0.0001). Within the TNBC group, there were 12 (29%) patients who tested positive for BRCA1,2 mutation and 23 (8%) who tested positive for BRCA 1,2 mutations in the non-TNBC group. Interestingly, BRCA1 was not associated with TNBC (p = 0.40) and BRCA2 was significantly associated with TNBC (p &lt; 0.0001). We also found a higher proportion of TNBC in women who had a PHBC (p = 0.01). Conclusions: In our study, women with TNBC were similar in age to women who did not have TNBC. We found that the women with TNBC in our cohort had elevated rates of BRCA2 mutations. We also found that women with a personal history of breast cancer were at risk for developing TNBC. This may be related to the use of hormonal therapy that reduces the risk of ER/PR-positive tumors. Women of all ages are at risk for developing TNBC and older age at TNBC should not deter from genetic testing.