Androgen Receptor In Triple-negative Breast Cancer Research Articles

Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy

Extensive exploration of the molecular subtypes of triple-negative breast cancer (TNBC) is critical for advancing precision medicine. Notably, the luminal androgen receptor (LAR) subtype has attracted attention for targeted treatment combining androgen receptor antagonists and CDK4/6 inhibitors. Unfortunately, this strategy has proven to be of limited efficacy, highlighting the need for further optimization. Using our center’s comprehensive multiomics dataset (n = 465), we identified novel therapeutic targets and evaluated their efficacy through multiple models, including in vitro LAR cell lines, in vivo cell-derived allograft models and ex vivo patient-derived organoids. Moreover, we conducted flow cytometry and RNA-seq analysis to unveil potential mechanisms underlying the regulation of tumor progression by these therapeutic strategies. LAR breast cancer cells exhibited sensitivity to chidamide and enzalutamide individually, with a drug combination assay revealing their synergistic effect. Crucially, this synergistic effect was verified through in vivo allograft models and patient-derived organoids. Furthermore, transcriptomic analysis demonstrated that the combination therapeutic strategy could inhibit tumor progression by regulating metabolism and autophagy. This study confirmed that the combination of histone deacetylase (HDAC) inhibitors and androgen receptor (AR) antagonists possessed greater therapeutic efficacy than monotherapy in LAR TNBC. This finding significantly bolsters the theoretical basis for the clinical translation of this combination therapy and provides an innovative strategy for the targeted treatment of LAR TNBC.

MicroRNAs Associated with Androgen Receptor and Metastasis in Triple-Negative Breast Cancer

It is crucial to identify novel molecular biomarkers and therapeutic targets for triple-negative breast cancer (TNBC). The androgen receptor (AR) is a regulator of TNBC, acting partially via microRNA molecules (miRNAs). In this study, we used PCR arrays to profile the expression of 84 miRNAs in 24 TNBC tissue samples, which were equally classified according to AR expression and/or metastasis. Several bioinformatics tools were then utilized to determine the potentially affected protein targets and signaling pathways. Seven miRNAs were found to be significantly more highly expressed in association with AR expression, including miR-328-3p and miR-489-3p. Increased expression of miR-205-3p was found to be significantly associated with metastasis. Certain miRNAs were specifically found to be differentially expressed in either metastatic or non-metastatic AR-positive tumors. A gene ontology (GO) analysis indicated biological roles in the regulation of transcription, cellular response to DNA damage, and the transforming growth factor-beta (TGF-beta) signaling pathway. The GO analysis also showed enrichment in kinase and transcription factor activities. The TGF-beta and a number of kinase-dependent pathways were also retrieved using the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. This study offers an understanding of the role of AR in TNBC and further implicates miRNAs in mediating the effects of AR on TNBC.

Estrogen receptor beta suppresses the androgen receptor oncogenic effects in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer associated with poor prognosis and limited treatment options. The androgen receptor (AR) has emerged as a potential therapeutic target for luminal androgen receptor (LAR) TNBC. However, multiple studies have claimed that anti-androgen therapy for AR-positive TNBC only has limited clinical benefits. This study aimed to investigate the role of AR in TNBC and its detailed mechanism. Immunohistochemistry and TNBC tissue sections were applied to investigate AR and nectin cell adhesion molecule 4 (NECTIN4) expression in TNBC tissues. Then, in vitro and in vivo assays were used to explore the function of AR and estrogen receptor beta (ERβ) in TNBC. Chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (co-IP), molecular docking method, and luciferase reporter assay were performed to identify key molecules that affect the function of AR. Based on the TNBC tissue array analysis, we revealed that ERβ and AR were positive in 21.92% (32/146) and 24.66% (36/146) of 146 TNBC samples, respectively, and about 13.70% (20/146) of TNBC patients were ERβ positive and AR positive. We further demonstrated the pro-tumoral effects of AR on TNBC cells, however, the oncogenic biology was significantly suppressed when ERβ transfection in LAR TNBC cell lines but not in AR-negative TNBC. Mechanistically, we identified that NECTIN4 promoter -42bp to -28bp was an AR response element, and that ERβ interacted with AR thus impeding the AR-mediated NECTIN4 transcription which promoted epithelial-mesenchymal transition in tumor progression. This study suggests that ERβ functions as a suppressor mediating the effect of AR in TNBC prognosis and cell proliferation. Therefore, our current research facilitates a better understanding of the role and mechanisms of AR in TNBC carcinogenesis.

High phosphorylated cyclin-dependent kinase 2 expression indicates poor prognosis of luminal androgen receptor triple-negative breast cancer.

High phosphorylated cyclin-dependent kinase 2 expression indicates poor prognosis of luminal androgen receptor triple-negative breast cancer.

Abstract 711: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability

Abstract Targeted therapy for triple-negative breast cancer (TNBC) remains challenging due to poor understanding of its molecular etiology. Previous studies have shown that androgen receptors (AR) are widely expressed in approximately one-third of TNBC, and are referred to as luminal androgen receptors (LAR). AR has recently emerged as a prognostic and treatment-predictive biomarker in breast cancer. However, the role of AR in TNBC remained elusive. Treatment with AR-antagonist have been shown to suppress AR activation and signaling in TNBC and results in a corresponding reduction in tumor cell proliferation and survival due to the initiation of apoptosis in these tumor cells. γ-Tocotrienol (γT3) is a natural isoform of vitamin E that displays potent anticancer activity. Results show that treatment with 0-7 μM γT3 induces a dose-responsive significant decrease, whereas treatment with 35 nM dihydrotestosterone (DHT) results in a significant increase of AR expression and corresponding increase in growth of MDA-MB-453 TNBC cells. Combined treatment of 7 μM γT3 with 35 nM DHT significantly inhibits DHT-induced proliferation and AR activation in MDA-MB-453 cells. Western blot analysis shows that combined treatment with γT3 and DHT induced a significant decrease in phosphorylated ERK1/2 (active form) expression, but had no effect on total ERK1/2 levels in MDA-MB-453 TNBC cells. Flow cytometry analysis shows that γT3 treatment was also found to induce G0/G1 cell cycle arrest and significantly increased the number of apoptotic and necrotic cells, as compared to MDA-MB-453 TNBC cells in the vehicle-treated control group. Western blot analysis shows that γT3 treatment also inhibited DHT-induced elevations in CDK2, CDK4, CDK6 and cyclin D1 expression (cell cycle progression biomarkers) and induces a significant increase in the expression of tumor suppressor protein, p53, in MDA-MB-453 TNBC cells. Similar treatment with 7 µM γT3 induces a corresponding significant increase in apoptosis biomarker expression of cleaved caspase-3 and Bax in MDA-MB-453 TNBC cells. In summary, results from these studies demonstrated that γT3 treatment inhibits AR expression, activation and signaling, as well as DHT-dependent cell proliferation and viability in TNBC. These finding also suggest that the LAR may be a potential therapeutic target for the treatment TNBC. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability [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 711.

Endoplasmic reticulum stress inhibits AR expression via the PERK/eIF2\u03b1/ATF4 pathway in luminal androgen receptor triple-negative breast cancer and prostate cancer

Androgen receptor (AR) is an important prognostic marker and therapeutic target in luminal androgen receptor triple-negative breast cancer (LAR TNBC) and prostate cancer (PCa). Endoplasmic reticulum (ER) stress may activate the unfolded protein response (UPR) to regulate associated protein expression and is closely related to tumor growth and drug resistance. The effect of ER stress on AR expression and signaling remains unclear. Here, we focused on the regulation and underlying mechanism of AR expression induced by ER stress in LAR TNBC and PCa. Western blotting and quantitative RT-PCR results showed that AR expression was markedly decreased under ER stress induced by thapsigargin and brefeldin A, and this effect was dependent on PERK/eIF2α/ATF4 signaling activation. Chromatin immunoprecipitation-PCR and luciferase reporter gene analysis results showed that ATF4 bound to the AR promoter regions to inhibit its activity. Moreover, ATF4 overexpression inhibited tumor proliferation and AR expression both in vitro and in vivo. Collectively, these results demonstrated that ER stress could decrease AR mRNA and protein levels via PERK/eIF2α/ATF4 signaling in LAR TNBC and PCa. Targeting the UPR may be a treatment strategy for AR-dependent TNBC and PCa.

QNBC Is Associated with High Genomic Instability Characterized by Copy Number Alterations and miRNA Deregulation.

Triple-negative breast cancer (TNBC) can be further classified into androgen receptor (AR)-positive TNBC and AR-negative TNBC or quadruple-negative breast cancer (QNBC). Here, we investigated genomic instability in 53 clinical cases by array-CGH and miRNA expression profiling. Immunohistochemical analysis revealed that 64% of TNBC samples lacked AR expression. This group of tumors exhibited a higher level of copy number alterations (CNAs) and a higher frequency of cases affected by CNAs than TNBCs. CNAs in genes of the chromosome instability 25 (CIN25) and centrosome amplification (CA) signatures were more frequent in the QNBCs and were similar between the groups, respectively. However, expression levels of CIN25 and CA20 genes were higher in QNBCs. miRNA profiling revealed 184 differentially expressed miRNAs between the groups. Fifteen of these miRNAs were mapped at cytobands with CNAs, of which eight (miR-1204, miR-1265, miR-1267, miR-23c, miR-548ai, miR-567, miR-613, and miR-943), and presented concordance of expression and copy number levels. Pathway enrichment analysis of these miRNAs/mRNAs pairings showed association with genomic instability, cell cycle, and DNA damage response. Furthermore, the combined expression of these eight miRNAs robustly discriminated TNBCs from QNBCs (AUC = 0.946). Altogether, our results suggest a significant loss of AR in TNBC and a profound impact in genomic instability characterized by CNAs and deregulation of miRNA expression.

Clinicopathological Characteristics and Androgen Receptor Expression of Triple Negative Breast Cancer: A Retrospective Study in Upper Egypt

Background: Breast cancer is a big challenging problem for public health. Triple negative breast cancer (TNBC) is a subtype of breast cancer which is more aggressive; with no specific treatment guidelines are available. It was noted that androgen receptor (AR) is expressed in TNBC, suggesting that anti-androgens are a promising strategy for treatment. Recently, it was found racial differences in the biologic behavior of TNBC. Objectives: This study aims to assess the clinicopathological features of TNBC in Upper Egypt, and to evaluate the immunohistochemical (IHC) expression of AR in TNBC, in comparison to hormone receptor positive (HR+) invasive duct carcinomas (IDC) as a control group. Patients and Methods: IHC expression of AR was examined in 45 TNBC specimens and 15 HR+ IDC (ER+ and PR+/-). Results: Most of the TNBC cases were ≤50 years and their tumors had high histological grade, presence of lymphovascular emboli, lymph node metastasis and tumor necrosis while few cases revealed duct carcinoma in situ (DCIS) and most patients were stage I & II.TNBC group had significantly younger age, DCIS, higher nodal metastasis and necrosis compared to control group. AR expression was significantly higher in the control group.  Conclusion: TNBC occurs at young age and is associated with poor clinicopathological criteria, suggesting the presence of underlying BRCA mutations in our population. AR was expressed in a subset of TNBC, this makes AR an interesting drug target for those patients. Further molecular studies are recommended to uncover pathways of TNBC pathogenesis and improve its therapeutic options.

AR Is Not an Independent Marker for TNBC: The Lesson We Learn From Two PDX Models

Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. On the other hand, patient-derived xenografts (PDXs) are generally considered more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient’s tumor, but only a few have been reported to be AR-positive TNBC. In our study, we identified and molecularly characterized two new, AR-positive TNBC PDX models and assessed the impacts of AR agonist (DHT) and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry (IHC), western blots, and RNA-Seq and TNBC subtyping analyses. Two PDX models, termed TN1 and TN2, were derived from two grade 3 TNBC tumors, each containing 1~5% of AR positive tumor cells. DHT activated AR in both PDX tumors by increasing AR nuclear localization and protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while the DHT promoted gene expression in TN2 (673 up-regulated genes versus192 down-regulated genes). TNBC subtyping analyses based on RNA-Seq data predicted distinct molecular subtypes of TN1 and TN2: TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models that expand our current knowledge of AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.

Functional characterization of androgen receptor in two patient-derived xenograft models of triple negative breast cancer

Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. Considering that patient-derived xenografts (PDXs) are more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient’s tumor, we have identified and molecularly characterized two new AR-positive TNBC PDX models and assessed the impacts of AR agonist [dihydrotestosterone (DHT)] and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry, western blots, and RNA-Seq analyses. Two PDX models, termed TN1 and TN2, were derived from two grade-3 TNBC tumors, each harboring 1∼5% of AR nuclear positive cancer cells. DHT activated AR in both PDX tumors by increasing nuclear localization and AR protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT mainly suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while DHT promoted gene expression in TN2 (673 up-regulated genes versus 192 down-regulated genes). RNA-Seq data predicted distinct TNBC molecular subtypes for TN1 and TN2. TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models. The molecular characterization of these PDX models expands our current knowledge on AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.

Abstract PR18: Prognostic role of androgen receptor in triple-negative breast cancer: A global multi-institutional experience

Abstract Background: African American (AA) triple-negative breast cancer (TNBC) patients experience worse clinical outcomes and exhibit 40% higher mortality rate than their European Americans (EAs) counterparts. There are currently no distinctions in inherent tumor biology between the ethnically distinct TNBC patients that serve as risk-predictive markers allowing new tailored treatments. Recently, androgen receptor (AR) has emerged as a new target for treating TNBC. However, the prognostic value of AR in TNBC remains controversial. To reconcile conflicting reports about the impact of AR loss on prognosis of TNBC and uncover the molecular pathways that may underlie the racial disparity in outcomes among AR-negative TNBCs, we determined the prognostic value of AR in diverse TNBC cohorts. Since loss of AR is associated with worse clinical outcome and African and AA women are more prone to aggressive disease course, we hypothesized that AR loss may underlie the global disparate burden in TNBC. Methods: We evaluated AR expression in well-annotated formalin-fixed, paraffin-embedded TNBC resection samples (n=1351) obtained from multiple hospitals from US, UK, Norway, Ireland, Nigeria and India. Samples with ≥1% nuclei staining positive for AR were deemed to be AR-positive. Associations between AR status, clinicopathologic variables and overall survival (OS) were evaluated. We performed gene set enrichment analysis for AR low and high group in AA and EA TNBCs. In vitro experiments were performed to examine whether AR loss increased the metastatic potential of TNBC cells. Results: We observed a significant difference in AR expression among EA and AA TNBCs (p=0.02) with AR loss associated with women of African ancestry (>90%, p<0.05). AR loss was significantly associated with poor OS in TNBC patients from US cohort (p=0.0324; n=316 for AR-negative, n=104 for AR-positive) and Nigerian cohort (p=0.0251; AR-negative=164, AR-positive=16). AR-negative was associated with poor OS in adjuvant-treated high Ki67 (>14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). Furthermore, AR status retained its significant prognostic value (HR=1.549, p=0.036) after controlling for age, grade, Ki67, race and chemotherapy status. Gene set enrichment analysis revealed that Wnt/β-catenin signaling was the top-enriched gene ontology in the AR-low TNBC subgroup. Moreover, β-catenin protein levels are higher in AA AR-low TNBCs compared with AA AR-high TNBCs (p<0.05), suggesting Wnt signaling upregulation in AA women with AR-negative TNBC. In TNBC cell lines, loss of AR was significantly associated with higher cell proliferation, migration and invasion (p<0.05). Conclusion: Our study suggests a striking association of AR loss in TNBC with women of African ancestry. Our data offer compelling evidence that oncogenic Wnt/β-catenin signaling may link AR loss to more aggressive disease course and represent actionable biology in AA AR-negative TNBCs for whom no targeted treatments are currently on the horizon. This abstract is also being presented as Poster C102. Citation Format: Shristi Bhattarai, Sergey Klimov, Karuna Mittal, Uma Krishnamurthi, Xiaoxian Bill Li, Deepika Wali, Ceyda Sonmez Wetherilt, Ansa Riaz, Mohammad A. Aleskandarany, Andrew R. Green, Ian O. Ellis, Meenakshi Gupta, Lauren E. McCullough, Upender Manne, Johnson Agboola, Brett Baskovich, Emiel A. Janssen, Grace Callagy, Anurag Mehta, Tanuja Shet, Rakha A. Emad, Padmashree C.G. Rida, Ritu Aneja. Prognostic role of androgen receptor in triple-negative breast cancer: A global multi-institutional experience [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr PR18.

PO-446 Targeting co-regulators of hormone-receptors as a novel therapeutic approach for prostate and breast cancer

IntroductionCurrent treatments for prostate cancer mainly target the Androgen Receptor (AR), however despite an initial response these treatments fail. Among the multiple mechanisms of resistance, aberrations in AR co-factors and co-regulators represent a promising therapeutic approach since co-factors and co-regulators are not susceptible to AR resistance mechanisms. Triple negative breast cancer (TNBC) is an aggressive form of breast cancer (BC) which lacks oestrogen receptor, progesterone receptor, and HER2 amplification. Due to the fact that a proportion of TNBC express AR and that AR expression has been associated with poor prognosis, targeting AR in TNBC is attracting increasing interest. We previously identified Serum Response Factor (SRF) as an important transcription factor in an in vitro model of castrate-resistant prostate cancer (CRPC). We showed SRF association with CRPC and survival using TMAs of CRPC tissues. A cross-talk between AR and SRF in vitro and in clinical samples was also demonstrated. Using the SRF inhibitor CCG1423, we showed that combination of CCG1423 and Enzalutamide, a new-generation AR-targeting agent, is significantly more effective than monotherapies. The aim of this study was to extend our discoveries from prostate to breast cancer.Material and methodsThree TNBC cell lines with different expression levels of SRF and AR were selected to investigate the effect of SRF inhibition on cell viability and migration. CCG1423 was used to inhibit SRF. Cell viability was assessed by MTT assay. Scratch assays were used to analyse cell migration. Immunohistochemistry (IHC) was used to assess SRF expression in 3 BC TMAs: TMA1 (n=144) and TMA2 (n=512) with different subtypes of BC and TMA3 with 138 TNBC patients.Results and discussionsMTT assays showed response to CCG1423 in the two cell lines positive for SRF (MDA-MB-231 and HS578t) with IC50 values of 20 uM, while MDA-MB-453 (SRF negative) did not respond (IC50 >80 uM). Scratch assays showed a slower gap closure in the cells treated with CCG1423 compared to controls for the SRF positive cell lines, while there was no difference in the MDA-MB-453. IHC staining for SRF for the 3 TMAs is currently being performed.ConclusionOur preliminary data suggest that SRF inhibition is effective in reducing cell viability and migration in BC cell lines. Due to SRF cross-talk with AR, its inhibition in combination with current anti-androgens represents a promising therapeutic approach not only for CRPC but also for TNBC.

Abstract P1-09-05: Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (INO-464), a selective CYP17 lyase and AR inhibitor

Abstract Background: Increased rates of locoregional recurrence (LR) have been observed in TNBC despite chemotherapy and radiation (RT). A novel radiosensitizer screen nominated the AR as a promising target for treatment of radioresistant breast cancer, including TNBC. We assessed the activity of seviteronel (Sevi), a selective CYP17 lyase and AR inhibitor in Phase 2 clinical development for advanced breast and prostate cancer, as a potential radiosensitizer in AR+ TNBC model. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 breast cancer cell (BCC) lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq or qRT-PCR and protein expression was measured using RPPA arrays. AR function was assessed using functional inhibition with Sevi in MDA-MB-453, ACC-422, ACC-460, SUM-185 (all four AR+ TNBC), MDA-MB-231 (AR- TNBC), and T47D (AR- ER+) BCC lines. Double-stranded DNA (dsDNA) break repair was assessed with γH2AX foci counting. In vivo tumor growth was measured with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control. A Cox proportional hazards model and multi-variate analysis (MVA) were used to determine variables associated with LRF survival. Results: Our novel radiosensitizer screen identified the activity of anti-androgen therapy as a potentially effective strategy for radiosensitization in RT-resistant BCC lines (R2 =0.46, p-value < 0.01) (Speers et al, J Clin Oncol 35, 2017 (suppl; abstr e12102). Heterogeneity in AR expression was identified in human BCC lines and TNBC samples from patients (N=2098). There was a strong correlation between AR RNA expression and protein expression across all BC intrinsic subtypes. AR inhibition using Sevi induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.24-1.69 in four different AR+ TNBC lines. No such radiosensitization was seen in AR(-) TNBC or ER+, AR(-) BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair with significant delays in dsDNA break repair at 16 and 24 hours in all AR+ TNBC lines examined (p-value < 0.01). AR inhibition with Sevi significantly radiosensitized AR+ TNBC xenografts in mouse models and markedly delayed tumor-volume tripling time (TTT) and tumor growth (MDA-MB-453: median TTT 16.1 days for RT alone vs. not reached after 45 days for Sevi+RT, p-value <0.001). Similar delays were seen in tumor growth, weight, and tumor doubling. Clinically, TNBC patients whose tumors had higher than median expression of AR had higher rates of LR after RT (HR for LR ˜3, p-value <0.01, 2 independent datasets). In MVA, high AR expression was the variable most significantly associated with worse LR survival after RT in TNBC patients, outperforming all other variables (HR of 3.42; p-value < 0.01). Conclusions: Our results implicate the AR as a mediator of radioresistance in breast cancer and support the rationale for developing Sevi as a novel radiosensitizing agent in AR+ TNBC. Citation Format: Speers CW, Chandler B, Olsen E, Wilder-Romans K, Moubadder L, Nyati S, Rae J, Hayes DF, Spratt DE, Wahl DR, Eisner J, Feng FY, Pierce LJ. Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (INO-464), a selective CYP17 lyase and AR inhibitor [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-05.

Androgen Receptor-Targeted Therapy for Breast Cancer

Emerging evidence has identified the androgen receptor (AR) pathway as a potential driver for breast cancer (BC) carcinogenesis. The prevalence of AR expression differs across breast cancer subtypes, and its prognostic role in BC is not clear. Triple-negative breast cancer (TNBC) is a heterogeneously diverse disease, which includes a subset that may be androgen driven. This review will discuss the role of AR across the differing subtypes of BC and summarize the most recent clinical trial data for the use of androgen-directed therapy in the treatment of AR+ breast cancer, with a particular emphasis on TNBC. Preclinical and clinical data support the activity of the anti-androgen bicalutamide and more recent, next-generation, AR-targeted agents such as enzalutamide in targeting the AR in TNBC. In addition, novel agents which reduce androgen production such as abiraterone acetate and seviteronel are now being tested in BC. As our understanding of the interplay between AR and signaling pathways involving ER and HER2 grows, dual pathway inhibition may prove to be beneficial through informative, combinatorial approaches such as AR antagonism with CDK4/6 pathway inhibitors or PI3K inhibitors. Over the recent years, there is accumulating evidence that AR signaling is a relevant driver for some subsets of breast cancer. Several agents have now consistently demonstrated a signal of activity when targeting the androgen receptor in TNBC in particular. We need to better define which patients are most likely to benefit from an AR-directed approach. Ongoing and future trials aim to clarify the role of targeting AR with single agent and combinatorial therapies in both the metastatic and adjuvant setting. It is hoped that these trials, which incorporate informative correlative components, will further our understanding of the biology of AR+ BC and will show the way to an increased number of treatment options and improved outcomes for our patients.

Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (SEVI), a selective CYP17 lyase and AR inhibitor.

e12102 Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). A novel radiosensitizer screen nominated the AR as a promising target for treatment of radioresistant breast cancer, including TNBC. We assessed in vitro activity of SEVI (VT-464), a selective CYP17 lyase and AR inhibitor, as a potential radiosensitizer in AR+ TNBC model. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 breast cancer cell (BCC) lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq or qRT-PCR and protein expression was measured using RPPA arrays. AR function was assessed using functional inhibition with SEVI in MDA-MB-453, ACC-460, SUM-185 (all three AR+ TNBC), MDA-MB-231 (AR- TNBC), and T47D (AR- ER+) BCC lines. Double-stranded DNA (dsDNA) break repair was assessed with γH2AX foci counting. Results: Our novel radiosensitizer screen identified the activity of bicalutamide, an AR antagonist, in RT-resistant BCC lines (R2 = 0.46, p-value < 0.01). Heterogeneity in AR expression was identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression across all BC intrinsic subtypes. AR inhibition using SEVI induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.24-1.62 in three different AR+ TNBC lines. No such radiosensitization was seen in AR(-) TNBC or ER+, AR(-) BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair with significant delays in dsDNA break repair at 16 and 24 hours in all AR+ TNBC lines examined (p-value < 0.01). Conclusions: Our results implicate the AR as a mediator of radioresistance in breast cancer and support the rationale for developing seviteronel as a novel radiosensitizing agent in AR+ TNBC.

The Androgen Receptor Promotes Cellular Proliferation by Suppression of G-Protein Coupled Estrogen Receptor Signaling in Triple-Negative Breast Cancer

Background/Aims: The targeted therapy for triple-negative breast cancer (TNBC) is still challenging due to poor understanding on its molecular etiology. The androgen receptor (AR) has recently emerged as a prognostic and treatment-predictive marker in breast cancer. However, the role of AR in TNBC remained elusive. Methods: Immunohistochemistry (IHC) was used to detect AR and G-protein coupled estrogen receptor (GPER) expression in tissue microarrays of 165 TNBC patients. Microarray analysis of mRNAs was performed to identify downstream regulators of AR. TNBC cells were cultured with dihydrotestosterone (DHT) alone or in combination with AR knockdown performed with AR shRNA. Cell viability and colony formation were assessed. Western blotting and qRT-PCR were used to examine protein and mRNA expression, respectively. The potential mechanism of AR-mediated GPER suppression was identified by Chromatin immunoprecipitation (ChIP) assay. AR and GPER expressions were also assessed in nude mouse xenografts by IHC. Results: IHC staining showed that the expression of AR was positively associated with tumor size, lymph node metastasis and high-grade tumor in TNBC patients. AR activation triggered by DHT suppressed GPER expression, to promote cell growth of TNBC. G-1, a GPER agonist, inhibited DHT-stimulated proliferation. Further experiments illustrated that AR suppressed GPER activation via binding directly to the promoter of GPER. Moreover, a negative correlation between AR and GPER was observed in MDA-MB-231 tumor cell xenografts and TNBC patient samples. Conclusions: The suppression of GPER via AR may be involved in the positive actions towards the TNBC progression, making it a promising therapeutic target for TNBC treatment.

Ki-67 and AR expression and its relationship with recurrence in triple-negative breast cancer

Objective To investigate the expressions of cellular proliferative antigen (Ki-67) and androgen receptor (AR) in triple-negative breast cancer (TNBC) and their relationships with recurrence. Methods Sixty-six cases of TNBC and two hundred and fifteen cases of non triple-negative breast cancer (NTNBC) tissues between January 2006 and December 2010 in Jiangmen Hospital Affiliated to Sun Yat-sen University were collected, the expressions of Ki-67 and AR were detected by immunohistochemical, and through follow-up the relationships between TNBC recurrence and the expressions of Ki-67 and AR were analyzed. SPSS 19.0 was used to analyze the results. Results The positive rate of Ki-67 in TNBC (75.76%, 50/66) was significantly higher than that in NTNBC (62.33%, 134/215, χ2=4.031, P=0.045), and Ki-67 expression was correlated with histological grade (χ2=6.031, P=0.049), tumor diameter (χ2=6.630, P=0.036) and lymph node metastasis (χ2=5.440, P=0.020). The positive rate of AR in TNBC (31.82%, 21/66) was significantly lower than that in NTNBC (76.28%, 164/215, χ2=44.382, P<0.001), and AR expression was correlated with menopausal status (χ2=5.341, P=0.021) and body mass index (χ2=4.369, P=0.037). TNBC recurrence was related to Ki-67 expression (χ2=4.125, P=0.042), and was not related to AR expression (χ2=1.257, P=0.262). Conclusion High expression level of Ki-67 and low expression level of AR are characteristics of TNBC. Ki-67 positive patients are more likely to relapse, and it can be used as an indicator to predict TNBC recurrence. Key words: Breast neoplasms; Immunohistochemistry; Recurrence

Antiproliferative Effect of Androgen Receptor Inhibition in Mesenchymal Stem-Like Triple-Negative Breast Cancer

Background/Aims: Androgen receptor (AR), a steroid hormone receptor, has recently emerged as prognostic and treatment-predictive marker in breast cancer. Previous studies have shown that AR is widely expressed in up to one-third of triple-negative breast cancer (TNBC). However, the role of AR in TNBC is still not fully understood, especially in mesenchymal stem-like (MSL) TNBC cells. Methods: MSL TNBC MDA-MB-231 and Hs578T breast cancer cells were exposed to various concentration of agonist 5-α-dihydrotestosterone (DHT) or nonsteroidal antagonist bicalutamide or untreated. The effects of AR on cell viability and apoptosis were determined by MTT assay, cell counting, flow cytometry analysis and protein expression of p53, p73, p21 and Cyclin D1 were analyzed by western blotting. The bindings of AR to p73 and p21 promoter were detected by ChIP assay. MDA-MB-231 cells were transplanted into nude mice and the tumor growth curves were determined and expression of AR, p73 and p21 were detected by Immunohistochemistry (IHC) staining after treatment of DHT or bicalutamide. Results: We demonstrate that AR agonist DHT induces MSL TNBC breast cancer cells proliferation and inhibits apoptosis in vitro. Similarly, activated AR significantly increases viability of MDA-MB-231 xenografts in vivo. On the contrary, AR antagonist, bicalutamide, causes apoptosis and exerts inhibitory effects on the growth of breast cancer. Moreover, DHT-dependent activation of AR involves regulation in the cell cycle related genes, including p73, p21 and Cyclin D1. Further investigations indicate the modulation of AR on p73 and p21 mediated by direct binding of AR to their promoters, and DHT could make these binding more effectively. Conclusions: Our study demonstrates the tumorigenesis role of AR and the inhibitory effect of bicalutamide in AR-positive MSL TNBC both in vitro and in vivo, suggesting that AR inhibition could be a potential therapeutic approach for AR-positive TNBC patients.

Androgen Receptor Biology in Triple Negative Breast Cancer: a Case for Classification as AR+ or Quadruple Negative Disease.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) amplification. Due to the absence of these receptors, TNBC does not respond to traditional endocrine or HER2-targeted therapies that improve patient prognosis in other breast cancer subtypes. TNBC has a poor prognosis, and currently, there are no effective targeted therapies. Some TNBC tumors express androgen receptor (AR) and may benefit from AR-targeted therapies. Here, we review the literature on AR in TNBC and propose that TNBC be further sub-classified as either AR+ TNBC or quadruple negative breast cancer since targeting AR may represent a viable therapeutic option for a subset of TNBC.

A targetable androgen receptor-positive breast cancer subtype hidden among the triple-negative cancers.

Triple-negative breast cancer (TNBC) is a subgroup of breast cancers that by definition lack expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). A diverse group of tumors, TNBC shares some morphologic and molecular features with basal-like breast cancer, a category of breast cancer defined by gene expression profiling. More likely to occur in young women and African Americans, TNBCs may exhibit aggressive behavior and are associated with poor prognosis despite their initial response to conventional chemotherapy. Because hormonal or HER2-targeted therapies are ineffective for these tumors, the main therapeutic option is systemic chemotherapy. Therefore, identification of new targets for therapy is urgently needed for this group. To review and present recent literature along with our own experience regarding the clinical and morphologic characteristics and the prevalence of androgen receptor (AR) expression in TNBC, and to discuss the potential use of AR as a therapeutic target for AR(+) TNBC. Data sources are published articles from peer-reviewed journals in PubMed (US National Library of Medicine). AR is the most commonly expressed hormone receptor among all breast carcinomas, with a prevalence of 25% to 75% among TNBCs. Therefore, we strongly support the routine assessment of AR in TNBC, and preferably in all breast carcinomas.