Molecular docking and MD simulation approach to identify potential phytochemical lead molecule against triple negative breast cancer.

Background Triple-negative breast cancers are defined as tumors that lack the expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It exhibits unique clinical and pathological features, is highly aggressive, and has a relatively poor prognosis and poor clinical outcome. Objective To identify a novel drug target protein against triple-negative breast cancer (TNBC) and potential phytochemical lead molecules against novel drug targets. Methods In this study, we retrieved TNBC samples from NGS and microarray datasets in the Gene Expression Omnibus database and employed a combination of differential gene expression studies, protein-protein interaction analysis, and network topology investigation to identify the target protein. Using molecular docking and molecular dynamics simulation studies, followed by Molecular Mechanics with Generalised Born Surface Area solvation, a potential lead molecule was identified. Result The androgen receptor (AR) was found to be the target protein, and 2-hydroxynaringenin was discovered to be a possible phytochemical lead molecule to combat TNBC. Upregulated genes with LogFC > 1.25 and P-value < 0.05 from the TNBC gene expression dataset were given to STRING tool to investigate the network topology, and androgen receptor (AR) was found to be an appropriate hub gene in the protein-protein interaction network. Phytochemicals that inhibit breast cancer were retrieved from the PubChem database and virtual screening was performed using PyRx against the AR protein. Based on Lipinski’s rule and ADMET properties, molecular interaction studies were analyzed using induced fit docking, wherein significant binding interactions were displayed by 2-hydroxynaringenin. Molecular dynamics studies and MM-GBSA of AR and the 2-hydroxynaringenin complex revealed strong and stable interactions. Conclusion AR was identified as a hub protein that is highly expressed in breast cancer and 2-hydroxynaringenin efficacy of counter TNBC needs to be investigated further in vitro and in vivo.

BackgroundTriple-negative breast cancers (TNBC) are defined as tumors that lack the expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It exhibits unique clinical and pathological features, demonstrates high aggressiveness, and has a relatively poor prognosis and clinical outcome.ObjectiveTo identify a novel drug target protein against TNBC and potential phytochemical lead molecules against the identified target.MethodsIn this study, we retrieved TNBC samples from NGS and microarray datasets in the Gene Expression Omnibus database. We employed a combination of differential gene expression studies, protein-protein interaction analysis, and network topology investigation to identify the target protein. Additionally, the molecular docking and molecular dynamics (MD) simulation studies followed by Molecular Mechanics with Generalised Born Surface Area salvation was used to identify potential lead molecule.ResultThe upregulated genes with LogFC > 1.25 and P-value < 0.05 from the TNBC gene expression dataset were identified. Androgen receptor (AR) was found to be an appropriate hub target in the protein-protein interaction network. Phytochemicals that inhibit breast cancer target were retrieved from the PubChem database and virtual screening was performed using PyRx against the AR protein. Thereby, the AR was found to be the target protein and 2-hydroxynaringenin was discovered to be a possible phytochemical lead molecule for combating TNBC. Moreover, the AR and the 2-hydroxynaringenin complex showed structural stability and higher binding affinity through molecular dynamics and MM-GBSA studies.ConclusionAR was identified as a hub protein that is highly expressed in breast cancer and 2-hydroxynaringenin efficacy of counter TNBC requires further investigation both in vitro and in vivo.

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 &amp;lt; 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 &amp;lt; 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 &amp;lt;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 &amp;lt;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 &amp;lt; 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.

Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR as a target for radiosensitization. In addition, 70-95% of all estrogen receptor (ER) positive (ER+) breast cancers also have coexpression of AR, suggesting extended utility of AR inhibition in the radiosensitization of these AR+, ER+ tumors. Here we assessed the efficacy of AR inhibition in ER+, AR+ breast cancers to better understand the role of AR signaling across breast cancer models. Further, we also investigated the effect of ER inhibition on radiosensitization of ER+ breast cancer models. Methods: IC50 values were determined for MDV3100 (enzalutamide), ARN-509 (apalutamide), and ODM-201 (darolutamide) in TNBC cell lines (AR+ TNBC: MDA-MB-453, ACC-422, and SUM-185PE, and AR- TNBC: MDA-MB-231) and ER+ breast cancer cell lines (AR+, ER+: ZR-75-1, BT-474, CAMA-1, and AR-, ER+: MCF-7). IC50 values for tamoxifen were determined for ER+ breast cancer cell lines (MCF-7, T47D, ZR-75-1), and ER- (SUM-159) cells. Clonogenic survival assays were performed to assess radiosensitization with ER or AR inhibition with tamoxifen or second generation anti-androgens, respectively, in TNBC and ER+ breast cancer models. Results: AR inhibition with enzalutamide, apalutamide, and darolutamide showed limited single agent growth inhibition efficacy in AR+ TNBC and AR+, ER+ breast cancer cell lines (IC50 &amp;gt; 10 μM). AR inhibition with enzalutamide did not induce radiosensitivity in vitro. In AR+, ER+ CAMA-1 cells, AR blockade with enzalutamide had a radioprotective effect with enhancement ratios (enhR) of 0.76-0.83. No radiosensitization was observed in BT-474 (enhR: 0.92-1.01) or ZR-75-1 cells (enhR: 0.94-1.00). Radiosensitization was also assessed with anti-androgens apalutamide and darolutamide in AR+ breast cancer models. Inhibition of ER with tamoxifen, however, induced radiosensitization in MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells. No radiosensitization was observed with tamoxifen in ER- SUM-159 cells. Conclusion: Although AR is a mediator of radioresistance in AR+ TNBC, AR inhibition does not provide comparable radiosensitization in AR+, ER+ models and may actually confer a radioprotective effect. In contrast, our results demonstrate ER inhibition is an effective radiosensitizing strategy in ER+ breast cancers, independent of AR status. This work highlights the complexities of androgen and estrogen receptor signaling in AR+, ER+ breast tumors and underscores the necessity for understanding context dependent effects when translating into patients with AR+ breast cancer. Citation Format: Anna R. Michmerhuizen, Amanda Zhang, Rachel Schwartz, Andrea M. Pesch, Benjamin C. Chandler, Cassandra L. Ritter, Meilan Liu, Kari Wilder-Romans, Daniel E. Spratt, Daniel R. Wahl, Shyam Nyati, Lori J. Pierce, Corey Speers. Hormone receptor inhibition as a strategy for radiosensitization of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6271.

Background The androgen receptor (AR) is an emerging biomarker and favourable therapeutic target in breast cancer (BC), especially in Triple negative BC (TNBC) which lacks a targeted treatment and shows high molecular heterogeneity. Proportion of AR positive tumours are known to vary (10 to 43%) within TNBC and recent multi-institutional study on AR positive TNBC, showed AR protein status alone is not a reliable prognostic marker for therapeutic response. A deeper examination of AR regulated pathways is necessary to derive AR regulated gene signatures which could identify TNBC tumours truly driven by AR and amenable to anti androgen therapies. In this study, we attempted to develop AR regulated gene signature using gene expression data derived from cell lines under the influence of androgens using systematic bioinformatic approach and tested the ability of the signature to identify AR regulated tumours within TNBC. Methods Microarray data of AR positive BC cell lines (GSE61368 &amp; GSE28305) was used to derive AR regulated genes by a bioinformatic pipeline, using integrated method by incorporating protein interaction networks, topological parameters, and semantic similarity to arrive at final set of 16 genes. Correlation of these genes with AR protein status by reverse phase protein array (RPPA) was evaluated in 447 tumors of TCGA dataset. A 3rd quartile cut-off in the AR expression (by RPPA) was used to classify them as AR positive and negative and a logistic regression model was constructed using positive AR as determinant to generate AR probability score with minimum set of genes. 3rd quartile cut-off in the probability score was used to classify tumors as AR high and low. Distribution of AR probability score was examined in 144 and 187 TNBC tumors in TCGA and METABRIC, respectively. Comparisons between AR high and low tumors were done for clinical parameters, mutational spectrum, expression of ER regulated genes (FOXA1, GATA3 and TFF1) and EMT gene signatures (Byers et al., 2013, Tan et al., 2014). Luminal androgen receptor (LAR) subtype identification by TNBCtype (Lehmann et. al) was done in both datasets to examine the overlap with our method of identification of AR driven tumors within TNBC. Results Logistic regression model identified 5 (SOCS2, ID1,PIP,GADD45 and ZNF689) of the 16 AR regulated genes as predictors for AR probability score which ranged from 0.009 to 0.68 in 144 TNBC tumors within TCGA data. 41/144 (29%) TNBC tumors were categorized as having AR high at cut off of 0.13. The AR probability score ranged from 0.04 to 0.64 in 187 TNBC tumors within METABRIC dataset and 47/187 (25%) were classified as AR driven tumors at cut off of 0.22. Though differences in the clinical variables like age , tumor size and lymph node(LN) status were not different between the AR high and low groups in the TCGA dataset, a significant increase in age, LN positive tumors was observed in the AR high groups in METABRIC. A significant higher PIK3CA mutations was observed in the AR high tumors both in the TCGA and METABRIC (p=0.012 and p&amp;lt;0.0001 respectively) with a proportionate lower p53 mutations in the AR high tumors in both data sets (p=0.03 and p=0.35 respectively). Further, AR high tumors showed significant higher expression of ER regulated genes (TCGA-p&amp;lt;0.05 &amp; METABRIC, p&amp;lt;0.05), were more epithelial by EMT score (TCGA-p&amp;lt;0.05 &amp; METABRIC, p&amp;lt;0.05). Analysis by TNBCtype showed that 30% of AR high tumors in TCGA and 40% of AR high tumors in METABRIC were identified as belonging to LAR subgroup (Lehmann et al). Conclusion Identification of AR driven tumors within TNBC has both prognostic and predictive utility. Methods using limited number of AR regulated genes could be easily applied to larger BC cohorts to identify TNBC tumours driven by AR signalling and may respond well to anti-androgen therapies. Citation Format: Savitha Rajarajan, Snijesh V.P, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Identification of AR driven tumors within TNBC using a novel gene signature [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 P5-04-04.

Androgen receptor-positive, triple-negative breast cancer.

Background: Androgen receptor (AR) protein is expressed across breast cancer (BC) subtypes, including up to 50% of triple negative breast cancers (TNBC) and it is often maintained in metastases. Our research demonstrated that TNBC cell lines rely on increased AR for anchorage independent survival, mammosphere formation, tumor initiation, and other stem-cell like properties. Further, AR mRNA levels increased in circulating tumor cells in the blood as compared to levels in the primary tumors of mice harboring human patient derived xenografts (PDX). Seviteronel (SEVI), a CYP17 lyase and AR inhibitor that blocks androgen and estrogen biosynthesis and AR activation, is in phase 2 clinical development for men and women with AR+ solid tumors, including advanced BC and has clinical activity in both basal and LAR subtypes (Innocrin data on file). Hypothesis: We hypothesized that targeting both androgen biosynthesis and AR activation with an agent such as SEVI would decrease tumor growth in preclinical models of AR+ TNBC. Methods: Genes regulated by AR under anchorage independent conditions with and without 10 nM dihydrotestosterone (DHT) were identified by AR chromatin immunoprecipitation (ChIP)-seq, RNA-seq, and Ingenuity Pathway Analysis. In vitro activity of SEVI was assayed in AR+ TNBC cell lines (MDA-MB-453 and SUM159). The IC50 of SEVI was determined via crystal violet. Soft agar colony formation assays and growth on poly-HEMA coated plates measured anchorage independent growth. In vivo, an AR+ TNBC patient derived xenograft (HCI-009, generated by Dr. Alana Welm) was grown in NSG mice given either DHT vs. vehicle control or, in a separate experiment, SEVI (150 mg/kg/day PO) vs. vehicle control. Results: ChIP-seq and RNA-seq in MDA-MB-453 demonstrated that AR chromatin binding and gene regulation increased under anchorage independent conditions in a ligand dependent manner and showed an increase in mTOR signaling, aryl hydrocarbon receptor signaling and metabolism. SEVI inhibited proliferation of AR+ TNBC in a dose-dependent manner and growth on soft agar. In vivo, the AR+ TNBC HCI-009 PDX exhibited a significant increase in tumor volume with DHT indicating AR dependence. SEVI treatment also decreased HCI-009 tumor volume (p=0.0054) and rate of growth (p=0.0146). Nuclear AR protein and classical AR-regulated genes such as KLK3 and FKBP5 were increased with DHT in the HCI-009 xenograft tumors and decreased in mice treated with SEVI and were confirmed at the protein level. Conclusion: These results further demonstrate the activity of SEVI, a CYP17 lyase and AR inhibitor, in preclinical models of AR+ TNBC. Use of AR targeting agents may be a rational treatment approach for a subset of patients with AR+ TNBC since such tumors may respond less well to chemotherapy than other TNBC molecular subtypes. We continue to identify markers of AR dependence/responsiveness to AR-targeted therapy. Citation Format: Reese JM, Babbs BL, Christenson JL, Spoelstra NS, Elias A, Eisner JR, Baskin-Bey ES, Gertz J, Richer JK. Targeting the androgen receptor with seviteronel, a CYP17 lyase and AR inhibitor, in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-05.

Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&amp;D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p&amp;lt;0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

Objective To investigate androgen receptor(AR) expression in triple negative breast cancer(TNBC) patients and its predictive value for clinical outcomes. Methods Five hundred and sixty-eight primary breast cancer patients who received surgery treatment from January 2008 to January 2010 were included in this study.Tissue sections of surgical resection specimens were underwent estrogen receptor(ER), progesterone receptor(PR), and human epidermal growth factor receptor 2(HER2) immunohisto chemical staining, according to the judgment for triple negative breast cancer and non triple negative breast cancer.Data on age, family history, menstruation, tumor size, tumor stage, tumor grade, pathological type and lymph node metastasis were collected for analysis.Immunohistochemical analysis of AR was done in tissue sections of triple negative breast cancer patients, Kaplan-Meier method was used to compare survival between AR(+ ) and AR(-) groups, and Log-rank method was used to determine the association between AR- positive expression and 5-year overall survival as well as disease free survival. Results Among 568 breast cancer patients, 174(30.6%) TNBC patients were identified, including 84 patiens with AR(-) expression and 90 patients with AR(+ ) expression.During 5 years' follow-up, reccurrence rate was 35.7%(30/84) in AR(-) group and 20.0%(18/90) in AR(+ ) group; 5-year overall survival rate was 73.8% in AR(-) group and 81.1% in AR(+ ) group.There was no significant association between AR(+ ) expression and 5-year death or recurrence rate, while after adjusting for age, menstruation, tumor size and lymph node metastasis, AR(+ ) expression was significantly associated with decreased 5-year death and recurrence rate, adjusted hazard ratio(HR) was 0.794, 95CI(0.430-1.021) and 0.722, 95CI(0.451-0.965) respectively. Conclusion AR(-) expression is associated with increased risk of death and recurrence and is a promising predictive marker for poor clinical outcomes. Key words: Triple negative breast cancer; Androgen receptor; Estrogen receptor; Progesterone receptor; Human epidermal growth factor receptor 2; Survival rate

Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior, poor prognosis and still remains as a difficult disease since treatment options are limited. Despite some success in PARP inhibition in BRCA gene mutation patients or platinating agents that may offer superior outcomes in a subset of TNBC patients (pts), currently, there are no targeted therapies for TNBC available. Specific biomarkers are urgently needed for developing effective treatments to predict which patients will respond to the given therapy. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Since TNBC is closely related to epithelial-mesenchymal transition (EMT), a stem cell phenotype and, in addition, androgen receptor (AR) expression has been detected in up to a third of TNBC pts, we here established a multi-marker gene panel for the characterization of CTCs in TNBC pts and compared these findings with CTC characteristics in non-TNBC pts. Methods: 2x5 ml blood of 30 TNBC pts before and/or after neoadjuvant therapy and 30 non- TNBC pts (E+/PR+: n=23; ER+/PR-: n=4; HER2+: n=1; HER2+/ER+: n=1; HER2+/ER+/PR+: n=1) before therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)]. Results: In general, the distribution of the markers across all patients was highly variable. However, different expression patterns were found when CTCs of TNBC pts were compared with those of non-TNBC pts. In TNBC pts, SRC1 was the gene that was predominantly expressed, followed by c-Kit, HER3, BRCA1 and AURKA expression, before as well as after therapy. Interestingly, AKT2, EGFR, ERCC1 and PARP1 expression could not be detected at any time point studied. In addition, ALK, AR, c-Met, HER2 and KRT5 were only detected before but not after therapy. All other genes were expressed below 15%. In contrast, in non-TNBC pts, AKT2 was the gene that was predominantly expressed, followed by c-MET, HER3 and PI3K whereas c-KIT, ERCC1, mTOR and NOTCH1 were never found. All other genes were expressed below 10%. Conclusion: We successfully established a gene panel for the detection of the heterogeneous CTC population and demonstrated that CTCs in TNBC pts and non-TNBC pts show different genetic profiles. Although these data have to be confirmed in a bigger patient cohort, the knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future. Citation Format: Bittner A-K, Hoffmann O, Hauch S, Kimmig R, Kasimir-Bauer S. Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-05.

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 &lt; 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 &lt; 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.

Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton of TNBC are critically needed. We have previously characterized the radiation response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We now pair this data with high-throughput drug screen data available through cancer cell line encyclopedia studies to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were performed to determine the intrinsic RT sensitivity of 21 BCC lines (0-8 Gy RT). IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values (for drug sensitivity) and SF-2Gy (for radiation sensitivity). Gene expression was measured using Affymetrix microarrays and protein expression was measured using reverse-phase protein lysate arrays (RPPA) of human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or inhibition with MDV3100 (enzalutamide). Kaplan-Meier analysis was performed to determine the clinical impact of AR expression on local control and survival. A Cox proportional hazards model was constructed to identify potential factors of survival, and multivariate analysis was used to determine variables most significantly associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as one of the most effective drugs in treating radioresistant BCC lines (R2= 0.46, p-value &amp;lt;0.001). Recognizing that a subgroup of TNBC includes AR expressing tumors, we interrogated the expression of AR in &amp;gt;2000 human breast tumor samples and found signifi[not]cant heterogeneity in AR expression with an increase in TNBC (35% of tumors) compared to non-TNBC (28% of tumors). This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p &amp;lt;0.0001). Inhibition of AR using both siRNA and MDV3100 induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. In vivo assessment of tumor growth inhibition with RT and anti-AR strategies are currently underway. Clinically, analyses of patients with TNBC showed that patients whose tumors had high expression of AR had markedly higher rates of LR after RT than patients with low expression of AR (HR for LR 2.9-3.2, p-value &amp;lt;0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression status. In multivariate analysis, AR expression was the variable most significantly associated with worse LRF survival after RT with a HR of 3.58 (p-value &amp;lt; 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Corey Speers, Shuang G Zhao, Meilan Liu, Joseph Evans, Prasanna Alluri, Daniel F Hayes, Felix Y Feng, Lori J Pierce. Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-08.

Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We paired this with high-throughput drug screen data to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 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 and protein expression was measured using RPPA arrays in human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or functional inhibition with MDV3100 (enzalutamide). We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control and survival. A Cox proportional hazards model was used to identify factors of survival, and MVA was used to determine variables associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as a most effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value &amp;lt;0.01). We interrogated the expression of AR in &amp;gt;2000 human breast tumor samples and found significant heterogeneity in AR expression with enrichment of expression at the protein and RNA level in TNBC. This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p &amp;lt;0.01). Inhibition of AR using both siRNA and MDV3100 (enzalutamide) induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. AR inhibition either with MDV3100 significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor tripling time and tumor growth (median tumor tripling time 17.4 days for RT alone vs. not reached after 50 days for MDV3100 + RT, p-value &amp;lt;0.001). Biomarker analysis identifies DNAPKcs as a potential biomarker of response. Clinically, analyses of patients with TNBC showed that patients whose tumors had higher than median expression of AR had markedly higher rates of LR after RT (HR for LR 2.9-3.2, p-value &amp;lt;0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression. In MVA, AR expression was the variable most significantly associated with worse LRF survival after RT (HR of 3.58;p-value &amp;lt; 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Speers C, Zhao SG, Liu M, Rae JM, Hayes DF, Feng FY, Pierce LJ. Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-05.

Background: A novel genomic signature that identifies androgen receptor (AR)-driven disease (PREDICT AR) is being developed for its ability to select patients (pts) who may benefit from enzalutamide (ENZA), a potent AR inhibitor.1 In a phase 2 study of triple-negative breast cancer (TNBC) pts treated with ENZA (NCT01889238) ≈ half of pts had PREDICT AR+ TNBC, with improved clinical outcomes in this sub-population.2 Because pts with hormonally driven (estrogen-receptor/progesterone-receptor[ER/PR]+) BC are thought to have a better prognosis, we investigated whether the outcomes observed in pts with PREDICT AR+ TNBC could be explained by prognosis alone. Methods: PREDICT AR was assessed on advanced TNBC from 118 ENZA-treated pts.2 Because the duration of treatment (Tx) typically decreases with each subsequent Tx, exploratory analyses included median progression-free survival (mPFS) on ENZA vs duration of Tx prior to ENZA (used as a PFS surrogate) in pts who had received ≤1 prior Tx (data cutoff 24Mar15). Baseline characteristics by PREDICT AR status were also evaluated. An independent non-ENZA-Tx TNBC data set (after neo/adjuvant Tx)3 was probed for PREDICT AR status and clinical outcomes. Results: ENZA-Tx pts with PREDICT AR+ TNBC (n=56; 47%) were older (66 years vs 52 years), had a longer disease-free interval (DFI) (37 vs 20 months), more bone involvement (57% vs 24%), were more likely to have had prior ER/PR+ BC (30% vs 8%), and a longer median duration on the first-line Tx (29 vs 19 weeks[wks]) vs pts with PREDICT AR- TNBC (n=62;53%). Table 1. mPFS from ENZA-Tx pts and duration of prior Tx by PREDICT AR status and line of Tx.PREDICT AR StatusTx prior to ENZAmPFS on ENZA (wks)Median duration of prior Tx (wks)PREDICT AR+ (n=26)No prior Tx (n=10)32-- 1 prior Tx (n=16)4034PREDICT AR- (n=36)No prior Tx (n=11)8-- 1 prior Tx (n=25)1317 The prevalence of PREDICT AR+ primary TNBC from the non-ENZA-Tx dataset was 51% (n=182). DFI after neo/adjuvant therapy was not statistically different (p=0.605) between pts with PREDICT AR+ vs PREDICT AR- TNBC, and pathologic complete response rates were 23% vs 38% in PREDICT AR+ vs PREDICT AR- TNBC, respectively. Conclusion: mPFS in pts with PREDICT AR+ TNBC who received first-line ENZA was longer than first-line Tx duration in those who received prior Tx (32 vs 29 weeks). In pts with PREDICT AR+ TNBC who received second-line ENZA, mPFS was longer than the median duration of their first-line Tx (40 vs 34 weeks). This trend was not observed in pts with PREDICT AR- TNBC. Key demographic differences did not explain these outcomes, suggesting benefit from ENZA. Duration of first-line non-ENZA-Tx was shorter in pts with PREDICT AR- TNBC vs pts with PREDICT AR+ TNBC who received second-line ENZA (17 vs 34 weeks), suggesting a better prognosis in pts with PREDICT AR+ advanced TNBC; however, PREDICT AR status was not associated with DFI following neo/adjuvant treatment for primary TNBC. PREDICT AR may identify a hormonally driven subset of pts with advanced TNBC who have a better prognosis and who may benefit from ENZA. 1. Parker J et al. J Clin Oncol 2015;33(suppl): abstr 1083. 2. Traina TA et al. J Clin Oncol 2015;33(suppl): abstr 1003. 3. Hatzis C et al. JAMA 2011;305:1873-1881. Citation Format: Miller K, Krop I, Schwartzberg L, Abramson V, Garcia-Estevez L, Eakle J, Nanda R, Yardley D, Ademuyiwa F, Chan S, Crown J, Danso M, Gelmon K, Ma L, Martinez-Janez N, Gradishar W, Steinberg J, Tudor IC, Uppal H, Paton VE, Parker J, Hudis CA, Traina TA. Improved clinical outcomes on enzalutamide observed in patients with PREDICT AR+ triple-negative breast cancer: prognosis or prediction?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-25.

Abstract Background Triple negative breast cancer (TNBC) is defined by the lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Prognostic immunohistochemical biomarkers in TNBC have been studied in recent years such as the androgen receptor (AR) which is expressed in 10-40% of TNBC. However the prognostic value of AR expression is not clear. Here, we studied the prognostic significance of AR expression in combination with the presence of a ductal carcinoma in situ (DCIS). Considering DCIS is a precursor of invasive ductal carcinoma, we hypothesize that TNBC with co-existing DCIS and presence of AR expression is less aggressive and patients are older at diagnosis. Methods We analyzed data retrospectively from all patients with stage 1-3 TNBC who underwent primary surgery and adjuvant chemotherapy in the University hospitals Leuven, between 01-01-2000 and 31-12-2017. Patient and tumor related characteristics were compared between two subgroups, one with co-existing DCIS and one with pure invasive carcinoma, without co-existing DCIS. AR expression was assessed by immunohistochemistry (IHC). We used AR expression in ≥1% and ≥10% of cells as cut-off scores. The prognostic role of the expression of AR in combination with a co-existing DCIS was analyzed, using the distant metastasis rate as primary endpoint. Results are presented as hazard ratios (HR) with 95% confidence intervals (CI). Secondary endpoints were associations of AR expression with clinical-pathological characteristics, time between diagnosis and metastasis, and disease specific mortality. Results In the 426 included patients with TNBC, co-existing DCIS was present in 66.7%; AR expression was expressed ≥1% in 29.3% and ≥10% in 21.4% of cases. Median age at diagnosis was 51 years (range: 22-85y). Age at diagnosis was independent of DCIS, dependent of AR expression; in DCIS positive cases, median age was 49 years if AR negative (IHC &amp;lt; 1%), 53 years if AR positive (IHC ≥1%), and 56 years if AR positive (IHC ≥10%) (p=0.006). In contrast in DCIS negative cases, median age was 51 years if AR negative (IHC &amp;lt; 1%), 51 if AR positive (IHC ≥1%), and 51 years if AR positive (IHC ≥10%) (p=0.895). AR expression was DCIS dependent and was ≥1% in 34.9% and ≥10% in 25.0% of patients in the DCIS group compared to 18.3% and 14.1% in the non-DCIS group (p=0.001 and p&amp;lt; 0.001 respectively). In both subgroups there was no significant difference for AR positive versus AR negative cases in lymph node involvement, tumor grade, tumor size and Nottingham Prognostic Index. Patients with a coexisting DCIS and AR expression did not have a different incidence of distant relapse compared to AR negative cases (AR ≥1%: p=0.2803 and AR ≥10%: p=0.5527). Of patients with coexisting DCIS, 12.0% (95% CI: 7.8; 17.1) in the AR negative group, 8.2% (95% CI: 3.8; 14.6) in the AR ≥ 1% group and 7.1% (95% CI: 2.6; 14.7) in the AR ≥ 10% group, had distant relapse within 2 years. Conclusion In patients with TNBC, AR expression is associated with older age in case of co-existing DCIS and patients with co-existing DCIS are more frequently AR positive. In patients with co-existing DCIS, there was no significant difference in distant relapse between AR negative and AR positive cases. Citation Format: Micaëlle Merckx. The prognostic role of androgen receptor status in patients with triple negative breast cancer with an associated ductal carcinoma in situ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-03-10.