Significance of prostate specific antigen as a predictor of androgen receptor activity in breast cancer

Abstract BACKGROUND: Preclinical data suggest that androgen receptor (AR) activity plays a role in breast cancer (BC). Clinical trials testing AR-targeted therapies in BC have shown clinical benefit and additional trials are currently underway. However, a method is needed to predict which patients may benefit the most. Prostate specific antigen (PSA) is the secreted product of the androgen-responsive gene kallikrein related peptidase 3 (KLK3). Serum PSA (sPSA) is an established indicator of tumor burden in prostate cancer. Recently, we found that sPSA can be detected in BC patients and is positively correlated with serum testosterone levels, AR expression in the primary tumor, and aromatase inhibitor resistance (Hanamura, et al. SABCS 2018). HYPOTHESIS: Serum PSA may reflect AR activity in BC and have utility as a predictive marker for identifying patients that would benefit from AR-targeted therapy. OBJECTIVE: To establish that PSA is regulated by AR in BC, we examined the relationship between AR and PSA in BC cell lines, BC patient derived xenograft (PDX), and publically-available datasets of patient gene expression profile. METHODS: Nine BC cell lines representing three clinical subtypes (ERα+, HER2+ and TNBC) were analyzed for response to dihydrotestosterone (DHT) and the AR antagonist enzalutamide (Enza) by crystal violet assay and further analyzed by western blot for PSA. Next, PSA was measured in conditioned media from BT474 BC cells by chemiluminescence immunoassay. Finally, HCI-009, an AR+ TNBC PDX model, grown in NOD-scid gamma mice were given vehicle control versus DHT and were used to identify AR regulated genes by RNA-sequencing, followed by verification at the protein level by IHC. Genes increased in HCI-009 from mice given DHT treatment as compared to vehicle were defined as “PDX androgen-responsive gene set” and imported into gene set enrichment analysis (GSEA). To test whether PSA reflects AR activity, two publically available datasets, The Cancer Genome Atlas (TCGA) (n=1100) and the population-based Multi center Sweden Cancerome Analysis Network (n=405), were mined for correlation between KLK3 andAR or ESR1mRNA (for comparison), and with GSEA with regard to known androgen- or estrogen-responsive gene sets. RESULTS: Although the proliferative response to DHT varied among cell lines, Enza decreased proliferation of the majority of AR+ BC lines regardless of the proliferative response to DHT. Western blot analysis showed PSA to be regulated in an AR-dependent manner (induced by DHT and suppressed by Enza). In BT474 cells, PSA secretion into the media was DHT-responsive and suppressed by Enza. Gene expression analysis of the HCI-009 PDX model showed KLK3 as one of most DHT responsive genes. IHC analysis showed PSA and other AR regulated proteins are significantly higher in tumors from mice given DHT as compared to control and that tumor growth was increased in these mice compared to control mice. In the TCGA and Sweden cohort, a stronger positive correlation was found between KLK3 and ARmRNA expression compared to KLK3 and ESR1 mRNA. An androgen-responsive gene set was significantly enriched in patients with a high KLK3, but the estrogen-responsive gene set was enriched in patients with a low KLK3. The PDX androgen-responsive gene set was also significantly enriched in patients with a high KLK3. CONCLUSIONS: Results of in vitro studies suggest that PSA reflects AR activity and the responsiveness of BC cells to AR antagonism. In comprehensive analysis of gene expression profiling, KLK3 was positively correlated with AR activity, but negatively correlated with ER activity. Together with our previous findings, these data suggest the need for further studies to determine whether sPSA or other AR-regulated secreted proteins are useful as a clinical predictive marker of response to AR targeted therapies. Citation Format: Toru Hanamura, Jessica L Christenson, Kathleen I O’Neill, Emmanuel Rosas, Nicole S Spoelstra, Michelle M Williams, Jennifer K Richer. Significance of prostate specific antigen as a predictor of androgen receptor activity in 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 P6-05-06.