Transcriptomic Analysis to Uncover the Mechanism of Radiosensitization of AR-Positive Triple Negative Breast Cancers with AR Inhibition

Abstract

The androgen receptor (AR) has been shown to drive tumor growth in triple negative breast cancers (TNBC), and previous work demonstrated AR inhibition as a strategy for radiosensitization in AR-positive (AR+) TNBC. Despite its role in radioresistance, the mechanistic role of AR in response to radiation therapy (RT) remains unknown, as does the benefit of 2nd generation anti-androgens in this context. We hypothesized that all 2nd generation anti-AR therapy would radiosensitize similarly and that canonical AR transcriptional function was responsible for radioresistance in these models. Radiosensitization was assessed using 2nd generation AR antagonists (apalutamide, enzalutamide, and darolutamide) using clonogenic survival assays in MDA-MB-453, SUM185, MFM-223, and MDA-MB-231 cells at 2-6Gy. Cellular fractionation experiments were performed and quantitated to determine the location of the AR protein in cells treated with AR agonists +/- RT. RNA Seq was performed and transcriptomic approaches were used (Advaita iPathway analysis) to investigate AR-mediated effects in response to RT. Inhibition with the 2nd generation anti-androgens enzalutamide and apalutamide is sufficient to radiosensitize AR+ TNBC models (rER: 1.34-1.41); while darolutamide had no effect on radiosensitivity (rER: 0.96-1.11). Additionally, TNBC cells with low AR expression were not radiosensitized by AR inhibition with any drug (rER: 0.96-1.03). While stimulation with the synthetic androgen methyltrienolone R1881 is sufficient to induce nuclear translocation of AR in AR+ TNBC cells, AR inhibition with enzalutamide, apalutamide, or darolutamide blocked AR nuclear translocation under growth conditions with charcoal stripped serum or fetal bovine serum. When cells are treated with R1881+RT, nuclear translocation of AR was induced at similar or greater levels compared to R1881 alone in AR+ TNBC cells. Combination treatment of RT with enzalutamide in the presence of hormones reduced nuclear localization of AR (32-39% reduction) compared to RT alone. RNA-sequencing after RT identified transcriptional changes potentially regulated by AR+RT, including changes in the NHEJ pathway genes. Additionally, pathway analyses in these models demonstrated changes in the MAPK/ERK signaling pathway, among others, that may regulate RT resistance in AR+ TNBC models. Most 2nd generation anti-androgens confer radiosensitization in AR+ TNBC models with cellular localization changes of AR noted after RT. The known structural differences amongst 2nd generation anti-androgens may account for differences in radiosensitization noted. Furthermore, AR-mediated radioresistance may be due, at least in part, to downstream MAPK/ERK signaling. This work builds on the mechanistic understanding of AR-mediated radioresistance in AR+ TNBC and may expose vulnerabilities to overcome resistance to combination treatment with AR inhibition and RT.