Abstract P6-03-12: Characterization of anti-androgen resistant androgen receptor-positive triple negative breast cancer cells
Authors: Saswati Bhattacharya, Noah Siegel, Ruth O'Regan
Journal: Cancer Research (2020)

Abstract Triple negative breast cancer (TNBC) has a poor prognosis in part due to a lack of targeted therapeutics that have improved outcome for other breast cancer subtypes. Gene expression analysis has identified several subtypes of TNBC, including subtypes that express androgen receptor (AR). However, to date the use of AR inhibitors (ARI) in AR+ TNBC has produced disappointing results, likely due to inherent resistance. Additionally, patients that initially benefit from ARIs ultimately develop acquired resistance with resultant progressive disease. In order to better understand the mechanisms of resistance to ARIs in AR+ TNBC, we developed cell lines with acquired resistance to standard ARI therapy. Methods: Bicalutamide (Bica) and enzalutamide (Enza)-resistant cell lines (Bica-R and Enza-R) were generated via continuous passaging of the wild type MDA-MB- 231 (Wt-231) cells in drug-containing media. Cell viability assays were conducted over a 72-hour period using respective anti androgen drugs at varying concentrations. IC-50 for respective drugs was determined via linear regression on normalized control values. Immuno-histochemical analysis and Western blotting for AR and other proteins were compared between the sensitive and resistant lines. Real time PCR was used to detect AR variants (AR-Vs). Additionally we evaluated the efficacy of a CDK inhibitor abemaciclib (Abe) in the resistant cell lines in comparison to the Wt-231 cells. Results: The ARI-resistant cell lines showed higher IC-50 values compared to the Wt cells. For the Bica-R and Enza-R cells the values were at 729 μM and 496μM with Bica and Enza respectively, whereas the values are at 134 μM and 91μM with Bica and Enza respectively for the Wt-231 cells. The resistant lines also exhibited a different morphology, compared to the sensitive cell lines with a more mesenchymal appearance. Expression of AR-Vs differed between the Wt and resistant AR+ TNBC cells. Confocal microscopy revealed different AR localization in the resistant cells along with changes in cell size: compared to Wt cells, Bica-R cells show more diffuse distribution of AR while still maintaining high nuclear levels; Enza-R cells are 3-4 fold size larger than Wt cells and show predominantly nuclear AR localization. Chromogranin A, a protein known for its protective role in regulating endothelial barrier function against increased production of tumor necrosis factor during cancer progression, was found to be down regulated in the resistant cells compared to the sensitive cells. The CDK inhibitor Abe inhibited the growth of the Bica-R cells (IC50 5.74 μM) and was equally effective in inhibiting growth of Wt cells (IC50 5.4 μM). Enza treatment inhibited growth of the Bica-R cells with IC-50 values of 173 μM compared to 91 μM in Wt cells. Interestingly, synergistic effects on cytotoxicity were observed between Abe and Enza in the Bica-R cells, where combination of Enza and 0.1 μM Abe inhibited the growth of the bicalutamide-resistant cells at even lower Enza concentrations (71 μM) than when used as single agents (173 μM). Conclusion: ARI-resistant AR+ TNBC is distinct from AI-sensitive AR+ TNBC at the protein and genomic level. CDK inhibition appears effective in AR+ TNBC, even in cells with acquired-resistance to ARIs and the combination of CDK inhibition and AR inhibition is currently being evaluated in several ongoing clinical trials for patients with AR+ TNBC. Citation Format: Saswati Bhattacharya, Noah Siegel, Ruth O'Regan. Characterization of anti-androgen resistant androgen receptor-positive triple negative breast cancer cells [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 P6-03-12.