Molecular Analysis of Triple Negative Breast cancer

Abstract

Triple negative breast cancer (TNBC) is a heterogeneous and aggressive subtype making up 10-20% of newly diagnosed invasive breast cancers and currently has no targeted agents approved for systemic therapy. However up to 41% of TNBC are positive for the androgen receptor (AR) leading to a possible new avenue for treatment of these patients. This study investigates mechanisms that may dictate future applicability of the anti-androgen drug enzalutamide (ENZ) in the treatment of AR positive TNBC.

A panel of 9 TNBC cell lines was selected that had a wide representation of intrinsic TNBC subtype. AR expression status was determined via western blot. A representative cell line was chosen from two AR positive subtypes, MDAMB453 from the Luminal androgen receptor (LAR) subtype and BT549 from the mesenchymal like (ML) subtype. Functionalty of the AR was investigated in both cell lines via hormone starvation, stimulation with the androgen dihydrotestosterone (DHT) and AR specific inhibition with Enzalutamide (ENZ). Whole transcriptome profiling by RNA-Seq was performed in MDAMB453 and BT549 cells with and without ENZ treatment to identify candidate residual oncogenic signalling mechanisms in the LAR and ML subtypes.

Stimulation of MDAMB453 cells with DHT for 7 Days resulted in a 2.1 fold increase (P=0.0006) in proliferation relative to vehicle control, This growth was abrogated by 34% (P=0.005) in the presence of 10-fold excess ENZ. In contrast, BT549 cells showed no significant difference in proliferation in the presence of DHT but their growth was slowed by 17 percent (P=0.0047) with the addition of ENZ. These data suggest AR is functional in both LAR and ML TNBC, however only LAR cell lines indicate a dependence on androgens as a mitogen. We observed no correlation between 1) TNBC intrinsic subtype and 2) levels of AR expression with susceptibility to ENZ suggesting intrinsic subtype or AR protein level are both poor predictors of response to anti-androgens.

Analysis of transcriptomes from ENZ treated cells revealed candidate pathways up-regulated in response to the ENZ treatment. Upregulation of hormone signalling mechanisms were observed in the LAR cell line, and upregulation of Wnt/Beta-catenin and Interleukin signalling were two of several pathways observed in the ML cell lines.

This study highlights the significant complexity in sensitivity to anti-androgen therapy in AR positive TNBC, and reveals novel subtype-specific vulnerabilities that may prove clinically effective if targeted alongside androgen signalling.