Abstract P6-03-07: Targeting multiple pathways in breast cancer: Androgen receptor, HER2, and mTOR
Authors: Gordon Michael A , D'Amato Nicholas , Gu Haihua , Wong Darren , Elias Anthony , Richer Jennifer K 
Published: May 2015

Background: Androgen receptor (AR) is expressed in ∼60% of breast cancers (BC) and current evidence indicates that some BC become reliant on AR signaling for growth. AR activation affects numerous cellular functions and signaling cascades, including the HER2/PI3K/mTOR pathway. Conversely, the HER2/PI3K/mTOR pathway regulates steroid hormone receptors including AR. Therapeutics targeting this pathway such as the anti-HER2 trastuzumab (TRAS) and the anti-mTOR everolimus (EVE) have shown significant clinical benefit; however the impact that AR inhibition may have on enhancing the efficacy of these agents and a potential mechanism of synergy have not been explored in BC. The anti-androgen enzalutamide (ENZ) inhibits nuclear entry of AR, is clinically effective in castrate-resistant prostate cancer, and is currently being tested in clinical trials for BC.

Hypothesis: Given the overlap in these pathways, simultaneous inhibition of AR pathway with agents targeting HER2/PI3K/mTOR may be more effective in BC therapy.

Methods: HER2+ and triple-negative BC (TNBC) cell lines were treated with ENZ, TRAS, and EVE, either alone or in combination, at three clinically relevant doses per drug. Cell proliferation was measured either by crystal violet (MDAMB453, SUM225) or on an IncuCyte live cell imager (Essen Bioscience) with nuclear-red labeled cells (BT474, SKBR3, BT549). Drug synergy was calculated using Calcusyn (Biosoft, Inc.) from IncuCyte proliferation data for selected cell lines (BT474, SKBR3, BT549) by comparing combinations of multiple drug concentrations where a combination index (CI)&lt;1 indicated synergy. Protein expression of pathway components was measured by western blot, and gene expression was measured by RT-qPCR.

Results: ENZ inhibited proliferation of HER2+ BC cells, and a combination of ENZ plus TRAS inhibited proliferation more effectively than either agent alone. The effect was synergistic in SKBR3 cells (CI&lt;0.1), and additive in BT474 cells. Treatment of a HER2+/ER+ cell line (BT474) as well as an ER-, HER2-non-amplified but overexpressing cell line (MDAMB453) with dihydrotestosterone (DHT) caused increased expression of pHER3 and total HER3 protein, which was attenuated by addition of ENZ. However, in HER2+/ER- cell lines (SKBR3 and SUM225), pHER3 and total HER3 levels did not change, but rather pHER2 levels increased in response to DHT and were attenuated with ENZ treatment. Additionally, ENZ inhibited proliferation of TRAS-resistant SKBR3 cells. However, unlike the parental SKBR3 cells, ENZ did not affect pHER2 levels in the TRAS-resistant cells. ENZ plus EVE treatment resulted in a significant decrease in proliferation compared to single agent; this effect was synergistic in two HER2+ cell lines (SKBR3 CI&lt;0.1, BT474 CI&lt;0.1) as well as one TNBC cell line (BT549 CI&lt;0.5). Treatment with EVE alone caused a compensatory increase in AR protein and downstream AR gene expression in all cell lines. This increase was attenuated with ENZ plus EVE combination treatment.

Conclusion: ENZ synergizes in vitro with FDA-approved BC therapies TRAS and EVE through distinct mechanisms. Combination therapies containing ENZ may provide benefit for multiple BC subtypes, including HER2+ and triple negative BC.

Funded by DOD BCRP Clinical Translational Award BC120183 to JKR.

Citation Format: Michael A Gordon, Nicholas D'Amato, Haihua Gu, Darren Wong, Anthony Elias, Jennifer K Richer. Targeting multiple pathways in breast cancer: Androgen receptor, HER2, and mTOR [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 P6-03-07.