Abstract P3-05-05: Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer
Authors: Nikitorowicz-Buniak J , Ribas R , Rani A , Pancholi S , Guest SK , Cutler RE , Lalani A , Dowsett M , Johnston SR , Martin L-A 
Published: May 2016

Abstract
AIM To target tumor re-wiring by combined mTORC1 inhibition plus hormonal treatment with or without co-blockade of ERBB signaling in endocrine resistant models of human breast cancer (BC).
BACKGROUND Around 80% of BCs are estrogen receptor positive (ER+). Endocrine therapies target estrogenic stimulation of tumor growth but resistance remains problematic. Several strategies have shown that resistance often depends on the acquisition of enhanced cross-talk between ER and growth-factor pathways, allowing the disease to circumvent the need for steroid hormones. We have previously reported the antiproliferative effects of the combination of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signaling were observed. We hypothesised that combined targeting of three signaling pathways, namely ER, ERBB and mTORC1 may provide enhanced anti-tumor activity.
METHODS ER+ BC cell lines (MCF7, SUM44 and HCC1428) adapted to long term estrogen-deprivation (LTED) which model relapse on an aromatase inhibitor, along with their wild-type (wt) cell lines were treated with neratinib, a pan-ERBB tyrosine kinase inhibitor, in combination with RAD001 ± estradiol (E2), tamoxifen or fulvestrant. End points included proliferation, cell signaling, cell cycle and effect on ER-mediated transactivation and recruitment by ChIP.
RESULTS All cell lines showed a concentration-dependent decrease in proliferation in response to RAD001 (IC50 0.6-50nM in absence of E2 and 1-10nM in presence of E2). A wide range of IC50 values (300-1000nM) was observed with neratinib treatment in the presence of E2. However, in the absence of E2, wt cell lines showed IC50 values in excess of 1800nM with hormetic dose response curves, in which lower concentrations induced cell proliferation. In contrast, LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in all wt cell lines and their LTED derivatives, but the maximum effect was observed when a triple combination of RAD001, neratinib and ER-blockade was used. Expression of pS6 was dramatically suppressed by RAD001 ± neratinib in all cell lines tested, whilst neratinib caused a cell line specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines following treatment with RAD001, indicating both inhibitors were effectively suppressing their respective targets. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and significantly reduced cell cycle progression. In the absence of E2, RAD001 caused a reduction in ER-mediated transcription and decrease in recruitment of ER and the CREB-binding protein (CBP) to the TFF1 promoter. In contrast, neratinib induced a marked increase in ER-recruitment and concomitant rise in ER-mediated transactivation, which was reduced by the addition of RAD001.
CONCLUSION Targeting tumor re-wiring by triple blockade of ERBB, ER and mTORC1 signaling pathways significantly reduces cell proliferation supporting the potential combination in patients who have relapsed on endocrine therapy and retain a functional ER.
Citation Format: Nikitorowicz-Buniak J, Ribas R, Rani A, Pancholi S, Guest SK, Cutler Jr RE, Lalani A, Dowsett M, Johnston SR, Martin L-A. Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-05.