Abstract 4157: Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity
Authors: Ribas Ricardo , Pancholi Sunil , Guest Stephanie K. , Rani Aradhana , Nikitorowicz-Buniak Joanna , Simigdala Nikiana , Thornhill Allan , Cutler Richard E. , Lalani Alshad S. , Avogadri-Connors Francesca , Dowsett Mitch , Johnston Stephen R. , Martin Lesley-Ann 
Published: July 2017

Abstract
Introduction The majority of breast cancers (BC) are estrogen (E) receptor positive (ER+). Endocrine therapies target E stimulation of tumour growth but resistance remains problematic, often a result of enhanced crosstalk between ER and growth factor pathways. Previously we reported the antiproliferative efficacy of combining everolimus (RAD001, mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB and mTORC1) may target tumour rewiring and provide enhanced long-term clinical utility in endocrine resistant BCs.
Methods Several ER+ BC lines adapted to long term E deprivation (LTED), modelling relapse to an aromatase inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan ERBB inhibitor) in the presence or absence of estradiol (E2), tamoxifen or fulvestrant. Effects on proliferation, cell signalling, cell cycle and transcription were assessed. Additionally, an in vivo model of AI resistance was treated with monotherapies or combinations to evaluate efficacy in delaying tumour progression.
Results All cell lines showed dose dependent decreases in proliferation in response to RAD001 (IC50 0.6-50nM without E2; 1-10nM with E2). Neratinib showed a wide range of IC50 values in the presence of E2 (300-1000nM). In the absence of E2, wild type (wt) cell lines showed IC50 values in excess of 1800nM with hormetic response curves, whilst in the LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in both wt and LTED lines but the maximum effect was observed with a triple combination of RAD001, neratinib and endocrine therapy. Expression of pS6 was suppressed by RAD001 in all cells tested, whilst neratinib caused a cell specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines upon treatment with RAD001. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and reduced cell cycle progression. In the absence of E2, RAD001 reduced ER mediated transcription and recruitment of ER and CREB binding protein to the TFF1 promoter, contrasting with neratinib, which caused a marked increase. In vivo study using LTED tumour xenografts showed the triple combination of RAD001, neratinib and fulvestrant was the most effective at reducing tumour volume (89%, p&lt;0.001). Antitumor effects persisted after therapy removal with exception of RAD001 alone or in combination with neratinib where a slow increase in tumour growth was observed.
Conclusion The combination of RAD001, neratinib and endocrine agents may be effective in patients who have relapsed on endocrine therapy but retain a functional ER by combating ERBB pathway upregulation.
Citation Format: Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, Aradhana Rani, Joanna Nikitorowicz-Buniak, Nikiana Simigdala, Allan Thornhill, Richard E. Cutler Jr, Alshad S. Lalani, Francesca Avogadri-Connors, Mitch Dowsett, Stephen R. Johnston, Lesley-Ann Martin. Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2017-4157