Effectiveness and molecular interactions of the clinically active mTORC1 inhibitor everolimus in combination with tamoxifen or letrozole in vitro and in vivo

Lesley-Ann Martin,Dean B Evans,Stephanie Guest,Heidi A Lane,Sunil Pancholi,Allan M Thornhill,Mitch Dowsett,Roger A'Hern,Zara Ghazoui,Stephen R Johnston,Ricardo Ribas,Marion T Weigel,Ian Farmer

doi:10.1186/bcr3330

Abstract

IntroductionStrategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results.MethodsBC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation.ResultsEverolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies.ConclusionsThe results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

Highlights

Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1, which regulate cell-cycle progression and are supported by recent clinical results
We report xenograft studies of the combination of everolimus (RAD001) with the aromatase inhibitor (AI) letrozole and parallel studies in the ER+ BT474 cell line, whose endocrine resistance depends on human epidermal growth factor receptor 2 (HER2) amplification that is associated with response to rapalogs [26]
Effect of RAD001 alone or in combination with endocrine therapy on cell growth To enable the study of an AI in combination with everolimus, we used our MCF-7 and BT474 cells that had been genetically engineered to express aromatase (MCF7-AROM1 and BT474-AROM3) [27,28] and provided 10 nM androstenedione as growth support

Summary

Introduction

Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results. In BC, the PI3K/AKT pathway modulates responses to signals, communicated through the ER and the HER family of receptors [2]. This pathway is important in the clinical sensitivity of BC to antiendocrine therapy [3,4,5,6]. Elevated levels of AKT have been shown to change the genome-wide binding pattern of ER, effectively altering the ER program [9]. These data suggest that signaling partners downstream of PI3K/AKT may provide potential therapeutic targets

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