Abstract P4-03-04: Targeting the mevalonate pathway in HER2-positive breast cancer to overcome resistance to anti-HER2 therapy
Authors: Sethunath V , Hu H , De Angelis C , Veeraraghavan J , Qin L , Rimawi MF , Osborne KC , Schiff R 
Published: February 2018

Abstract
Background: Despite the advent of HER2-targeted therapies for HER2+ breast cancer (BC), including the monoclonal antibody trastuzumab (T) either alone or in combinations, resistance still poses a major clinical challenge. Using our broad panel of HER2+ cell lines made resistant (R) to T alone (TR), and to lapatinib plus T (LTR), we observed that in resistant models where HER2 remains inhibited, especially the LTR derivative, the mevalonate (MVA) pathway is activated and provides an alternative proliferative signal, including the activation of mTOR, to drive resistance. While these resistant cell models were hypersensitive to the widely-used cholesterol-lowering statins, the role of other MVA pathway inhibitors such as zoledronic acid (ZA), which is in clinical use to treat bone metastasis, in overcoming resistance to HER2-targeted therapy has not been explored. Based on recent reports and our preliminary data using reverse phase protein array (RPPA) analysis, the YAP/TAZ transcription factor (TF) emerged as a potential mediator of MVA pathway signaling to mTOR. Here, we investigated the therapeutic efficacy of additional MVA pathway inhibitors and the role of YAP/TAZ in mediating resistance to HER2-targeted therapy.
Methods: HER2+ SKBR3 and AU565 BC cells and their LTR derivatives were used. Changes in cell growth upon genetic and pharmacologic inhibition of the MVA pathway were quantified by methylene blue staining. Luciferase reporter assays and western blots (WB) measured changes in total and phosphorylated (S127 and S381/inactive) YAP protein levels to examine activity of the YAP/TAZ TF complex. To validate the function of YAP/TAZ in resistance, we performed YAP/TAZ knockdown (siRNA), overexpression of dominant-active YAP constructs (S381A, S381/127A), and qRT-PCR assessment of YAP/TAZ target gene expression.
Results: ZA, like simvastatin (Sim), selectively inhibited the growth of resistant cells in a dose-dependent manner. This inhibition was rescued by geranyl geranyl pyrophosphate (GGPP), a downstream metabolite, but not by MVA, an upstream metabolite, indicating the on-target effect of ZA. Further, ZA and Sim combination showed a synergistic growth-inhibitory effect in R but not in parental (P) cells. YAP/TAZ luciferase reporter assays and phosphorylated YAP and total TAZ levels by WB, confirmed the increased activity of YAP/TAZ in R models, which was selectively inhibited by Sim or ZA and was rescued by the corresponding downstream metabolites. YAP/TAZ knockdown selectively inhibited resistant cell growth and mTOR signaling in R vs. P cells, and dominant-active YAP/TAZ rescued the mTOR inhibition by Sim. YAP/TAZ inhibition by siRNA or by Sim significantly decreased the expression of YAP/TAZ target gene survivin in R vs. P cells, and the Sim inhibition was rescued by MVA.
Conclusions: The MVA pathway plays a critical role in mediating resistance to anti-HER2 therapy, which was overcome by Sim and ZA either alone or in combination. Given the synergistic effect of Sim and ZA, their combination may offer a therapeutic strategy to overcome HER2-targeted therapy resistance. Our results also reveal the role of YAP/TAZ in MVA pathway-mediated HER2-targeted therapy resistance, which could suggest new biomarkers and therapeutic targets.
Citation Format: Sethunath V, Hu H, De Angelis C, Veeraraghavan J, Qin L, Rimawi MF, Osborne KC, Schiff R. Targeting the mevalonate pathway in HER2-positive breast cancer to overcome resistance to anti-HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-04.