Abstract 1201: Disrupted endoplasmic reticulum-mitochondria contacts promote multidrug resistance
Authors: Coku Jorida , Pedrotty Madison C. , Booth David M. , Kim Sharon , Vu Annette , Reynolds C. Patrick , Hajnóczky György , Hogarty Michael D. 
Published: July 2017

Abstract
Background: Most high-risk neuroblastoma patients succumb to lethal therapy resistant disease acquired during the course of intensive multimodality treatment. This acquired therapy resistance is largely attributed to insensitivity to drug-induced apoptosis, however the exact mechanisms remain unknown. Apart from integrating death signals, mitochondria (mito) interact with the endoplasmic reticulum (ER) at close contact sites known as mitochondria associated membranes (MAM) of the ER to regulate calcium and lipid transfer and apoptotic sensitivity, a process often derailed in therapy resistant cancers. ER-mito contact sites are juxtaposed by various tethering protein complexes that include MFN2 and PACS2. Pathologic deregulation of these contact sites has been implicated in the genesis of neurodegenerative and metabolic disorders. Here, we show that disruption of bona fide ER-mito tethering proteins in therapy sensitive neuroblastomas induces apoptotic insensitivity and a drastic shift towards a resistance-like phenotype.
Methods and Results: Previously we showed that isolated mitochondria from tumors at relapse resist induction of mitochondrial apoptosis when primed with the terminal death effectors that are downstream of therapeutic stress (tBid and Bim-BH3 peptide). We isolated mitochondria from seven matched isogenic tumor pairs obtained at diagnosis (DX) and relapse (REL) and quantified their apoptotic response to tBid and Bim by measuring cytochrome C release by ELISA. Electron microscopy (EM) image analyses of ER-mito contact sites revealed that REL tumors contain up to 70% fewer ER-mito interactions than their matched DX tumors, as confirmed by IB for organelle-specific proteins. Here, we recapitulate the post-therapy resistance phenotype in DX therapy sensitive cells by shRNA silencing of MFN2 or PACS2 and confirm their apoptotic resistance by BH3 profiling and drug response in vitro. A 60% decrease in MFN2 protein in DX cells partially phenocopied the resistance profile of isogenic REL cells. The degree of apoptotic resistance correlated with the extent of protein knockdown for MFN2. Treatment of shMFN2 and shPACS2 DX cells with ABT-737, a BH3 mimetic, or carboplatin, increased their IC50s multiple-fold compared to control cells, paralleling their blunted mitochondrial apoptotic response.
Conclusions: Our data implicate ER-mito contact sites as positive regulators of apoptosis, whose disruption may be necessary for apoptotic attenuation in therapy resistant cancer cells to enable apoptosis evasion and survival. We present a potential mechanism for broad therapy resistance arising under therapeutic stress that selects for reduced communication of ER with mitochondria.
Citation Format: Jorida Coku, Madison C. Pedrotty, David M. Booth, Sharon Kim, Annette Vu, C. Patrick Reynolds, György Hajnóczky, Michael D. Hogarty. Disrupted endoplasmic reticulum-mitochondria contacts promote multidrug resistance [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 1201. doi:10.1158/1538-7445.AM2017-1201