Abstract 443: The anion channel GPHR/GPR89 regulates protein-folding homeostasis by regulating the IRE1α pathway in breast cancer

Abstract

Abstract In order to identify novel tumor drivers and gene addictions in breast cancer we have recently completed an integrated in silico/in vitro analysis of gene copy-number alterations and gene-expression profiles in a Triple Negative Breast Cancer-enriched cohort. An amplified and overexpressed gene that appeared to be a driver required for breast cancer cell proliferation is GPHR/GPR89. Several lines of evidence have recently suggested GPHR to be an anion channel, resident in the Golgi, where it controls intraluminal pH. Perturbation of GPHR function leads to altered secretory protein modification and impairment of intracellular transport. Additional data obtained in non-mammalian systems suggest that the localization of GPHR is wider and includes the endoplasmic reticulum (ER). Here, we show that GPHR is amplified and highly expressed across the breast cancer subtypes in our cohort, which we validate in the publicly available TCGA and METABRIC datasets. Using orthogonal characterization by both siRNA-shRNA and CRISPR/Cas9 gene editing, we demonstrate that GPHR down regulation impairs breast cancer cell proliferation and clonogenic ability. To further exclude the possibility that the observed phenotype was due to an “off target” effect, we also performed a rescue of function experiment overexpressing a GPHR protein resistant to GPHR shRNA that confirmed specificity. Importantly, breast cancer spheroids inducibly overexpressing GPHR are shown to have an increased area compared to control spheroids, demonstrating an important role of GPHR in breast cancer cell proliferation. Furthermore, by gradient sucrose subcellular fractionations and immunofluorescence analysis, we show that GPHR is localized in the ER of breast cancer cells. Unfolded protein response (UPR) signatures based on first-degree protein-protein interactions indicate that GPHR is positively correlated with IRE1α and negatively correlated with ATF4/CHOP networks. GPHR is also positively correlated with the XBP1 gene signature. We then further demonstrate that over expression or down regulation of GPHR are able to modulate the IRE1α/XBP1 network of proteins. In summary, in order to sustain uncontrolled growth, breast cancer cells undergo significant stress due to accelerated metabolism, deficient nutrient provision and oxidative stress. UPR signaling and GPHR expression are then essential to protect cells and to allow tumor growth by reducing protein synthesis and favoring chaperonine functions. Here we demonstrate that GPHR is over expressed across the breast cancer subtypes where it controls IRE1α/XBP1 network, and, therefore, can be a novel target for breast cancer therapy as malignant breast cells overexpressing GPHR are selectively dependent on GPHR expression. Citation Format: Riccardo Ferro, Pierfrancesco Marra, Ana Mendes Pereira, Virinder Reen, Cynthia Prince, Sumi Mathew, Alessandra Facchetti, Priyanka Ghongane-Salpe, Sedigeh Kareemaghay, Patrycja Gazinska, David Robertson, Farzana Noor, Fara Braso' Maristany, Daniel Weekes, Dragomir Krastev, Tencho Tenev, Hasan Mirza, Nirmesh Patel, Anita Grigoriadis, Andrew Tutt. The anion channel GPHR/GPR89 regulates protein-folding homeostasis by regulating the IRE1α pathway in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 443.