Abstract 444: ER Stress marker GRP78 is <i>not</i> expressed on ER-/PR-/Her2- human breast cancer cell surface <i>or</i> secreted
Authors: Jesus E. Serrano, Andrea Rivera-Ruiz, Krishna Baksi, Dipak K. Banerjee
Journal: Cancer Research (2018)

Abstract GRP78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr (i.e., N-linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER-/PR-/HER2+ breast tumor in athymic nude mice with the N-glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N-glycan level, and reduced angiogenesis but increased GRP78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER-/PR-/HER2- (MDA-MB-231) and non-metastatic ER+ (MCF-7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose-dependent manner. Interestingly, GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 µg/ml) treated MDA-MB-231 and in MCF-7 cells and supported by both Western blotting and qPCR. Since GRP78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) or with anti-GRP antibody. There were expression of N-glycans but not GRP78. GRP78 however became detectable after permeabilization of cells with a brief exposure to ice-cold methanol. GRP78 was not detected in the conditioned media of the cancer cell but a high level of MMP-1 did. We have therefore, concluded that GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. This is obviously, contradicted the current dogma that GRP78 expression on the tumor cell surface interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti-angiogenic and anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors. Supported in part by the university funds and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Andrea Rivera-Ruiz, Krishna Baksi, Dipak K. Banerjee. ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted [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 444.