Endoplasmic Reticulum (ER) Stress is Anti‐angiogenic and Anti‐tumorigenic

Abstract

IntroductionThe functions of endoplasmic reticulum (ER) are significantly influenced by cellular microenvironment. The availability of oxygen (i.e., the hypoxia) or glucose (i.e., the hypoglycemia), hyperthermia, acidosis, calcium levels, the redox milieu, energy levels etc. influence and disturb proper functioning of the ER, resulting in ER stress and impacting protein folding in the lumen of the ER. The result is accumulation of unfolded or misfolded proteins. If the balance between accumulation and degradation of unfolded or misfolded proteins is shifted towards their accumulation, cells a suicidal pathway through unfolded protein response (upr) signaling. Our laboratory has recently shown that inhibiting asparagine‐linked (N‐linked) protein glycosylation with an Mr860Da glucosamine containing pyrimidine nucleoside tunicamycin [a natural product, a cytokine mimic, and a competitive inhibitor of N‐acetylglucosaminyl 1‐phosphate (GlcNAc‐1P) transferase activity] induces ER stress in capillary endothelial cells as well as in breast cancer cells. The treatment causes cell cycle arrest and induction of apoptosis in capillary endothelial cells. Since, angiogenesis is essential for normal growth and development as well as for the progression and metastasis of breast tumor, we have observed inhibition of ER−/PR−/Her2+ (double negative) and ER−/PR−/Her2− (triple negative) breast cancers in athymic balb/c (nu/nu) mice following tunicamycin treatment.HypothesisWe have therefore hypothesized that tumor microvessles are not only the unique target for ER stress in breast tumor; tumor epithelial cells are also equally vulnerable to tunicamycin‐induced ER stress.MethodsTo test the hypothesis, we have used human breast cancer cell lines as models and tunicamycin as a tool.ResultsWhen treated with tunicamycin, both MDA‐MB‐231(ER−/PR−/Her2−; triple negative) and MCF‐7 (ER+) breast cancer cells exhibited a dose and time dependent inhibition of cell proliferation. There were inhibition of surface expression of N‐glycans as well. Immunofluorescence microscopy of unfixed cells stained for FITC‐conjugated Concanavalin A (FITC‐Con A) and Texas Red‐conjugated Wheat germ agglutinin (Texas Red‐WGA). To measure the ER stress, we have monitored the expression of ER chaperone GRP78/Bip by immunofluorescence microscopy as well as by SDS‐PAGE followed by western blotting. Western blotting detected higher GRP78/Bip expression but none by immunofluorescence microscopy in unfixed tumor cells. Higher expression of GRP78 in tunicamycin treated cells is expected to support the presence of ER stress in breast cancer cells. So, the GRP78/Bip expression was tested by immunofluorescence microscopy after permeabilization of cells with ice‐cold methanol. There was a robust intracellular expression of GRP‐78/Bip. To evaluate if the induction of ER stress mediates its effect through upr signaling, we have analyzed the expression of IRE‐1, ATF4/ATF6 and PERK by western blotting.ConclusionInduction of ER stress both in endothelial cells and in tumor cells by targeting their asparagine‐linked (N‐linked) protein glycosylation machinery is unique and the concept could be used to develop new generation therapeutics for breast cancer..Support or Funding InformationSupported in part by the NIH‐NIDDK STEP‐UP program (AR) and NIH/NIMHD G12MD007583 (KB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.