GRP78 is Not Expressed on the Surface of Triple Negative Human Breast Cancer Cells

Abstract

GRP78, an Mr78 kDa glucose responsive protein is located in the lumen of the endoplasmic reticulum (ER). It functions as ER chaperone in translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn‐X‐Ser/Thr (N‐linked glycosylation). This is an important biochemical event in protein folding. When N‐glycosylation is impaired with an N‐glycosylation inhibitor tunicamycin, angiogenesis, a hallmark for tumor progression and metastasis is inhibited. The result is regression of breast tumor. Reduced N‐glycan expression correlates with thinning and narrowing of the tumor microvessels. On the other hand, GRP78 expression in microvasculature and in tumor tissue is upregulated, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)‐mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127–29138, 2011; Pure Appl. Chem. 84, 1907–1918, 2012). This contradicts the current dogma that GRP78 expresses on the cell surface, interferes with the therapeutic approach(es) and becomes a tumor promoter. To evaluate the GRP78 localization in our study, we have used a triple negative (ER−/PR−/HER2−) human breast cancer cell line MDA‐MB‐231 cultured normally or without serum as well as before after inducing ER stress, and immunofluorescence microscopy. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α‐D‐Mannose, α‐D‐Glucose, Branched mannose), Wheat germ agglutinin (WGA; Specificity = (GlcNAc‐β‐(1,4)‐GlcNAc)1‐4>β‐GlcNAc‐NeuAc) as well as with anti‐GRP78 antibody followed by their detection either with Rhodamin or Alexa–conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining have provided images of N‐glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of the cancer cell. The results were the same whether the cells were cultured in the absence of serum and/or in the presence of 1μg/mL of tunicamycin. GRP78 fluorescence however was detectable in cells after either fixing them with ice‐cold methanol or permeabilizing them with digitonin. We, therefore, conclude that GRP78 is not expressed on the outer‐leaflet of the cell surface of the triple negative human breast cancer cells MDA‐MB‐231. But, its intracellular expression is anti‐tumorigenic.Support or Funding InformationSupported in part by grants from NIH U54‐CA096297 and Susan G. Komen for Cure BCRT58206 (DKB) and NIH/NIMHD 2G12MD007583 (KB).