Abstract 4509: GRP78 is neither expressed on ER-/PR-/Her2- human breast cancer cell surface nor secreted in the culture media

GRP78 (Mr78 kDa) is a glucose regulated protein. It is a member of the heat-shock protein (HSP) family but located in the lumen of the endoplasmic reticulum (ER). Its function as ER chaperone 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) is well recognized. This important biochemical event is essential for glycoprotein folding and function. For example, when the protein N-glycosylation is impaired with tunicamycin a protein N-glycosylation inhibitor, angiogenesis, a hallmark for tumor progression and metastasis is inhibited due to an induction of ER stress. Under such condition, GRP78 is overexpressed in microvasculature and in tumor tissue, 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 supports GRP78 expression on the tumor cell surface interfering with the therapeutic(s) and making them as tumor promoters instead. To evaluate the GRP78 localization, we have used ER-/PR-/Her2- (i.e., triple negative) human breast cancer cell line MDA-MB-231 (Caucasian) cultured normally or without serum as well as before or after inducing the ER stress. After establishing the expression of GRP78 mRNA by qPCR and protein by western blotting, we focused on its cell surface expression. 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 by immunofluorescence microscopy using either Rhodamin or Alexa-conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining 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 these cancer cells. Similar results were also obtained irrespective of cells cultured in the absence of serum and/or in the presence of 1µg/mL of tunicamycin. GRP78 fluorescence however was detected in cells after either fixing them with ice-cold methanol or after permeabilization with digitonin. In addition, western blotting also failed to detect G