Upregulated Expression of GRP78 Supports ER Stress‐induced Apoptosis in Tunicamycin Treated Triple Negative Breast Tumor

Abstract

Breast cancer affects nearly 1.5 million women (5–10%) worldwide per year. Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer with a disproportionate share of mortality. The patients are younger and pre‐menopausal. The cancers are poorly differentiated, and most fall into the basal subgroup of breast cancers. The long‐term survival is extremely low because specific treatment guidelines are lacking. The patients are managed with standard treatment causing a high rate of local and systemic relapse. This make them resistant to existing targeted therapies (endocrine/biologics/adjuvant/neo‐adjuvant). Anthracycline/taxane combination therapy and PARP inhibitor are currently being explored but not known if they would achieve a pathological complete response (pCR). Protein glycosylation has been claimed as a universal feature of helping cancer cells escaping immune surveillance, facilitate tumor invasion, and increase malignancy with increased tumor burden and poor prognosis as well as enhanced angiogenesis. Based on the glycomics profile of human breast cancer cells as well as tumor specimen, we have hypothesized that targeting asparagine‐linked (N‐linked) protein glycosylation would evolve a new generation therapeutic preventing breast tumor progression and eliminating the disease. To test our hypothesis, we have used tunicamycin a natural product that competitively inhibits the N‐acetyl glucosaminyl 1‐phosphate transferase activity in the ER and consequently the N‐linked protein glycosylation. When tested in a triple negative breast tumor in nude mice, the tumor progression is inhibited by ~65% in one week at tunicamycin concentration of 0.25 mg/Kg administered orally twice a week. Human triple negative breast cancer cells (MDA‐MB‐231) are also equally susceptible to tunicamycin action. The cells are arrested in G1 and exhibit ER stress followed by induction of apoptosis mediated by unfolded protein response (upr) signaling. Western blotting and qRT‐PCR support increased expression of the ER stress master regulator GRP78. But, the immunofluorescence microscopy could not detect GRP78 on the surface of control or tunicamycin treated cells. The result is the same in cells cultured in the absence of serum. GRP78 fluorescence however, is detectable in cells after either they are fixed with ice‐cold methanol or permeabilized 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 NIH U54‐CA096297 and Komen for the Cure BCTR06582 (DKB) and NIH/NIMHD 2G12MD007583 (KB).