Cell Surface GRP78 Accelerated Breast Cancer Cell Proliferation And Migration By Activating STAT3

Aim:Theincidence of breast cancer has been increased year by year, and it has becomeone ofthe most common femalemalignancies. Recurrent breast cancer remains a mostly incurable disease with drug resistance, tumor latency and distant metastases driving breast tumor recurrence and mortality. Understanding drug resistance is a critical component of combating breast cancer. High level of cell surface glucose regulated protein 78 (sGRP78) has been implicated in cancer growth, survival, metastasis, and chemotherapy resistance. However, the underlying mechanism remains largely unknown. Here we explore the relationship between the level of sGRP78 expression in human breast tumor and the pathological stage. And further studies on the mechanism of sGRP78 accelerating breast cancer cell proliferation and migration was performed in cultured breast cancer cells.Methods:We detected the expression of cell surface glucose regulated protein 78 in breast cancer tissue by double-labeling immunofluorescence method, andthen detected the expression of sGRP78 protein in breast cancer tissues and non-tumor tissues by western blot method, and then the relationship between sGRP78expression and clinical pathological characteristics of patients with breast cancer were analyzed.The construction of recombinant adenoviral vectors expressinghuman GRP78 (Ad/GRP78) were performed, then infected MCF-7 and MDA-MB-453 cells. GRP78 expression was detected by immunofluorescence staining and western blotting, we next tested whether high sGRP78 expression is a consequenceof ER stress resulting in GRP78 overexpression. MTT and TUNEL assays were performed to measure cell proliferation and apoptosis. Wound migration assay were used to measure the cell migration. We investigated STAT3 phosphorylation in sGRP78 positive tumor tissues by western blot. MCF-7 and MDA-MB-453 cells were infected with Ad/?-gal and Ad/GRP78, and then the protein phosphorylation of JAK2 and STAT3 was determined by western blot. To su ppress STAT3 phosphorylation, we used a specific STAT3 inhibitor NSC74859 or transd uced with human STAT3/shRNA. Then the protein phosphorylation of STAT3 was determined by western blot.Finally, Cell proliferation, apoptosis, and migration were assessed by M TT, TUNEL, and wound healing assay, respectively.Results:(1).sGRP78 is highly expressed in a group of tumors with a high pathological stage. (2).sGRP78 was dramatically increased in the Ad/GRP78-infected MCF-7 cells when compared to the Ad/?-gal-infected cells. (3).Ectopically expressed GRP78 did not induce ER stress, however, ER stress can upregulate the membrane distribution of GRP78. (4). sGRP78 overexpression significantly increased MCF-7 cell proliferation and that GRP78-enhanced proliferation was mitigated by treatment with GRP78 antibody. Ad/GRP78-infected MCF-7 cells were against TNFa-induced cell death, which is reversed by anti-GRP78 antibody. The wound healing abilities of GRP78-infected MCF-7 cells were markedly increased, compared with the control cells. (5). sGRP78 stimulates JAK2/STAT3 pathway. (6).STAT3 phosphorylation could be suppressed by treatment with NSC74859 or by silencing of STAT3. STAT3 inhibitor and STAT3/shRNA significantly attenuated cell proliferation, increased apoptosis, suppressed wound-closure, and reduced the number of migrated cells in both MCF-7 and MDA-MB-453 cells.Conclusions:Our results demonstrated for the first time that 1) sGRP78 is highly expressed in a group of tumors with a high pathological stage; 2) sGRP78 stimulates JAK2/STAT3 pathway leading to proliferation and migration of breast cancer cells.