Paper Title
INVESTIGATION OF THE ROLE OF THE ENDOPLASMIC RETICULUM CHAPERONES GRP78 AND GRP94 IN THE REGULATION OF DIFFERENT HALLMARKS OF CANCER IN LUNG ADENOCARCINOMA AND BREAST CANCER CELLS

Abstract
The glucose-regulated proteins (GRPs) GRP78 and GRP94 are endoplasmic reticulum (ER) proteins with chaperone activity playing important roles in folding, assembly, and degradation of de novo synthesised proteins. In cells undergoing ER stress triggered by a variety of stimuli GRP78 and GRP94 -as stress-inducible proteins- can translocate to the cell surface where they regulate metastasis and immune response signalling. Given their role in immunity, this study hypothesized that cell surface GRP78 and GRP94 differentially modulate the breast versus lung cancer tumour microenvironment via their interaction with the immune checkpoint proteins programmed death ligand-1 (PD-L1) and human leucocyte antigen G (HLA-G). To explore this hypothesis the PD-L1 and HLA-G expression was followed in lung adenocarcinoma A549 and breast cancer MCF-7 cells in which the cell surface expression of GRP78 and GRP94 was induced by treating them with either tunicamycin or thapsigargin. The cellular levels of these immune checkpoint proteins were also investigated in A459 and MCF-7 cells in which the GRP78 and GRP94 gene expression had been silenced. GRP94 was upregulated in GRP78 silenced A549 and MCF-7 cells and vice versa.GRP78 and GRP94 negatively associated with PD-L1 levels in A549 and MCF-7 cells treated with thapsigargin but positively correlated with PD-L1 levels in tunicamycin treated cells. Low GRP78 protein levels were associated with decreased HLA-G levels in MCF-7 cells, while low levels of both GRP78 or GRP94 protein levels were associated with increased HLA-G levels in A549 and MCF-7 cells treated with either one of the ER stress inducers. PD-L1 and HLA-G surface levels were elevated in GRP78-silenced A549 and MCF-7 cells regardless of the type of ER stress induction. Reduced migratory potential was observed in silenced A549 cells, while increased migration was seen in silenced MCF-7 cells. High levels of GRP78 correlate with shorter overall survival in breast cancer patients, whereas in lung adenocarcinoma patients high GRP78 levels correlate with longer overall survival. In addition, high GRP78 levels positively associated with the infiltration of immune cells including dendritic cells, monocytes,Tcm CD8, Tgdand T regular cells in breast cancer, whereas negatively associated with the infiltration of these immune cells in lung adenocarcinoma. Altogether results presented in this study indicate that GRP78 and GRP94 by regulating the levels of the PD-L1 and HLA-G immune checkpoint proteins differentially affect the infiltration of immune cells in the lung versus breast tumor microenvironment.