Clinical And Basic Research On Oncogenic Role Of Molecular Chaperone Grp94

Glucose regulated protein 94(grp94)is an endoplasmic reticulum stress protein that functions as a molecular chaperone and assist protein folding,transportation,secretion,and degradation.Many tumor endogenous and exogenous factors can cause endoplasmic reticulum stress in tumor cells,activate unfolded protein responses,up-regulate the expression of grp94 and other chaperone proteins,maintain tumor homeostasis,and promote tumor cell survival.The molecular chaperone grp94 can still assist in the folding and membrane trafficking of multiple receptor proteins,including Toll-like receptors,LRP6,GARP,IGF,integrin,HER2,etc.These proteins are involved in the immune microenvironment,angiogenesis and invasion and metastasis of tumors.In addition,cell surface expression of grp94 protein plays a role in anti-tumor immune response.In summary,the molecular chaperone grp94 can help maintain the stability of the endoplasmic reticulum and tumor environment by participating in the folding and transportation of multiple cancer-promoting proteins,participate in the anti-tumor immune response,and play an important role in the early initiation and malignant progression of tumors.The over-expression of grp94 in various tumors is closely related to invasive clinical phenotype and poor prognosis,making grp94 likely to become a new tumor molecular marker.Preclinical studies of tumor vaccines,monoclonal antibodies,and small molecular inhibitors have made grp94 a promising therapeutic target for cancer.This study aims to investigate the expression of molecular chaperone grp94 in human common tumors,evaluate its potential as a tumor marker and clinical predictor,and further investigate the carcinogenic effects of grp94 and targeted small molecular inhibitors of grp94 through in vitro and in vivo studies.Study methods1.Clinical study of molecular chaperone grp94Multiple gene databases were used to investigate the expression of grp94 in human common tumors at the gene level,and the correlation between grp94 gene expression,clinical phenotype and prognosis was further analyzed.Multiple tumor tissue microarray were constructed to verify the expression of grp94 protein in human common tumors at the protein level,and to further analyze the correlation between the expression of grp94 protein and the clinical phenotype.2.Carcinogenesis and mechanism of molecular chaperone grp94grp94 down-expression plasmid was constructed and the grp94 down-expression and control cell lines were established by lentivirus transfection.The effect of grp94 on cell proliferation and apoptosis was examined using cell function test(CCK-8 and flow cytometry).Western blot was used to detect the expression of apoptosis protein caspase-7,CHOP,and JNK phosphorylation protein.JNK inhibitor was used to treat grp94 down-expressing cell lines.Cell function experiments and Western blot were used to examine the effect of JNK phosphorylation on the tumor-promoting function of grp94 protein.3.In vitro and in vitro studies on anti-cancer effects of smaller molecular inhibitor targeting grp94Smaller molecular inhibitor targeting grp94 was used to treat tumor cells.The effects on cell proliferation and apoptosis were observed by CCK-8 kit and flow cytometry.Western blot was used to detect grp94,HSP70 and apoptosis protein caspase-7 expressions.The model of subcutaneous xenografts in immunodeficient mice was constructed and treated with small molecular inhibitor targeting grp94 to observe its effect on tumor growth.The expressipns of grp94,HSP70 and apoptosis-related protein PARP in vivo was examined by Western blot.Study results1.The grp94 encoding genes and proteins are over-expressed in human common tumors,and grp94 over-expression is closely related to clinical invasive phenotype and poor prognosis.In breast cancer,grp94 over-expression is significantly associated with triple negative breast cancer,high HER2 expression,lymph node metastasis,poor histological differentiation,disease progression,and poor survival.In colorectal and lung cancer,grp94 over-expression is significantly associated with lymph node metastasis,poor histological differentiation,disease progression,and poor survival.2.The down-expression of grp94 reduced tumor cell proliferation,increased apoptosis,increased the expression of activated caspase-7 and CHOP protein,and enhanced the phosphorylation of JNK protein.JNK inhibitors could reverse the cell proliferation and apoptosis induced by down-regulation of grp94,and decreased expression of activated caspase-7 and CHOP proteins.3.Small molecular inhibitor targeting grp94 can inhibit cell proliferation,promote apoptosis,increase the expression of activated caspase-7,reduce the expression of survivin protein,not affecting the expression of grp94 and HSP70 protein.4.Small molecular inhibitor targeting grp94 can inhibit tumor growth in mice,promote apoptosis-related protein PARP expression,not affecting the expression of grp94 and HSP70 protein.Conclusion1.Over-expression of grp94 in human common tumor indicates invasive phenotype and poor prognosis.2.grp94 plays a cancer promoting role by inhibiting JNK phosphorylation.3.Small molecular inhibitor targeting grp94 play anti-cancer effects in vitro and in vivo.