Treatment of several types of cancer such as lung, breast, prostate, and pancreas has shown notable progresses in the past decades. However, after an initial response, tumors eventually became resistant to chemotherapy. This phenomenon, known as chemoresistance, accounts for the death of most cancer patients. Several studies in patients refractory to therapy have revealed the upregulation of the molecular chaperone GRP78/Binding Protein, BiP (BiP) both at the RNA and protein expression level. Furthermore GRP78/BiP relocates to the cell membrane in malignant but not in benign cells. In this communication we review studies on the role and the mechanism of action of GRP78/BiP during development of chemoresistance in cancer cells. In addition we discuss the possible role of GRP78 as a biomarker and as a target in cancer therapy.

BackgroundThe Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function.MethodsBag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses.ResultsOur results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death.ConclusionsWe have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.

Purpose of the study: the purpose of this study is to investigate the role of the Bcl-2 associated athanogene 5 (Bag5) protein in stress tolerance and chemoresistance development in prostate cancer cells. Introduction: the Bcl-2 associated athanogene (Bag) family of proteins consists of six members sharing a common Bag domain that mediates the interaction with the molecular chaperone Hsp70. Bag family members like Bag1 and Bag3 have been already reported to be involved in the development of several cancers like breast prostate and lung cancer. However, the role of Bag5 in cancer has not been described yet. Considering the involvement of the Bag proteins in stress tolerance, we therefore investigated the role of Bag5 upon stress induction and its ability to induce tolerance to stress-inducing chemotherapeutic agents. Experimental procedures: for gene expression analysis real-time PCR assay was used. Immunofluorescence and cell fractionation were carried to analyze subcellular protein localization. Co-immunoprecipitation and GST-pull down assays were employed for protein-protein interaction analysis. GRP78 enzymatic activity was measured by in vitro ATPase assay and by intracellular refolding experiment. Clonogenic assay was used to determine cell growth upon chemotherapeutic agents treatment. Western blot experiment and Caspase-3 cleavage measurement were used to analyze stress induced cell death and activation of the unfolded protein response. Results: In order to determine if Bag5 plays a role in stress response, Bag5 gene and protein expression levels were analyzed upon treatment with the stress inducers thapsigargin and tunicamycin in 22Rv.1 and PC3 prostate cancer cell lines. In both cases we could appreciate a significant increase of Bag5 levels indicating its stress inducibility. Intriguingly we could observe that Bag5 accumulates into the endoplasmic reticulum once stress is induced. Moreover we could determine that Bag5 binds to the ER chaperone GRP78/BiP, enhancing its enzymatic activity. Since GRP78 plays a major role in the regulation of the unfolded protein response (UPR), we wanted to determine if Bag5 levels could influence UPR activation. Stable transfection of Bag5 in 22Rv.1 cells attenuated UPR activation as well as apoptosis compared to the control upon stress induction. Consistently with this observation, when Bag5 gene levels were reduced, cells became more sensitive to stress induction. Since UPR activation and stress tolerance represent the basic mechanism for chemoresistance development, we analyzed the effect of Bag5 overexpression when cells were treated with a range of chemotherapeutic agents including fenretinide, casodex, (−)epigallocatechingalle and taxol. Colony forming assay and Caspase-3 cleavage measurement showed in all the treatments an increased survival in Bag5 overexpressing cells compared to the vector control, indicating that Bag5 indeed plays a role in prostate cancer cell survival and might be responsible for chemoresistance development. Conclusions: In this work we showed for the first time that Bag5 can interact with the molecular chaperone GRP78/BiP and influence UPR activation. In addition, we provided the very first evidence that Bag5 could play a role in development of stress tolerance in prostate cancer cells.