Abstract A62: The Bcl-2 associated athanogene 5 binds the molecular chaperone GRP78/BiP and increases chemoresistance in prostate cancer cells
Authors: Anja Bruchmann, Corinna Roller, Tamara Vanessa Walther, Andrew Cato, Danilo Maddalo
Journal: Clinical Cancer Research (2012)

Abstract 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.