The Molecular Mechanism Of PC-1Coregulating AR Protein With CHIP In Prostate Cancer Cells

Prostate cancer(Pca) is a common malignant tumor in males. People who arediagnosed with Pca in the world has been increasing in recent years. The role thatandrogens play in PCa was first discovered by Huggins in1941, who described thatupon getting rid of androgens, prostate tumors shrink. Since then,androgen-depravated therapy has been the main treatment for advanced PCa.Although apoptosis results in a decreased mass of the tumor, PCa usually reoccurswithin18–36months, and ends up with castration-resistant prostate cancer (CRPC).along with castrated levels of androgens in CRPC, AR becomes activated. There isoften an elevated AR expression in CRPC. And AR is believed to be either activatedby non-canonical pathways, highly sensitive to androgens, or constitutively active.Until now, there’s no efficient clinical treatment. So it’s urgent to make it clear thathow the PC-1work mocularly in the progression of Pca cells and find validly targeteddiagnostic and therapeutic methods.The androgen receptor (AR) belongs to the steroid hormone receptor family;other family members consist of the mineralocorticoid, progesterone, glucocorticoid,and estrogen receptors. By regulating secretion, cellular proliferation, apoptosis andsurvival, AR plays a very important role in the development progression of theprostate, benign prostate hyperplasia as well as prostate cancer. AR is a protein with919-amino-acid, and it encodes from a~180kb gene that is located at chromosomeXq11-12. AR consists of three major functional domains. The highly unstructuredN-terminal domain (NTD) comprising of over1/2of the receptor is the largestdomain, where one of the two activation function (AF1) motifs lies. The DNA bindingdomain (DBD) is the second functional region in the AR, which contains two zincfingers. And the ligand binding domain (LBD) is connected to DBD by a shortflexible peptide sequence called the hinge region.PC-1was identified to be down-regulated in androgen sensitive parent cell lineLNCaP, compared with androgen-refractory prostate cancer cell line C4-2, which wasfirst cloned by professor Zhou. PC-1is a member of Tumor Protein D52family, manymembers of which contribute to a series of proliferation and cancer. The PC-1protein is made up of224amino acids, the180amino acids of the C-terminal is highlyhomologous with D52protein, while the46amino acids of the N-terminal is unique.Androgen can induce PC-1gene expression, and PC-1can predominantly expresses inprostate tissue and cells. These results all indicate that PC-1has some potentialoncogene characteristics, and may play some role in. So far, the biologicalmechanisms of PC-1gene in prostate cancer progression are not clearly understood.In this article, a series of investigation are performed to make the molecularmechanism mediating the functions of PC-1gene clear in prostate cancer progression.1) At first, plasmids of PC-1and AR are transiently transfected in293T cells,which don’t belong to prostate cancer cells,and the correlation between AR proteinand PC-1was detected by western blot. It comes out that AR protein decrease if PC-1is overexpressed in dose-dependent manner. Then, a prostate cancer LNCaP cell linestably overexpressing PC-1and a C4-2cell line in which PC-1expression is stablyknocked down are constructed to verify the correlation between AR protein and PC-1again. There are a negative correlation between the expression level. Here, aftertheating the cells with CHX, the Cycloheximide, a protein synthesis inhibitor, toinhibit synthesis of AR protein, we find that the half-life of AR protein is shortened inthe presence of PC-1. Therefore, in perspective from degradation of proteins in theclassical pathway of ubiquitin-proteasome pathway, AR protein ubiquitinationexperiments demonstrate that levels of AR protein is regulated by PC-1in proteasomepathway.2) To explore whether the direct correlation between PC-1and AR is caused byinteraction between the two proteins, GST-pull down experiments andco-immunoprecipitation experiments are performed to find out that there is aprotein-protein interaction between PC-1and AR which are expressed exogenously orendogenously, and it’s the section629-634aa of the hinge region which interacts withPC-1.3) The above results show that, it’s the section629-634aa of the hinge regionwhich interacts with PC-1, and ultimately AR protein levels are regulated in theproteasome pathway. some related vitro experiments also confirm that CHIP playsthe role of E3ubiquitin ligase. To study whether PC-1regulates AR through CHIP,this new E3ubiquitin ligase, GST-pull down experiments and exogenous,endogenous co-immunoprecipitation experiments are performed which turns out thatthhere is protein-protein interactions among PC-1,CHIP and AR, and overexpression of PC-1can enhance the interaction between AR and CHIP, thereby enhancing thethe degradation of AR in ubiquitin-proteasome way mediated with CHIP.4) By exogenous transfection of PC-1, AR and CHIP plasmids in293T cells,western blot experiments are conducted to find out that AR protein level goes lower inthe presence of PC-1and CHIP than they exist alone. And knocking down theendogenous CHIP protein in293cells by RNAi technology, the PC-1overexpressioncan not make AR protein decreased. AR protein ubiquitination experiments areperformed to prove that PC-1can promote the CHIP-mediated degradation of AR inubiquitin-proteasome way.5) Our previous study find that PC-1is highly expressed in the cell cycle G2/Mphase. When the cellular processes of cell cycle is arrest in G2/M phase by usingNocodazole and2-ME, overexpression of PC-1can increase the degree of reduce inAR and CHIP protein. It indicates that PC-1can promote CHIP-mediated ARdegradation in G2/M phase. However, the specific mechanism is still unclear, pendingfurther study.6) Using LNCaP and PC3as a prostate cancer models, dual luciferase reportergene assay is conducted to study the biological significance of PC-1. The results showthat PC-1can inhibitAR’s role of transcriptional activation.In conclusion, This study investigats the role of PC-1gene and its mechanism indevelopment of prostate cancer in the cellular level, which provides a molecularbiologic basis for PC-1gene to be a new therapeutic target for prostate cancer.