Androgen Receptor Splicing Variant 7 Promotes Docetaxel Resistance Of Prostate Cancer Cells Via Regulating The Spindle Assembly Checkpoint

Background:Docetaxel(DTX)has been the first-line chemotherapeutic drug for treating metastatic CRPC patients since 2004 by significantly benefits the clinical outcomes of many patients.However,the clinical efficacy of DTX in prostate cancer treatment is barely satisfactory due to diverse responses of the patients and rapid development of resistance.As we all know,the growth of prostate cancer cells depends on the pathways regulated by androgens and their receptors.However,castration therapy cannot effectively inhibit the Androgen receptor(AR)pathway for a long time,which means that the activation of AR-related pathways is still the key to promoting the survival of CRPC.Recently,research reports aberrant AR signaling,including expression of the constitutively active ARV7,is reported to contribute to DTX resistance.However,the underlying molecular mechanism remains largely unknown.Accumulating studies have confirmed that ARV7 is capable of activating canonical AR downstream targets,mediating androgen-independent cell growth and resistance to AR-targeting therapy.Of note,many studies have highlighted that ARV7 might also control a transcriptional program which is largely different from the one regulated by AR,especially by promoting the expression of some genes involved in the mitotic progression.Thus,we were motivated to further explore this unique role of ARV7 based on those studies mentioned above.Since DTX mainly targets microtubule dynamic and mitosis,we investigated the specific role of ARV7 in mitotic regulation and its relationship with cellular response to DTX.Although many factors such as multidrug resistance,drug absorption,and combination have an important impact on the clinical therapeutic effect of DTX,we believe that the target link of drugs acting on cells is still a non-negligible part of drug tolerance research.After the treatment of DTX,its primary role is to inhibit the depolymerization of microtubules,thereby activating the spindle assembly checkpoint(SAC)to block cells in mid-mitosis and trigger apoptosis.However,tumor cells can also withdraw from mitosis prematurely through the process of mitotic slippage,that is,without the correct separation of chromosomes and cytokinesis.To a certain extent,mitotic slip promotes the escape of cancer cells from DTXinduced apoptosis,which leads to the development of drug resistance and genome instability.Therefore,deciphering the precise regulation of SAC is crucial to understanding the underlying mechanism of DTX resistance.This study is based on the lentiviral system to change the expression level of ARV7 in a variety of PCa cells.By studying the changes in the mitosis process and drug resistance of PCa cells under the action of DTX,the effect of ARV7 on the DTX sensitivity of PCa cells and the regulation of mitotic arrest after DTX treatment,To provide new mechanism exploration and potential targets for overcoming DTX tolerance in the clinical treatment of prostate cancer.Methods:1.In this study,we first used lentiviral vectors to establish prostate cancer cell lines C4-2,PC-3(ARV7 overexpression),22RV-1(silent endogenous ARV7)with constant changes in ARV7 expression.2.After the model is established,through the analysis of cell survival rate,apoptotic protein expression level,clone formation and other indicators,to explore whether ARV7 participates in regulating the sensitivity of PCa cells to DTX,and at the same time,to explore whether ARV7 affects endogenous cell apoptosis.3.Secondly,use Western-blotting,flow cytometry,immunofluorescence and other techniques to explore whether ARV7 affects the degree of mitotic block and mitotic slippage.4.Subsequently,using IP and other experiments to further test whether ARV7 regulates the stability of the mitotic checkpoint complex,so as to determine the degree of freeness of the key protein Cdc20 that regulates the premature exit of mitosis.5.With the help of bioinformatics and literature reports,analyze the important mitotic genes that may be regulated downstream of ARV7,such as UBE2 C,to explore whether ARV7 mediates mitotic slippage and DTX tolerance by regulating UBE2 C.6.Finally,use the mitotic exit inhibitor in combination with DTX to explore whether blocking mitotic slippage can enhance the sensitivity of PCa cells to DTX through in vitro cell experiments.Result:1.Overexpression of ARV7 can reduce DTX-induced apoptosis,while silencing endogenous ARV7 promotes the sensitivity of cells to DTX,but the endogenous apoptosis pathway is not affected by ARV7.2.After DTX treatment and release,ARV7 overexpression group cells Cyclin B1 and mitotic marker p-H3 degraded faster,the proportion of p-H3 protein-positive cells was significantly reduced,containing more DNA content greater than 4N Cdc20 has a higher level of polyubiquitination,and the binding capacity between Cdc20 and the two key mitotic checkpoint complex components Mad2 and Bub R1 is reduced,indicating that ARV7 promotes the mitotic slippage of PCa cells under the action of DTX.3.The proportion of cells in the G2/M phase of sh UBE2 C group was significantly increased,the expression level of apoptotic protein was increased,the degradation rate of Cyclin B1 and p-H3 protein was slower,the polyubiquitination level of Cdc20 was significantly reduced,and the level of Cdc20 and Mad2/ The enhanced binding between Bub R1 indicates that UBE2 C is a key protein that regulates the degree of mitotic arrest.4.Transcriptome analysis and WB results prove that ARV7 promotes the expression of UBE2 C,and silencing UBE2 C in ARV7-overexpressing cells can greatly reverse the mitotic slippage and drug resistance phenotype,indicating that ARV7 mainly promotes UBE2 C by regulating UBE2 C.Mitotic slip is tolerated by DTX.5.The combination of DTX and mitotic exit inhibitors can synergistically inhibit the growth of cells that express ARV7.Conclusion:1.The expression level of ARV7 is closely related to the sensitivity of PCa cells to DTX,but the endogenous apoptosis pathway is not regulated by ARV7.2.ARV7 mediates the reduction of PCa cell sensitivity to DTX through the mitotic slippage pathway and the regulation of UBE2 C levels.3.The combination therapy of DTX and mitotic exit inhibitor can play a synergistic effect in PCa cells that highly express ARV7.In sum,our work identified a novel role of ARV7 in promoting DTX resistance,not only offering a potential path to combat DTX resistance related to abnormal activation of the AR signaling and mitotic dysregulation,but also providing possible synergistic treatment options and novel targets for clinically overcoming drug resistance of CRPC patients.