Functional And Mechanistic Studies On Deubiquitinases Of MCL1 In Chemoresistance Of Gynaecological Malignancy

JOSD1 Inhibits Mitochondrial Apoptotic Signalling to Drive Acquired Chemoresistance in Gynaecological Cancer by Stabilizing MCL1 expression of JOSD1 stabilizes MCL1 protein,but its enzymatic mutant JOSD1C36A lost this ability;consistently,depletion of JOSD1 shortened the half-life of MCL1.The subcutaneous xenograft assays showed that JOSD1-overexpressing cells grow much more rapidly than the control cells after the treatment of CBP.However,knockdown of MCL1 reversed the chemoresistance effect caused by JOSD1 overexpression.Taken together,these data indicated that JOSD1 exerts its effects on drug resistance by stabilizing MCL1.Furthermore,immunochemical staining was performed to detect the expression of JOSD1 and MCL1 in 150 ovarian cancer patient samples.We found that the expression level of JOSD1 and MCL1 are highly positively correlated,and the high expression of JOSD1/MCL1 is associated with chemotherapy insensitivity and poor prognosis.Excitingly,the secreted JOSD1 levels in the supernatant of the chemoresistant cell lines were extremely higher than those in the supernatant of the parental cell lines.To further confirm this result in clinical samples,we performed both IB and IHC experiments to detect the serum and intracellular JOSD1 expression levels in 20 ovarian cancer patients and identified that the serum JOSD1 levels are positively correlated with intracellular JOSD1 expression,indicating that intracellular JOSD1 expression levels could be adopted as a marker for clinical diagnosis and prognosis evaluation.Gynaecological cancer is a main subtype of cancer in women,among which ovarian and cervical cancers are the most common.Ovarian cancer is the leading cause of death among gynaecologic malignancies,and cervical cancer is the second most common cancer among women.The high incidence and high mortality of gynaecological cancer have seriously endangered the quality of life and health of women.And acquired chemoresistance is a major contributor to the poor prognosis of gynaecological cancer,but its underlying mechanism remains ill-defined.E3 ligases and deubiquitinases regulate the expression of many important oncoproteins and tumor suppressing proteins at the post-translational level,which has become a hot topic in the field of tumor research in recent years.Therefore,it is expected to find a new strategy to solve the acquired chemoresistant issue by revealing the abnormal regulation of deubiquitinases in acquired chemoresistance of gynaecologic malignancies.Here we established a chemoresistant xenograft model and acquired chemoresistant cell lines to mimic the establishment of acquired chemoresistance.We identified that JOSD1 is the most significantly upregulated DUB during the development of chemoresistance by qPCR analysis,indicating the important role of JOSD1 in this process.To further explore its role in gynaecological cancer,we first knocked down JOSD1 in gynaecological cancer cells.The results showed that JOSD 1 depletion triggers severe cell death under normal culture conditions.The subsequent experiments also confirmed that JOSD1 depletion inhibits the growth of ovarian cancer cells and cervical cancer cells both in vivo and in vitro.Moreover,adenoassociated virus(AAV)-mediated short hairpin RNA(shRNA)interference significantly suppresses the growth of ovarian cancer patient-derived xenografts(PDXs).Mechanistically,the mass spectrometry,Co-IP analysis and in vivo and in vitro deubiquitination assays demonstrated that MCL1 is a substrate of JOSD1.And the immunofluorescence results showed that MCL1 and JOSD1 are co-localized in the cytoplasm.The interaction intensity between JOSD1 and MCL1 after the CBP treatment increased in a time-dependent manner,indicating that JOSD I might exert its antiapoptotic function by binding MCL1.The CHX pulse chase assays showed that ectopicCollectively,our study first revealed that JOSD1 is a novel and critical oncogene that contributes to the acquisition of chemoresistance by inhibiting mitochondrial apoptotic signalling via deubiquitinating and stabilizing MCL1 in gynaecological malignancies.We also suggest that JOSD1 is an ideal therapeutic target and a promising diagnostic marker.MGMT-activated DUB3 Stabilizes MCL1 and Drives Chemoresistance in Ovarian CancerOvarian cancer is one of the most common gynecological malignancies.The incidence of ovarian cancer ranks second,which is the leading cause of death among gynecological tumors.Most ovarian cancer patients are diagnosed at advanced stage.The combination of surgery and chemotherapy is a common treatment for those patients,but most of them develop drug resistance during the course of chemotherapy,which leads to tumor recurrence and metastasis.Chemoresistance is one of the key factors leading to the treatment failure of ovarian cancer patients,which seriously threatens the quality of life and health of women.Chemoresistance has become a major obstacle during the treatment of ovarian cancer patients.Therefore,it is of great significance to investigate the mechanism underlying drug resistance of ovarian cancer,explore effective targets and drugs,and increase the sensitivity of ovarian cancer cells to chemotherapeutics for improving the prognosis of ovarian cancer patients.Chemoresistance is a multifactorial and complex process,in which apoptosis escape plays a very important role.MCL1 is a pivot member of anti-apoptotic BCL-2 proteins,which is highly expressed in various malignant tumors,including ovarian cancer,and leads to the resistance of radiotherapy and chemotherapy.Therefore,to reveal the mechanism underlying MCL1 overexpression in tumors is expected to develop new therapeutic targets and reverse the resistance of ovarian cancer to chemotherapy drugs.And E3 ligases and deubiquitinases regulate the expression of many important oncogenes and tumor suppressor genes at post-translational level,thereby widely involving in tumorigenesis and development.Therefore,UPS regulation has become a hot topic of tumor research in recent years.The protein level of MCL1 is tightly regulated by the ubiquitin proteasome system,and we planned to deeply investigate the regulation of deubiquitinases on MCL1.In this study,we performed an unbiased screening with a cDNA library containing 66 DUBs,and identified that DUB3 is the DUB with the most significant upregulation of MCL1 protein level.DUB3 elevated endogenous MCL1 protein but not its mRNA level in a dose-dependent manner,indicating that DUB3 regulates MCL1 at the post-translational level.The CHX pulse-chase assay showed that ectopic expression of DUB3 could stabilize MCL1 protein.Consistently,the half-life of MCL1 is significantly shortened after knocking down DUB3.The Co-IP assays showed that DUB3 and MCL1 could interact with each other.Further investigation revealed that the N-terminal domains of both DUB3 and MCLI are responsible for this interaction.The ubiquitination assay showed that overexpression of DUB3 could significantly decreased the ubiquitination level of MCL1,but its inactive mutant DUB3C895 lost this effect,indicating that the stabilizing function of DUB3 on MCL1 depends on its enzymatic activity.Next,we detected the expression of MCL1,DUB3,USP9X and USP13 in nine ovarian cancer cell lines.The correlation analysis showed a strong positive correlation between DUB3 and MCLI expression,whereas no obvious positive correlation between either USP13 and USP9X was observed.In addition,the CHX pulse-chase assays and ubiquitination assays showed that the stabilizing effect and deubiquitinating ability of DUB3 on MCLI were stronger than that of USP9X and USP13.The ubiquitination assays with MCLI deletion mutants demonstrated that DUB3 and USP9X mainly modifies the N terminus of MCL1,while USP13 mainly modifies the C terminus.Next,we constructed MCLI mutants and further identified that DUB3 mainly deubiquitinates the 40th lysine within MCL1.Additionally,the in vitro and in vivo functional assays showed that DUB3 depletion increases apoptosis and sensitivity of ovarian cancer cells to carboplatin,which could be rescued by ectopic expression of MCL1;on the other hand,stable overexpression of DUB3 could significantly increase survival rate and resistance to chemotherapeutics.The above results indicated that DUB3 is the primary deubiquitinase stabilizing MCL1 in ovarian cancer.Next,we screened several SMIs and found that patrin-2,a MGMT inhibitor,is the most effective SMI in repressing DUB3 mRNA expression.Moreover,there was a positive correlation between MGMT protein level and DUB3 mRNA level in nine ovarian cancer cell lines,which further indicated that MGMT transcriptionally activates DUB3.The in vilro and in vivo assays also confirmed that Patrin-2 significantly inhibits the growth of ovarian cancer cells with high MGMT/DUB3/MCL1 expression,but has no such effect in low expressing cell lines.Interestingly,we found that HDACis could markedly upregulate the mRNA level of DUB3 in the screening results.And the functional assays confirmed that HDACis could increase the sensitivity of ovarian cancer cells to Patrin-2.Finally,we detected the expression of MGMT,DUB3 and MCL1 in 150 cases of ovarian cancer patients using IHC staining.There was a significantly positive correlation between the expression of MGMT,DUB3 and MCL1,and high MGMT/DUB3/MCL1 expression was associated with chemotherapy insensitivity and poor prognosis.In conclusion,our study revealed the important role of MGMT/DUB3/MCL1 regulatory axis in driving ovarian cancer chemoresistance,and proposed that the combination of Patrin-2 and HDACis could be an effective strategy to overcome ovarian cancer chemoresistance,which has great potential in clinical treatment.