The Role And Mechanism Of UBC13 In Regulating Paclitaxel Sensitivity In Ovarian Cancer

Ovarian cancer has still presented the highest lethality among all gynecological tumors for the decades,with 46%of the overall 5-year survival rate and only 26%for advanced stages.Primary cytoreductive surgery followed by combined paclitaxel and carboplatin chemotherapy is recognized as the first-line treatment strategy.In this strategy,chemotherapy is an indispensable element because surgery can not completely resect all tumor tissues,especially in advanced ovarian cancer.Although more than 80%o of the patients initially respond to the standard chemotherapy,most of them relapse and require further therapy.Unfortunately,almost all of recurrent ovarian cancers are chemoresistant and the disease persistently progresses.Chemoresistance remains the critical cause for treatment failure and death in ovarian cancer patients.Paclitaxel,as a first-line antineoplastic agent for ovarian cancer,is used for a wide range of solid tumors,but the overall response rate is only 20-40%.Palclitaxel resistance is still an unresolved issue though some mechanisms have been uncovered,such as overexpressed multidrug transporter P-glycoprotein,changed microtubule dynamics or the composition of tubulin isotypes,inhibited apoptosis by BCL-2 family proteins,weakened spindle checkpoint triggered by BUB IB downregulation,enhanced autophagy induced by TXNDC17,and others.However,the progress of reversing paclitaxel resistance according as above mechanisms appears still slow.Recent findings reveal that ubiquitination functions in regulating the sensitivity of tumor cell to chemotherapy agents,including paclitaxel.As an important Post Translational Modification(PTM),ubiquitination widely exists in diverse cellular processes,such as protein degradation,cell cycle and signalling transduction.Ubiquitin could combines with substrates by covalent bonds,and one substrate could be combined with multiple ubiquitins and form ubiquitin chains,which is called polyubiquitination,and then lead subtrates to be degraded.Ubiquitination involves three consecutive process:1.an ubiquitin activating enzyme E1 for attaching Ub to the active site Cys via a thioester bond in an ATP-dependent manner;2.an ubiquitin conjugating enzyme E2 for receiving Ub from E1 to the active site Cys;3.an ubiquitin ligase E3 for aiding transfer of Ub from E2 to specific Lys residues of substrate proteins.Different polyubiquitin chains consisted with different Lys residues ubiquitins make their functions variable.The canonical ubiquitination mediated by Lys-48-linked polyubiquitin chain participates in protein degradation,and the non-canonical ubiquitination mediated by Lys-63-linked polyubiquitin chain involves in other non-degradation processes,such as intracellular signaling transduction,DNA repair.Until now,there are 2 E1 at least 38 E2,and more than 600 E3,encoded by human genome.As E3 colud regulates variable cell processes by ubiquitinating different subtrates due to its substrate specificity,so that present researches about ubiquitination and chemoresistance focus on exploring the role of E3 in regulating chemosensitivity of tumor cells.However,there are still lots of problems to be resolved between ubiquitination and chemoresistance.We used a DIGE quantitative proteomic analysis to search differentially expressed proteins between the ovarian cancer cell line SKOV3 and SKOV3-TR30,a cell line with a 27-fold increase in paclitaxel resistance than its parental SKOV3,and found a remarkably decreased expression of UBC13(UBE2N,ubiquitin conjugating enzyme E2 N)in SKOV3-TR30 cells.UBC13 is one of the ubiquitin conjugating enzyme(E2)family members and plays a central role in canonical and non-canonical ubiquitination.Moreover,UBC13 has been also reported to be closely related to the development of various cancers,even related to chemoresistance.But the relationship between UBC13 and paclitaxel is still unclear.Thus,there may be a link among UBC13 down-regulation,ubiquitination and paclitaxel resistance in ovarian cancer.In this study,we firstly found that UBC13 expression decreased in paclitaxel-resistant ovarian cancer cells and paclitaxel induced downregulationg of UBC13,synchronously reduced CHFR expression,using Western Blot detection.UBC13 abrogation with siRNA reduced paclitaxel sensitivity in ovarian cancer cells,increased DNMT1 and Aurora A expression and decreased CHFR expression,synchronously.Overexpressed UBC13 with plasmid made the results above reverse.Moreover,we found that UBC13 regulated DNMT1 ubiquitination for degradation,DNMT1 regulated CHFR expression by DNA methlylation,and CHFR inhibited Aurora A,so that UBC13 regulated paclitaxel sensitivity in the end.UBC13 expression was detected in 71 ovarian cancer tissues by immunohistochemical staining.Low-expression of UBC13 was significantly correlated with chemoresistance,high risk clinicopathological parameters and poorer prognosis.So UBC13 regulates paclitaxel sensitivity via DNMT1-CHFR-Aurora A pathway and may be used as predictor of chemotherapy response and prognosis in ovarian cancer.Part ?:UBC13 regulates paclitaxel sensitivityin ovarian cancer cellsObjectives:To confirm the effect of regulating UBC13 on paclitaxel sensitivity and the potential molecules participate in regulating paclitaxel sensitivity in ovarian cancer cells.Methods:To evaluate the UBC13 expression between paclitaxel-sensitive and paclitaxel-resistant ovarian cancer cells,and the influence of paclitaxel on UBC13 and CHFR expression,Western Blot detection was used.Paclitaxel sensitivity was assessed by MTS and DNMT1,CHFR and Aurora A were detected by Western Blot after UBC13 was genetic regulated by siRNA and plasmid.Results:1.The amount of UBC13 was decreased in A2780-TR and SKOV3-TR30 cells.2.The amounts of UBC13 and CHFR were substantially reduced after treated with paclitaxel at various concentrations in A2780 and SKOV3 cells.3.Paclitaxel sensitivity was decreased,DNMT1 and Aurora A were increased,and CHFR was decreased after UBC13 expression was reduced using siRNA.4.Paclitaxel sensitivity was increased,DNMT1 and Aurora A were decreased,and CHFR was upregulated after UBC13 expression was upregulated using plasmid.Conclusions:1.Paclitaxel induces UBC13 down-regulation,and regulating UBC13 alters paclitaxel sensitivity in ovarian cancer cells.2.DNMT1,CHFR and Aurora A may participate in regulating paclitaxel sensitivity mediated by UBC 13 in ovarian cancer cells.Part IIUBC13 regulates paclitaxel resistance via DNMT1-CHFR-Aurora A pathway in ovarian cancer cellsObjectives:To confirm that UBC13 modulates paclitaxel sensitivity via DNMT1-CHFR-Aurora A pathway in ovarian cancer cells.Methods:Altered ubiquitination was detected by Western Blot and immunoprecipitation after UBC13 was genetic regulated with siRNA or plasmid.Paclitaxel sensitivity was assessed by MTS and UBC13,DNMT1,CHFR and Aurora A were detected by Western Blot when reduced DNMT1 with siRNA on the basis of UBC13 knockdown with shRNA.CHFR promotor DNA methylation status and CHFR mRNA expression were analysied by Bisulfite sequencing and qRT-PCR after DNMT1 knockdown.Paclitaxel sensitivity and UBC13,DNMT1,CHFR and Aurora A were detected when CHFR was genetic regulated with siRNA or plasmid on the basis of UBC13 genetic regulated.The amounts of UBC13,DNMT1,CHFR and Aurora A were detected by Western Blot after explored to paclitaxel at various concentrations and times.Results:1.Up-regulating UBC13 increased DNMT1 ubiquitination and down-regulating UBC13 decreased DNMT1 ubiqutination.The sensitivity to paclitaxel,the decreased CHFR and increased Aurora A induced by UBC13 down-regulation were partially reversed by DNMT1 knockdown,but didn't affect UBC13 expression.2.Knockdown of DNMT1 with siRNA reduced CHFR promotor DNA methylation,increased mRNA expression of CHFR in A2780 and SKOV3 cells.3.The sensitivity to paclitaxel,aa well as the alteration of Aurora A,due to UBC 13 regulation were partially reversed by CHFR genetic regulation,but neither UBC 13 nor DNMT1 were affected.4.Paclitaxel induced UBC 13 down-regulation,DNMT1 increased,CHFR reduction and Aurora A amplification in A2780 and SKOV3 cells.Conclusions:1.UBC 13 regulates DNMT1 via ubiquitination.2.DNMT1 regulates CHFR by DNA methylation.3.CHFR modulates paclitaxel sensitivity via Aurora A in ovarian cancer cells.4.DNMT1-CHFR-Aurora A pathway participates in regulating paclitaxel sensitivity in ovarian cancer cells.Part ?The low-expression of UBC13 in ovarian cancer tissues is associated with poorer patient prognosisObjectives:To establish the relationship between UBC13 expression and clinical prognosis in ovarian cancer.Methods:We detected UBC13 expression in 71 ovarian cancer tissues by immunohistochemical staining.The relationships between the staining of UBC13 and clinicopathological parameters,such as age,FIGO stage,tumor grade,ascetic fluid volume,serum CA125,primary surgery,chemoresistance,PFS,OS,were analysed.Results:Low-expression of UBC13 was related with chemoresistance,high grade tumor,larger volume of ascitic fluid and suboptimal primary surgery significantly.Ovarian cancer patients with UBC13 low-expression had significantly poorer PFS and OS than those with UBC13 high-expression.Conclusions:Low-expression of UBC13 was significantly correlated with chemoresistance,high risk clinicopathological parameters and poorer prognosis.UBC13 may be used as a predictor for poorer prognosis and potentially used as a therapeutic molecule drug for reversing paclitaxel resistance in ovarian cancer patients.