OTUB2 Epigenetic Silence Contributes To The Tumorigenesis And Chemoresistance Of Malignancies

Squamous cell carcinoma(SCC)is a main subtype of malignancies,mainly happening on skin,mucous and appendages,such as oral cavity,esophagus,skin,vagina and uterus cervix which are covered with squamous cells;SCC also happens on bladder,lung and renal pelvis.Esophageal squamous cell carcinoma(ESCC)is associated with high incidence and mortality in China,yet molecular mechanisms underlying this malignancy are largely unknown.OTUB2(obtain 2),which belongs to deubiquitinases OTU superfamily,affects the stability,function,interaction and localization via altering the ubiquitination level of substrates,so as to participate in influencing cell biological processes.In contrast to previous researches which show that OTUB2 promotes the metastasis of breast cancer and progression of lung cancer,we found that OTUB2 inhibits the tumorigenesis and chemo-resistance of ESCC.Here we established 4-nitroquinoline 1-oxide(4NQO)induced SCC mouse models with Otub2 knockout,and the inducing results showed that OTUB2 deficiency promotes the tumorigenesis and chemo-resistance of oral and esophageal squamous cell carcinomas(SCCs).Besides,we found that OTUB2 is ubiquitously lost in ESCC samples,which indicates the tumor suppressive function of OTUB2 in SCCs.To further explore the function of OTUB2 in human context,we knockdown OTUB2 in normal squamous cells,which then subcutaneously xenografted into NPI mice.The results showed that OTUB2 depletion significantly promotes the tumorigenesis.Next,we identified the tumor suppressive function of OTUB2 in SCCs via in vitro and in vivo functional assays.Mechanistically,we found that epigenetic silence contributes to OTUB2 low expression in SCCs.After that,we found that CALML3 is a critical downstream effector which mediates the tumor suppressive function of OTUB2 via the analysis of combining RNA-seq and proteomics,and STAT1 is the specific substrate of OTUB2 via mass-spectrometer.Through researching,we demonstrate that epigenetic silencing of OTUB2,a previously recognized oncogene,promotes deubiquitylation and phosphorylation of signal transducer and activator of transcription 1(STAT1),then activates transcriptional regulation of calmodulin-like protein 3(CALML3).Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation and glycerophospholipid synthesis,among which phosphatidylserine(PS)is the most obviously affected.Orally administered soybean-derived PS dramatically inhibits low-OTUB2-expressed SCC initiation and increased sensitivity to chemotherapy in mouse models.Gynecological cancer is another main subtype of malignancies,including ovarian cancer,endometrial cancer,cervical cancer,vaginal cancer,vulvar cancer,among which,ovarian cancer and cervical cancer are most common and associated with high mortality.Difficult early diagnosis and late stage chemoresistance contributes to that ovarian cancer is a leading cause of death among patients with gynecological malignancies,which has severely threatened the health and life of female in China.Recently,instead of gene mutation,epigenetic regulation has been the hot topic research area that reveals the mechanism of tumorigenesis.Deubiquitinases(DUBs)play a vital role in malignancies via regulating the expression of specific substrates.Therefore,demonstrating the epigenetic regulation of deubiquitinases is conductive to reveal the process of tumorigenesis and chemoresistance in ovarian cancer from transcriptional level and post-translational level.Here we detected the methylation level of total 45 deubiquitinases via RT-qPCR assays after treating ovarian cancer cells with 5-aza and found that the methylation level of OTUB2 is significantly higher than other 44 DUBs.And the protein level of OTUB2 in ovarian cancer cells is ubiquitously low,indicates high methylation level of OTUB2 promote region contributes to OTUB2 low expression.Then we established DMBAinduced tumorigenesis mice models with Otub2 knock out and found that OTUB2 depletion promotes the tumorigenesis of ovarian cancer,indicating that OTUB2 functions as a tumor suppressor in ovarian cancer.Through studying,we found that the methylation of OTUB2 promoter region leads to OTUB2 deficiency in ovarian cancer,which corelates with poor prognosis of patients.Mechanistically,we found that SNX29P2 is the downstream target of OTUB2 via combining both omics and demonstrated that OTUB2 stabilizes the protein level of SNX29P2 via removing the ubiquitin modification of SNX29P2.SNX29P2 promotes PIK3IP1 expression transcriptionally.and we performed mass-spectrum and found HCE interacts with both OTUB2 and SNX29P2,which promotes the mRNA stability by adding the m7G cap to the 5’end of PIK3IP1 mRNA.Summarily,our study indicates that the OTUB2/STAT1/CALML3/PS axis plays a critical tumor-suppressive role in SCCs,clarifies the low incidence of oral and esophageal SCCs in western countries with a high-fat diet,and demonstrates the potential of food supplementation with PS as a strategy for treatment and prevention of SCCs.Besides,this research reveals that OTUB2/SNX29P2/HCE functions as a booster complex to promote PIK3IP1 expression via accelerating the transcription of PIK3IP1 and delaying the degradation of PIK3IP1 mRNA,therefore inhibiting PI3K/Akt signaling,which inhibits the tumorigenesis and chemoresistance of ovarian cancer.