TIPE1 Promotes Cervical Cancer Progression And Its Underlying Mechanisms

Cervical cancer is the fourth most frequently occurring carcinoma in women worldwide.There are about 530,000 new cases each year in the world,and the number of deaths can reach 270,000 per year.More than 95%of malignant cervical cancer patients suffer from human papillomavirus(HPV)infection,and approximately 85%of the deaths occur in underdeveloped or developing countries.The 3 to 5 year survival rates of cervical cancer in underdeveloped countries were less than 50%.Epidemiological studies have found that the occurrence of cervical cancer is closely related to various types of human papillomavirus(HPV)infection,and 96.6%of cervical cancer patients have HPV infection.Based on the high incidence and high mortality of cervical cancer in our country,it is urgent to find new diagnostic and therapeutic targets for cervical cancer.The TIPE(tumor necrosis factor-α-induced protein 8-like,TNFAIP8L)family is a recently described protein family involved in cell proliferation,immunomodulation,and carcinogenesis.This group consists of four highly homologous mammalian members:TIPE,TIPE1,TIPE2 and TIPE3.Thus far,the role of TIPE1 has not been fully elucidated.Recent studies showed that downregulation of TIPE1 is positively correlated with TNM staging and patient survival in hepatocellular carcinoma(HCC),and it serves as a novel prognostic indicator by reducing cell growth and metastasis in lung cancer and gastric cancer.However,The Cancer Genome Atlas(TCGA)dataset revealed that TIPE1 mRNA is upregulated in cervical cancer tissues,and increased expression of TIPE1 was observed in HeLa cells.The evidence indicates that TIPE1has comprehensive roles in carcinogenesis and exerts important functions in cervical cancer.Hitherto,however,the role of TIPE1 was unclear,indicating the urgent need to explore its underlying mechanism,especially in tumorigenesis.Previous studies in our group have found that TIPE1 is highly expressed in cervical cancer cell lines compared with the normal cervical epithelial cell line H8;and TIPE1 was highly expressed in cervical cancer tissues compared with adjacent tissues.More importantly,the expression of TIPE1 was positively correlated with proliferation index Ki67 and negatively correlated with the three-year survival rate of cervical cancer patients.These results suggested that TIPE1 is involved in the progression of cervical cancer and may promote the malignant biological behavior of cervical cancer.Based on the above experiments,the subject will demonstrate the mechanisms of TIPE1 for malignant biological behaviors in cervical cancer in vivo and in vitro by a variety of molecular biology methods,and will provide theory and practice for discovering new functions of TIPE family and new targets for treatment of cervical cancer.Chapter 1 Introduction:An Overview of the Role of the TIPE FamilyThe TIPE(tumor necrosis factor-α-induced protein 8-like,TNFAIP8L)family is a recently described protein family.This group consists of four highly homologous mammalian members:TIPE,TIPE1,TIPE2 and TIPE3.Recent studies have found that the family is closely related to tumors and inflammation,suggesting that they may play a key biological function in tumors and inflammatory diseases.Chapter 2:the expression of TIPE1 in cervical cancer cell lines and tissues2.1 TIPE1 expression in cervical cancer cell linesIn this study,the expression of TIPE1 in the normal cervical epithelial cell line H8 and cervical cancer cell lines,including HeLa,SiHa and CaSki were detected by real-time PCR and Western Blot.The results showed that both mRNA and protein levels of TIPE1,Compared with the normal cervical epithelial cell line H8,were highly expressed in cervical cancer cell lines.2.2 TIPE1 expression in cervical cancer tissuesTo further evaluate the expression of TIPE1 in cervical cancer tissues,we next measured the pathophysiological significance of TIPE1 expression in human cervical cancer tissues and the matched adjacent non-tumor tissues by immunohistochemical staining.TIPE1 proteins were strongly expressed in cancer tissues compared to the paired adjacent non-tumor tissues.Importantly,TIPE1 expression was positively correlated with Ki67,and TIPE1 expression in cervical cancer tissues was negatively correlated with the 3-year overall survival rate of patients.These results suggest that TIPE1 is involved in the progression of cervical cancer and may promote the malignant biological behavior of cervical cancer.Chapter 3:Effects of TIPE1 on biological behaviors(proliferation)of cervical cancer cells3.1 TIPE1 promotes the growth of cervical cancer cellsTo further verify the effect of TIPE1 on the growth of cervical cancer cells in vitro,and based on the expression pattern of TIPE1 in cervical cancer tissues and cell lines,we evaluated its role in human cervical cancer cells in vitro.A shRNA vector that targets TIPE1(TIPE1-sh)and control vector(Scr-sh)were transfected into human cervical cancer cell lines(SiHa and CaSki).The TIPE1 lentivirus was overexpressed in the HeLa cell line.CCK8 method was used to detect the growth curve;PI staining method was used to detect cell cycle by flow cytometry;BrdU method was used to detect the cell proliferation.The results showed that TIPE1 knockdown significantly reduced the cell growth of these cells.In addition,cell cycle analysis showed that there was a reduction in cells in S phase after TIPE1-sh transfection.Conversely,TIPE1 overexpression significantly increased the cell growth and accelerated cell cycle progression,as shown by an increased proportion of cells in S phase,in HeLa cells.These data indicate that TIPE1 promotes cervical cancer cell growth and proliferation in vitro.3.2 TIPE1 significantly enhances the cloning ability of cervical cancer cellsAs above described,a shRNA vector that targets TIPE1(TIPE1-sh)and control vector(Scr-sh)were transfected into human cervical cancer cell lines(SiHa and CaSki),and the TIPE1 lentivirus was overexpressed in the HeLa cell line.After 24hours of transfection,the cells were digested and seeded in a six-well plate at a cell density of 1000 cells/well.After about 10 days,stained with 0.1%crystal violet and photographed.The results showed that after SiHa and CaSki interfered with TIPE1,the ability of colony formation was significantly compromised,and the colony forming ability of HeLa cells after TIPE1 overexpression was significantly increased.3.3 Tumor formation experiments show that TIPE1 significantly enhances tumor growth in BALB/c miceTo further clarify the role of TIPE1 in cervical cancer in vivo,SiHa cell line transfected with TIPE1-sh was transplanted into the BALB/c nude mice subcutaneously for tumor formation.In detail,four-to-six-week-old male BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd.(Shanghai,China).The mice were housed in the animal facilities under specific pathogen-free conditions according to the Animal Care and Use Committee of Jiangsu University,following approval.SiHa cells containing TIPE1-sh and control vectors in PBS were subcutaneously injected into the nude mice.The tumor volume and tumor weight were measured every three days for all groups.The mRNA expression in tumor xenografts was detected by real-time PCR,and the protein expression was assayed by immunohistochemical staining and western blotting.The tumor growth curve indicated that TIPE1 significantly promoted cancer cell growth compared to that of the control group.Consistently,the average tumor weight of the TIPE1-sh group at the time of killing was substantially lower than that of the control group.Furthermore,as Ki67 is a robust biomarker for cell proliferation,we detected Ki67expression in tumors with TIPE1-sh transfection and found it was much lower in the transfected group than the control group.Taken together,these data,consistent with our in vitro results,strongly suggest that TIPE1 enhances cervical cancer development.Chapter 4:Mechanisms of TIPE1 for Promoting Malignant Biological Behavior of Cervical Cancer4.1 TIPE1 suppresses the expression of p53 downstream target genes using Gene chip technologyBased on the phenomenon that TIPE1 promotes the proliferation of cervical cancer cell lines in vitro and in vivo,we performed gene expression and pathway analysis using Affymetrix GeneChip?Human Gene 2.0 ST arrays with HeLa cells transfected with TIPE1 or control vector.The results showed that p53 downstream target genes,especially GADD45,were downregulated compared to those of the control group.For further validation,we then determined the expression of these candidate genes using western blot and qRT-PCR analyses of SiHa cells transfected with TIPE1-sh and HeLa cells overexpressing TIPE1.The results showed that TIPE1significantly increased both the mRNA and protein levels of well-characterized p53target genes,and the gene with the strongest changes was GADD45,which is a key cell cycle checkpoint gene and is responsible for stress-induced apoptosis.These findings suggested that TIPE1 may have a negative regulatory role in the p53 pathway and may regulate cell proliferation and stress-induced apoptosis in cervical cancer.4.2 Verify the interaction between TIPE1 and p53Based on our previous results,we hypothesized that TIPE1 may bind to p53 to affect the expression of p53 downstream target genes.As expected,western blot analysis of Flag/TIPE1 immunoprecipitates from cell extracts revealed that p53 was specifically detected in TIPE1-associated immunoprecipitates in human embryonic kidney 293T(HEK293T)cells.Since p53 is mostly mutant in other tumors,and about5%of mutant p53 is also presented in cervical cancer,the mutant p53 plasmids(R175H,R248W,and R273H)were constructed at the same time.Co-IP method showed that TIPE1 can interact with Wild-type p53 rather than mutant p53.4.3 TIPE1 suppresses the levels of p53 acetylationFurthermore,our results showed that TIPE1 significantly decreased p53acetylation,whereas the total p53 levels were slightly changed.P53 activity is regulated by post-translational modifications.As acetylation of p53 is normally required for its transcriptional activity,TIPE1 may therefore suppress the p53 pathway by reducing the level of p53 acetylation.4.4 TIPE1 promotes proliferation of cervical cancer cells in a p53-dependent mannerGiven our findings that TIPE1 promotes the proliferation of cervical cancer cells along with decreased p53 acetylation levels,we investigated whether TIPE1 affected the proliferation of cervical cancer in a p53-dependent manner.SiHa and CaSki cells stably expressed either a scramble(p53+)or p53-directed shRNA(p53-)and were then transfected with TIPE1-sh or controls.The CCK-8 and colony forming assays showed that depletion of p53 blunted the effect of TIPE1 on cervical cancer proliferation,indicating that TIPE1 enhances cervical cancer proliferation in a p53-dependent manner.4.5 P53 binds to the intronic p53-binding region in TIPE1 promoter to upregulate its transcriptionA scan for potential p53 response elements(REs)confirmed that there has a noncanonical p53 binding sequence composed of?sites that named p53 half-site REs in the intronic region of TIPE1.Binding of p53 to this site was validated with p53ChIP-PCR in SiHa cells,with binding to the GADD45A promoter region serving as a positive control.We following constructed a~500 bp region encompassing the p53RE or mutant one upstream of a luciferase reporter.Consequently,the control vector,the p53RE or mutant vectors were individually transfected into SiHa cells stably expressing either a scrambled(p53+)or p53-directed shRNA(p53-)with the administration of Dox.The result showed that luciferase activity was significantly increased in p53RE group and was blunted when knockdown of p53.These results confirm that the p53RE in the TIPE1 intron is functional as p53-dependent transactivation ability.The phenomenon revealed the site may possibly be an intragenic enhancer.To address this possibility,we use SiHa and CaSki cells stably expressing either a scramble(p53+)or p53-directed shRNA(p53-)to detect the expression of TIPE1.As expected,the levels of TIPE1 both in mRNA and protein were decreased by p53-directed short hairpin RNAs.These results proved that,apart from TIPE1 can suppress the p53 expression;p53 can positively regulate TIPE1transcription through binding to its intragenic region as well,by which comprises a negative feedback loop.Chapter 5:TIPE1 inhibites Cisplatin induced apoptosis in cervical cancer cell lines and its mechanisms5.1 Up-regulation of TIPE1 and MDR1 expression in Cisplatin resistant cervical cancer cell lines and tissuesThe CDDP-resistant cervical cancer cell lines(SiHa/CDDP,HeLa/CDDP)were successfully established.It has significantly higher IC50 value than the mached parental cells.qPCR and western blot experiments showed that the expression of TIPE1 and MDR1 in drug-resistant cell lines were up-regulated.Meanwhile,TIPE1expression in CDDP-resistant cervical cancer tissues was also up-regulated using immunohistochemistry mothed.More interestingly,the expression of MDR1 was negatively correlated with the prognosis of patients.These results suggested that TIPE1 may participate in the process of cervical cancer drug resistance.5.2 TIPE1 inhibits CDDP-induced apoptosis in CDDP-resistant cervical cancer cell linesAfter transfected with TIPE1 interference lentivirus in cervical cancer resistant cell lines,the IC50 values of SiHa/CDDP and HeLa/CDDP were down-regulated,and the apoptotic rate was increased when treated with CDDP using flow cytometry.The above results indicated that TIPE1 inhibits apoptosis of cervical cancer resistant cell lines.5.3 Tumor formation experiment in nude mice shows that TIPE1 inhibits Cisplatin induced apoprosis in CDDP-resistant cervical cancer cell linesTo further clarify the role of TIPE1 in SiHa/CDDP and HeLa/CDDP resistant cell lines,SiHa/CDDP cells were transfected with TIPE1-sh and control virus,and then were transplanted into BALB/c nude mice at a density of 1*10~7.The mice were randomly divided into four groups:control group,TIPE1-sh group,control+CDDP group,and TIPE1-sh+CDDP group.Two groups of which were injected with CDDP intraperitoneally.The long diameter(a)and width(b)of the tumor were measured every 2 day,and the volume of the tumor was calculated as v=a×b2/2.The growth curve of the tumor was drawed.The expression of TIPE1 and MDR1 was detected by immunohistochemistry.The results showed that TIPE1 inhibits Cisplatin induced apoprosis in cervical cancer drug resistant cells when administrated with CDDP.The expression of TIPE1 was positively correlated with the expression of MDR1,indicating TIPE1 inhibits apoprosis by promoting MDR1 expression.5.4 TIPE1 promotes MDR1 expression through inhibiting WTp53 activityPrevious experiments in our group showed that TIPE1 can interact with wild-type p53(WTp53)and thus affect its activity.WTp53 can suppress the transcription of multidrug resistance gene 1(MDR1)promoter,thereby increasing the sensitivity of resistant cell lines to chemotherapeutic drugs.Therefore,we speculate whether TIPE1also promotes the development of cervical cancer resistance by inhibiting WTp53activity.The results showed that interfering with WTp53 expression can compromise the resistance of SiHa/CDDP and HeLa/CDDP cell lines to CDDP after transfected with TIPE1,suggesting that TIPE1 participates in the process of cervical cancer drug resistance by inhibiting wild-type p53 activity.Moreover,downregulation of TIPE1enhanced cisplatin-induced apoptosis in the p53-proficient(p53+)but not p53-deleted(p53-)SiHa cells,suggesting TIPE1 promotes resistance to cisplatin-induced apoptosis in a p53-dependent manner as well.Interestingly,we cotransfected with the TIPE1 and MDR1 promoter plasmids to SiHa cell line that cantaining wild-type p53and the C33A cell line that cantaining mutant p53,dual-luciferase assays showed that TIPE1 can activate MDR1 promoter activity in SiHa,whereas the effect of TIPE1disappeared in C33A.Meanwhile,overexpression of TIPE1 could promote the expression of MDR1 in SiHa cells when treated with CDDP,and this function disappeared in C33A cells.These results suggest that TIPE1 participates in the process of cervical cancer drug resistance by inhibiting wild-type p53 activity.In sum,our study reveals an important insight into the new functions of TIPE1.On the basis of clinical findings,our results unravel a distinct function for TIPE1 that promotes cervical carcinogenesis.Mechanistically,TIPE1 serves as an important factor disables p53 acetylation and activity.TIPE1 drives cervical cancer proliferation and hyposensitivity to CDDP-susceptibility in a p53-dependant manner.This study also identifies p53 directly binds to the promoter of TIPE1 to upregulate its transcription.Thus,therapeutically targeting TIPE1 might is a promising strategy for cancer therapy and TIPE1 as a biomarker to predict the prognosis of cervical cancer.