The Regulation Of TGF-?1-induced CK17 In Cervical Cancer Stem Cell-like Properties

BACKGROUND AND PURPOSEDespite the initial potency of conventional strategies,tumor relapse and metastasis are still the major causes of therapeutic failure and bad prognosis in late-stage cervical cancer(CC)patients.Studies have shown that traditional therapy can kill most differential and proliferative tumor cells in tumor tissue.However,a small population of tumor cells,because of its unlimited self-renewal and anti-chemotherapy and tumorigenic capabilities,can successfully escape from the traditional therapy.This powerful sub-population of cells,called "cancer stem cells".The population of cells,similar with normal stem cells and keeping in a quiescent stage from cell division activity,is the root cause of tumor recurrence and metastasis.For cervical cancer patients,patients with early-stage cervical cancer with surgical resection or locally advanced disease with chemoradiation,can be cured.However,once people suffer from a local invasion or distant metasitasis of CC(bulkly neoplasm or carcinoma extending beyond cervix,involves vaginal and pelvic metastasis,usually meant FIGO stages IB2 to IV)or recurrent cervical cancer,the prognosis becomes poor and better treatments are required.Therefore,to find treatment programs targeted cervical cancer stem cells,or supplemented by comprehensive treatment program of traditional therapy,is of great significance the treatment for patients with late-stage cervical cancer.Currently,there's not one opinion about unique and reliable marker of CSCs and relevant signal pathways regulating stemness in cervical cancer.In-depth study of cervical cancer stem cell marker and relevant signaling pathway is essential for development of treatment targeting CCSCs.Recent studies indicate that the tumor microenvironment and cancer stem cells have a close relationship.Inflammatory microenvironment can recruit myeloid mesenchymal stem cells,which fused with tumor cells inducing a genetic recombination transforming into tumor stem cells;Immune cells in inflammatory microenvironment activate the stemness signal pathways in tumor cells through secretion of cytokines,leading to the conversion of non-CSCs to CSCs.Transforming growth factor-?1(transforming growth factor-?1 TGF-?1),is one of the most important cytokines in HPV-related cervical cancer microenvironment.Recent years,scholars have found that TGF-?1 can promotes stemnesss in tumor cells as well as EMT.In the study of cervical cancer,no one has reported the relationship between TGF-?1 and cervical cancer stem characteristic.Therefore,the role of TGF-?1 in cervical cancer sternness-transformation and metastasis causes us great interest.In this study,we used TGF-?1 to demonstrate the relationship and regulatory mechanism among tumor microenvironment,CSCs and EMT and to describe its role in preservation of stemness of cervical cancer stem cells.This study will be divided into 4 parts.METHODS1.Effect of TGF-?1on cancer stem cell-like properties and the screening and identification of its target in cervical cancer cells1.1 Human cervical cancer cell line SiHa was used in all our in vitro experiments.Oncosphere-forming assays and SP detections were used to examine the number of oncospheres and percentage of Side Population cells with or without recombinant human TGF-?1 treatment in SiHa cells,respectively.1.2 Fluorescence Quantitative PCR were used to test the mRNA level of a promising marker of cervical cancer stem cells(NANOG,NESTIN,SOX2,OCT4,CK17,ALDH1,CD133)with or without recombinant human TGF-?1 treatment in SiHa cells in Oncosphere-forming assay and SP detection were used to elevate the stemness properties in cervical cancer cells.1.3 Used the method of cationic liposomes to transiently transfect SiHa with three commercial CK17 SiRNAs fragment,used fluorescence quantitative PCR and Western blot to test CK17 expression change and got Si3 efficient interference fragment.Oncosphere-forming assays and SP detections were used to examine the number of oncospheres and percentage of Side Population cells with or without recombinant human TGF-?1 treatment in SiHa cells after transfecting with nc or SiCK17,respectively.2.The Correlation between CK17 and epithelial-mesenchymal transition(EMT)induced by TGF-?12.1 The morphological changes associated with EMT were analyzed by phase contrast microscopy,Western blot and immunofluorescence.2.2 Western blot was used to test the changes of relative marker of EMT induced by TGF-?1 after the interference of CK17.3.Study and identification of the signal pathway involved in TGF-?1 induced upregulation of CK173.1 Western blot was performed to examine a set of relevant protein of TGF-?1-involved signal pathways,including phospho-Smad3,Smad3,phospho-ERK1/2,ERK1/2,phospho-p38,p38,phospho-JNK,JNK,phospho-PI3K and PI3K after the stimulation of TGF-?1.3.2 Used a specific MEK inhibitor(U0126),Smad3 inhibitor(SIS3)or JNK inhibitor(SP600125)to inhibit the phosphorylation of corresponding protein,respectively.Western blot and dual-luciferase reporter assays were conducted to test the protein level of CK17 or luciferase activity of CK17 promoter after the pretreatment of inhibitor with or without the stimulation of TGF-?1.3.3 The CK17 truncated promoters were amplified by PCR and then cloned into the pGL3-basic vector as pGL3-CK17-1366,pGL3-CK17-1013,pGL3-CK17-797,pGL3-CK17-661,pGL3-CK17-183,pGL3-CK17-34.To test the target sites of TGF-?1 on CK promoter,we performed luciferase reporter assays to conform it with or with the stimulation of TGF-?1.3.4 Potential molecular targets of TGF-?1 on the segment-661?-183 of CK17 promoter were analysed by bioinformatics algorithms in the two commonly used databases including TFSEARCH and CONSITE.Luciferase reporter assays were conducted to screen the predicted transcriptional factors.3.5 Used inhibitor U0126 and fluorescent quantitative PCR to verify the relation among TGF-?1,ERK1/2 and CK17.3.6 Used siRNA and fluorescent quantitative PCR to verify the relation among TGF-?1,E2F4 and CK17.4.Correlation between TGF-?1 and CK17 expression in human SCCImmunohistochemistry waa used to examine the expression of TGF-?1 and CK17 in 70 cases of human SCC in cervical tissue microarrays,respectively.RESULTS1.Effect of TGF-?1on cancer stem cell-like properties and the screening and identification of its target in cervical cancer cells1.1 Oncosphere formation assays showed that the number of oncpspheres in the control group was 35.33 ± 2.52 cells/1000 cells,while the number of oncospheres in the TGF-?1 treatment group was 53.33 ± 3.51 cells/1000 cells.Compared with the control group,the oncosphere forming ability of TGF-?1-treated group was significantly enhanced(P<0.05);The SP detection showed that increased proportion of side population cells in TGF-?1 treatment group:the proportion of SP cells in the control group was only 0.5%,while the proportion of cells in TGF-?1 treatment group was 1.9%,the increase is statistically significant(P<0.001).1.2 Fluorescent quantitative PCR showed that CK17 was significantly forced up(X±SD=7.367±0.949,P<0.001)among all possible markers of CCSCs after the stimulation of TGF-?1.1.3 After CK17 instantaneous interference,used the fluorescent quantitative PCR to detect CK17 expression quantity change in SiHa cell lines,used NC group as control(X±SD=1±0.000),one-way analysis of variance result showed that CK17 expression has significant difference in group 3 compared with NC group(X±SD=0.181±0.001)and has no significant difference between group 2 or group 1 and Nc group(X±SD =0.713±0.035,X±SD =0.623±0.021),and the Nc group is significantly higher than interference and group 3(F=327.387,P<0.001).Selected the interference fragment 3 to conduct on oncosphere formation assays and SP detections.Oncosphere formation assays showed that the number of oncospheres in the nc+TGF-?1 was significantly increased compared with which in the Nc groups,while the SiCK17+TGF-?1 groups significantly decreased compared with the nc+TGF-?1 groups(P=0.002).Only silence of CK17 decreased the number of oncospheres compared with the nc group while there is no statistical difference between the two groups.The SP detection showed the similar results.2.The Correlation between CK17 and epithelial-mesenchymal transition(EMT)induced by TGF-?12.1 After 72 hours' exposure of TGF-?1 most CC cells acquired long spindle,bulkly mesenchymal alteration.Western blot showed that There was a readily reduction in E-Cadherin expression,whereas enhanced expression of vimentin and fibronectin observed in TGF-?1-treated cells.IC had showed similar results as well.2.2 Western blot showed that whether CK17 was knocked down or not,the EMT makers,including Ecadherin,Vimentin and Fibronectin,was not change.In addition,the effect of TGF-?1 on EMT markers were not change even with the deletion of CK17 expression,either.3.Study and identification of the signal pathway involved in TGF-?1 induced upregulation of CK173.1 Western blot showed that the activation of Smad3,ERK1/2,JNK pathway,as indicated by the increase of phosphorylated smad3 and p42/p44(p-ERK1/2)at 30 min-1 hour,p-JNK at 6h after stimulation,which then rapidly declined to basal level.Other signaling pathways including c-Jun NH2-terminal kinase(JNK),p38 MAPK,and Akt were not activated up to 4 h after TGF-?1 treatment.3.2 Western blot analysis showed that TGF-?1-induced expression of CK17 in SiHa cells was greatly reduced by U0126 but was not affected by SIS3 or SP600125.3.3 Luciferase reporter assays showed that transient transfection of-34/+1,-183/+1 constructs only stimulated luciferase activity by 0.3-1.1 fold after TGF-?1,while-661/+1,-799/+1,-1013/+1,-1366/+1,-1999/+1 significantly stimulated luciferase activity by 1.6-4.5 fold.3.4 Five TFs for putative binding sites were predicted,including MZF1,CREB,E2F,SP1,Snail(As E2F superfamily contains eight known E2F family members,we toke two standard E2F members,E2F1 and E2F4 instead).Luciferase reporter assays showed that transient transfection of MZF1(FsiHa=48.998,PSiHa=0.008,F293T=213.113,P293T=0.001)and E2F4(FSiHa=482.022,PSiHa<0.001,F293T=367.496,P293T=0.001)could significantly increase the transactivation of the CK17 promoter(-661?-+1)in both SiHa and 293A cells.While E2F1 could decrease the the transactivation of the CK17 promoter(-661?+1)in both SiHa and 293A cells significantly.The other TFs did not have significant influence on the the transactivation of the CK17 promoter.3.5 Fluorescent quantitative PCR showed that the expression of E2F4 was increased during the stimulation of TGF-?1(P<0.05),while cells were pretreated with the inhibitor(U0126),such upregulation of by TGF-?1 dropped.3.6 Fluorescent quantitative PCR showed that interference of E2F4 expressin could block the transactivation of CK17 by TGF-?1.4.Correlation between TGF-?1 and CK17 expression in human SCCImmunohistochemistry analysis results showed that High expression of TGF-?1 was detected in 62.9%(44 of 70)of the samples,and high expression of CK17 detected in 65.7%(46 of 70).The immunostaining analysis revealed a strong correlation of CK17 expression with TGF-?1 expression(r=0.441,P<0.001).With regard to clinical parameters,Both TGF-?1 and CK17 was positive correlated with International Federation Of Gynecology And Obstetrics(FIGO)stage(PTCF-?1<0.001,PCK17=0.001)and lymphatic metastasis(PTGF-?1=0.002,PCK17=0.025).However,other clinical parameters,including age and pathology grade were not significant related to either expression of CK17 or TGF-?1(all P>0.05).For further investigation of the association between the expression levels of TGF-?1 and CK17 and the clinical parameters in SCC patients,we put the age and pathology grade(the FIGO stage was excluded as it is influenced by lymphatic metastasis)were entered into the logistic regression model,only the subgroup of patients with a TGF-?1high/CK17high expression profile but not the CK17 expression,TGF-?1 expression or other clinical parameters,showed a significantly higher risk of developing lymphatic metastasis.CONCLUSIONS1.CK17 is essential for cancer stem cell-like properties induced by TGF-?1 in SiHa cells but not essential for TGF-?1-induced EMT.2.Proposed a new regulation mechanism,that is TGF-?1 can activated ERK1/2 signal pathway by phosphorylation and which increased the expression of the transcriptional factor E2F4 promoting the transactivation of CK17.3.There is a significantly positive correlation between TGF-?1 and CK17 expression in SCC.The combined expression of CK17 and TGF-?1 was a more powerful predictor in predicting CC lymphatic metastasis.THE INNOVATION OF THIS PROJECT1.The first demonstration of cervical cancer stem cells CK17 markers in the aspect og mechanism;2.Proposed a new regulation mechanism participated in the regulation of cervical cancer stem cell-like properties,which provides us a promising target and relevant pathway for the treatment of cervical cancer relapse and metastasis.