The Study On Regulatory Mechanism Of Radiosensitivity In Cervical Squamous Cell Carcinoma

Part Ⅰ Effect of HPV16 E6 Regulating EGFR Signaling Pathway on Radiosensitivity of Cervical Squamous Carcinoma cellsBackground Cervical cancer is the second most common malignant tumor and the fourth major cause of death in women worldwide.More than 90% of patients are cervical squamous cell carcinomas(CSCC).It is estimated that there are 570,000 new cases in 2018,accounting for 6.6% of the total number of female cancers(WHO report,2018)and serious threaten to womens’ health.Most patients with locally advanced cervical cancer(LACC)are treated with concurrent chemoradiotherapy(CCRT)based on radiotherapy and platinum-containing regimens in recent 20 years.The 5-year survival rate is 16% higher than that of radiotherapy alone.However,the treatment responses are not satisfactory,more than 270,000 women died every year in the world every year and about 50% of patients have local recurrence and distant metastasis within 2 years of initial concurrent chemoradiotherapy(CCRT).Therefore,it is very important for patients with LACC to clarify the radiosensitivity and intervene insensitive factors to improve the curative effect and prolong the survival time.Human papillomavirus(HPV)is a non-enveloped DNA virus that infects human epithelial tissue.HPV infection includes over 90% of cervical cancers and high-risk HPV genotype infection is considered the important reason of cervical cancer.High-risk HPV,including HPV16 and HPV18,expresses oncogenes E6 and E7,which bind to p53 and Rb tumor suppressor genes,respectively,resulting in rapid degradation of p53 and loss of Rb products.HPV16 causes about 55% of cervical cancer,HPV18 causes about 15% of cases,and the rest is caused by other HR-HPVs.It has been known epidermal growth factor receptor(EGFR)mostly overexpressed in cervical cancer.EGFR expression in the tumors leads to poor therapeutic efficacy and prognosis.Both HPV infections and EGFR pathways have been identifified as targets for cervical cancer therapy.Recently,there has been great interest in the role of EGFR in HPV infection.In 1995,Peto et al first reported that the interaction between EGFR signaling pathway and HPV16 oncogene expression[1].HPV16 E6 is the target of EGF-induced signal.Its activity is amplified by EGF in cervical cancer cells,which establishes the basis of the correlation between HPV16 E6 expression and EGFR level [2].Other studies have shown that not only E6 oncoprotein activates EGFR signal transduction pathway,but also EGFR expression level affects HPV16 E6 oncoprotein expression level,and ultimately affects the growth rate of cervical cancer cell lines [3,4].Therapy strategy toward EGFR inhibition in cervical cancers either by anti-EGFR antibodies or by tyrosine Kinase Inhibitor(TKIs)has been ongoing.HPV16-infected cervical squamous cell carcinoma is sensitive to radiotherapy,but its mechanism is still unclear.It has been pointed out that HPV-related head and neck squamous cell carcinomas are highly sensitive to radiotherapy with high apoptotic rate.It may be related to AKT,which is the downstream signaling pathway of EGFR regulated by E6 protein.Therefore,it is assumed that the radiosensitivity of HPV16 infection to cervical squamous cell carcinoma may be related to AKT inactivation of EGFR downstream signaling pathway.The hypothesis is verified by cell proliferation,apoptosis and animal experiments.Objectives To establish a HPV-E6 transfected C33 A cervical squamous cells and investigate the proliferation of these cells,we would confirm that the oncogene characteristic of E6 gene in cervical squamous carcinoma.To investigate the radiosensitivity of HPV16 E6 overexpressed C33 A cells compared with the control group,and to clarify that the apoptosis of E6 overexpressed C33 A cells for radiotherapy is independent of the apoptosis induced by p53.To further clarify the effect of radiosensitivity on E6 gene through knocking down E6 gene.To analysis the correlation between HPV6 E6,EGFR and radiotherapy through being treated with nituzumab, nituzumab plus radiotherapy and radiotherapy respectively.To detect the possible regulatory mechanisms of E6 overexpressed C33 A cells were in downstream signaling pathway of EGFR.Finally,we constructed a nude mice model of HPV16 E6 overexpressed C33 A cells and control cells.Radiotherapy and anti-EGFR were used as treatment factors to further explore the effect of AKT,which was a regulatory factor of HPV16 E6 regulating EGFR downstream signaling pathway and affected on radiosensitivity of cervical squamous cell carcinoma.Methods 1.CCK-8 assay was used to detect the effects of radiotherapy and radiotherapy combined with Nituzumab on proliferation of the C33 A cells grouped to the normal control group,HPV16 E6 overexpression group and sh E6 gene group.2.Flow cytometry was used to detect the effects of radiotherapy combined with or without Nituzumab on apoptosis rate and cell cycles of the cells in those three groups.3.EGFR,AKT,ERK1/2,P53,Factor associated suicide(Fas)and Caspase-3 in HPV16 E6 overexpression and the knocking down E6 gene of C33 A cells were detected by q RT-PCR and Western blot,respectively.4.Establishing tumor-bearing models of cervical squamous cell carcinoma in nude mice is to observe the relationship between radiotherapy and anti-EGFR therapy on HPV16 E6 overexpression and control cells and to further find the tumorigenic rate and tumor volume.HE staining was used to observe the histomorphology of the tumors.Immunohistochemical techniques were used to detect the expression levels of cycle and apoptosis-related proteins.Results 1.The results of CCK-8 experiment show:The optical density(OD)of HPV16 E6 overexpressing C33 A cells was significantly higher than that of the control cells,and the difference began to be statistically significant at 48 h(P<0.05).The OD value of the RT group was significantly lower than that of the control cells,the difference was statistically significant at 72h(P<0.05).The OD value of RT+Ni group was significantly lower than that of control group and the difference was statistically significant at 48h(P<0.05).In addition,the OD value of RT+Ni group was significantly lower than that of RT group and the difference was statistically significant at 72h(P<0.05).The OD value of HPV16 E6 knockdown C33 A cells was significantly lower than that of the control cells,and the difference was statistically significant from 48h(P<0.05).The OD value of the RT group was significantly higher than that of the control group and the difference was statistically significant at 48 h(P<0.05).The OD value in the RT+Ni group was significantly higher than that in the control group and the difference was statistically significant at 48h(P<0.05).In addition,the OD value of the RT+ Ni group was significantly lower than that of the RT group and the difference was statistically significant at 48h(P<0.05).The OD value of C33 A cells was not significantly different from that of the same group of E6 knockdown cells(P>0.05).2.The results of flow cytometry experiments show: From the perspective of apoptosis,the apoptosis rate of HPV16 E6 overexpressing C33 A cells in the RT group was significantly higher than that in the control group(P<0.05).The apoptosis rate in the RT+Ni group was significantly higher than that in the control group(P<0.05).In addition,the apoptotic rate of RT+Ni group was significantly higher than that of RT group,and the difference was statistically significant(P<0.05).The apoptotic rate of HPV16 E6 knockdown C33 A cells in the RT group was lower than that in the control group(P<0.05),and the apoptosis rate in the RT+Ni group was significantly lower than that in the control group(P< 0.05);In addition,the apoptotic rate of RT+Ni group was higher than that of RT group,the difference was statistically significant(P<0.05).The apoptotic rate of C33 A cells was not significantly different from that of the same group of E6 knockdown cells(P>0.05).From the cell cycle,the proportion of G0/G1 phase cells in the HPV16 E6 overexpressing C33 A cells was significantly lower than that in the control group(P<0.05),while the proportion of G0/G1 cells in the RT+Ni group was lower than that in the RT+Ni group(P<0.05).In addition,the proportion of G2/M phase cells in the RT group was significantly higher than that in the control group(P<0.05).The proportion of G2/M phase cells in the RT+Ni group was higher than that in the control group,and the difference was statistically significant(P<0.05).The proportion of G0/G1 phase cells in HPV16 E6 knockdown C33 A cells was significantly higher than that in control group(P<0.05).The proportion of G0/G1 cells in RT+Ni group was significantly higher than that in control cells(P<0.05).There was no significant difference in the proportion of cells in the G2/M phase of the RT group compared with the control cells(P>0.05).The proportion of G2/M phase cells in the RT+Ni group was significantly higher than that of the control cells(P<0.05),also higher than the RT group(P <0.05).The proportion of G0/G1 phase cells in C33 A cells was not significantly different from that in the knockdown group(P>0.05).3.RT-PCR and Western blot results show: The levels of EGFR,AKT and ERK1/2 in HPV E6 overexpressing cells were significantly higher than those in the control group(P<0.05).The expression of AKT in the RT group was significantly lower than that in the control group(P<0.05);The expression levels of EGFR,AKT and ERK1/2 in RT+Ni group were significantly lower than those in control group(P<0.05)and further decreased compared with RT group,the difference was statistically significant(P<0.05).The levels of EGFR,AKT and ERK1/2 in HPV16 E6 knockdown cells were significantly lower than those in the control group(P<0.05).There was no significant difference in the expression of EGFR,AKT and ERK1/2 between C33 A cells and HPV16 E6 knockdown cells(P>0.05).Apoptotic proteins were detected by Western blot and found:in the HPV16 E6 overexpressing cells,the expression of Fas and Caspase 3 in the RTgroup were significantly higher than that in the control group(P<0.05).The expression of Fas and Caspase 3 in the RT+Ni group was significantly higher than that of the control group,and the difference was statistically significant(P<0.05).In addition,the expression levels of Fas and Caspase 3 in RT+Ni group were significantly higher than those in RT group(P<0.05).In HPV16 E6 knockdown cells,the expression levels of Fas and Caspase 3 in the RT group were significantly lower than those in the control group(P<0.05).The RT+Ni group was significantly lower than the control group,and the difference was statistically significant(P<0.05)and Fas and Caspase 3 in RT+Ni group were significantly higher than those in RT group.There was no significant difference in the levels of Fas and Caspase 3 between C33 A cells and E6 knockdown cells(P>0.05).4.Establish tumor-bearing models of cervical squamous cell carcinoma in nude mice:As the tumor cell planting time prolongs,the tumor tissue gradually increases.The tumor volume of over-expressed HPV16 E6 was significantly higher than that of the control group from the 4th day(P<0.05).The tumor volume of the RT group was significantly lower than that of the control group from the 7th day.The difference was statistically significant(P<0.05);the tumor volume in the RT+Ni group was significantly lower than that in the control group from the 7th day(P<0.05),and the tumor volume in the RT+Ni group was significantly lower than that in the RT group from the 7th day(P < 0.05).The relationship between Caspase 3 and Cleaved-caspase 3 and apoptosis in tumor-bearing models of nude mice was detected by immunohistochemistry.Caspase 3 and Cleaved-caspase 3 were mainly expressed in the cytosol by microscopic observation.In HPV E6 overexpressing tumors the expression levels of caspase 3 and Cleaved-caspase 3 in the RT group were significantly higher than those in the control group(P<0.05).The expression levels of caspase 3 and Cleaved-caspase 3 in the RT+Ni group were significantly higher(P<0.05),and the expression levels of caspase 3 and Cleaved-caspase 3 in RT+Ni group were significantly higher than those in RT group(P<0.05).Conclusion 1.HPV16 E6 is an oncogene of CSCC,and E6 expression is positively correlated with the levels of EGFR,AKT and ERK1/2 protein expression.2.Overexpression of HPV16 E6 in C33 A cells is more radiosensitive,and mechanism of apoptosis induced by radiotherapy is independent of p53 protein expression,which is related to the regulation of EGFR signal pathway by E6 protein.3.EGFR inhibitor,combined with radiotherapy,fully confirmed that the radiosensitive regulatory mechanism of HPV16 E6-positive cervical squamous cell carcinoma was related to AKT,which is one of downstream EGFR signaling pathway.AKT combined with E6 affected AKT phosphorylation,thus inhibiting EGFR-PI3K-AKT signaling pathway and enhenceing the efficacy of radiotherapy.PartⅡ Correlation between expression of aldehyde dehydrogenase-1 in cervicalsquamous cell carcinoma and efficacy of Chemoradiation therapyAldehyde dehydrogenase 1(ALDH-1)is a cancer stem cell marker whose overexpression can predict the prognosis of several human cancers.In this study,we assessed ALDH-1 expression in cervical squamous cell carcinoma tissues and investigated the usefulness of ALDH-1 expression levels for the prediction of patient response to chemoradiation therapy and survival.Tissue samples from 74 patients were examined via immunostaining with ALDH-1 antibody before and after chemoradiation therapy.Twenty-nine of the 74 cervical cancer cases exhibited ALDH-1 expression(39.19 %)in the pre-treatment tissue samples;however,only 19 of these 39 cases exhibited ALDH-1 expression(48.72 %)in post-treatment tissue samples,indicating that chemoradiotherapy eliminated the ALDH-1 positive tumor stem cells in cervical cancer.However,patient disease-free survival was not associated with ALDH-1 expression(11.78 ± 1.95 vs.12.50 ± 1.91 months in ALDH-1-positive and-negative tumors,respectively).This study indicates that the reduction of the cancer stem cell population via chemoradiotherapy may not be suffificient to improve patient survival. Thus,the clinical value of ALDH-1 expression in cervical squamous cell carcinoma may need to be re-examined.