Role Of Human Papillomavirus 16 E2 In Mediating The Activation Of Apoptosis And Cervical Cancer’s Pathogenic Reversal

Background and objective: In developing countries, cervical cancer is one of the most frequent neoplastic diseases among women for poor screening and therapeutics procedures. Cervical carcinomas are frequently associated with infection by human papillomaviruses (HPVs). It would be an important work to study the relationship between HPV infection and cervical carcinogenesis (CC) and to reverse CC’s development by targeting HPV oncogenic keypoint. Integration of HPV DNA into the host genome is a major mechanism of cervical carcinogenesis. The HPV E2 open reading frame (ORF) has been identified as the preferential site of integration because it has been found to be disrupted or deleted more frequently than other sites. Therefore, the disruption of E2 dependent negative feedback controlling E6 and E7 transcription is considered a selective event in tumor development and progression. Furthermore, several observations have suggested that other E2 functions are necessary to mediate cellular growth arrest. Recent studies have shown that HPVE2 can induce apoptosis through activation of caspase 8, the Fas/FasL extrinsic pathway effector, but its exact mechanism has not been explored. Since c-FLIP has shown to act as dominant negative inhibitors of FADD, a crucial mediator of Fas/FasL signaling, it was suggested to inhibit death receptor-mediated apoptosis by displacing DED-containing caspase-8 from the death-inducing signaling complex (DISC). This study is designed to explores the relationship among HPV16E2, cFLIP and caspase8 and the regulation between HPV16E2 and the Fas/FasL immune monitoring mediated by CTL,and to set up a series of selective proliferating adenovirus to validate the aforesaid mechanisms in vivo. The aim of this study is to realize efficacious biotherapy for cervical cancer targeting the key carcinogenic factor of HPV and to make a great contribution to cut down the incidence and mortality of cervical cancer.Methods:1. A series of archival samples, including squamous cervical carcinomas, cervical intraepithelial neoplasia (CIN) lesions and normal cervical tissues, were subjected to for HPV HC-Ⅱanalysis. Typing HPV-16 infection was analysed by the polymerase chain reaction (PCR) and immunohistochemical staining, and its infection status was assessed with the integrity and disruption of the HPV-16 E2 gene, which was amplified in three overlapping fragments.2. Three vectors, which express HPV16 E2, its DNA binding domain and transcriptional domain respectively, were constructed. RT-PCR、WB、Confocal、MTT、FCM、Transwell methods were used to study the role of these vectors in reversing the malignant phenotype of cervical carcinoma cells.3. Construct HPV16 E2 expression vector containing GFP or His tag. RT-PCR、WB、Confocal、MTT、FCM methods were used to study the role of these two vectors in inducing apoptosis and the relationship between HPV E2 and FLIP.4. Construct selective replicating adenovirus E2-Ad with gene recombination method, transfect it into cervical carcinoma cells to observe CPE effect, adenovirus replication and apoptosis after treated with DDP and irradiation. In vivo antitumor activity of E2-Ad was investigated in human SiHa xenografts.Results:1. HPV16 E2 disruption was associated closely with cervical lesion severity and its progression.2. HPV16 E2 can decrease the expression of E6/E7 gene in SiHa, and inhibit cell growth, promote cell apoptosis, decrease cell transfer activity.3. HPV16 E2 can induce apoptosis not only in HPV(+) SiHa cell, but also in HPV(-) C33A cell through activating caspase-8 and caspase-3. In SiHa, other than C33A, HPV16 E2 can decrease the expression of E6/E7、FADD、FLIPS gene. And there exits a direct interaction between HPV16 E2 and FLIP protein.4. Transfect E2-Ad into different cervical carcinoma cell can induce selective replication and CPE effect, block E6/E7 expression, and excessively increase the role of irradiation and DDP. In vivo, E2-Ad can significantly retard the growth of the SiHa xenografts.Conclusions:1. The loss of HPV16 E2 function during viral integration has been an activation mechanism for progression from high grade CINs to invasive CC.2. HPV16 E2 would be an effective target for cervical carcinoma treatment.3. HPV16 E2 can induce apoptosis through activating caspase-8/3. The mechanism is not only related to the bloke of E6/E7 expression, but also associated with the direct interaction between HPV16 E2 and FLIP protein.4. E2-Ad has significant antitumor effect in vivo and in vitro.