The Mechanism Of High-Risk HPV Early Oncoprotein E6 And E7 Regulate Cell Cycle

Human papillomaviruses (HPVs) are small double-strands DNA virus with a 8kb genome. More than 150 HPV types have been identified so far. The genital HPV types are subdivided into a low-risk subgroup that are predominantly found in benign lesions such as HPV6,11 and a high-risk subgroup, including the most common types HPV16 and HPV18, which are associated with more than 70% of all premalignant and malignant disease of the cervix.The epidomologic study shows that, HPV16 is the dominant HPV type worldwide, besides HPV16 and HPV18, other HPV types such as HPV31,33,45 and 58 also are highly related with cervical cancer. Of which HPV58 is the third most common HPV type in cervical cancer from Eastern Asia, accounting for 9.8% of cervical cancer, following HPV16,18. HPV58 is the second common HPV type in cervical cancer in Southern China, including Taiwan and Hongkong, whose prevalence is close to HPV16.The genome of HPV can be divided into three regions:the non-coding region, the early region which encodes 7 early genes and late region which encodes 2 late genes. The transforming ability of HPV resides in the E6 and E7 oncogenes, of which E6 binds and degrades p53, a tumor suppressor. E7 from high-risk HPV types binds to and degrades Rb protein, releasing E2F from Rb/E2F complexes. E2F then activates genes such as cyclins which are essential for S phase entry. My research focus on 3 parts:(1) HPV16E6 has functions independent of degradation of p53.16E6 can induce polyploidy which is an important early event in carcinogenesis by abrogating post-mitotic checkpoint and this occurs without degrading p53. (2) We infected human keratinocytes-HPV natural host cell with HPV58E7 and found that 58E7 degraded pRb and activated cyclin-dependent kinase Cdk2, by which accelerated cell proliferation. (3) Inteferon-inducible protein IFI16 can remarkably slow down cell growth of cervical cancer cell line SiHa and decrease the size of tumor in nude mice, which suggests that IFI16 could be a potential inhibitor of HPV related cervical cancer.The mechanism of HPV16E6 induced polyploidyGenomic instability in the form of polyploidy has been implicated in a causal role in carcinogenesis. Polyploidy not only occurs as an early event during cervical carcinogenesis but also predisposes cervical cells to aneuploidy, an important hallmark of human cancers.Previously, it was hypothesized that E6 induce polyploidy by abrogating the spindle checkpoint or disrupting cytokinesis and that E6 degrades the tumor suppressor p53 to induce polyploidy. Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint. Instead, it abrogates the post-mitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy.1. To test whether 16E6 induces polyploidy depending on its p53 degradation, recombinant retroviruses expressing high-risk 16E6 alone and F2V-a 16E6 mutant with intact p53 activity were used to infect a spontaneously immortalized keratinocytes cell line (NIKS). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Our data shows that both 16E6 and F2V induce polyploidy, which means 16E6 can induce polyploidy without degrading p53.2. Upon nocodazole treatment, cells underwent spindle checkpoint, however cells didn't arrest in M phase. Our previous data showed that cells adapted to this checkpoint and slippage to next cell phase-G1 like phase. Vector NIKS will arrest in this phase since the exist of post-mitotic checkpoint, while both 16E6 and F2V can abrogate this checkpoint and progress into S phase with a tetrapoloidy genome. Since Cdks are the core kinase to drive cell cycle, we then tested the expressions of all cyclins and Cdks. Our data showed the expressions of Cdk1 and Cdk2 were increased dramatically.3. Since Cdk itselft doesn't have kinase activitity, we then determined the kinase activities of all cyclins and Cdks. We found that Cdkl and Cdk2 have higher enzyme activities which proved that cells abrogated post-mitotic checkpoint by activating Cdk1 and Cdk2.These mechanisms underscore the nature of the important viral proteins E6 to redirect host cells machineries and promote tumorigenesis. Our research investigates under-studied cell cycle processes and challenges existing dogmas. These studies will shed light on mechanisms by which HPV induces genomic instability and hold promise for the identification of targets for drug development.The biological activities of HPV58 oncoprotein E7Although we have already learned a lot about the mechanism by which several high-risk HPV types such as HPV16 and 18 to induce cellular transformation, little is known about how other high-risk HPV types like HPV58 induce carcinogenesis.1.We choose primary human keratinocytes (PHKs)-the host cell of HPV infection as our target cell. Stable cell lines were created by infection of PHKs with retroviruses containing HPV-58 E7 (PHK/58E7) or an empty vector (PHK-Vector). PHKs expressing HPV-16 E7 (PHK/16E7) were included in the study as a control. E7 expression in the stable cell lines was confirmed by RT-PCR～. As antibody against HPV-58 E7 protein is not available, we also established a PHKs line expressing HPV-58 E7 fused at the N-terminal to hemagglutinin epitope tag (HA tag, PHK-HA-58E7). HPV-58 E7 promoted the proliferation of PHKs. PHK/58E7 proliferated as efficiently as PHK/16E7 and significantly more rapidly than the vector control cells. HPV-58 E7 significantly extended the life-span of PHKs. While the vector control PHKs stop divide at passage 5 or after approximately 20 population doublings (PDs), PHKs expressing all E7s continue to proliferate at passage 20 (～110 PDs)2. We examined the expression and kinase activity of Cdk2 in PHK/58E7. The regulatory partners of Cdk2, cyclin A and cyclin E, were also up-regulated in these cells. In contrast, there was no significant change in the steady-state levels of Cdk6 in E7 expressing PHKs and the vector control PHK. What is more important, the Cdk2-associated kinase activity is higher in E7-expressing PHKs. In contrast and consistent with the expression levels, the kinase activity of Cdk6 were similar in E7 and vector containing PHKs. p53 controls these processes mainly by regulating transcription of target genes. One of the most important downstream targets of p53 for cell cycle regulation is p21, which is a universal inhibitor of cycliri and cyclin-dependent kinase. We therefore examined the expression of p53 and 21 in HPV-58 E7 expressing PHKs, the steady-state levels of both p53 and p21 were increased in E7 expressing PHKs. These results are consistent with previous observation with HPV-16 E7 and suggesting that HPV-58 E7, like HPV-16 E7, overrides the tumor suppressor activity of p53 and p21.3. It is well established that HPV-16 E7 abrogates the G1 cell cycle checkpoint. To examine the ability of HPV-58 E7 to modulate the G1 checkpoint, we treated PHKs expressing HPV-58 E7 with bleomycin, a radiomimetic agent capable of inducing both single-stranded and double-stranded DNA breaks and G1 and G2 cell cycle arrest. As expected, upon bleomycin treatment, a reduction of number of S phase cells occurred in the vector control PHK. In contrast, PHK/58E7 showed a significantly decreased number of cells in the G1 phase of the cell cycle. These results demonstrate that HPV-58 E7 can abrogate the DNA damage-induced G1 checkpoint. Polyploidy has been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy. Accordingly, we treated cells with the microtubule poison nocodazole, which disrupts microtubule polymerization. After treating with nocodazole, PHK/58E7 showed a significant increase in polyploidy compared to vector controls.4. It was suggested that the ability of high-risk HPV E7 proteins to bind and distablize pRb family members is necessary for abrogating the G1 checkpoint. We therefore examined the steady-state levels of pRb family members in HPV-58 E7 expressing PHKs by Western blot. The overall steady-state levels of pRb are reduced in HPV-58 E7 expressing PHKs as compared with the vector control PHKs. These results suggest that degradation of pRb is a mechanism by which HPV-58 E7 to regulate cell cycle checkpoints. Surprisingly, the steady-state levels of p1 07 did change significantly in all three PHKs expressing HPV E7 as compared with the vector control PHKs. On the other hand, the steady-state levels of p130 were significantly reduced in PHK/58E7. Notably, the steady-state levels of p130 in PHK/58E7 are consistently and significantly lower than that of PHK/16E7.It is the first time that we investigate the molecular basis of 58E7. These studies will shed light on mechanisms by which HPV induces genomic instability and hold promise for the identification of targets for drug development and vaccine.IFI16 inhibites HPV associated cervical carcinogenesisInterferons (IFNs) are vertebrate cytokines which perform important functions within the cell, including the regulation of cell growth and differentiation, as well as the inhibition of virus-based processes. IFN-inducible proteins, which are expressed downstream of IFN signaling, are thought to be mediators of antitumor and antivirus actions of IFNs. The p200 family is one such family and IFI16 is a human member of p200.1. We first detected the levels of IFI16 in normal cervical tissue and cervical cancer, and found that the expression of IFI16 was significantly decreased in human cervical cancer tissues compared with normal cervical tissues. We next generated stable cell lines overexpressing IFI16 in SiHa cervical cancer cells. The overexpression of IFI16 inhibited cell proliferation and reduced the number of apoptotic cells. In addition, significant higher P-galactosidase activity was observed in IFI16-expressing stable lines than in corresponding control cells. We injected nude mice subcutaneously with stable cells overexpressing IFI16 in SiHa cells as well as control cells. Tumor growth was substantially inhibited in the IFI16 group as compared with the control group. 2. On the other hand, we suppressed IFI16 gene expression in H8 cells using an siRNA approach. Stable lines bearing either pSUPER-IFI16 or pSUPER vector were generated. Both an MTT assay and cell number counting revealed that reduction in IFI16 resulted in enhanced cell proliferation; in addition, reduced IFI16 significantly increased the number of apoptotic cells. Stable H8 cells bearing siIFI16 and control cells were injected into female nude mice. As expected, none of the mice injected with the control cells developed tumors within 5 weeks, whereas mice injected with H8 cells stably transfected with pSUPER-IFI16 developed tumors of considerable size within the same time, demonstrating that IFI16 is a critical modulator of the transforming action of HPV16 in cervical carcinogenesis.3. To elucidate the molecular events underlying IFI16-controlled transforming activity of HPV16 in cervical cancer, we examined the effect of IFI16 on tumor suppressor p53 and Rb, apoptosis-related enzymes caspase3 and caspase8, oncogene myb,c-myc,HPV16 16E6,16E7. We found that the levels of caspase3,aspase8,myb,c-myc,HPV16 E6,16E7 were dramatically reduced when IFI16 was overexpressed. While pRb and p53 were increased about 3 fold.In conclusion, IFI16, a member of the p200 family of IFN-inducible genes, was singled out as a critical mediator of HPV carcinogenesis in cervical cancer. Understanding its role could yield important discoveries that may lead to the development of novel therapies in the treatment of HPV-related disorders, such as cervical cancer.