Examination of the pRb-Dependent and pRb-Independent Functions of E7 In Vivo

Abstract

High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRb ⌬LXCXE ) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb ⌬LXCXE mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19 ARF . These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.Human papillomaviruses (HPVs) are small DNA viruses and the causative agents of epithelial warts. The so-called "high-risk" HPVs, including HPV-16, infect the anogenital tract epithelium and are associated with almost all cases of cervical cancer, a leading cause of cancer mortality in women worldwide (64,69). Although the HPV genome usually exists extrachromosomally, many HPV-associated cancers contain HPV genomes integrated into the host DNA (20). These integrated genomes invariably contain intact viral E6 and E7 genes, and integration causes their increased expression (35). These data suggest that E6 and E7 contribute to the development of cervical cancers.HPV-16 E7 is a small nuclear phosphoprotein with potent transforming and tumorigenic properties. Coexpression of E6 and E7 is necessary and sufficient to transform primary human keratinocytes (48), and E7 acts robustly in a number of other in vitro transformation assays (6,44,52,62,63,65). E7-expressing cells exhibit genomic instability in culture (17,18,56,66,67) and have impaired cell cycle arrest responses to DNA damage (12,60). Additionally, expression of E7 in primary human keratinocytes or in transgenic mice results in abnormal centrosome synthesis, with associated multipolar mitoses and aneuploidy (3,(15)(16)(17). Previously, our laboratory generated mice transgenic for HPV-16 E7 under the control of the keratin 14 promoter, targeting E7 expression to the basal layer of stratified squamous epithelia such as the skin and cervical epithelium (30). Th...

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

“…The p53 and Rb tumor-suppressor genes are crucial in cell cycle regulation checkpoints and are common targets of inactivation by viral oncogenes, including large T antigen of SV40 virus (De Luca et al, 1997) and E6 and E7 of human papilloma virus (Balsitis et al, 2005). However, functional interactions of EBV oncogenes with tumor-suppressor genes during NPC development are unclear.…”

Role of Rb-Dependent and Rb-Independent Functions of Papillomavirus E7 Oncogene in Head and Neck Cancer

“…In these mice, the HPV oncogenes are directed in their expression from the keratin 14 (K14) promoter, which targets expression of the viral oncogenes to the same basal compartment of stratified epithelia as observed in natural HPV infections. Furthermore, extensive analyses of these mice have shown that the HPV16 E6 and E7 oncoproteins display the same capacity to inactivate relevant cellular targets, including but not limited to p53 and pRb, respectively, as has been documented to occur in HPV-infected human epithelia (10,(21)(22)(23)(24)(25). From our studies, we have learned that these oncogenes not only possess properties that distinguish one from the other, but also that those properties may vary in a tissue-dependent manner.…”

Cell Cycle Regulators p105, p107, Rb2/p130, E2F4, p21CIP1/WAF1, Cyclin A in Predicting Cervical Intraepithelial Neoplasia, High-Risk Human Papillomavirus Infections and Their Outcome in Women Screened in Three New Independent States of the Former Soviet Union

“…In line with previous studies, we could confirm an intimate relationship between Ki-67 and p21 CIP1/WAF1/SDI1 expression (P = 0.0001; refs. 5,6,25,35,[44][45][46]. 12 On the other hand, AI and MI do not seem to be as sensitive markers of p21 CIP1/WAF1/SDI1 upregulation, albeit related to it with a lower statistical power (P = 0.05 and 0.013, respectively).…”

“…Earlier studies on cell cycle proteins and their associations with HPV infections in cervical lesions were focused on interactions between E7 and p105 (7,37,(40)(41)(42) followed by the recent interest in p107 and p130 (11)(12)(13)(14)(15)(16)43). The HPV interactions with p21 CIP1/WAF1/SDI1 have received some attention only recently (5,6,(44)(45)(46)(47)(48), and even less reports are available on cyclin A expression and its eventual prognostic value in cervical cancer (8,(49)(50)(51). Thus, it was of interest to elucidate how the differential expression of these cell cycle regulators relates to the different intermediate end point markers in cervical carcinogenesis based on immunohistochemical staining of the baseline biopsies from Pap smear -positive and/or HCII-positive women followed-up as part of the original New Independent States Cohort study (26)(27)(28)(29)(30)(31).…”

“…The different oncogenic potential of low-risk and high-risk human papillomaviruses (HPV) in inducing cervical cancer and its precursor [cervical intraepithelial neoplasia (CIN)] lesions is attributable to the different interactions of the two viral oncoproteins, E6 and E7, with several cellular proteins of which the most well characterized are the two key regulatory proteins of the cell cycle, p53 and pRb (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The Rb gene family includes three members: p105, p107, and Rb2/p130 (11)(12)(13)(14)(15)(16).…”