The Mechanism Of Rrm2Promotes Angiogenesis By Human Papillomavirus E7Oncoprotein In Cervical Cancer

Cervical cancer is the second leading cause of cancer-related mortalities in women worldwide. Epidemiological studies have shown that persistent infection by specific types of human papillomaviruses (HPVs), such as types16and18, is a causal factor for human cervical cancer. The transforming activity of HPVs is dependent on functions of two viral oncoproteins, E6and E7, both of which are consistently expressed in cervical tumors. E7is the major oncoprotein of the high-risk human papillomavirus (HPV), and contributes to carcinogenesis of HPV-positive cervical cancer. The transforming activity of E7is mainly dependent on its interaction with pRb and degradation of pRb, leading to release of active E2F and activation of E2F target genes. However, the precise regulatory mechanisms downstream of E7and E2F activation in cervical cancer are not fully elucidated. The focus of this thesis project is to identify a molecular mechanism by which oncoprotein E7promotes oncogenesis.To identify novel potential downstream genes of HPVE7, I analyzed four publicly available microarray datasets and detected a set of overlapping genes whose mRNA expression was upregulated at least two-fold in cervical cancer tissues and cell lines in these datasets. Then I searched the DNA sequence upstream of these genes for the core binding sequence for E2F with the TFSEARCH(ver.l.3) program. RRM2, encoding the ribonucleotide reductase small subunit M2, is one of the genes shown to be significantly up-regulated. RRM2contains two putative E2F-binding sites within its promoter region, which suggested that RRM2might be a potential target gene activated by HPVE7.Interestingly, I found that RRM2was a novel downstream target gene for E7, but not for E6(another oncoprotein of HPVs). RRM2protein is a component of ribonucleotide reductase (RNR), a key enzyme that reduces ribonucleoside diphosphate (NDP) to deoxyribonucleoside diphosphate (dNDP), and is required for DNA synthesis. It is well known that RNR activity is positively correlated with cancer cell division. A substantial body of evidences demonstrated that RRM2was tightly linked to the biological behaviour of cancers. For example, overexpression of RRM2correlated with cellular invasiveness, metastasis, tumour angiogenesis. However, the molecular mechanisms for malignant phenotypes caused by a high level of RRM2expression are not clear. RRM2overexpression may result in increased radical generation and formation of reactive oxygen species, ROS plays a critical role in tumor growth, metastasis and angiogenesis. ROS induces activation of mitogen-activated protein kinases, NF-κB, and activator protein1which are related to tumor progression. However, it is unclear whether ROS is involved in a malignancy potential mediated by RRM2.In our study, bioinformatic analysis identified two E2F conservative binding sites within RRM2promoter region. Luciferase assays showed that high-risk HPV E7upregulates RRM2transcription through interaction with pRb and direct binding of E2F to the RRM2promoter region.Western blot and immunohistochemical analysis showed that the protein expression level of RRM2positively correlated with the E7protein level in human cervical cancer. These data indicate that high-risk HPV E7specifically had a critical role in transcriptional activation of the RRM2promoter and regulated the expression of RRM2.In this study, we first demonstrated that the molecular mechanisms by which RRM2overexpression leads to the activation of the ERK1/2pathway, and overexpression of HIF-1α and VEGF. Previous studies have shown that RRM2can promote tumour angiogenesis by upregulating VEGF expression, but did not elucidate the underlying molecular mechanism. We therefore hypothesised that RRM2, as a downstream target gene of HPV E7might be involved in the progression of cervical cancer. Interestingly, we found that RRM2overexpression in cervical cancer cells, produces ROS, which leads to the activation of the ERK1/2signalling pathway, increased expression of HIF-1α and VEGF, increased angiogenesis and eventually cervical carcinogenesis. Furthermore, NAC, a ROS inhibitor, prevented the effect of RRM2on activation of the ERK1/2pathway and upregulation of VEGF expression. In vitro, HPV E7stimulated RRM2-dependent capillary tube formation by HUVECs, and RRM2-enhanced angiogenesis was VEGF-dependent. In a xenograft mouse model, overexpression of RRM2in C33A cells enhanced tumor growth as well as microvessel densities. In conclusion, RRM2is the downstream target gene for E7, and E7induces up-regulation of RRM2, which results in increases of ROS, activating the ERK1/2signaling pathway, and inducing expression of HIF-1α and VEGF, leading to cervical angiogenesis and carcinogenesis. These findings identify a novel HPVE7-RRM2-ROS-ERK-HIF-la-VEGF pathway involved in HPV-associated cervical cancer development. The data suggest that the inhibition of RRM2activity may be a novel therapeutic strategy for human cervical cancer.