Increased expression of RRM2 by human papillomavirus E7 oncoprotein promotes angiogenesis in cervical cancer

Background: The purpose of this study was to confirm that RRM2 as a novel target of HPVE7 involved in cervical cancer angiogenesis. Methods: Gene expression was analysed by RT-qPCR, western blot and immunohistochemistry in cervical cancer tissue and cell lines. Luciferase reporter assay was used to determine the activities of various RRM2 promoters. Secreted VEGF was measured by ELISA. RRM2-mediated capillary tube formation induced by HPVE7 in cervical cancer cells were evaluated using human umbilical vein endothelial cells in vitro. ROS induced by RRM2 in cercal cancer cells was confirmed by flow cytometry. The growth of cervical cancer cell overexpression RRM2 was examined by nude mouse xenograft. Results: RRM2 as a novel downstream target for HPVE7 was upregulated by it at the transcriptional level through the E7-pRb interaction and binding of E2F to the RRM2 promoter region. Immunohistochemical analysis showed that the level of RRM2 positively correlated with the HPVE7 level in human cervical cancer. Functionally, overexpression of RRM2 enhanced the expression of HIF-1 alpha and VEGF via activation of the ERK1/2 signalling pathway in cervical cancer cells, and significantly associated with increased microvessel densities in cervical cancer tissues. In vitro, HPVE7 stimulated RRM2-dependent capillary tube formation by HUVECs, and RRM2-enhanced angiogenesis was VEGF dependent. RRM2-activated ERK1/2 pathway was mediated through production of ROS. In the xenograft mouse model, overexpression of RRM2 in cervical cancer cells enhanced tumour growth as well as microvessel densities. Conclusion: HPVE7 induces upregulation of RRM2, which then promotes cervical carcinogenesis via ROS-ERK1/2-HIF-1 alpha-VEGF-induced angiogenesis. Thus, the inhibition of RRM2 activity may be a novel therapeutic strategy for human cervical cancer.