HPV16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases

Production of matrix metalloproteinases (MMPs) for degradation of extracellular matrix is a vital step in cancer metastasis. We investigated the effects of HPV16 oncoproteins (16E6, 16E6*I and 16E7), either individually or combined, on the transcription of 7 MMPs implicated in cervical cancer invasiveness. The levels of 7 MMPs reported to be increased in cervical cancer were determined in C33A stably expressing different HPV16 oncoproteins using quantitative RT-PCR and compared with invasion ability of cell lines using in vitro invasion and wound healing assays. Overexpression of MMP-2 and MT1-MMP was detected in HPV16E6E7 expressing cells which correlated with increased cell invasion. Combination of HPV oncoproteins always showed greater effects than its individual form. Inhibition of cell invasion using a specific MMP-2 inhibitor, OA-Hy, and anti-MT1-MMP antibody confirmed that invasion in these cells was dependent on both MMP-2 and MT1-MMP expression. Depletion of HPV16E6E7 by shRNA-mediated knock-down experiments resulted in decreased MMP-2 and MT1-MMP expression levels as well as reduced invasion ability which strongly suggested specific effects of HPV oncoproteins on both MMPs and on cell invasion. Immunohistochemistry study in invasive cervical cancers confirmed the enhanced in vivo expression of these two MMPs in HPV16-infected cells. In addition, possible sites required by HPV16E6E7 on the MMP-2 and MT1-MMP promoters were investigated and PEA3 (at -552/-540 for MMP-2, -303 for MT1-MMP) and Sp1 (at -91 for MMP-2, -102 for MT1-MMP) binding sites were shown to be essential for mediating their transactivation activity. In conclusion, our study demonstrated that HPV16E6 and E7 oncoproteins cooperate in promoting cervical cancer invasiveness by specifically upregulating MMP-2 and MT1-MMP transcription in a similar manner.