Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter

SIMPLE SUMMARY: Transcription factor homeobox D9 (HOXD9) was previously reported to bind to the P97 promoter of HPV16 to induce viral E6/E7 oncogenes. In this article, we investigated whether HOXD9 regulated the P105 promoter of HPV18 and examined the role of HOXD9 in intracellular signaling of cervical cancer (CC). HOXD9 was directly bound to the P105 promoter and regulated the expression of E6/E7 genes of HPV18. The HOXD9 knockdown suppressed the E6/E7 gene expression in HPV18-positive cervical cancer cells. It decreased the expression of E6, activated the p53 pathway, and induced apoptosis. In addition, downregulation of the E7 gene expression activated the Rb pathway, causing G1 arrest in the cell cycle and markedly suppressing cell proliferation. Our results indicate that HOXD9 has pivotal roles in the proliferation and immortalization of HPV18-positive cervical cancer cells through activating the P105 promoter. ABSTRACT: Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells.