The role of human papillomavirus (HPV)-related molecular markers in cervical neoplasia, with emphasis on p-21 activated kinase type 1 (PAK 1)

Abstract

Since high-risk Human Papillomavirus (HPV) plays a critical role in cervical carcinogenesis, it is essential to improve our understandings on the role of HPV-related molecular markers in cervical neoplasia. The aim of this study was to investigate the expression and prognostic significance of selected HPV-related markers, including HPV16/18 E2 binding sites (E2BS) methylation, Pak 1, c-FLIP, Notch 1 and Brd4 expressions, as well as the potential functions of Pak 1 in cervical neoplasia. First, the differential expressions of these markers among clinical samples of normal cervical epithelium, low-grade and high-grade cervical intraepithelial neoplasia (CINs) and cervical cancer were studied. Methylation status of E2BS 1, 2 and 4 was determined by pyrosequencing. Expressions of the target proteins were assessed by immunohistochemistry. Both HPV16/18 E2BS 1&2 and E2BS4 methylation progressively increased from normal cervix through CINs to cancer. More importantly, HPV16 E2BS1&2 (for transcriptional repression of E6/E7 oncoproteins) became more heavily methylated than E2BS4 (for transcriptional activation of E6/E7) in cervical cancer, favouring the differential binding of E2 protein to E2BS4. Pak 1, c-FLIP, Notch 1 and Brd4 were all overexpressed in cervical cancer. Their expressions increased progressively from normal cervix to low-grade +/- high-grade CINs. Pak 1 and c-FLIP expression was positively correlated with HPV18 E6 and HPV16 E7 expression respectively. Notch 1 expression was inversely correlated with HPV16 E7 and HPV18 E6 expressions. Brd4 expression was positively correlated with HPV16 E2 and inversely correlated with HPV16 E7 expressions. The prognostic significance of the molecular markers was investigated by correlation with clinical parameters. Heavier methylation at E2BS1&2 relative to E2BS4 was associated with better overall and disease-free survival in cervical cancer patients. HPV16 E2BS1&2 hypermethylation, weak cytoplasmic Brd4 expression, strong c-FLIP or Notch 1 expression were associated with higher risk of recurrent abnormal smears after treatment of CINs. The role of Pak 1 in cervical cancer was further explored by comparing its functions between cervical cancer cell lines with and without transient knock-down of Pak 1 by siRNAs. It was demonstrated that the significant functions of Pak 1 in cervical cancer were on promoting cell proliferation and inhibiting apoptosis. Transient HPV16 E6 inhibition showed no effect on total / phosphorylated Pak 1 expressions. Lastly, the function of Pak 1 on the regulation of cervical cancer cell radiosensitivity was also investigated. Pak 1 inhibition increased cell sensitivity in response to irradiation by enhancing apoptosis and inhibiting cell proliferation. Conclusively, differential methylation status at HPV16/18 E2BS, as well as Pak 1, c-FLIP, Notch 1 and Brd4 proteins contribute to cervical carcinogenesis, and are potential prognostic markers in cervical cancer and CIN patients. Pak 1 inhibitor may be a potential adjunctive agent to improve radiotherapy.