PAX6: A POTENTIAL EPIGENETICALLY-REGULATED TUMOR SUPPRESSOR IN HPV-ASSOCIATED CANCERS
Denise Wong*, Abul Elahi, Leah Hendrick, Abidemi Ajidahun, Syed M. Hussain, Irina Getun, Rita G. Kansal, Evan S. Glazer, David Shibata
University of Tennessee Health Science Center, Memphis, TN
Background
The carcinogenic effects of human papillomavirus (HPV) infection are thought to be mediated in part by methylation of host DNA. By methylomic array analyses, we have identified differentially methylated genes in the progression of HPV-associated anal and cervical cancers. PAX6, a known lineage-specific tumor suppressor, was identified as one common target of interest. We sought to examine the potential biologic role of PAX6 in anal and cervical cancer cell lines.
Methods
PAX6 expression was analyzed by reverse transcriptase-PCR and Western Blot analyses in HPV16+ anal (ACC) and cervical (SiHa) cancer cell lines, as well as in an HPV-negative cervical cancer (C33) cell line. Quantitative methylation-specific PCR (qMSP) was used to analyze the methylation status of PAX6. Re-expression of PAX6 was confirmed following demethylating treatment with 5’Aza-2-deoxycitidine (5AzaDC). Non-expressing cell lines were then transiently transfected with full-length PAX6 and subsequent impact on proliferation was examined by MTT assay.
Results
PAX6 expression was significantly downregulated in ACC and SiHa and associated with corresponding high levels of promoter DNA methylation. The C33 cell line demonstrated minimal downregulation of PAX6 with mild levels of promoter methylation. 5AzaDC treatment resulted in PAX6 re-expression in the non-expressing cell lines. Restoration of PAX6 expression by transient transfection resulted in a significant reduction in proliferation in both ACC (p=0.0001) and SiHa (p=0.0017) cell lines as compared to empty vector controls. Transfection of PAX6 did not yield any significant impact in the C33 cell line (p=0.21).
Conclusion
We have demonstrated that PAX6 is transcriptionally repressed by DNA methylation and its loss may play a pro-proliferative role in HPV-associated anal and cervical cancer cell lines. Further investigation into the specific PAX6-associated mechanisms of growth suppression in HPV-associated carcinogenesis are warranted.
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