5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells

Abstract

High-risk human papillomaviruses (hr HPVs) may cause various human cancers and associated premalignant lesions. Transformation of the host cells is triggered by overexpression of the viral oncogenes E6 and E7 that deregulate the cell cycle and induce chromosomal instability. This process is accompanied by hypermethylation of distinct CpG sites resulting in silencing of tumor suppressor genes, inhibition of the viral E2 mediated control of E6 and E7 transcription as well as deregulated expression of host cell microRNAs. Therefore, we hypothesized that treatment with demethylating agents might restore those regulatory mechanisms. Here we show that treatment with 5-aza-2′-deoxycytidine (DAC) strongly decreases the expression of E6 and E7 in a panel of HPV-transformed cervical cancer and head and neck squamous cell carcinoma cell lines. Reduction of E6 and E7 further resulted in increased target protein levels including p53 and p21 reducing the proliferation rates and colony formation abilities of the treated cell lines. Moreover, DAC treatment led to enhanced expression of tumor the suppressive miRNA-375 that targets and degrades E6 and E7 transcripts. Therefore, we suggest that DAC treatment of HPV-associated cancers and respective precursor lesions may constitute a targeted approach to subvert HPV oncogene functions that deserves testing in clinical trials.

Down‐regulation of p16 and MGMT promotes the anti‐proliferative and pro‐apoptotic effects of 5‐Aza‐dC and radiation on cervical cancer cells

“…When cells transfected with siRNAs targeting p16 or/and MGMT as well as cells stimulated with 5‐Aza‐dC, the expression of p53, p21, and Rb was up‐regulated more significantly, and E6 and E7 expression down‐regulated significantly. Stich et al also found that treatment with 5‐aza‐2′‐deoxycytidine strongly decreases the expression of E6 and E7 in a panel of HPV‐transformed cervical cancer and head and neck squamous cell carcinoma cell lines . Zhang et al showed that upon demethylation treatment with 0.5‐μM 5‐aza‐dc for 96 hours, the expression of E6 and E7 mRNA was significantly decreased in UM‐SCC47 and CaSki cells .…”

“…Stich et al also found that treatment with 5-aza-2′deoxycytidine strongly decreases the expression of E6 and E7 in a panel of HPV-transformed cervical cancer and head and neck squamous cell carcinoma cell lines. 31 Zhang et al showed that upon demethylation treatment with 0.5-μM 5-aza-dc for 96 hours, the expression of E6 and E7 mRNA was significantly decreased in UM-SCC47 and CaSki cells. 32 33 Rb, p53, and p21 protein expression was increased in the p16-silenced cells compared to control siRNA-treated cells.…”

Demethylation Therapy as a Targeted Treatment for Human Papillomavirus–Associated Head and Neck Cancer

“…Other statistical analyses were done using Fisher exact and t test. Restoring p53 protein expression and tumor suppressor functions has been suggested as a promising strategy to combat HPV þ cancer, and the 5-aza structural analogue 5-aza-2 0 -deoxycytidine (decitabine) was recently found to increase p53 protein levels in HPV þ cervical and head and neck cancer cells (32). p53 is a powerful transcription factor able to transactivate proapoptotic and growth-inhibitory genes.…”

Aberrant methylation‐induced dysfunction of p16 is associated with osteoblast activation caused by fluoride

“…The methylation status of genes can be reversed by a methyltransferase inhibitor, 5‐AZA‐dC, a deoxycytidine analog that has been approved by the U.S. Food and Drug Administration for the treatment of myelodysplastic syndromes. It has shown promising effects on treating certain solid cancers . 5‐AZA‐dC can reactivate silenced suppressor genes by promoter demethylation .…”