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.
Highlights
High-risk human papillomavirus (HR-HPV) infections may cause various human cancers in particular of the female anogenital tract and of the oropharynx and other epithelial sites [1]
In order to analyze the effect of demethylating agents on the expression of the HPV oncogenes E6 and E7, we treated cervical carcinoma and head and neck squamous cell carcinoma (HNSCC) cell lines for 72 hours with different concentrations of DAC ranging from 0.1 μM to 1.0 μM
We assessed the demethylating effect of DAC treatment by measuring DNA methylation in the retrotransposon Long Interspersed Nuclear Element 1 (LINE1), which is heavily methylated in most tissues and cell lines and often used as a surrogate marker for global DNA methylation levels [34]
Summary
High-risk human papillomavirus (HR-HPV) infections may cause various human cancers in particular of the female anogenital tract and of the oropharynx and other epithelial sites [1]. HPV-associated cancer cells are addicted to the consistent expression of two HPV-encoded oncogenes referred to as E6 and E7 [2, 3]. Their gene products interfere with a variety of host proteins resulting in the deregulation of the cell cycle and chromosomal instability upon expression in replicating cells [4, 5]. Key oncogenic functions of these proteins are the E6-mediated degradation of the p53 protein driving the abrogation of proapoptotic pathways and the E7mediated degradation of the retinoblastoma protein (pRB) causing an excess release of E2F transcription factors and activation of cell cycle progression [6,7,8,9,10]. Despite intense research activities on the biochemistry and molecular biology of the viral oncogene products and their functions in HPV-transformed cells no targeted therapies www.impactjournals.com/oncotarget interfering with the activity of E6 and E7 yet reached the level of broader clinical testing
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