Epitenetic mechanisms of arsenic-induced transformation in human keratinocytes

Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide, however the biomolecular mechanism is yet to be fully elucidated. Genomewide epigenetic repatterning occurs with arsenic exposure ‚Äö- a process which is associated with altered gene expression and activity of epigenetic regulators. Whether this mechanism is a direct, or indirect, consequence of arsenic toxicity, and whether dysregulated maintenance of the epigenetic landscape drives arsenic-induced cancer, are questions which remain unanswered. SIRT1 is a lysine deacetylase with a well-characterised role in mediating cellular adaptation to metabolic stress; in part, by regulating activation of the tumour suppressor p53, and also by maintaining patterns of gene expression by regulating the activity of chromatin remodelling complexes. SIRT1 is overexpressed in numerous cancer subtypes, therefore the primary hypothesis guiding this thesis was that aberrant SIRT1 activity mediates the epigenetic events which initiate and promote arsenicinduced skin cancer. The overarching aim for this study was to characterise the response of SIRT1 and its targeting microRNA, miR-34a in cultured human keratinocytes when exposed to arsenic over an extended time frame. For the first part of this investigation, wild-type (primary) and p53-mutated (HaCaT) keratinocytes were used as an in vitro model to determine the role of the p53/SIRT1/miR-34a axis during cell death signalling. Using a SIRT1 siRNA targeting pool, and with a targeted SIRT1:miR-34a binding site block, this study found that microRNA biogenesis and maturation is dysregulated in HaCaT keratinocytes, causing overexpression of microRNA regulatory targets. Consequently, inhibition of the miR-34a target, SIRT1, was effective in overcoming apoptotic resistance in the HaCaT cell line by restoring p53 transcriptional activation. Second, the role of SIRT1 in arsenic-induced transformation of cultured keratinocytes was determined by exposing primary keratinocytes to 0.5 ˜í¬¿M arsenite in culture medium for 10 weeks. By analysing these cells for changes in gene expression, chromatin condensation and DNA methylation, this investigation determined that arsenic-induced acetylation of H4K16 in keratinocytes was associated with remodelling of the pri-miR-34a promoter and up-regulation of miR- 34a expression, which was sustained by DNA hypomethylation with extended arsenic exposure. SIRT1 initially accumulated in arsenic-exposed cells, however this effect was transient, indicating that arsenic not only inhibits SIRT1 activity, but also down-regulates expression over time. Finally, although arsenic treated primary keratinocytes display epigenetic changes consistent with dysregulation of SIRT1 activity and expression, these cells were unable to escape senescence during sustained exposure to arsenic in vitro. These data reveal that dysregulation of the p53/SIRT1/miR-34a axis contributes significantly to keratinocyte carcinogenesis by blocking efficient cell death signalling. Consequently, treatment of skin cancers may be enhanced by using SIRT1 inhibitors as an adjuvant to pro-apoptotic chemotherapy. Although skin cancerinducing concentrations of arsenic altered epigenetic patterns of miR-34a gene regulation by interfering with SIRT1 activity and expression in cultured human keratinocytes, extended exposure was insufficient to induce transformation in the absence of a second hit such as those provided by characteristic UV-induced p53 mutations.