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Epitenetic mechanisms of arsenic-induced transformation in human keratinocytes


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Herbert, KJ 2014 , 'Epitenetic mechanisms of arsenic-induced transformation in human keratinocytes', PhD thesis, University of Tasmania.

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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 – 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 μM
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

Item Type: Thesis - PhD
Authors/Creators:Herbert, KJ
Keywords: Keratinocytes, Epigenetics, p53, SIRT1 miR-34a, arsenic, cancer
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Copyright 2014 the author

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