Abstract
In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.
Highlights
Sirtuin-1 (SIRT1) is an NAD-dependent class III histone deacetylase belonging to the protein family of sirtuins
Since SIRT1 participates on stress response [18, 19], has elevated expression, increased Mxd1 association in melanocytes submitted to stress condition, and is usually involved in gene silencing, as expected for histone deacetylase, we suggest that SIRT1 could participate in Mxd1 repression along melanocyte malignant transformation
Our in vitro melanocyte malignant transformation model based on repeated cycles of anchorage blockade allows studying different stages of melanoma progression from normal melanocytes to non-metastatic and metastatic melanoma cells [27]
Summary
Sirtuin-1 (SIRT1) is an NAD-dependent class III histone deacetylase belonging to the protein family of sirtuins. The induction of a DNA double strand break in an exogenous E-cadherin promoter CpG island resulted in the relocation of SIRT1 to the site of damage, along with other www.impactjournals.com/oncotarget components of the epigenetic machinery, such as DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3B (DNMT3B), and Enhancer of zeste homolog 2 (EZH2) [17]. In this regard, it was reported that SIRT1 can relocate from regions non-rich in CGs to areas with high levels of CGs to increase their methylation [18]. SIRT1 relocation in response to oxidative stress might have a crucial role in the establishment of aberrant epigenetic patterns associated with the development of pathologies, including cancer
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