Search

Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5μM; >5weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Together with p53, the NAD-dependent lysine deacetylase SIRT1 and the microRNA miR-34a form a feedback loop which self-regulates SIRT1 expression and modulates p53-dependent responses. In addition to its well-described role in mediating transcriptional responses to genotoxic stress, p53 may also regulate microRNA processing and maturation., Objective: This study explored the functional relationship among p53, SIRT1 and miR-34a, and the influence of p53 and SIRT1 on microRNA biogenesis and maturation in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines., Methods: RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors were used to modulate activity and expression of SIRT1 and p53. Changes in microRNA and mRNA were analysed by qRT-PCR and protein expression was determined by immunoblotting., Results: Mature miR-34a decreased in p53-suppressed NHEK cells, whereas ablation of SIRT1 reduced the primary transcript (pri-miR-34a). When either SIRT1 expression or activity was inhibited in combination with p53 ablation, pri-miR-34a levels increased and mature miR-34a levels decreased. Under these same conditions, additional p53-regulated microRNAs (miRs 16-1/15, 145 and 107) also failed to mature. In HaCaT cells, primary microRNA transcripts for miR-16-1/15, miR-145 miR200c/141 and miRNA-107, but not miR-34a, were approximately 8-fold higher than in NHEK cells. However, the levels of mature microRNA sequences in HaCaT cells were only 1.5-2 fold higher (miR-16-1, miR-145), unchanged (miR-107) or decreased (miR-200c/141, miR-34a) compared to NHEK cells., Conclusions: Our results suggest that p53 mutations interfere with efficient microRNA biogenesis in keratinocytes, and that SIRT1 functions in combination with p53 in this process., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)