Abstract 4244: Epigenetic regulation by SET protein in HNSCC: histone acetylation versus DNA methylation.
Authors: Almeida Luciana , Tannous Maryna , Matsumoto Camila , Sobral Lais , Curti Carlos , Leopoldino Andréia 
Published: August 2014

Abstract
Background: Epigenetic regulation is essential in the control of both normal cellular processes and cancer development. DNA methylation and histone acetylation are involved in chromatin remodeling and gene transcription control. Recently, we reported that SET protein, a member of the acetyltransferase inhibitor complex, is strongly accumulated in HNSCC. However, SET action in gene expression is unknown, so we assessed SET role in the control of epigenetic modifications.
Materials and Methods: HNSCC (HN6, HN12 and HN13), HEK293 and NOK-SI cell lines were used. SET overexpression was obtained in HEK293 and NOK-SI cells using pcDNA3.1 with SET cDNA, and SET knockdown in HNSCC cells using siRNA or shRNA. Human transcription factors RT² Profiler™ PCR Array profile (TF) and human tumor suppressor genes (TSG) EpiTect® DNA Methyl qPCR Array System (SABioscience-Qiagen) were used to determine the SET action in gene transcription and DNA methylation. Quantitative real time PCR (qPCR), chromatin immunoprecipitation (ChIP), immunofluorescence, Western blotting and DNA methyltransferases (DNMT) activities (EpiSeeker DNMT Activity Quantification Kit; Abcam) assays were used. Trichostatin A (TSA, 100 ng/mL), which increases histone acetylation, or 5-aza-2-deoxycytidine (5-aza, 1ug/mL), which decreases DNA methylation, were used for epigenetic modifications.
Results: TFs mRNA analysis showed only 15 genes up-regulated and 65 down-regulated in HEK293/SET cells. Eight down-regulated (ATF2, CTNNB1, HIF-1A, NFATC3, RELA and STAT1) and two up-regulated (ATF3 and MYB) genes were also assessed by qPCR and similar mRNA profile was obtained. HNSCC SET knockdown cells presented opposite mRNA profile. ChIP analysis was performed for nine genes and HIF-1A, NFATC3 and STAT1 promoters showed interaction with SET. However, NFATC3 and STAT1 protein levels were not altered. DNA methylation profile of TSG showed that SET overexpression in HEK293 and NOK-SI cells promoted hypomethylation whereas SET knockdown in HN12 and HN13 cells promoted hypermethylation. HN6, HN12 and HN13 control cells, which present constitutive SET accumulation, presented a methylation loss pattern similar to the observed in HEK293 and NOK-SI cells with SET overexpression. GSTP1, PTEN and TP16 mRNAs levels decreased in SET-overexpressing cells despite the DNA demethylation status. To assess which modification is the main SET mechanism in the gene expression regulation, we performed qPCR in cell lines treated with TSA or 5-aza. TSA treatment reversed the SET overexpression effect in GSTP1, HIF-1A, NFATC3, PTEN, TP16 and STAT1 mRNA whereas 5-aza did not reverse. SET overexpression promoted increase in DNMT activities accompanied by increase in DNMT-1 protein.
Conclusions: SET accumulation promotes transcriptional repression through histone hypoacetylation regulation and DNA demethylation is not associated with loss of DNMTs activities.
Citation Format: Luciana Almeida, Maryna Tannous, Camila Matsumoto, Lais Sobral, Carlos Curti, Andréia Leopoldino. Epigenetic regulation by SET protein in HNSCC: histone acetylation versus DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2013-4244