Chromatin , Gene , and RNA Regulation Prognostic Potential of DNA Methylation and Transcript Levels of HIF 1 A and EPAS 1 in Colorectal Cancer

Hypoxic conditions during the formation of colorectal cancer may support the development of more aggressive tumors. Hypoxia-inducible factor (HIF) is a heterodimeric complex, composed of oxygen-induced HIFa and constitutively expressed HIFb subunits, which mediates the primary transcriptional response to hypoxic stress. Among HIFa isoforms, HIF1a (HIF1A) and endothelial PAS domain–containing protein 1 (EPAS1) are able to robustly activate hypoxia-responsive gene signatures. Although posttranslational regulation of HIFa subunits is well described, less is known about their transcriptional regulation. Here, molecular analysis determined that EPAS1 mRNA was significantly reduced in primary colonic adenocarcinoma specimens compared with histopathologically nonneoplastic tissue from 120 patients. In contrast, no difference in HIF1A mRNA levels was observed between cancerous and noncancerous tissue. Bisulfite DNA sequencing and high-resolution melting analysis identified significant DNA hypermethylation in the EPAS1 regulatory region from cancerous tissue compared with nonneoplastic tissue. Importantly, multivariate Cox regression analysis revealed a high HR for patients with cancer with low EPAS1 transcript levels (HR, 4.91; 95% confidence interval, CI, 0.42–56.15; P 1⁄4 0.047) and hypermethylated EPAS1 DNA (HR, 33.94; 95% CI, 2.84–405.95; P 1⁄4 0.0054). Treatment with a DNA methyltransferase inhibitor, 5-Aza-20-deoxycytidine (5-aza-dC/Decitabine), upregulated EPAS1 expression in hypoxic colorectal cancer cells that were associated with DNA demethylation of the EPAS1 regulatory region. In summary, EPAS1 is transcriptionally regulated by DNA methylation in colorectal cancer. Implications:DNAmethylation andmRNA status of EPAS1 have novel prognostic potential for colorectal cancer. Mol Cancer Res; 12(8); 1112–27. 2014 AACR. Introduction Immense proliferation of tumor cells and their inadequate perfusion results in hypoxia, which is a hallmark of many solid tumors, including colorectal cancer (1). Mechanisms by which tumor cells alter their expression profile to adjust to low oxygen tension involve hypoxia-inducible factor (HIF; ref. 1). HIF is a heterodimeric transcription factor assembled from a and b subunits. It recognizes the hypoxia response element (HRE) and promotes expression of many genes involved in glucose metabolism, angiogenesis, or metastasis (1). The b subunit of HIF is constitutively expressed, whereas HIFa is mainly controlled by a posttranslational mechanism (1, 2). In normoxic conditions, HIFa is hydroxylated at specific residues, which results in proteasomal degradation (2). There are three a isoforms referred to as: HIF1a, HIF2a (officially designated as endothelial PAS domain–containing protein 1—EPAS1) and HIF3a, which are encoded by the HIF1A, EPAS1, and HIF3A genes, respectively (2). Among them, HIF1a or EPAS1 may bind together with b and other coactivators to HRE and activate HIF-dependent gene transcription (2). Many articles describe aberrant HIF1a or EPAS1 protein levels and their association with colorectal cancer prognosis (3–10). However, there are only few articles about the same issues on the HIF1A and EPAS1 mRNA level (8, 11, 12). Moreover, relatively little is known about transcriptional regulation of HIF1A and EPAS1. It should be noted that both of them possess a CpG island in their promoter region. DNA methylation within the CpG island associates the gene transcriptional repression, and aberrant DNA methylation patterns are observed during colorectal tumorigenesis (13). To date, only one article indicates DNA methylation of HIF1A in colorectal cancer (14), and there is no scientific reports about this type of epigenetic regulation of EPAS1 expression. Therefore, we aimed to examine DNA methylation and mRNA levels of the HIF1A and EPAS1 genes in Authors' Affiliations: Departments of Biochemistry and Molecular Biology and General and Colorectal Surgery, Pozna n University of Medical Sciences, Poland Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Agnieszka Anna Rawłuszko-Wieczorek, Pozna n University of Medical Sciences, S ́więcickiego 6 Street, Pozna n 60-781, Poland. Phone: 486-1854-6516; Fax: 48618546510; E-mail: arawluszko@ump.edu. pl doi: 10.1158/1541-7786.MCR-14-0054 2014 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(8) August 2014 1112 on May 4, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst May 13, 2014; DOI: 10.1158/1541-7786.MCR-14-0054