Abstract

BackgroundColorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH).MethodsPHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n = 90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines.ResultsWe found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p = 0.00026; p < 0.00001; p < 0.00001) and proteins (p = 0.004164; p = 0.0071; p < 0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p = 0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p < 0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2’-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells.ConclusionWe demonstrated that reduced PHD3 expression in cancerous tissue was accompanied by methylation of the CpG rich region located within the first exon and intron of the PHD3 gene. The diminished expression of PHD1 and PHD2 and elevated level of FIH protein in cancerous tissue compared to histopathologically unchanged colonic mucosa was not associated with DNA methylation within the CpG islands of the PHD1, PHD2 and FIH genes.

Highlights

  • Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies

  • We found significantly lower levels of PHD1, PHD2 and PHD3 transcript (p = 0.00026; p < 0.00001; p < 0.00001) and protein (p = 0.004164; p = 0.0071; p < 0.00001) in primary cancerous than in histopathologically unchanged tissues in ninety patients with CRC (Figure 1A, B; Figure 2)

  • We found a significantly higher level of factor inhibiting HIF (FIH) protein in cancerous tissue in the male patient group (p = 0.0210), and in patients aged above 60 (p = 0.0257), with CRC localized in the rectum (p = 0.031) and G2 histologic grade (p = 0.0226) (Additional file 4)

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Summary

Introduction

Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Colorectal cancer (CRC) belongs to one of the most extensively studied types of cancers due to its high mortality and severity It is the third and second leading cause of death from malignant disease among adults in the US and Europe, respectively [1]. The PHD enzymes catalyze the hydroxylation of two conserved proline residues in the oxygen dependent degradation domain of the HIF-α protein. FIH hydroxylates the asparagine residue within the C-terminal transactivation domain of HIF-α [5,6]. This results in the prevention of HIF-α interaction with its coactivators. Under normoxic conditions, there is a dual mechanism of HIF inhibition by its degradation or inactivation by PHDs and FIH enzymes, respectively

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