Karolina Horbacka

Prognostic potential of DNA methylation and transcript levels of HIF1A and EPAS1 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 HIFα and constitutively expressed HIFβ subunits, which mediates the primary transcriptional response to hypoxic stress. Among HIFα isoforms, HIF1α (HIF1A) and endothelial PAS domain–containing protein 1 (EPAS1) are able to robustly activate hypoxia-responsive gene signatures. Although posttranslational regulation of HIFα 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 = 0.047) and hypermethylated EPAS1 DNA (HR, 33.94; 95% CI, 2.84–405.95; P = 0.0054). Treatment with a DNA methyltransferase inhibitor, 5-Aza-2′-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: DNA methylation and mRNA status of EPAS1 have novel prognostic potential for colorectal cancer. Mol Cancer Res; 12(8); 1112–27. ©2014 AACR.

Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

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.

Effect of DNA methylation profile on OATP3A1 and OATP4A1 transcript levels in colorectal cancer.

Epidemiological studies indicate that 17β-estradiol (E2) prevents colorectal cancer (CRC). Organic anion transporting polypeptides (OATPs) are involved in the cellular uptake of various endogenous and exogenous substrates, including hormone conjugates. Because transfer of estrone sulfate (E1-S) can contribute to intra-tissue conversion of estrone to the biologically active form -E2, it is evident that the expression patterns of OATPs may be relevant to the analysis of CRC incidence and therapy. We therefore evaluated DNA methylation and transcript levels of two members of the OATP family, OATP3A1 and OATP4A1, that may be involved in E1-S transport in colorectal cancer patients. We detected a significant reduction in OATP3A1 and a significant increase in OATP4A1 mRNA levels in cancerous tissue, compared with histopathologically unchanged tissue (n=103). Moreover, we observed DNA hypermethylation in the OATP3A1 promoter region in a small subset of CRC patients and in HCT116 and Caco-2 colorectal cancer cell lines. We also observed increased OATP3A1 transcript following treatment with 5-aza-2-deoxycytidine and sodium butyrate. The OATP4A1 promoter region was hypomethylated in analyzed tissues and CRC cell lines and was not affected by these treatments. Our results suggest a potential mechanism for OATP3A1 downregulation that involves DNA methylation during colorectal carcinogenesis.

Preliminary study with SprayShield™ Adhesion Barrier System in the prevention of abdominal adhesions

Introduction Peritoneal adhesions, the fibrotic bands that form between the surfaces in the peritoneal cavity following surgery, still pose a difficult clinical challenge. Aim To evaluate the SprayShield™ Adhesion Barrier System (PEG ester amine solution and a buffer solution) in reducing post-operative adhesion formation. Material and methods This was a prospective, multi-center, randomized, single blind study. A total of 11 subjects diagnosed with ulcerative colitis (UC) or familial adenomatous polyposis (FAP) were randomized: 8 to the SprayShield™ arm and 3 to the control arm. SprayShield™ was applied on the viscera directly under the midline peritoneal incision and at the site of ileostomy. During the follow-up surgery, the incidence, extent, and severity of post-operative adhesion formation were evaluated, as well as the time required to mobilize the ileal loop. Results In patients who received SprayShield™ the time required to mobilize the ileal loop at the ileostomy closure was slightly shorter and the incidence and severity of adhesions were somewhat lower vs. control subjects (NS). Conclusions SprayShield™ was found to be easy to use, safe, and quick to apply, and performed well in adherence and conformity. The incidence and severity of adhesions were lower for SprayShield™ subjects vs. control subjects, but due to the limited number of patients there are not enough data to confirm the effectiveness of the SprayShield™ Adhesion Barrier System in prevention of adhesions.

Reduced expression of steroid sulfatase in primary colorectal cancer

Colorectal cancer (CRC) is considered an estrogen-dependent malignancy, and intratissue estrogen concentration can be controlled by steroid sulfatase (STS). Little is known about changes in the expression of STS during the development of CRC. Therefore, we analysed the STS mRNA levels in primary colonic adenocarcinoma tissues and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical colon resection (n=90). We found a statistically significant decrease in STS transcript levels in CRC (P=0.0453). Moreover, we found that sodium butyrate (NaBu) significantly upregulated STS transcript levels in DLD-1 and HCT116 CRC cells. Our results suggest that STS expression can be decreased in the process of large intestinal carcinogenesis. Moreover, we observed that NaBu might increase STS expression in CRC cells.

Usefulness of Magnetic Resonance Enterography in Diagnosis of Crohn's Disease

UNLABELLED The numbers of patients with diagnosed Crohns disease in Poland continue to be on the rise. It may be assumed that it is associated not with an increased incidence but with significant advancements in diagnostic techniques which in an increasingly better manner solve problems of abdominal pain. One of such methods is magnetic resonance enterography, which gives high hope in the diagnostics of Crohns disease. The aim of the study was the evaluation of the results of magnetic resonance enterography (MREG) and their comparison with the results of histopathological examination o perioperative specimens. MATERIAL AND METHODS The clinical material comprised 48 patients with suspected Crohns disease. Colonoscopy was performed in all the patients, followed by magnetic resonance enterography, which evaluated the lesion localisation, large intestine wall thickening, small intestine stenosis, mesenteric vessel proliferation, infiltration of surrounding adipose tissue, lymph node enlargement, presence of enteroenteral, enterovesical and enterocutaneous fistulas. Next, a surgical procedure was performed, with collection of specimen for histopathology. The examination results were compared with those of magnetic resonance enterography. RESULTS MREG was performed in 48 individuals. Suspected Crohns disease based on the above examination was diagnosed in 35 cases, isolated small intestine inflammation--in 5, and fibrosis in the remaining 5 patients. No significant differences were found between the lesion localisation done by MREG or perioperativelly. Crohns disease was confirmed by histopathology in 36 cases. The sensitivity of MREG with histopathology was 91.6%, and the specificity--77.8%. CONCLUSIONS Magnetic resonance enterography is a highly effective and sensitice method in the diagnostics of Crohns disease, free of adverse effects and possible to be performed even in pregnant female patients.

Significance of intratissue estrogen concentration coupled with estrogen receptors levels in colorectal cancer prognosis

Dysregulation of estrogen related pathways is implicated colorectal cancer (CRC) development. However, significance of intratissue concentration of estrone (E1) and 17β-estradiol (E2) in relation to estrogen receptor (ESR) expression level was not addressed so far. Herein, we measured E1 and E2 intratissue concentration using liquid chromatography electrospray ionization tandem mass spectrometry (ESI LC/MS) and mRNA levels of ESR1 and ESR2 using RT-qPCR in cancerous and histopathologically unchanged tissue from 75 and 110 CRC patients, respectively. The obtained results were associated with clinicopathological factors, expression of estrogen dependent genes (CTNNB1, CCND1) and prognostic significance. We found no statistically significant differences in E1 or E2 concentration between cancerous tissue and histopathologically unchanged counterparts. Moreover, mRNA levels of ESR1 and ESR2 were significantly decreased in cancerous tissue compared with histopathologically unchanged (p=0.00001). Log rank analysis revealed no benefit of low E1 to E2 ratio, high E1, E2 concentration or ESR1, ESR2 mRNA level for patients’ overall (OS) and disease free survival (DFS). Interestingly, we have observed that patients with low ESR1 mRNA level coupled with low E1 intratissue concentration had a significant decrease in DFS compared with group of patients with high ESR1 mRNA level and high E1 concentration (HR=0.16, 95% CI 0.02-1.05; p=0.06). Furthermore, patients with low E1 concentration and low ESR1 transcript had significantly higher CTNNB1 and CCND1 mRNA level compare with subgroup with high level of both grouping factors. Our study indicates a potential value of estrogen intratissue concentration and its receptor expression level for CRC patients’ prognosis.