<i>Cysteine dioxygenase type 1 (CDO1)</i> gene promoter methylation during the adenoma-carcinoma sequence in colorectal cancer

<div><p>Background</p><p>Progression of colorectal cancer (CRC) has been explained by genomic abnormalities along with the adenoma-carcinoma sequence theory (ACS). The aim of our study is to elucidate whether the promoter DNA methylation of the cancer-specific methylation gene, <i>cysteine dioxygenase 1</i> (<i>CDO1</i>), contributes to the carcinogenic process in CRC.</p><p>Methods</p><p>The study group comprised 107 patients with CRC who underwent surgical resection and 90 adenomas treated with endoscopic resection in the Kitasato University Hospital in 2000. We analyzed the extent of methylation in each tissue using quantitative TaqMan methylation-specific PCR for <i>CDO1</i>.</p><p>Results</p><p>The methylation level increased along with the ACS process (<i>p</i> < 0.0001), and statistically significant differences were found between normal-appearing mucosa (NAM) and low-grade adenoma (<i>p</i> < 0.0001), and between low-grade adenoma and high-grade adenoma (<i>p</i> = 0.01), but not between high-grade adenoma and cancer with no liver metastasis. Furthermore, primary CRC cancers with liver metastasis harbored significantly higher methylation of <i>CDO1</i> than those without liver metastasis (<i>p</i> = 0.02). As a result, the area under the curve by <i>CDO1</i> promoter methylation was 0.96, 0.80, and 0.67 to discriminate cancer from NAM, low-grade adenoma from NAM, and low-grade adenoma from high-grade adenoma, respectively.</p><p>Conclusions</p><p><i>CDO1</i> methylation accumulates during the ACS process, and consistently contributes to CRC progression.</p></div

Analysis of <i>CDO1</i> TaqMeth V derived from NAM, adenoma, and cancerous tissue.

<p>A: Adenoma is classified into three categories: mild atypia, moderate atypia, and severe atypia. B: Adenomas were divided into low-grade adenoma and high-grade adenoma.</p

Univariate and multivariate prognostic analysis of clinicopathological factors for OS in colorectal cancer.

<p>Univariate and multivariate prognostic analysis of clinicopathological factors for OS in colorectal cancer.</p

Results of CDO1 immunostaining.

<p>A: IHC score = 0. B: IHC score = 1. C: IHC score = 2. D: Results of IHC scoring in cancer tissue and NAM are shown. Intense staining was observed in NAM, and less intense staining was seen in cancer tissues. A significant difference in scoring was seen between the groups.</p

Standard light microscopy findings on hematoxylin-eosin-stained specimens of colorectal adenoma (40×, magnification) and ROC curves for distinguishing between the two tissues.

<p>A: mild atypia. B: moderate atypia. C: severe atypia. D: ROC curve of <i>CDO1</i> promoter methylation for distinguishing between cancer tissue and NAM. When the cut-off value was 15.6, the AUC was 0.96, sensitivity was 95%, and specificity was 90%. E: ROC curve for distinguishing between low-grade adenoma and NAM. F: ROC curve for distinguishing between low-grade adenoma and high-grade adenoma.</p

Relationship with TaqMeth V of CDO1 and clinicopathological factors at cancer tissue.

<p>Relationship with TaqMeth V of CDO1 and clinicopathological factors at cancer tissue.</p

Associations of clinicopathological factors with <i>CDO1</i> TaqMeth V in cancerous tissue.

<p>The relationship between <i>CDO1</i> and A: age, B: histological type, C: tumor diameter, D: liver metastasis, E: pT, F: v, G and H: Dukes classification.</p

Log-rank plot analysis and Kaplan-Meier survival curves for OS and RFS in CRC patients according to <i>CDO1</i> TaqMeth V.

<p>A, B: Log-rank plot analysis for OS. C: Kaplan-Meier survival curves for OS comparing CRC patients with <i>CDO1</i> TaqMeth V equal to or below 20.5 and those with <i>CDO1</i> TaqMeth V over 20.5. D, E: Log-rank plot analysis for RFS. F: Kaplan-Meier survival curves for RFS comparing CRC patients with <i>CDO1</i> TaqMeth V equal to or below 44.8 and those with <i>CDO1</i> TaqMeth V over 44.8.</p

Clinical significance of CD133 expression in primary colon cancer tissues.

(a) In semi-quantitative RT-PCR, CD133 transcripts were seen in Colo205 and HCT116 among 8 CRC cell lines (lower panel). Quantitative PCR is consistent with the semi-quantitative RT-PCR (upper panel). (b) CD133 expression was significantly reduced in 60 colon cancer tissues as compared to in 60 non-cancerous mucosa tissues (p = 0.048). (c) CD133 expressions in colon cancer tissues were significantly associated with those in non-cancerous mucosa tissues (R = 0.26, p = 0.026). (d) Public database of microdissection followed by microarray revealed that CD133 expression is rather higher in non-cancerous tissues than in cancer tissues. N.E., normal epithelia N.S., normal stroma C.E., cancer epithelia C.S., cancer stroma (e) CD133 expression in colon cancer tumor tissues was not different according to T factors (upper panel), whereas CD133 expression in non-cancerous mucosa tissues was significantly higher in T4 than in non-T4 (p = 0.048). (f) CD44T expression was not significantly different between primary colon cancer and liver metastasis (p = 0.24, left panel). On the other hand, CD44V expression was significantly lower in liver metastasis than in primary colon cancers (p = 0.0006, middle panel). CD133 expression was significantly lower in liver metastasis than in primary colon cancers, either (p = 0.0005, right panel).</p

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BackgroundCD44 and CD133 are stem cell markers in colorectal cancer (CRC). CD44 has distinctive isoforms with different oncological properties like total CD44 (CD44T) and variant CD44 (CD44V). Clinical significance of such markers remains elusive.MethodsSixty colon cancer were examined for CD44T/CD44V and CD133 at mRNA level in a quantitative PCR, and clarified for their association with clinicopathological factors.Results(1) Both CD44T and CD44V showed higher expression in primary colon tumors than in non-cancerous mucosas (pCD133 was expressed even in non-cancerous mucosa and rather decreased in the tumors (p = 0.048). (2) CD44V expression was significantly associated with CD44T expression (R = 0.62, pCD133 at all in the primary tumors. (3) CD44V/CD44T expressions were significantly higher in right colon cancer than in left colon cancer (p = 0.035/p = 0.012, respectively), while CD133 expression were not (p = 0.20). (4) In primary tumors, unexpectedly, CD44V/CD44T/CD133 mRNA expressions were not correlated with aggressive phenotypes, but CD44V/CD44T rather significantly with less aggressive lymph node metastasis/distant metastasis (p = 0.040/p = 0.039, respectively). Moreover, both CD44V and CD133 expressions were significantly decreased in liver metastasis as compared to primary tumors (p = 0.0005 and p = 0.0006, respectively).ConclusionOur transcript expression analysis of cancer stem cell markers did not conclude that their expression could represent aggressive phenotypes of primary and metastatic tumors, and rather represented less demand on stem cell marker-positive cancer cells.</div