Epigenetic silencing of HOPX is critically involved in aggressive phenotypes and patient prognosis in papillary thyroid cancer.

HOPX is involved in multiple organ development and acts as a tumor suppressor in various cancers. Epigenetic silencing of HOPX via its promoter methylation has been shown frequent and cancer-specific in human cancers. The proliferation of thyroid cancer cells and cancer progression are strongly influenced by epigenetic alterations as well as genetic changes. Papillary thyroid cancer (PTC) comprises the vast majority of thyroid cancers and exhibits slow progression. However, ~10% of patients still show disease recurrence and refractoriness to treatment. Accordingly, it is important approach to research epigenetic mechanisms in PTC progression to find useful biomarkers. Here, we aimed to seek into the roles and clinical impact of epigenetic silencing of HOPX in PTC. The promoter methylation of HOPX was observed in five of six human thyroid cancer cell lines. Down-regulation of HOPX was seen in three cell lines including PTC line K1, and demethylating agents restored HOPX expression. The promoter methylation was observed with high sensitivity and specificity in human PTC tissues. HOPX promoter methylation independently predicted disease recurrence in PTC patients. Epigenetic silencing of HOPX was associated with Ki-67 expression. Of note, HOPX promoter methylation was dramatically associated with worse prognosis especially in patients with stage I PTC. Forced HOPX expression suppressed cell proliferation, invasive activities, and anchorage-independent growth in vitro. HOPX promoter methylation is frequent and cancer-specific event, leading to aggressive phenotype in PTC. Epigenetic silencing of HOPX may be a clue to tackle cancer progression and have clinical impact as a novel biomarker in PTC.

Comprehensive Exploration to Identify Predictive DNA Markers of ΔNp63/SOX2 in Drug Resistance in Human Esophageal Squamous Cell Carcinoma.

BACKGROUND: OBP-801 is a novel histone deacetylase inhibitor being developed as an anticancer drug. In this study, we explored genes to predict drug resistance in human cancer. METHODS: OBP-801 resistance was assessed in 37 strains of human cancer cell lines. Expression microarrays harboring 54,675 genes were used to focus on candidate genes, which were validated for both functional and clinical relevance in esophageal squamous cell carcinoma (ESCC). RESULTS: OBP-801 is sensitive to esophageal, gastric, and thyroid cancer, and resistant to some esophageal and colorectal cancers. We therefore used ESCC to explore genes. Comprehensive exploration focused on ΔNp63/SOX2, which were both genetically and epigenetically overexpressed in ESCC. Genomic amplifications of ΔNp63/SOX2 were tightly correlated each other (r = 0.81). Importantly, genomic amplification of ΔNp63/SOX2 in the resected tumors after neoadjuvant chemotherapy was significantly associated with histological grade of response (G1). Forced expression of either of these two genes did not induce each other, suggesting that their functional relevances were independent and showed robust drug resistance in OBP-801, as well as 5-fluorouracil. Furthermore, ΔNp63 could exert a potent oncogenic potential. RNA interference of ΔNp63 supported its oncological properties, as well as drug resistance. CONCLUSION: Comprehensive exploration of genes involved in anticancer drug residence could identify critical oncogenes of ΔNp63/SOX2 that would predict chemotherapy response in ESCC.

Prediction of onset of remnant gastric cancer by promoter DNA methylation of CDO1/HOPX/Reprimo/E-cadherin.

BACKGROUND: Early detection of remnant gastric cancer (RGC) is required to reduce the risk of death, but long-term endoscopic surveillance is difficult after gastrectomy. In this study, data for the methylation status of 4 methylation genes (CDO1, HOPX, Reprimo, and E-cadherin) to predict the onset of RGC are presented. RESULTS: The 4 genes showed hypermethylation in RGC tumors in contrast to the corresponding non-cancerous mucosa tissues. The methylation level in the non-cancerous mucosa tissues of the initial surgery was obviously high in initial malignant disease for CDO1 (P = 0.0001), while in initial benign one for E-cadherin (P = 0.003). Promoter DNA methylation status in the remnant non-cancerous mucosa tissues together with the basic clinical data in turn predicted either initial malignant disease or initial benign disease with a high AUC score of 0.94, suggesting that methylation events are differentially recognized between the initial malignant and benign disease. We then finally confirmed that 4 genes hypermethylation of the non-cancerous tissues by biopsy prior to onset of RGC could predict terms until RGC occurred (P < 0.0001). METHODS: A total of 58 RGC patients were used to establish the model. The 4 genes promoter methylation were analyzed for DNA obtained from the patient's specimens using quantitative methylation specific polymerase chain reaction. CONCLUSIONS: This risk model would help provide guidance for endoscopic surveillance plan of RGC after gastrectomy.

Comprehensive Genetic Search to Clarify the Molecular Mechanism of Drug Resistance Identifies ASCL2-LEF1/TSPAN8 Axis in Colorectal Cancer.

BACKGROUND: Treatment-resistance genes limiting anticancer therapy have not been well clarified in colorectal cancer (CRC). We explored gene expression profiles to identify biomarkers for predicting treatment resistance to an anticancer drug in CRC. METHODS: Six CRC cell lines were treated with phenylbutyrate (PB). The gene expression profiles were then compared using microarrays (harboring 54,675 genes), and genes associated with PB resistance were identified. Candidate genes were functionally examined in cell lines and clinically validated for treatment resistance in clinical samples. RESULTS: Both DLD1 and HCT15 cells were PB resistant, while HCT116 cells were identified as PB sensitive. On microarray analysis, among the PB resistance-related genes, the expression of the genes ASCL2, LEF1, and TSPAN8 was clearly associated with PB resistance. PB-sensitive cells transfected with one of these three genes exhibited significant (P < 0.001) augmentation of PB resistance; ASCL2 induced expression of both LEF1 and TSPAN8, while neither LEF1 nor TSPAN8 induced ASCL2. RNA interference via ASCL2 knockdown made PB-resistant cells sensitive to PB and inhibited both genes. ASCL2 knockdown also played a critical role in sensitivity to treatment by 5-fluorouracil and radiotherapy in addition to PB. Finally, ASCL2 expression was significantly correlated with histological grade of rectal cancer with preoperative chemoradiation therapy. CONCLUSIONS: ASCL2 was identified as a causative gene involved in therapeutic resistance against anticancer treatments in CRC.

Clinical significance of cancer specific methylation of the CDO1 gene in small bowel cancer.

Although small bowel cancer (SBC) is extremely rare, its prognosis is poor, and molecular mechanism of the SBC development remains unclear. The aim of our study is to elucidate whether DNA methylation of the promoter region of the cancer-specific methylation gene, cysteine dioxygenase 1 (CDO1), contributes to the carcinogenic process in SBC. The study group comprised patients with 53 patients with SBC, 107 colorectal cancer (CRC), and other rare tumors of the small intestine such as 4 malignant lymphomas, 2 leiomyosarcomas, and 9 gastrointestinal stromal tumors. We analyzed the extent of methylation in each tissue using quantitative TaqMan methylation-specific PCR for CDO1. Significantly higher CDO1 methylation was observed in cancer tissues compared with non-cancerous mucosa of the small intestine (ROC = 0.96). Among the various clinicopathological factors, positive correlation of CDO1 methylation with tumor diameter was observed (R = 0.31, p = 0.03), and the CDO1 methylation level was a possible prognostic factor for relapse-free survival (p = 0.09). Compared with CRC, SBC had a significantly poorer prognosis (p = 0.007) and displayed a significantly higher CDO1 methylation level (p < 0.0001). Intriguingly, especially in pStage I/II, there were robust prognostic difference between SBC and CRC (p = 0.08 / p < 0.0001), which may reflect CDO1 methylation status (p = 0.02 / p = 0.001). Among small bowel tumors, CDO1 methylation in SBC was higher in order of malignant lymphoma, cancer, and leiomyosarcoma/GIST (p = 0.002) by ANOVA. The CDO1 gene shows extremely cancer-specific hypermethylation, and it can be a prognostic marker in SBC.

Cysteine dioxygenase type 1 (CDO1) gene promoter methylation during the adenoma-carcinoma sequence in colorectal cancer.

BACKGROUND: 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, cysteine dioxygenase 1 (CDO1), contributes to the carcinogenic process in CRC. METHODS: 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 CDO1. RESULTS: The methylation level increased along with the ACS process (p < 0.0001), and statistically significant differences were found between normal-appearing mucosa (NAM) and low-grade adenoma (p < 0.0001), and between low-grade adenoma and high-grade adenoma (p = 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 CDO1 than those without liver metastasis (p = 0.02). As a result, the area under the curve by CDO1 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. CONCLUSIONS: CDO1 methylation accumulates during the ACS process, and consistently contributes to CRC progression.

Prognostic significance of promoter DNA hypermethylation of the cysteine dioxygenase 1 (CDO1) gene in primary gallbladder cancer and gallbladder disease.

BACKGROUND: Aberrant promoter DNA methylation of the cysteine dioxygenase 1 (CDO1) gene is found in various human cancers and is associated with clinical outcome. In this study, we assessed for the first time the clinicopathological significance of CDO1 methylation in primary gallbladder cancer (GBC) in comparison with non-malignant gallbladder disease. METHODS: CDO1 DNA methylation was quantified using quantitative TaqMan methylation specific PCR (Q-MSP) in 99 primary GBC patients together with the 78 corresponding non-tumor tissues and 26 benign gallbladder disease (including 7 patients with xanthogranulomatous cholecystitis) who underwent surgical resection between 1986 and 2014. RESULTS: The average CDO1 TaqMeth value of primary GBCs was 23.5±26. These values were significantly higher than those of corresponding non-tumor tissues (average 8±13, p < .0001) and diseased gallbladder tissues from patients with benign gallbladder diseases (average 0.98±1.6, p < .0001). CDO1 hypermethylation is also found in xanthogranulomatous cholecystitis. Using a cut-off value of 17.7, GBC cases with CDO1 hypermethylation (n = 47) showed significantly poorer prognosis than those with CDO1 hypomethylation (n = 52) (p = 0.0023). Multivariate Cox proportional hazards analysis identified that CDO1 hypermethylation was an independent prognostic factor. Notably, CDO1 hypermethylation showed prognostic relevance, especially in stage II GBC, in which it is highly anticipated to work as a predictive marker for candidates of adjuvant therapy. CONCLUSIONS: Promoter NA methylation of CDO1 was demonstrated for the first time to be a cancer-associated methylation in primary GBC, and it has the potential to be a prognostic biomarker of GBC for high-risk patients with stage II GBC.