Therapeutic potential of PRL-3 targeting and clinical significance of PRL-3 genomic amplification in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Phosphatase of regenerating liver-3 (PRL-3) has deserved attention as a crucial molecule in the multiple steps of metastasis. In the present study, we examined the mechanisms regulating PRL-3 expression, and assessed the clinical potential of PRL-3-targeted therapy in gastric cancer.</p> <p>Methods</p> <p>PRL-3 genomic amplification was analyzed using quantitative-polymerase chain reaction and/or fluorescence in situ hybridization in 77 primary gastric tumors. The anticancer activity of PRL-3 inhibitor (1-4-bromo-2-benzylidene rhodanine) treatment was evaluated against cancer cells with different genetic and expression status.</p> <p>Results</p> <p>PRL-3 genomic amplification was closely concordant with high level of its protein expression in cell lines, and was found in 20% (8/40) among human primary tumors with its expression, which were all stage III/IV disease (40%, 8/20), but in none (0/37) among those without expression. Additionally, PRL-3 genomic amplification was associated with metastatic lymph node status, leading to advanced stage and thereby poor outcomes in patients with lymph node metastasis (<it>P </it>= 0.021). PRL-3 small interfering RNA robustly repressed metastatic properties, including cell proliferation, invasion, and anchorage-independent colony formation. Although neither PRL-3 genomic amplification nor expression level was responsible for the sensitivity to PRL-3 inhibitor treatment, the inhibitor showed dose-dependent anticancer efficacy, and remarkably induced apoptosis on all the tested cell lines with PRL-3 expression.</p> <p>Conclusions</p> <p>We have for the first time, demonstrated that PRL-3 genomic amplification is one of the predominant mechanisms inducing its expression, especially in more advanced stage, and that PRL-3-targeted therapy may have a great potential against gastric cancer with its expression.</p

Epigenetic Silencing of HOPX Promotes Cancer Progression in Colorectal Cancer

AbstractHomeodomain-only protein X (HOPX)-β promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to investigate the mechanistic roles of HOPX-β promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-β promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and protein levels were measured in relation to HOPX-β promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-β promoter methylation and clinical relevance of CRC patients was determined. HOPX-β promoter methylation is cancer-specific and frequently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, invasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-β promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio= 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-β promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients

Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.

BACKGROUND: Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction. METHODS: DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from "positive control" DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined. RESULTS: (1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%). CONCLUSION: We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients

Kaplan-Meier analysis of 5 year RFS and Stage distribution in pStage II/III gastric cancer in the <i>p53</i> aberrant and <i>p53</i> non-aberrant groups.

<p>(A) Survival curves for intestinal type were compared with those of diffuse type gastric cancer with pStage II/III. (B) Cancer stage distribution according to the <i>p53</i> aberration group and histological findings.</p

Distribution of clinical and pathological factors for correlation with gene & methylation status and univariable prognostic analysis in 163 pStageII/III gastric cancer with gastrectomy and subsequent S-1 treatment.

<p>*<i>p53</i> wild type with super-high methylation;</p><p>**<i>p53</i> wild type without super-high methylation;</p><p>***relapse free survival;</p><p>****overall survival;</p><p>^# vs. <i>p53</i> mutat, Mann-Whtney U test.</p><p>Distribution of clinical and pathological factors for correlation with gene & methylation status and univariable prognostic analysis in 163 pStageII/III gastric cancer with gastrectomy and subsequent S-1 treatment.</p

Immunohistochemical expression of PGP9.5, NMDAR2B, and CCNA1 in gastric cancer according to their methylation status.

<p>Immunohistochemical staining of PGP9.5, NMADR2B, and CCNA1 in primary tumor with or without hypermethylation of the promoter region of the corresponding gene (original magnification, X100, scale bars, 100 μm).</p

Epigenetic conversion in the <i>p53</i> pathway.

<p>Epigenetic conversion in the <i>p53</i> pathway.</p

TaqMeth value of <i>PGP9</i>.<i>5</i>, <i>NMDAR2B</i>, and <i>CCNA1</i> in pStage II/III gastric cancer.

<p>(A) Methylation of the indicated genes was analyzed using Q-MSP and TAqMeth values in tumors with <i>p53</i> wild type were compared with those with <i>p53</i> mutation. The threshold value for determination that a gene was methylated was determined as the maximum TaqMeth value of <i>p53</i> wild type. (B) Tumors with <i>p53</i> wild type or <i>p53</i> mutation were compared in terms of the presence of super-high methylation of the indicated genes. Super-high methylation was defined that at least one gene showed higher TaqMeth value than each threshold values among three genes.</p