Impact of GFRA1 gene reactivation by DNA demethylation on prognosis of patients with metastatic colon cancer.

BACKGROUND: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC). AIM: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target. METHODS: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo. The phosphorylation of AKT and ERK proteins was examined by Western blot analysis. RESULTS: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways. CONCLUSION: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential.

[Downregulation of chromobox protein homolog 7 expression in multiple human cancer tissues].

OBJECTIVE: To investigate the relationship between chromobox protein homolog 7 (cbx7) expression and the occurrence and development of colorectal carcinoma (CRC), gastric carcinoma (GC) and hepatocarcinoma (HCC) tissues. METHODS: The samples of neoplastic tissues and the corresponding cutting-edge normal tissues from 22 cases of CRC, 20 cases of GC, 30 cases of HCC were surgically collected. Level of cbx7 mRNA was detected with a fluorescent quantitative RT-PCR assay, and the correlationship among expression of cbx7 mRNA, the patients' clinicopathologic features and the surviving time after surgery was analyzed. RESULTS: The relative copy number of cbx7 mRNA in carcinomas and the normal tissues was 0.010 ± 0.015 vs 0.053 ± 0.042 for CRCs, 0.197 ± 0.195 vs 1.891 ± 1.254 for GCs, and 0.008 ± 0.008 vs 0.030 ± 0.021 for HCCs, respectively. Compared with the corresponding normal tissues, cbx7 expression was significantly downregulated in CRCs, GCs, and HCCs (t = -7.351, -5.417 and -6.680, respectively, P < 0.01). The expression of cbx7 mRNA in CRCs had significant differences not only between two age groups (the relative copy number of cbx7 mRNA in age > 55 group was 0.007 ± 0.015, but 0.017 ± 0.012 in age ≤ 55 group, t = -2.586, P = 0.022); but also between vascular embolus-positive and negative groups (the level of cbx7 mRNA in positive and negative group was 0.022 ± 0.021 vs 0.006 ± 0.011, t = -3.175, P = 0.010). The area under the receiver operating characteristics (ROC) curve is 0.769 (P = 0.033). when the Cut-off value of the relative copy number of cbx7 mRNA was 0.002 in CRCs. The values less-than 0.002 were defined as low expression. The CRC patients with low expression of cbx7 had a shorter overall survival time; whose 5 years survival rate was only 30.8% (4/13); while the rate was 77.8% (7/9) in high expression of cbx7 group. The difference had statistical significance (χ(2) = 4.329, P = 0.037). The similar differences could not be found among GC and HCC patients. CONCLUSION: Downregulation of cbx7 expression was very common among multiple carcinomas cases, and the downregulation influenced the prognosis of CRC patients.

Methylation of GATA-4 and GATA-5 and development of sporadic gastric carcinomas.

AIM: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis. METHODS: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed. RESULTS: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides). CONCLUSION: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

[Methylation specific binding activity of zinc finger protein Kaiso].

OBJECTIVE: To compare binding activity of different zinc finger domain of human Kaiso with methylated CpG. METHODS: pGEX constructs with different human Kaiso domain were generated and then corresponding fusion proteins were induced and purified. Electrophoretic mobility shift assays were applied to evaluate the binding activity of fusion proteins with methylated CpG. RESULTS: The purified GST-KaisoZF fusion protein (without the POZ protein binding domain) could bind with methylated CpG probe specifically, but not for three or two zinc fingers without flanking domains. CONCLUSION: Human zinc finger protein Kaiso could bind with methylated CpG specifically, only in the assistance of the neighboring flank sequence of the zinc finger domain.

[Effect of selenium-enriched garlic on chronic gastritis of the glandular stomach of Mongolian gerbils induced by H. pylori].

OBJECTIVE: To detect the therapeutic effect of selenium-enriched garlic (SeG) on chronic gastritis. METHODS: Chronic gastritis was induced of the glandular stomach of male Mongolian Gerbils via gastric instillation of H. pylori TN2 strain once every 4 days for 5 consecutive times followed by random classification into six groups. Fresh SeG suspension was administrated daily at dosages of 4.70, 1.5, 0.47, 0.15 g.kg(-1).d(-1) for four weeks. The gerbils in the positive control group were treated with omeprazole, clarithromycin, and amoxicillin for one week. The gerbils were killed for pathological examination four weeks after SeG-treatment. RESULTS: Chronic gastritis (CAG), low-grade dysplasia or gastric intraepithelial neoplasia (DYS/GIN) were observed among 77% and 38.5% of the 13 H. pylori-treated animals in the negative control group, respectively; whereas 40% and 26.7% in the positive control group (n = 15), respectively. The incidences of CAG and DYS/GIN in the SeG groups (n = 21 - 27) were reduced dose-dependently, 16.7% - 38.7% and 11.1% - 14.3% for CAG and DYS/GIN, respectively. CONCLUSION: SeG administration inhibits the development and progression of CAG induced by H. pylori remarkably.