Bisulfite sequencing confirmed the DHPLC results (Figure ?(Figure1B)
Bisulfite sequencing confirmed the DHPLC results (Figure ?(Figure1B).1B). biological assays and 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%% 55.8%, Chi-square test, = 0.002) and significantly Dutasteride (Avodart) higher in CC samples with distant metastasis than in samples without (77.8% 46.0%, = 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, Dutasteride (Avodart) 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, 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. overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways. CONCLUSION 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. GFRA1GFRA1reactivation by DNA demethylation is a frequent event in colon cancer (CC) development and that the high level of GFRA1 demethylation in CC tissues is correlated with high metastasis risk of CC and shorter overall survival of patients, especially patients with metastatic CC. We find that GFRA1 overexpression enhances the proliferation and growth of CC cells and by DNA demethylation is an important prognosis factor for CC and the cancer-related cell membrane protein GFRA1 may be a therapeutic target for CC patients. INTRODUCTION Although advances have been made in the treatment of colorectal carcinoma (CRC), it remains the most common gastrointestinal cancer worldwide[1]. CRC is annually diagnosed in 1.4 million individuals and leads to 700000 deaths worldwide[2]. Metastasis is the leading cause of CRC-related death[3]. Although the biological mechanisms associated with CRC metastasis have been extensively studied[4], the sensitivity and specificity of existing clinical biomarkers for predicting the prognosis of CRC patients are not satisfactory. Recognition of specific biomarkers is important for predicting prognosis, including metastasis, of colorectal cancer. Molecular therapy targets for patients with metastatic CRC are also urgently needed. GFRA1 is a cell surface membrane receptor for glial cell-derived neurotrophic factor (GDNF)[5,6]. The GDNF-GFRA1 complex binds to and activates the tran-smembrane RET proto-oncogene receptor, induces the phosphorylation of RET tyrosine residues, and activates the RAS-ERK and PI3K-AKT signaling pathways, thus mediating the development of the nervous system and kidneys, as well as cancer growth[6-9]. The gene is not only normally expressed in neural cells, especially in enteric neurons, but is also abnormally overexpressed in many cancers[9-17]. overexpression can affect multiple biological behaviors of cancer cells, including proliferation, metastasis, and perineural invasion. overexpression is associated with a poor overall survival (OS) of patients with pancreatic or breast cancer[12,16]. Anti-GFRA1 autoantibodies can be detected in patients with luminal A (hormone receptor-positive) breast cancer[18,19]. Apparently, the cancer-associated GFRA1 protein is a potential therapeutic target. Several preclinical anti-GFRA1 antibody-drug conjugates for breast cancer treatment have been developed[20,21]. It is well known that the methylation of CpG islands around the transcription start site (TSS-CGIs) epigenetically inactivates gene transcription, and Mouse monoclonal antibody to Beclin 1. Beclin-1 participates in the regulation of autophagy and has an important role in development,tumorigenesis, and neurodegeneration (Zhong et al., 2009 [PubMed 19270693]) TSS-CGI demethylation is essential for methylated gene reactivation. Our recent studies have shown that alleles are epigenetically silenced by the methylation of TSS-CGI (mGFRA1) in many normal non-nervous cells in adult tissues and are abnormally reactivated in cancer cells by the demethylation (or hypomethylation) of the TSS-CGI (dmGFRA1)[22]. The presence of dmGFRA1 was consistently found to be associated with an increased metastasis risk of gastric carcinoma and short OS in multiple Dutasteride (Avodart) cohorts in China, Japan, and Korea[22]. The results of our prospective study further showed that dmGFRA1 could be a potential biomarker for prediction of metastasis of gastric carcinoma in Chinese patients[23]. However, no studies have reported the impacts of the reactivation Dutasteride (Avodart) of expression by dmGFRA1 on CRC progression. In this study, we examined the demethylation status of TSS-CGIs in alleles in colon carcinoma (CC) and paired surgical margin (SM) tissue samples and normal colon mucosal biopsy samples from noncancer patients. We observed that was significantly demethylated in both CC.