Anti-C-Myc polyclonal antibody, Biotin

Simple Summary Esophageal adenocarcinoma has become a major clinical challenge in the western world due to its rapid increasing incidence and poor overall prognosis. Understanding the molecular events of its tumorigenesis is the key to better diagnosis and development of better therapeutic strategies. In the current study we aimed to identify epigenetic alteration targets in esophageal adenocarcinoma. We focused on a candidate gene,NDRG4(N-myc downregulated gene 4). We found thatNDRG4was frequent downregulated in esophageal adenocarcinoma through DNA hypermethylation of its promoter region. Re-expression of NRDG4 in cancer cells significantly suppressed tumor growth via inhibition of cell proliferation. These results will improve our understanding on how dysfunction ofNDRG4contributes to esophageal adenocarcinoma. DNA hypermethylation ofNDRG4may be a useful biomarker in clinical monitoring of esophageal adenocarcinoma patients. The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically in the past few decades in the United States and Western world. The N-myc downregulated gene 4 (NDRG4) belongs to the human NDRG family. In this study, we aimed to identify the expression levels, regulation, and functions ofNDRG4in EAC. Using an integrative epigenetic approach, we identified genes showing significant downregulation in EAC and displaying upregulation after 5-Aza-deoxycitidine. Among these genes, likely to be regulated by DNA methylation,NDRG4was among the top 10 candidate genes. Analyses of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) data sets and EAC tissue samples demonstrated thatNDRG4was significantly downregulated in EAC (p< 0.05). Using Pyrosequencing technology for quantification of DNA methylation, we detected thatNDRG4promoter methylation level was significantly higher in EAC tissue samples, as compared to normal esophagus samples (p< 0.01). A strong inverse correlation betweenNDRG4methylation and its gene expression levels (r= -0.4,p< 0.01) was observed. Treatment with 5-Aza restored theNDRG4expression, confirming that hypermethylation is a driving force forNDRG4silencing in EAC. Pathway and gene set enrichment analyses of TCGA data suggested thatNDRG4is strongly associated with genes related to cell cycle regulation. Western blotting analysis showed significant downregulation of Cyclin D1, CDK4 and CDK6 in EAC cells after overexpression of NDRG4. Functionally, we found that the reconstitution of NDRG4 resulted in a significant reduction in tumor cell growth in two-dimensional (2D) and three-dimensional (3D) organotypic culture models and inhibited tumor cell proliferation as indicated by the EdU (5-ethynyl-2 '-deoxyuridine) proliferation assay.

Background: Transmembrane 4 L six family member 1 (TM4SF1) is upregulated in several epithelial cancers and is closely associated with poor prognosis. However, the role of TM4SF1 and its potential mechanism in colorectal cancer (CRC) remain elusive. Methods: We investigated the expression of TM4SF1 in the Oncomine, the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and confirmed the results by immunohistochemistry (IHC), qPCR and Western blotting (WB) of CRC tissues. The effect of TM4SF1 on the epithelial-to-mesenchymal transition (EMT) and cancer stemness of CRC cells was investigated by Transwell, wound healing and sphere formation assays. A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which TM4SF1 modulates EMT and cancer stemness in CRC. Results: TM4SF1 expression was markedly higher in CRC tissues than in non-tumour tissues and was positively correlated with poor prognosis. Downregulation of TM4SF1 inhibited the migration, invasion and tumour sphere formation of SW480 and LoVo cells. Conversely, TM4SF1 overexpression significantly enhanced the migration, invasion and tumoursphere formation potential of CRC cells, Additionally, TM4SF1 silencing inhibited the EMT mediated by transforming growth factor-beta 1 (TGF-beta 1). Mechanistically, gene set enrichment analysis (GSEA) predicted that the Wnt signalling pathway was one of the most impaired pathways in TM4SF1-deficient CRC cells compared to controls. The results were further validated by WB, which revealed that TM4SF1 modulated SOX2 expression in a Wnt/beta-catenin activation-dependent manner. Furthermore, we found that knockdown of TM4SF1 suppressed the expression of c-Myc, leading to decreased c-Myc binding to the SOX2 gene promoter. Finally, depletion of TM4SF1 inhibited metastasis and tumour growth in a xenograft mouse model. Conclusion: Our study substantiates a novel mechanism by which TM4SF1 maintains cancer cell stemness and EMT via the Wnt/beta-catenin/c-Myc/SOX2 axis during the recurrence and metastasis of CRC.