Jingwan Zhang

Gut mucosal microbiome across stages of colorectal carcinogenesis.

Gut microbial dysbiosis contributes to the development of colorectal cancer (CRC). Here we catalogue the microbial communities in human gut mucosae at different stages of colorectal tumorigenesis. We analyse the gut mucosal microbiome of 47 paired samples of adenoma and adenoma-adjacent mucosae, 52 paired samples of carcinoma and carcinoma-adjacent mucosae and 61 healthy controls. Probabilistic partitioning of relative abundance profiles reveals that a metacommunity predominated by members of the oral microbiome is primarily associated with CRC. Analysis of paired samples shows differences in community configurations between lesions and the adjacent mucosae. Correlations of bacterial taxa indicate early signs of dysbiosis in adenoma, and co-exclusive relationships are subsequently more common in cancer. We validate these alterations in CRC-associated microbiome by comparison with two previously published data sets. Our results suggest that a taxonomically defined microbial consortium is implicated in the development of CRC.

Integrative Identification of Epstein–Barr Virus–Associated Mutations and Epigenetic Alterations in Gastric Cancer

BACKGROUND & AIMS The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. METHODS We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). RESULTS Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. CONCLUSIONS By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.

MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome.

Background Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. Methods MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. Results MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1–S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan–Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer. Conclusions MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP–WT1–TBL1 axis

Objective We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. Design The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. Results CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of β-catenin. CA4 interacted with Wilms’ tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms’ tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin β-like protein 1 (TBL1) and the degradation of β-catenin. Conclusions CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP–WT1–TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.

RNF6 Promotes Colorectal Cancer by Activating the Wnt/β-Catenin Pathway via Ubiquitination of TLE3

Gene amplification is a hallmark of cancer and is frequently observed in colorectal cancer. Previous whole-genome sequencing of colorectal cancer clinical specimens identified amplification of Ring finger protein 6 (RNF6), a RING-domain E3 ubiquitin ligase. In this study, we showed that RNF6 is upregulated in 73.5% (147/200) of patients with colorectal cancer and was positively associated with RNF6 gene amplification. Furthermore, RNF6 expression and its gene amplification were independent prognostic factors for poor outcome of patients with colorectal cancer. RNF6 promoted cell growth, cell-cycle progression, and epithelial-to-mesenchymal transition in colorectal cancer cells; RNF6 also promoted colorectal tumor growth and lung metastasis in mouse models. Mechanistic investigations revealed that RNF6 bound and ubiquitylated transducin-like enhancer of split 3 (TLE3), a transcriptional repressor of the β-catenin/TCF4 complex. RNF6-mediated degradation of TLE3 significantly suppressed the association of TLE3 with TCF4/LEF, which in turn led to recruitment of β-catenin to TCF4/LEF, triggering Wnt/β-catenin activation. Restoration of TLE3 expression abolished the oncogenic effects of RNF6. Taken together, these results demonstrate that RNF6 plays a pivotal oncogenic role in colorectal tumorigenesis.Significance: RNF6-mediated ubiquitination and degradation of TLE3 activates the Wnt/β-catenin pathway in colorectal carcinogenesis. Cancer Res; 78(8); 1958-71. ©2018 AACR.