Wai Yin Tsui

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1.

Lynch syndrome patients are susceptible to colorectal and endometrial cancers owing to inactivating germline mutations in mismatch repair genes, including MSH2 (ref. 1). Here we describe patients from Dutch and Chinese families with MSH2-deficient tumors carrying heterozygous germline deletions of the last exons of TACSTD1, a gene directly upstream of MSH2 encoding Ep-CAM. Due to these deletions, transcription of TACSTD1 extends into MSH2. The MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive but not in Ep-CAM negative normal tissues, thus revealing a correlation between activity of the mutated TACSTD1 allele and epigenetic inactivation of the corresponding MSH2 allele. Gene silencing by transcriptional read-through of a neighboring gene in either sense, as demonstrated here, or antisense direction, could represent a general mutational mechanism. Depending on the expression pattern of the neighboring gene that lacks its normal polyadenylation signal, this may cause either generalized or mosaic patterns of epigenetic inactivation.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer

Epimutations in the germline, such as methylation of the MLH1 gene, may contribute to hereditary cancer syndrome in human, but their transmission to offspring has never been documented. Here we report a family with inheritance, in three successive generations, of germline allele-specific and mosaic hypermethylation of the MSH2 gene, without evidence of DNA mismatch repair gene mutation. Three siblings carrying the germline methylation developed early-onset colorectal or endometrial cancers, all with microsatellite instability and MSH2 protein loss. Clonal bisulfite sequencing and pyrosequencing showed different methylation levels in different somatic tissues, with the highest level recorded in rectal mucosa and colon cancer tissue, and the lowest in blood leukocytes. This mosaic state of germline methylation with different tissue distribution could act as the first hit and provide a mechanism for genetic disease inheritance that may deviate from the mendelian pattern and be overlooked in conventional leukocyte-based genetic diagnosis strategy.

Downregulation of ID4 by promoter hypermethylation in gastric adenocarcinoma

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5′ CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix–loop–helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

RNF43 germline and somatic mutation in serrated neoplasia pathway and its association with BRAF mutation

Objective Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1me+). We investigate genetic alterations in the serrated polyposis pathway. Design We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies. Results In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1me+ (85%) versus MLH1me− (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1me+; APC and CTNNB1 in MLH1me−); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1me+ MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1. Conclusions These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.

Abstract 179: Regulation of stromal miR-125b on normal colonic epithelial cell renewal and its putative role in tumorigenesis

MicroRNAs (miRNAs) are small non-coding RNAs which exert their effects by post-transcriptionally silencing target mRNAs. Deregulation of miRNA expression is a frequent event in tumorigenesis. MiR-125b is a highly conserved miRNA among various species and is composed of three homologs: hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125-2. The tumorigenic roles of miR-125b have been studied in various cancers including prostate, colon, glioma etc, and studies have demonstrated that it can act as a tumor suppressor or an oncogene depending on the cellular context. It was characterized as an oncogene in prostate cancer and glioma through down-regulation of pro-apoptotic regulators BAK1 and Bcl-2 modifying factor (BMF), respectively. In colon cancer, a recent clinico-pathological study showed that high expression of miR-125b was associated with tumor invasion and poor prognosis. However, the localization of miR125b in normal colon and tumors is currently unknown. Therefore, we aimed to address the precise expression and the functional role of miR-125b in normal colon, which may provide insight on its potential oncogenic effects during carcinogenesis. We performed gene expression analysis of miR-125b in normal colon tissues from 16 pairs of colon top versus basal crypts and 4 pairs of crypts versus stroma by real-time RT-PCR. Colon top and basal crypts were microdissected from frozen sections, whereas pure normal crypts and stromal fractions were isolated from freshly resected human colon specimens. We found that miR-125b was significantly enriched in the basal crypts (p expression may contribute to carcinogenesis through disruption of this pathway. Note: This abstract was not presented at the meeting. Citation Format: Helen H N Yan, Jackie K Y Lau, Annie S Y Chan, Wai Yin Tsui, Tsun Leung Chan, Suet Yi Leung. Regulation of stromal miR-125b on normal colonic epithelial cell renewal and its putative role in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2015-179

Abstract 5016: Retinoic acid receptor responder 3 expression suppresses colorectal cancer cell growth in vitro and is a stage-independent prognostic marker in colorectal cancer patients in vivo

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. Whilst adjuvant chemotherapy can improve survival in Dukes’ C patients, its application on Dukes’ B patients is controversial. Identification of factors that can predict recurrence in the latter group may facilitate classification of high risk patients for receiving adjuvant therapy. Retinoic acid receptor responder 3 (Rarres3) was firstly identified as the retinoid induced class II tumor suppressor in human keratinocytes and psoriatic lesions. In breast, head and neck, lung and gastric cancer cell lines, induced expression of Rarres3 by retinoid can inhibit cell proliferation. Less is known of its effect on colon cancer. Using real-time quantitative RT-PCR, coupled with in vitro functional assays, we have characterized the role of Rarres3 in colorectal cancer development. By comparing the mRNA expression level of Rarres3 in 405 CRCs and 19 colon cancer cell lines with 63 paired normal colons, Rarres3 is down-regulated in most of the CRCs and cell lines. Loss of Rarres3 expression is strongly associated with Dukes’ D patients (p=0.009) and well or moderately differentiated tumor type (p=0.016). Kaplan-Meier analysis demonstrated an improved overall survival (p=0.001) for patients with high expression of Rarres3 by log-rank test. Specifically, there is a prolonged overall survival (p=0.007) and disease free (p=0.012) for Dukes’ B patients (n=158). In multivariate Cox Regression analysis, Rarres3 is an independent factor to predict overall survival (HR=0.473, p=0.001) and disease free (HR=0.557, p=0.047) apart from lymphovascular invasion and Dukes’ stages. Rarres3 protein expression can be induced in colon cancer cell lines by treatment with either interferon-γ (IFNγ) or all-trans retinoic acid (ATRA). Stable transfection of Rarres3 into SW480 colon cancer cells led to inactivation of Akt signaling pathway, suppression of cell growth and enhancement of cell apoptosis via capase-1 activated apoptotic pathway in response to 5-fluorouracil treatment. In addition, we found that Rarres3 is a Wnt target in which its expression is suppressed during Wnt activation and vice versa in colon cancer cell lines. In summary, Rarres3 expression can be induced by IFNγ and ATRA, causing suppression of cell growth and sensitization of cancer cells to apoptosis. Loss of Rarres3 expression in CRCs is predictive of tumor recurrence and poor prognosis especially in Dukes’ B patients, thus aids in molecular stratification of high risk patients. The results also raise the possibility for the use of IFNγ and ATRA in CRC treatment especially in the high risk Dukes’ B subgroup. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5016.