Escape from cell-cell and cell-matrix adhesion dependence underscores disease progression in gastric cancer organoid models.

OBJECTIVE: Cell-cell (CC) and cell-matrix (CM) adhesions are essential for epithelial cell survival, yet dissociation-induced apoptosis is frequently circumvented in malignant cells. DESIGN: We explored CC and CM dependence in 58 gastric cancer (GC) organoids by withdrawing either ROCK inhibitor, matrix or both to evaluate their tumorigenic potential in terms of apoptosis resistance, correlation with oncogenic driver mutations and clinical behaviour. We performed mechanistic studies to determine the role of diffuse-type GC drivers: ARHGAP fusions, RHOA and CDH1, in modulating CC (CCi) or CM (CMi) adhesion independence. RESULTS: 97% of the tumour organoids were CMi, 66% were CCi and 52% were resistant to double withdrawal (CCi/CMi), while normal organoids were neither CMi nor CCi. Clinically, the CCi/CMi phenotype was associated with an infiltrative tumour edge and advanced tumour stage. Moreover, the CCi/CMi transcriptome signature was associated with poor patient survival when applied to three public GC datasets. CCi/CMi and CCi phenotypes were enriched in diffuse-type GC organoids, especially in those with oncogenic driver perturbation of RHO signalling via RHOA mutation or ARHGAP fusions. Inducible knockout of ARHGAP fusions in CCi/CMi tumour organoids led to resensitisation to CC/CM dissociation-induced apoptosis, upregulation of focal adhesion and tight junction genes, partial reversion to a more normal cystic phenotype and inhibited xenograft formation. Normal gastric organoids engineered with CDH1 or RHOA mutations became CMi or CCi, respectively. CONCLUSIONS: The CCi/CMi phenotype has a critical role in malignant transformation and tumour progression, offering new mechanistic information on RHO-ROCK pathway inhibition that contributes to GC pathogenicity.

Mutational landscape of normal epithelial cells in Lynch Syndrome patients.

Lynch Syndrome (LS) is an autosomal dominant disease conferring a high risk of colorectal cancer due to germline heterozygous mutations in a DNA mismatch repair (MMR) gene. Although cancers in LS patients show elevated somatic mutation burdens, information on mutation rates in normal tissues and understanding of the trajectory from normal to cancer cell is limited. Here we whole genome sequence 152 crypts from normal and neoplastic epithelial tissues from 10 LS patients. In normal tissues the repertoire of mutational processes and mutation rates is similar to that found in wild type individuals. A morphologically normal colonic crypt with an increased mutation burden and MMR deficiency-associated mutational signatures is identified, which may represent a very early stage of LS pathogenesis. Phylogenetic trees of tumour crypts indicate that the most recent ancestor cell of each tumour is already MMR deficient and has experienced multiple cycles of clonal evolution. This study demonstrates the genomic stability of epithelial cells with heterozygous germline MMR gene mutations and highlights important differences in the pathogenesis of LS from other colorectal cancer predisposition syndromes.

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles.

OBJECTIVE: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. DESIGN: We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. RESULTS: We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. CONCLUSIONS: These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening.

Gastric cancer displays marked molecular heterogeneity with aggressive behavior and treatment resistance. Therefore, good in vitro models that encompass unique subtypes are urgently needed for precision medicine development. Here, we have established a primary gastric cancer organoid (GCO) biobank that comprises normal, dysplastic, cancer, and lymph node metastases (n = 63) from 34 patients, including detailed whole-exome and transcriptome analysis. The cohort encompasses most known molecular subtypes (including EBV, MSI, intestinal/CIN, and diffuse/GS, with CLDN18-ARHGAP6 or CTNND1-ARHGAP26 fusions or RHOA mutations), capturing regional heterogeneity and subclonal architecture, while their morphology, transcriptome, and genomic profiles remain closely similar to in vivo tumors, even after long-term culture. Large-scale drug screening revealed sensitivity to unexpected drugs that were recently approved or in clinical trials, including Napabucasin, Abemaciclib, and the ATR inhibitor VE-822. Overall, this new GCO biobank, with linked genomic data, provides a useful resource for studying both cancer cell biology and precision cancer therapy.

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.

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.

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.

Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms.

BACKGROUND & AIMS: Repulsive guidance molecule member A (RGMA) is a glycosylphosphatidylinositol-anchored glycoprotein and axon guidance molecule that signals through its receptor, neogenin (NEO1), a homologue of the deleted-in-colorectal cancer (DCC) gene. RGMA also functions as a bone morphogenetic protein (BMP) coreceptor. We studied the potential roles of RGMA and NEO1 in colorectal cancer (CRC) pathogenesis. METHODS: We analyzed expression of RGMA and NEO1, as well as their epigenetic and genetic changes, in a large series of CRC samples, normal colon tissues, adenomas, and cell lines. These studies were accompanied by in vitro functional assay. RESULTS: RGMA and NEO1 expression were significantly down-regulated in most CRCs, adenomas, and cell lines. RGMA was frequently silenced by promoter methylation in CRCs (86.7%), adenomas (90.9%), and CRC cell lines (92.3%) but not in normal colon tissues; allelic imbalance of RGMA and NEO1 was observed in 40% and 49% of CRCs, respectively. In CRC samples, reduced RGMA levels were significantly associated with mismatch repair deficiency or mutations in KRAS or BRAF. Exposure to 5-aza-2'-deoxycytidine restored RGMA expression in CRC cell lines. Transfection of RGMA into CRC cells suppressed cell proliferation, migration, and invasion and also increased apoptosis in response to DNA-damaging agent. CONCLUSIONS: The frequent genetic and epigenetic inactivation of RGMA in CRCs and adenomas along with its in vitro function collectively support its role as a tumor suppressor in colon cells. These findings add to the expanding list of axon guidance molecules with disrupted function during colon carcinogenesis and create new opportunities for early detection and drug development.

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.

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.

Reduced expression of EphB2 that parallels invasion and metastasis in colorectal tumours.

EphB2, a receptor tyrosine kinase regulated by the beta-catenin/Tcf4 complex, is expressed in the proliferative compartment of mouse intestine and regulates bidirectional migration of intestinal precursor cells in the crypt-villus axis through repulsive interaction with Ephrin-B ligands. Recently, it has been shown that reduction of EphB activity accelerates colon tumour progression in the Apc(Min/+) mice. In this study, we examined the expression of EphB2 in normal colon, adenomas, primary colorectal cancers (CRCs), lymph node metastases and liver metastases using immunohistochemistry on tissue microarrays. In addition, EphB2 was overexpressed in SW480 colon cancer cells to study its effect in vitro. We found that EphB2 was expressed in 100% of normal colon crypt base cells, 78% of adenomas, 55.4% of primary CRCs, 37.8% of lymph node metastases and 32.9% of liver metastases (all differences were statistically significant at P < 0.001 compared with primary CRCs). Patients with CRCs that lose EphB2 expression had more advanced tumour stage (P = 0.005), poor differentiation (P < 0.001), poor overall survival (P = 0.005) and disease-free survival (P = 0.001), with the latter being independent of tumour stage. In vitro studies showed that overexpression of EphB2 inhibited colon cancer cell growth in colony formation assay and activation of EphB2 receptor inhibited colon cancer cell adhesion and migration. Our data demonstrated a progressive loss of EphB2 expression in each critical step of colon carcinogenesis, including the onset of invasion, dedifferentiation and metastasis which are paralleled by adverse patient outcome. EphB2 may achieve its tumour suppressor function through regulation of cell survival, adhesion and migration.

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.