Chia Huey Ooi

Intrinsic Subtypes of Gastric Cancer, Based on Gene Expression Pattern, Predict Survival and Respond Differently to Chemotherapy

BACKGROUND & AIMS Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs. METHODS We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed. RESULTS Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Laurens histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil-based therapy. CONCLUSIONS Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.

Oncogenic pathway combinations predict clinical prognosis in gastric cancer.

Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-kappaB, and Wnt/beta-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms.

STAT3-Driven Upregulation of TLR2 Promotes Gastric Tumorigenesis Independent of Tumor Inflammation

Gastric cancer (GC) is associated with chronic inflammation; however, the molecular mechanisms promoting tumorigenesis remain ill defined. Using a GC mouse model driven by hyperactivation of the signal transducer and activator of transcription (STAT)3 oncogene, we show that STAT3 directly upregulates the epithelial expression of the inflammatory mediator Toll-like receptor (TLR)2 in gastric tumors. Genetic and therapeutic targeting of TLR2 inhibited gastric tumorigenesis, but not inflammation, characterized by reduced proliferation and increased apoptosis of the gastric epithelium. Increased STAT3 pathway activation and TLR2 expression were also associated with poor GC patient survival. Collectively, our data reveal an unexpected role for TLR2 in the oncogenic function of STAT3 that may represent a therapeutic target in GC.

Genomic Loss of miR-486 Regulates Tumor Progression and the OLFM4 Antiapoptotic Factor in Gastric Cancer

Purpose: MicroRNAs (miRNA) play pivotal oncogenic and tumor-suppressor roles in several human cancers. We sought to discover novel tumor-suppressor miRNAs in gastric cancer (GC). Experimental Design: Using Agilent miRNA microarrays, we compared miRNA expression profiles of 40 primary gastric tumors and 40 gastric normal tissues, identifying miRNAs significantly downregulated in gastric tumors. Results: Among the top 80 miRNAs differentially expressed between gastric tumors and normals (false discovery rate < 0.01), we identified hsa-miR-486 (miR-486) as a significantly downregulated miRNA in primary GCs and GC cell lines. Restoration of miR-486 expression in GC cell lines (YCC3, SCH and AGS) caused suppression of several pro-oncogenic traits, whereas conversely inhibiting miR-486 expression in YCC6 GC cells enhanced cellular proliferation. Array-CGH analysis of 106 primary GCs revealed genomic loss of the miR-486 locus in approximately 25% to 30% of GCs, including two tumors with focal genomic losses specifically deleting miR-486, consistent with miR-486 playing a tumor-suppressive role. Bioinformatic analysis identified the secreted antiapoptotic glycoprotein OLFM4 as a potential miR-486 target. Restoring miR-486 expression in GC cells decreased endogenous OLFM4 transcript and protein levels, and also inhibited expression of luciferase reporters containing an OLFM4 3′ untranslated region with predicted miR-486 binding sites. Supporting the biological relevance of OLFM4 as a miR-486 target, proliferation in GC cells was also significantly reduced by OLFM4 silencing. Conclusions:miR-486 may function as a novel tumor-suppressor miRNA in GC. Its antioncogenic activity may involve the direct targeting and inhibition of OLFM4. Clin Cancer Res; 17(9); 2657–67. ©2011 AACR.

Comprehensive genomic meta-analysis identifies intra-tumoural stroma as a predictor of survival in patients with gastric cancer

Objective Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors and reveal novel therapeutic targets. The authors aimed to produce a comprehensive inventory of gene expression programmes expressed in primary GCs, and to identify those expression programmes significantly associated with patient survival. Design Using a network-modelling approach, the authors performed a large-scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. The authors analysed a training set of 428 GCs and 163 non-malignant gastric samples, and a validation set of 288 GCs and 61 non-malignant gastric samples. Results The authors identified 178 gene expression programmes (‘modules’) expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs and clinicopathological parameters. Expression of a transforming growth factor β (TGF-β) signalling associated ‘super-module’ of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. The proportion of intra-tumoural stroma, quantified by morphometry in tissue sections from gastrectomy specimens, was also significantly associated with stromal super-module expression and GC patient survival. Conclusion Stromal gene expression predicts GC patient survival in multiple independent cohorts, and may be closely related to the intra-tumoural stroma proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting the GC stroma may merit evaluation.

Integrated epigenomics identifies BMP4 as a modulator of cisplatin sensitivity in gastric cancer

Objective Cisplatin is a widely used gastric cancer (GC) chemotherapy; however, genetic factors regulating GC responses to cisplatin remain obscure. Identifying genes regulating cisplatin resistance could aid clinicians in tailoring treatments, by distinguishing cisplatin sensitive patients from those who might benefit from alternative platinum therapies, and highlight novel targeted strategies for overcoming cisplatin resistance. Here integrated epigenomics is applied to identify genes associated with GC cisplatin resistance. Design 20 GC cell lines were subjected to gene expression profiling, DNA methylation profiling and drug response assays. The molecular data were integrated to identify genes highly expressed and unmethylated specifically in cisplatin-resistant lines. Candidate genes were functionally tested by several in vitro and in vivo assays. Clinical impact of candidate genes was also assessed in a cohort of 197 GC patients. Results Epigenomic analysis identified bone morphogenetic protein 4 (BMP4) as an epigenetically regulated gene highly expressed in cisplatin-resistant lines. Functional assays confirmed that BMP4 is necessary and sufficient for the expression of several prooncogenic traits, likely mediated through stimulation of the epithelial-mesenchymal transition. In primary tumours, BMP4 promoter methylation levels were inversely correlated with BMP4 expression, and patients with high BMP4-expressing tumours exhibited significantly worse prognosis. Therapeutically, targeted genetic inhibition of BMP4 caused significant sensitisation of GC cells to cisplatin. Notably, BMP4-expressing GCs also did not exhibit cross resistance to oxaliplatin. Conclusions BMP4 epigenetic and expression status may represent promising biomarkers for GC cisplatin resistance. Targeting BMP4 may sensitise GC cells to cisplatin. Oxaliplatin, a clinically acceptable cisplatin alternative, may represent a potential therapeutic option for BMP4-positive GCs.

Integrative Genomic, Transcriptional, and Proteomic Diversity in Natural Isolates of the Human Pathogen Burkholderia pseudomallei

Natural isolates of pathogenic bacteria can exhibit a broad range of phenotypic traits. To investigate the molecular mechanisms contributing to such phenotypic variability, we compared the genomes, transcriptomes, and proteomes of two natural isolates of the gram-negative bacterium Burkholderia pseudomallei, the causative agent of the human disease melioidosis. Significant intrinsic genomic, transcriptional, and proteomic variations were observed between the two strains involving genes of diverse functions. We identified 16 strain-specific regions in the B. pseudomallei K96243 reference genome, and for eight regions their differential presence could be ascribed to either DNA acquisition or loss. A remarkable 43% of the transcriptional differences between the strains could be attributed to genes that were differentially present between K96243 and Bp15682, demonstrating the importance of lateral gene transfer or gene loss events in contributing to pathogen diversity at the gene expression level. Proteins expressed in a strain-specific manner were similarly correlated at the gene expression level, but up to 38% of the global proteomic variation between strains comprised proteins expressed in both strains but associated with strain-specific protein isoforms. Collectively, >65 hypothetical genes were transcriptionally or proteomically expressed, supporting their bona fide biological presence. Our results provide, for the first time, an integrated framework for classifying the repertoire of natural variations existing at distinct molecular levels for an important human pathogen.

CD44-SLC1A2 Gene Fusions in Gastric Cancer

One partner of a fusion gene found in gastric cancer, CD44-SLC1A2, may contribute to the tumor’s abnormal metabolism. Bad Drivers Steer Scientists Toward New Drug Targets It’s ironic, but cancer cells are notoriously bad at cell division, losing bits and rearranging chunks of the genome in the process. One result of this chaos is the birth of chimeric genes, wherein one gene segment gets erroneously fused to part of another, sometimes forming peculiar hybrid proteins that contribute to the cancer cell phenotype. For example, the fused aberrant BCR-ABL gene drives chronic myelogenous leukemia and has proven to be a vulnerable target for therapy. Gene fusions in solid cancers are not so easy to spot, but have been located in prostate and small cell lung cancers. Now, Tao and her co-workers have documented a fusion gene that forms in a small percentage of gastric tumor cells and may contribute to the development of cancer. The authors analyzed copy number variations of genes in more than 100 primary gastric tumors and 27 established gastric tumor cell lines and pinpointed a common breakpoint in three and one, respectively. The resulting chimeric gene fused most of the coding region of SLC1A2/EAAT2 (which encodes a glutamate transporter) to what is probably the strong transcriptional promoter of its neighboring gene, CD44, likely the result of a chromosome inversion. The fusion gene generated a truncated SLC1A2 protein in the original tumors and in a new group of gastric cancers created by the authors through overexpression of the fusion gene in normal gastric cells. But an abnormal protein that lives in tumor cells can be an innocent bystander. So, the authors asked whether the truncated SLC1A2 contributes to gastric cancer development, and their evidence suggested that the answer is yes. Cells in which shortened SLC1A2 expression was silenced with small interfering RNA were less proficient at dividing and invading soft substrates—hallmarks of cancer cells—and overexpression of the pruned protein enhanced these traits. Consistent with the function of SLC1A2 as a transporter of glutamate, the amino acid—which can act as a growth regulator—existed in higher concentrations in gastric cancer cells and cell lines than in normal cells. And in a final set of incriminating evidence, tumor cells that sported the CD44-SLC1A2 fusion gene had higher amounts of SLC1A2 than did wild-type cells, suggesting that this aberrant protein may trigger a pro-oncogenic phenotype. Most other genes that are fused in cancers encode kinase enzymes or transcriptional regulatory proteins. The implication of an overexpressed metabolism-related gene in some gastric tumors may define a new class of cancer-driving genes, although the protein could also augment other cancer-promoting genetic aberrations. The utility of this fusion gene as a drug target or prognostic tool will require more studies, but this particular mistake made by a dividing cancer cell may act as a GPS that directs researchers down a new therapeutic avenue for gastric cancer. Fusion genes are chimeric genes formed in cancers through genomic aberrations such as translocations, amplifications, and rearrangements. To identify fusion genes in gastric cancer, we analyzed regions of chromosomal imbalance in a cohort of 106 primary gastric cancers and 27 cell lines derived from gastric cancers. Multiple samples exhibited genomic breakpoints in the 5′ region of SLC1A2/EAAT2, a gene encoding a glutamate transporter. Analysis of a breakpoint-positive SNU16 cell line revealed expression of a CD44-SLC1A2 fusion transcript caused by a paracentric chromosomal inversion, which was predicted to produce a truncated but functional SLC1A2 protein. In primary tumors, CD44-SLC1A2 gene fusions were detected in 1 to 2% of gastric cancers, but not in adjacent matched normal gastric tissues. When we specifically silenced CD44-SLC1A2, cellular proliferation, invasion, and anchorage-independent growth were significantly reduced. Conversely, CD44-SLC1A2 overexpression in gastric cells stimulated these pro-oncogenic traits. CD44-SLC1A2 silencing caused significant reductions in intracellular glutamate concentrations and sensitized SNU16 cells to cisplatin, a commonly used chemotherapeutic agent in gastric cancer. We conclude that fusion of the SLC1A2 gene coding region to CD44 regulatory elements likely causes SLC1A2 transcriptional dysregulation, because tumors expressing high SLC1A2 levels also tended to be CD44-SLC1A2–positive. CD44-SLC1A2 may represent a class of gene fusions in cancers that establish a pro-oncogenic metabolic milieu favoring tumor growth and survival.

A Densely Interconnected Genome-Wide Network of MicroRNAs and Oncogenic Pathways Revealed Using Gene Expression Signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA–pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes (“hubs”), most nodes in the miRNA–pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA–pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.

The molecular pathogenesis of STAT3-driven gastric tumourigenesis in mice is independent of IL-17

Chronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by their ability to produce interleukin (IL)‐17A (Th17), is associated with a host of inflammatory responses, including gastritis. However, the role of these Th17 cells in the pathogenesis of gastric cancer is less clear. To formally address this, we employed gp130F/F mice, which spontaneously develop gastric inflammation‐associated tumours akin to human intestinal‐type gastric cancer. At the molecular level, these tumours demonstrate hyper‐activation of the latent transcription factor signal transducer and activator of transcription (STAT)3 via the IL‐6 cytokine family member, IL‐11. In gp130F/F mice, the generation of Th17 cells, as well as the gastric expression of IL‐17a and other Th17‐related factors (Rorγt, IL‐23), were augmented compared to wild‐type gp130+/+ mice. Consistent with a role for IL‐6 and STAT3 in regulating IL‐17A, increased Th17 generation and gastric expression of Th17‐related factors in gp130F/F mice were reduced to wild‐type levels in gp130F/F:Stat3−/+ mice displaying normalized STAT3 activity, and also in gp130F/F:IL‐6−/− mice. Importantly, genetic ablation of IL‐17A in gp130F/F:IL‐17a−/− mice did not suppress the initiation and growth of gastric tumours. Furthermore, IL‐17A and RORC gene expression was strongly increased in human gastric biopsies from patients with gastritis, but not gastric cancer. Collectively, our data suggest that increased expression of Th17‐related factors does not correlate with the molecular pathogenesis of gastric tumourigenesis. # Copyright