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Das K.,National University of Singapore | Gunasegaran B.,National University of Singapore | Tan I.B.,National Cancer Center Singapore | Tan I.B.,Genome Institute of Singapore | And 5 more authors.
Cancer Letters | Year: 2014

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others ("mutual exclusivity"). Here, we sought to validate these findings for three RTK/RAS components (. FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained. © 2014 Elsevier Ireland Ltd.


Grabsch H.I.,University of Leeds | Grabsch H.I.,Leeds Teaching Hospitals NHS Trust | Tan P.,National University of Singapore | Tan P.,Cellular and Molecular Research
Digestive Surgery | Year: 2013

The development of gastric adenocarcinoma is a complex multistep process involving multiple genetic alterations. Based on pathology, four different macroscopic types and at least two major histological types, intestinal and diffuse, have been described. Most gastric cancer (GC) show genetic instability, either microsatellite instability or chromosomal instability, which is considered an early event in gastric carcinogenesis. Molecular studies of alterations of single genes have provided evidence that intestinal and diffuse type GC evolve via different genetic pathways. Recent results from high-throughput whole-genome expression or copy number studies have demonstrated extensive genetic diversity between cases and within individual GC. Sets of commonly up- or downregulated microRNAs have been identified in GC and might be useful in the near future to identify pathways of GC progression. Results from detailed molecular and/or pathological GC studies, although promising, still have limited clinical utility in predicting survival and stratifying GC patients for appropriate treatment. Copyright © 2013 S. Karger AG, Basel.


Zhu Y.,National University of Singapore | Das K.,National University of Singapore | Wu J.,Cellular and Molecular Research | Lee M.H.,Cellular and Molecular Research | And 3 more authors.
Oncogene | Year: 2014

Histone deacetylase inhibitors (HDACis) are a promising class of anticancer epigenetic drugs, however, molecular factors influencing the responses of individual tumors to HDACi therapies remain obscure. Here, we sought to identify genes associated with HDACi resistance in gastric cancer. Treating a panel of 17 gastric cancer cell lines with multiple HDACi compounds (trichostatin A, SAHA and MS275), we identified two distinct classes of lines exhibiting either HDACi sensitivity or resistance. Genomic comparisons between the sensitive and resistant classes using two independent microarray platforms identified RNH1, encoding a ribonuclease inhibitor, as a gene highly expressed in HDACi-resistant lines. Using genetic knockdown and overexpression assays, we show that RNH1 is both necessary and sufficient to induce HDACi resistance, and that RNH1 is likely to mediate this resistance through the dampening of HDACi-induced reactive oxygen species (ROS) in cancer cells. The discovery of RNH1 as a regulator of HDACi resistance in gastric cancer highlights a functional role for ROS induction in the cellular effects of this important drug class. © 2014 Macmillan Publishers Limited.


Das K.,National University of Singapore | Tan P.,National University of Singapore | Tan P.,Genome Institute of Singapore | Tan P.,Cellular and Molecular Research
Clinical Genetics | Year: 2013

Aneuploidy or alteration in chromosome numbers is a characteristic feature in cancer that is generally a consequence of defective chromosome segregation during cell division. Molecular cytogenetic analyses have conferred substantial evidence with regards to the chromosomal architectures in cancer. Most importantly, the fluorescence in situ hybridization (FISH) technique that plays a leading role in diagnostic pathology for its single-cell analysis has provided crucial information regarding genomic variations in malignant cells. Further development of molecular cytogenetic methodologies such as chromosome specific FISH karyotyping and comparative genomic hybridization have also helped in the detection of cryptic genetic changes in cancer. But, the recent advancement of high throughput sequencing technologies have provided a more comprehensive genomic analyses resulting in novel chromosome rearrangements, somatic mutations as well as identification of fusion genes leading to new therapeutic targets. This review highlights the application of early molecular cytogenetics and the recent high throughput genomic approaches in characterizing various cancers and their invaluable support in cancer therapeutics. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.


Chia N.-Y.,National University of Singapore | Tan P.,National University of Singapore | Tan P.,Agency for Science, Technology and Research Singapore | Tan P.,Cellular and Molecular Research
Annals of Oncology | Year: 2016

Gastric cancer (GC), a heterogeneous disease characterized by epidemiologic and histopathologic differences across countries, is a leading cause of cancer-related death. Treatment of GC patients is currently suboptimal due to patients being commonly treated in a uniform fashion irrespective of disease subtype. With the advent of next-generation sequencing and other genomic technologies, GCs are now being investigated in great detail at the molecular level. High-throughput technologies now allow a comprehensive study of genomic and epigenomic alterations associated with GC. Gene mutations, chromosomal aberrations, differential gene expression and epigenetic alterations are some of the genetic/epigenetic influences on GC pathogenesis. In addition, integrative analyses of molecular profiling data have led to the identification of key dysregulated pathways and importantly, the establishment of GC molecular classifiers. Recently, The Cancer Genome Atlas (TCGA) network proposed a four subtype classification scheme for GC based on the underlying tumor molecular biology of each subtype. This landmark study, together with other studies, has expanded our understanding on the characteristics of GC at the molecular level. Such knowledge may improve the medical management of GC in the future.


Tan P.,National University of Singapore | Tan P.,Agency for Science, Technology and Research Singapore | Tan P.,Cellular and Molecular Research | Yeoh K.-G.,National University of Singapore
Gastroenterology | Year: 2015

Gastric cancer (GC) is globally the fifth most common cancer and third leading cause of cancer death. A complex disease arising from the interaction of environmental and host-associated factors, key contributors to GC's high mortality include its silent nature, late clinical presentation, and underlying biological and genetic heterogeneity. Achieving a detailed molecular understanding of the various genomic aberrations associated with GC will be critical to improving patient outcomes. The recent years has seen considerable progress in deciphering the genomic landscape of GC, identifying new molecular components such as ARID1A and RHOA, cellular pathways, and tissue populations associated with gastric malignancy and progression. The Cancer Genome Atlas (TCGA) project is a landmark in the molecular characterization of GC. Key challenges for the future will involve the translation of these molecular findings to clinical utility, by enabling novel strategies for early GC detection, and precision therapies for individual GC patients. © 2015 AGA Institute.


Cheng L.L.,National University of Singapore | Itahana Y.,National University of Singapore | Lei Z.D.,National University of Singapore | Chia N.-Y.,National University of Singapore | And 15 more authors.
Clinical Cancer Research | Year: 2012

Purpose: DZNep (3-deazaneplanocin A) depletes EZH2, a critical component of polycomb repressive complex 2 (PRC2), which is frequently deregulated in cancer. Despite exhibiting promising anticancer activity, the specific genetic determinants underlying DZNep responsiveness in cancer cells remain largely unknown. We sought to determine molecular factors influencing DZNep response in gastric cancer. Experimental Design: Phenotypic effects of DZNep were evaluated in a panel of gastric cancer cell lines. Sensitive lines were molecularly interrogated to identify potential predictors of DZNep responsiveness. The functional importance of candidate predictors was evaluated using short hairpin RNA (shRNA) and siRNA technologies. Results: DZNep depleted PRC2 pathway components in almost all gastric cancer lines, however, only a subset of lines exhibited growth inhibition upon treatment. TP53 genomic status was significantly associated with DZNep cellular responsiveness, with TP53 wild-type (WT) lines being more sensitive (P < 0.001). In TP53-WT lines, DZNep stabilized p53 by reducing ubiquitin conjugation through USP10 upregulation, resulting in activation of canonical p53 target genes. TP53 knockdown in TP53-WT lines attenuated DZNep sensitivity and p53 target activation, showing the functional importance of an intact p53 pathway in regulating DZNep cellular sensitivity. In primary human gastric cancers, EZH2 expression was negatively correlated with p53 pathway activation, suggesting that higher levels of EZH2 may repress p53 activity. Conclusion: Our results highlight an important role for TP53 genomic status in influencing DZNep response in gastric cancer. Clinical trials evaluating EZH2-targeting agents such as DZNep should consider stratifying patients with gastric cancer by their TP53 genomic status. ©2012 AACR.


Ivanova T.,National University of Singapore | Zouridis H.,National University of Singapore | Wu Y.,Cellular and Molecular Research | Cheng L.L.,National University of Singapore | And 15 more authors.
Gut | Year: 2013

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.


Busuttil R.A.,Cancer Genetics and Genomics Laboratory | Busuttil R.A.,Royal Melbourne Hospital | George J.,Cancer Genetics and Genomics Laboratory | Tothill R.W.,Molecular Genomics Core Facility | And 14 more authors.
Clinical Cancer Research | Year: 2014

Purpose: Gene-expression profiling has revolutionized the way we think about cancer and confers the ability to observe the synchronous expression of thousands of genes. The use of putative genome-level expression profiles has allowed biologists to observe the complex interactions of genes that constitute recognized biologic pathways. We used gastric and ovarian datasets to identify gene-expression signatures and determine any functional significance. Experimental Design: Microarray data of 94-tumor and 45-benign samples derived from patients with gastric cancer were interrogated using Hierarchical Ordered Partitioning and Collapsing Hybrid analysis identifying clusters of coexpressed genes. Clusters were further characterized with respect to biologic significance, gene ontology, and ability to discriminate between normal and tumor tissue. Tumor tissues were separated into epithelial and stromal compartments and immunohistochemical analysis performed to further elucidate specific cell lineages expressing genes contained in the signature. Results: We identified a "stromal-response" expression signature, highly enriched for inflammatory, extracellular matrix, cytokine, and growth factor proteins. The majority of genes in the signature are expressed in the tumor-Associated stroma but were absent in associated premalignant conditions. In gastric cancer, this module almost perfectly differentiates tumor from nonmalignant gastric tissue and hence can be regarded as a highly tumor-specific gene-expression signature. Conclusions: We show that these genes are consistently coexpressed across a range of independent gastric datasets as well as other cancer types suggesting a conserved functional role in cancer. In addition, we show that this signature can be a surrogate marker for M2 macrophage activity and has significant prognostic implications in gastric and ovarian high-grade serous cancer. Clin Cancer Res © 2014 American Association for Cancer Research.


Miao L.,Nanjing University | Wang Y.,Nanjing University | Xia H.,Cellular and Molecular Research | Yao C.,Nanjing Medical University | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

Lung cancer is the leading cause of cancer related death worldwide and the prognosis is still poor with 5-year survival of approximately 15%. Metastasis is the leading cause of death by cancer. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Here, we identified that SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1) is a novel transforming growth factor-β1 (TGF-β) target gene that regulates lung cancer cell EMT. TGF-β has been reported as a major inductor of EMT. We observed that the expression of SPOCK1 in lung cancer tumor tissues is significantly higher than matched normal lung tissues. Moreover, the expression of SPOCK1 was also significantly higher in metastasis tumor tissues than non-metastasis tumor tissues. Levels of SPOCK1 mRNA were increased among patients with shorter disease-free survival times, indicating the potential role of SPOCK1 in lung cancer progression and metastasis. Silencing SPOCK1 expression with endoribonuclease-prepared small interfering RNA (esiRNA) in lung cells inhibits lung cancer cell growth, colony formation and invasion in vitro. Interestingly, ectopic expression of SPOCK1 in epithelial lung cancer cells induced EMT with increased expression of the mesenchymal marker Vimentin and decreased expression of epithelial marker E-cadherin. We also found that the expression of SPOCK1 was increased under treatment of TGF-β, indicating that SPOCK1 is a novel downstream target of TGF-β. Taken together, our study showed that SPOCK1 is a novel metastasis related biomarker in lung cancer and may be new diagnostic and therapeutic target for lung cancer. © 2013 Elsevier Inc. All rights reserved.

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