Cross-testing of major molecular markers indicates distinct pathways of tumorigenesis in gastric adenocarcinomas and synchronous gastrointestinal stromal tumors
ABSTRACT: Small subtype of the gastrointestinal stromal tumor (micro-GIST, MG) is usually asymptomatic and is frequently found incidentally in association with gastric adenocarcinoma (GAC). The background of this coincidence is still an open question. This study comprehensively characterized nine MGs and GACs present in the same surgical specimen by cross-testing the markers of the major pathogenetic pathways of both tumor types. All of the MGs were immunohistochemically positive for CD117/KIT, CD34, and DOG1. DOG1 was also detected in four GACs. Four MGs carried mutations in c-KIT (exons 9, 11, and 13) and two cases in PDGFR? (exon 18). None of the GACs carried activating mutations in c-KIT or PDGFR?. MMR immunopanel identified one GAC as microsatellite unstable tumor. No EBV-positive tumor was found. According to the TCGA molecular classification, one GAC was categorized in the MSI subgroup, three GACs in the genomically stable subgroup, and the rest into the chromosomal instability subgroup. Although a common carcinogenic effect cannot be ruled out, our data suggest a distinct molecular background in the evolvement of the synchronous MGs and GACs. The presence of a MG in gastric resection specimens may be indicative of the development of synchronous malignant tumors in or outside the stomach.
Project description:BACKGROUND: Yin Yang 1 (YY1) is a transcription factor that regulates diverse biological processes and increasing recognized to have important roles in carcinogenesis. The function and clinical significance of YY1 in gastric adenocarcinoma (GAC) have not been elucidated. METHODS: In this study, the functional role of YY1 in gastric cancer was investigated by MTT proliferation assays, monolayer colony formation, cell cycle analysis, signaling pathway analysis, Western blot analysis and in vivo study through YY1 knockdown or overexpression. Immunohistochemical study with YY1 antibody was performed on tissue microarray consisting of 247 clinical GAC samples. The clinical correlation and prognosis significance were evaluated. RESULTS: YY1 expression was up-regulated in gastric cancer cell lines and primary gastric cancers. Knocking down YY1 by siYY1 inhibited cell growth, inducing G1 phase accumulation and apoptosis. Ectopic YY1 expression enhanced cell proliferation in vitro and in vivo. Knocking down YY1 in gastric cancer cells suppressed proliferation by inhibiting Wnt/?-catenin pathway, whereas its overexpression exerted oncogenic property by activating Wnt/?-catenin pathway. In primary GAC samples, YY1 nuclear expression correlated with shorter survival and predicted poor prognosis in early stage GACs. CONCLUSION: Our data demonstrated that YY1 contributes to gastric carcinogenesis in gastric cancer. In early stage GACs YY1 might serve as a poor prognostic marker and possibly as a potential therapeutic target.
Project description:Gastrointestinal stromal tumor (GIST) is the most common tumor of mesenchymal origin in gastrointestinal tract. Immunohistochemical (IHC) staining combined with a typical morphology is used for the diagnosis of GIST. Typically, IHC staining for v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (KIT) and discovered on GIST-1(DOG1) is positive in almost all GISTs. However, imatinib mesylate, a specific inhibitor of KIT tyrosine kinase, frequently involves changes in the morphology and IHC staining of GIST, impeding the diagnosis. Recently, in situ hybridization (ISH) for E26 transformation-specific sequence variant 1 (ETV1) mRNA was introduced as a useful marker to diagnose GIST.We report 2 cases of gastric GIST, which expressed unusual phenotypes after imatinib therapy.The first patient was found to have a gastric subepithelial tumor in gastroduodenoscopy done for regular checkup. In biopsy of the tumor, it showed homogenous spindle cells that were positive to standard IHC markers for GIST. The second patient visited our hospital because of a palpable mass in the abdomen. In abdominal computed tomography (CT), a tumor arising from the stomach was found. A needle biopsy was done and the patient was diagnosed of gastric GIST because the biopsy showed spindle cells positive to typical IHC markers for GIST. After imatinib treatment, in both patients, the resected tumors were composed of heterogeneous spindle cells negative to KIT, DOG1, and CD34 IHC staining, which was unusual for GIST. However, ISH for ETV1 mRNA done for both biopsied and resected tumors was positive, even after imatinib treatment. A molecular analysis found a mutation in exon 11 of KIT gene before and after imatinib therapy in both patients, confirming the diagnosis of GIST.Both patients took neoadjuvant imatinib treatment, and afterwards, underwent a surgical resection.The patients remain on imatinib treatment and no progression or recurrence has been detected to date.ISH for ETV1 mRNA is a useful technique in diagnosing GIST when IHC with KIT, DOG1, or CD34 fail to stain positive after imatinib therapy.
Project description:Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor ?(PDGFRA), which can be therapeutically targeted by tyrosine kinase inhibitors (TKI) such as imatinib. Despite long-lasting responses, most patients eventually progress after TKI therapy. The calcium-dependent chloride channel DOG1 (ANO1/TMEM16A), which is strongly and specifically expressed in GIST, is used as a diagnostic marker to differentiate GIST from other sarcomas. Here, we report that loss of DOG1 expression occurs together with loss of KIT expression in a subset of GIST resistant to KIT inhibitors, and we illustrate the functional role of DOG1 in tumor growth, KIT expression, and imatinib response. Although DOG1 is a crucial regulator of chloride balance in GIST cells, we found that RNAi-mediated silencing or pharmacologic inhibition of DOG1 did not alter cell growth or KIT signaling in vitro. In contrast, DOG1 silencing delayed the growth of GIST xenografts in vivo. Expression profiling of explanted tumors after DOG1 blockade revealed a strong upregulation in the expression of insulin-like growth factor-binding protein 5 (IGFBP5), a potent antiangiogenic factor implicated in tumor suppression. Similar results were obtained after selection of imatinib-resistant DOG1- and KIT-negative cells derived from parental DOG1 and KIT-positive GIST cells, where a 5,000-fold increase in IGFBP5 mRNA transcripts were documented. In summary, our findings establish the oncogenic activity of DOG1 in GIST involving modulation of IGF/IGF receptor signaling in the tumor microenvironment through the antiangiogenic factor IGFBP5.
Project description:We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene FLJ10261 (DOG1, discovered on GIST-1), encoding a hypothetical protein, was specifically expressed in GISTs. The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays. The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the KIT and PDGFRA mutation status was known. Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs. All seven GIST cases with a PDGFRA mutation were DOG1-positive, while most of these failed to react for KIT. The immunohistochemical findings were confirmed with in situ hybridization probes for DOG1, KIT, and PDGFRA. Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1. Only 4 of 438 non-GIST cases were immunoreactive for DOG1. DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors. Reactivity for DOG1 may aid in the diagnosis of GISTs, including PDGFRA mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase.
Project description:The GacS histidine kinase is the membrane sensor of the major upstream two-component system of the regulatory Gac/Rsm signal transduction pathway. This pathway governs the expression of a wide range of genes in pseudomonads and controls bacterial fitness and motility, tolerance to stress, biofilm formation, and virulence or plant protection. Despite the importance of these roles, the ligands binding to the sensor domain of GacS remain unknown, and their identification is an exciting challenge in this domain. At high population densities, the GacS signal triggers a switch from primary to secondary metabolism and a change in bacterial lifestyle. It has been suggested, based on these observations, that the GacS signal is a marker of the emergence of nutritional stress and competition. Biochemical investigations have yet to characterize the GacS signal fully. However, they portray this cue as a low-molecular weight, relatively simple and moderately apolar metabolite possibly resembling, but nevertheless different, from the aliphatic organic acids acting as quorum-sensing signaling molecules in other Proteobacteria. Significant progress in the development of metabolomic tools and new databases dedicated to <i>Pseudomonas</i> metabolism should help to unlock some of the last remaining secrets of GacS induction, making it possible to control the Gac/Rsm pathway.
Project description:Most gastrointestinal stromal tumors (GISTs) are driven by KIT or PDGFRA-activating mutations, but a small subset is associated with loss of function of the succinate dehydrogenase (SDH) complex of mitochondrial inner membrane proteins. This occurs by germline mutations of the SDH subunit genes and hitherto unknown mechanisms. SDH-deficient GISTs especially include pediatric GISTs and those associated with Carney triad (CT) or Carney-Stratakis syndromes (CSSs); the latter 2 also include paraganglioma as a component. SDH-deficient GISTs were identified in this study on the basis of immunohistochemical loss of succinate dehydrogenase subunit B (SDHB), which signals functional loss of the SDH complex. We found 66 SDH-deficient GISTs among 756 gastric GISTs, with an estimated frequency of 7.5% of unselected cases. Nearly, all gastric GISTs in patients <20 years, and a substantial percentage of those in patients <40 years, but only rare GISTs in older adults were SDH deficient. There was a female predominance of over 2:1. Two patients each had either pulmonary chondroma or paraganglioma (CT), but none of the examined cases had SDH germline mutations (CSS) or somatic KIT/PDGFRA or BRAF mutations. SDH-deficient GISTs were often multiple and typically showed plexiform muscularis propria involvement and epithelioid hypercellular morphology. They were consistently KIT-positive and DOG1/Ano 1-positive and almost always smooth muscle actin negative. Tumor size and mitotic activity varied, and the tumors were somewhat unpredictable with low mitotic rates developing metastases. Gastric recurrences occurred in 11 patients, and peritoneal and liver metastases occurred in 8 and 10 patients, respectively. Lymph node metastases were detected in 5 patients, but lymphovascular invasion was present in >50% of cases studied; these 2 were not related to adverse outcome. Seven patients died of disease, but many had long survivals, even with peritoneal or liver metastases. All 378 nongastric GISTs and 34 gastric non-GIST mesenchymal tumors were SDHB positive. SDH-deficient GISTs constitute a small subgroup of gastric GISTs; they usually occur in children and young adults, often have a chronic course similar to that of pediatric and CT GISTs, and have potential association with paraganglioma, necessitating long-term follow-up.
Project description:OBJECTIVE:Peritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC's development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets. DESIGN:We performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs. RESULTS:We identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of 'clock-like' mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: 'mesenchymal-like' and 'epithelial-like' with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive 'mesenchymal-like' subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-? as potential therapeutic immune targets. CONCLUSIONS:We have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.
Project description:Pseudomonas sp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS. Mutation of gac reduced both the length of the lag phase and the generation time. Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS. The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB. We conclude that in Pseudomonas sp. strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the "wild-type" population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained.
Project description:BACKGROUND:Overexpression of Galectin-3 (Gal-3), a ?-galactoside binding protein, has been noted in many tumour types but its functional significance and clinical utility in gastric adenocarcinoma (GAC) are not well known. METHODS:We studied 184 GAC patients characterised by histologic grade, sub-phenotypes (diffuse vs intestinal), and ethnicity (Asians vs North Americans). Immunohistochemistry was performed to assess the expression of Gal-3 in human GACs and we correlated it to the clinical outcomes. Cell proliferation, invasion, co-immunoprecipitation and kinase activity assays were done in genetically stable Gal-3 overexpressing GC cell lines and the parental counterparts to delineate the mechanisms of action and activity of inhibitors. RESULTS:Most patients were men, Asian, and had a poorly differentiated GAC. Gal-3 was over-expressed in poorly differentiated (P=0.002) tumours and also in diffuse sub-phenotype (P=0.02). Gal-3 overexpression was associated with shorter overall survival (OS; P=0.026) in all patients. Although, Gal-3 over-expression was not prognostic in the Asian cohort (P=0.337), it was highly prognostic in the North American cohort (P=0.001). In a multivariate analysis, Gal-3 (P=0.001) and N-stage (P=<0.001) were independently prognostic for shorter OS. Mechanistically, Gal-3 induced c-MYC expression through increasing RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype. YAP1/BET bromodomain inhibitors reduced Gal-3-mediated aggressive phenotypes in GAC cells. CONCLUSIONS:Gal-3 is an independent prognostic marker of shorter OS and a novel therapeutic target particularly in diffuse type GAC in North American patients.
Project description:Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.