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Expression Data from Gastrointestinal Stromal Tumor (GIST) Cell Lines

ABSTRACT: Activating mutations in either KIT or PDGFRA are present in approximately 90% of gastrointestinal stromal tumors (GISTs). Although treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients, the beneficial effect is not durable. Here, we report that ligands from the FGF family reduced the effectiveness of imatinib in GIST cells, and FGF2 and FGFR1 are highly expressed in all primary GIST samples examined. The combination of KIT and FGFR inhibition showed increased growth inhibition in imatinib-sensitive GIST cell lines in the presence or absence of added FGF2 in vitro, and delayed tumor regrowth in vivo. In addition, inhibition of mitogen-activated protein kinase (MAPK) signaling by imatinib was not sustained in GIST cells. An extracellular signal-regulated kinase (ERK) rebound occurred through activation of FGF signaling, and was repressed by FGFR1 inhibition. Downregultation of Sprouty proteins played a role in the imatinib-induced feedback activation of FGF signaling in GIST cells. We used micorarrays to quantify the gene expression levels in GIST cell lines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE64762 | GEO | 2015/01/08

SECONDARY ACCESSION(S): PRJNA271809

REPOSITORIES: GEO

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Project description:GISTs are the most common mesenchymal tumors and they display neuromuscular features. Gain-of-function mutations in KIT or PDGFRA are the initiating event in most of the cases and KIT oncogenic signaling is essential throughout all the disease. Interestingly, KIT oncogenic signaling is driven by PI3K/mTOR and RAS/MAPK pathways regardless of KIT primary or secondary mutations. Hence, dissection of KIT-downstream pathways may result in the identification of novel KIT critical effectors and a new opportunity to overcome disease heterogeneity of resistance mechanisms in GIST. Using human clinically representative GIST cell models, we undertook pharmacological and functional screenings such as Cell titer glo, kinase activation, apoptosis induction and proliferation to identify key signaling nodes within PI3K/mTOR and RAS/MAPK pathways. In vitro and in vivo validations were undertaken to model novel therapeutic strategies. Transcriptomic analysis (RNAseq) found essential genes co-regulated by both pathways. Functional studies, such as immunofluorescence, kinase activation, flow cytometry and proliferation were performed on a viral gene overexpression and knock-down models to elucidate their regulation and potential role in GIST biology. PI3K/mTOR and MEK1/2 are the most essential KIT-downstream mediators. Single node inhibition did not yield sustained antiproliferative and apoptotic effect, but simultaneous intermittent ablation was synergistic in vitro and in vivo, supporting the critical role of these two pathways.Transcriptomic analyses underscored FBXO32 (a SCF E3 ubiquitin-ligase and the main effector of muscular atrophy) as the most differentially expressed gene co-regulated by PI3K/mTOR and RAS/MAPK pathways. Functional studies demonstrated that FBXO32 is transcriptionally activated by FOXO3a which, in turn, is regulated by PI3K/mTOR and RAS/MAPK pathways. Notoriously, FBXO32 proved to have a pro-survival role in GIST cells. Microarray studies hinted a participation of FBXO32 in cell cycle arrest, which has been previously described as an adaptive pro-survival mechanism in GIST cells. This mechanism was further confirmed by functional studies. Remarkably, FBXO32 was expressed upon KIT inhibition in a panel of GIST cell lines, regardless the mutational status of KIT, and in a gene set database from GIST patients treated with first-line treatment, imatinib. PI3K/mTOR and MEK1/2 are the critical mediators of KIT oncogenic signaling in GIST, and their intermittent co-inhibition is an effective and well-tolerated therapeutic strategy to treat GIST tumors, independently of their KIT mutational status. FBXO32 emerges as a common mediator of KIT-downstream PI3K/mTOR and RAS/MAPK pathways with a critical pro-survival role in GIST participating in cell cycle arrest as pro-survival mechanism.

Project description:Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC. Examination of mRNA levels in DMSO and gefitinib-resistant cultures of HCC4006 and HCC827. Each group has two replicates.

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Expression Data from Gastrointestinal Stromal Tumor (GIST) Cell Lines

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