We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p 5 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p 5 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. ' 2008 Wiley-Liss, Inc.Key words: MET; amplification; EGFR; gefitinib; lung cancer Acquired somatic alterations within the cancer genome such as mutations, gene amplification and deletions cause activation of oncogenes or inactivation of tumor suppressor genes, and form the genetic basis of malignancies.1 Cancer-specific somatic mutations have been identified in several protein kinases, 2-4 including mutations within the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). 5-7The finding of mutations in the tyrosine kinase domain of EGFR in lung adenocarcinoma is of great clinical interest, because many of these tumors are responsive to tyrosine kinase inhibitors (TKIs). 5,6,8 Although most EGFR mutant NSCLC initially respond to TKI, the vast majority of these tumors ultimately become resistant to the drug treatment. In approximately half of these cases, resistance is due to the occurrence of a second point mutation in EGFR exon 20 (T790M). [9][10][11][12] Recently Engelman et al. 13 reported that MET proto-oncogene (MET) amplification led to gefitinib resistance in lung cancers lacking the secondary T790M mutation. Bean et al.14 also reported that MET was amplified in lung tumors with acquired resistance more frequently than in untreated lung tumors and accounted for about 20% of cases of acquired resistance to TKIs. MET encodes a heterodimeric transmembrane receptor tyrosine kinase for the hepatocyte growth factor. ...

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This is the first report in vivo of the biological effects induced by a specific HSPG in the liver, with potential implications in both regenerative biology and molecular lipidology.

IntroductionSystemic chronic low-grade inflammation has been linked to insulin resistance (IR) and non-alcoholic steatohepatitis (NASH). NOD-like receptor protein 3 (NLRP3) inflammasome and its final product, interleukin (IL)-1β, exert detrimental effects on insulin sensitivity and promote liver inflammation in murine models. Evidence linking hepatic NLRP3 inflammasome, systemic IR and NASH has been scarcely explored in humans. Herein, we correlated the hepatic abundance of NLRP3 inflammasome components and IR and NASH in humans.Research design and methodsMetabolically healthy (MH) (n=11) and metabolically unhealthy (MUH) (metabolic syndrome, n=21, and type 2 diabetes, n=14) subjects were recruited. Insulin sensitivity (homeostatic model assessment of IR (HOMA-IR) and Oral Glucose Sensitivity (OGIS120)), glycemic (glycated hemoglobin), and lipid parameters were determined by standard methods. Plasma cytokines were quantified by Magpix. Hepatic NLRP3 inflammasome components were determined at the mRNA and protein levels by reverse transcription–quantitative PCR and western blot, respectively. Liver damage was assessed by histological analysis (Non-alcoholic Fatty Liver Disease Activity Score (NAS) and Steatosis, Inflammatory Activity, and Fibrosis (SAF) scores). IR and liver histopathology were correlated with NLRP3 inflammasome components as well as with liver and plasma IL-1β levels.ResultsBody Mass Index, waist circumference, and arterial hypertension frequency were significantly higher in MUH subjects. These patients also had increased high-sensitivity C reactive protein levels compared with MH subjects. No differences in the plasma levels of IL-1β nor the hepatic content of Nlrp3, apoptosis-associated speck-like (Asc), Caspase-1, and IL-1β were detected between MUH and MH individuals. MUH subjects had significantly higher NAS and SAF scores, indicating more severe liver damage. However, histological severity did not correlate with the hepatic content of NLRP3 inflammasome components nor IL-1β levels.ConclusionOur results suggest that NLRP3 inflammasome activation is linked neither to IR nor to the inflammatory status of the liver in MUH patients.

Objective: To study deregulation of the SWI/SNF chromatin remodeling complex in lung cancer tumors and cell lines with the emphasis on SMARCA2, SMARCA4 and ARID1A. Background: The DNA in each mammalian cell is compacted about 5000 fold into chromatin, and in the compacted state is unavailable for transcription. This compaction is controlled by three mechanisms: including the ATP-dependent chromatin remodeling complex SWI/SNF, consisting of 20 genes that code for about 12 protein subunits (some of which can substitute for each other). There are over 280 possible subunit permutations, influencing transcription, chromatin binding and remodeling, and tissue and gene specificity. Several subunit genes function as tumor suppressor genes (TSGs) including the interchangeable ATP catalytic components SMARCA2 (protein = BRM) and SMARCA4 (protein = BRG1) and the ARID1A (protein = BAF250a) accessory gene. The catalytic components are known to be inactivated in several cancers including lung, and ARID1A in ovarian cancer. Materials and Methods: Up to 58 cell lines and 60 non small cell lung cancer tumors arising in smokers and never smokers were studied. NextGen and Sanger sequencing of the SWI/SNF complex genes were performed on the cell lines. Western blots of nuclear extracts, methylation and qPCR were also performed on cell lines. Genome wide gene copy number (by SNP analyses) and microarray expression studies were performed on the tumors and cell lines. Immunostaining (for BRG1 and BAF250a) were performed on lung cancer microarrays. Results: Mutations rates in NSCLC lines were: SMARCA4 28% (mainly homozygous deletions), SMARCA2 0%, ARID1A 11% (mainly heterozygous point mutations). However, a marked decrease in nuclear protein expression was frequently present in NSCLC lines: BRG1 28%, BRM 28%, BAF250a 31%. Loss of BRG1, BRM, or BAF250a was present in 41% of the NSCLC lines, and 16% had loss of two or three. Combined analyses with microarray expression, immunostaining and qPCR studies of these three genes indicated frequent low expression in both NSCLC tumors and cell lines. In addition, DNA copy number by SNP analyses of NSCLC tumors indicated widespread loss of alleles of multiple members of the SWI/SNF complex. Evidence for epigenetic inactivation of one or more complex genes was present. Conclusions: Our data indicate frequent inactivation of one or more members of the SWI/SNF complex by a variety of mechanisms including deletions, mutations, epigenetic, transcriptional and translational control. The predicted downstream effects on transcription and histone regulation are likely to be widespread and possibly demonstrating cell type and gene specific effects. Although highly complicated, elucidation of the precise chromatin complex abnormalities, mechanisms and downstream effects indicate the possibility of multiple new therapeutic targets for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2011-LB-130