MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI

Mitochondrial Genome Profile in Demyelinating Diseases

Multiple sclerosis and neuromyelitis optica are chronic inflammatory diseases of the central nervous system. These pathologies share clinical similarities with Leber hereditary optic neuropathy, which is primarily due to mutations of mitochondrial DNA. Mitochondrial genetic variations may influence susceptibility to develop multiple sclerosis and neuromyelitis optica. In order to explore the possible correlation between mitochondrial DNA specific patterns and demyelinating diseases involving central nervous system, mitochondrial DNA from 13 patients with relapsing-remitting multiple sclerosis, 4 patients with neuromyelitis optica, 1 patient with myelitis, 2 patient with optic neuritis, and 7 healthy controls were analyzed by sequencing the full length 16 Kbs of the mitochondrial DNA genome. Common variants presence in healthy controls and patients showing no clinical impact on diseases development were not further explored. Analyzing 414 patient specific variants, six nonsense mutations, causing early stop-codon formation, and nine previously described variants, associated with demyelinating/degenerative disease of central nervous system were identified. Some of these variants are linked to disease development through known and previously described mechanisms. We report for the first time other truncating mutations leading to incomplete proteins involved in Oxidative Phosporilation complexes and we speculate their role in demyelinating diseases developmen

Characterization of epithelial-mesenchymal transition intermediate/hybrid phenotypes associated to resistance to EGFR inhibitors in non-small cell lung cancer cell lines

Increasing evidence points to a key role played by epithelial-mesenchymal transition (EMT) in cancer progression and drug resistance. In this study, we used wet and in silico approaches to investigate whether EMT phenotypes are associated to resistance to target therapy in a non-small cell lung cancer model system harboring activating mutations of the epidermal growth factor receptor. The combination of different analysis techniques allowed us to describe intermediate/hybrid and complete EMT phenotypes respectively in HCC827- and HCC4006-derived drug-resistant human cancer cell lines. Interestingly, intermediate/hybrid EMT phenotypes, a collective cell migration and increased stem-like ability associate to resistance to the epidermal growth factor receptor inhibitor, erlotinib, in HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis

Characterization of an EMT intermediate phenotype associated to resistance to EGFR inhibitors in non small cell lung cancer cell lines

Epithelial-mesenchymal transition (EMT) plays a critical role in development and adult tissue homeostasis as well as in cancer progression. Lung cancer is the leading cause of cancer related deaths worldwide with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Approximately 10-30% of NSCLC patients have activating mutations in the epidermal growth factor receptor (EGFR) gene. Targeting EGFR in these patients has shown initial and significant success in the clinic although acquired resistance invariably develops. Increasing evidence points to a key role played by EMT in cancer progression and drug resistance. In this study, we used wet and in silico approaches to investigate whether the EMT phenotype was associated to Erlotinib (ERL)-resistance in a NSCLC cellular model system. The combination of different analysis techniques allowed us to describe intermediate and complete EMT phenotypes respectively in HCC827- and HCC4006-derived ERL-resistant cell lines. Interestingly, EMT intermediate phenotypes and collective cell migration features associated to resistance to target therapy in all ERL-resistant HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small interesting EMT-related gene list, which should be otherwise overlooked by standard stand-alone methods for gene expression analysis

Drug effects on metabolic profiles of Schistosoma mansoni adult male parasites detected by 1H-NMR spectroscopy.

Schistosomiasis is one of the most devastating neglected tropical parasitic diseases caused by trematodes of the genus Schistosoma. Praziquantel (PZQ) is today the only drug used in humans and animals for the treatment of schistosomiasis but unfortunately it is poorly effective on larval and juvenile stages of the parasite. Therefore, it is urgent the discovery of new drug targets and compounds. We have recently showed that the anti-anginal drug perhexiline maleate (PHX) is very active on multiple developmental stages of Schistosoma mansoni in vitro. It is well known that PHX impacts the lipid metabolism in mammals, but the final target on schistosomes still remains unknown. The aim of this study was to evaluate the ability of 1H nuclear magnetic resonance (NMR) spectroscopy in revealing metabolic perturbations due to PHX treatment of S. mansoni adult male worms. The effects of PHX were compared with the ones induced by vehicle and gambogic acid, in order to detect different metabolic profiles and specificity of the PHX action. Remarkably a list of metabolites associated to PHX-treatment was identified with enrichment in several connected metabolic pathways including also the Kennedy pathway mediating the glycerophospholipid metabolism. Our study represents the first 1H-NMR metabolomic approach to characterize the response of S. mansoni to drug treatment. The obtained "metabolic fingerprint" associated to PHX treatment could represent a strategy for displaying cellular metabolic changes for any given drug and to compare compounds targeting similar or distinct biochemical pathways

INTEGRATED ANALYSIS OF DNA COPY NUMBER AND GENE EXPRESSION DATA IN LUNG CANCER MODELS OF RESISTANCE TO TARGETED THERAPY

Tyrosine Kinase inhibitors (TKIs) constitute the most promising frontier of cancer treatment. However, the development of resistance mechanisms often makes the tumour insensitive to TKI-targeted therapy. The present work aims to identify genes and pathways involved in TKI-resistance. To this end, we developed cellular models of NSCLC and designed cellular, molecular and bioinformatics analyses. Preliminary functional enrichment analysis of genes, found consistently altered in resistant cell lines, indicates their involvement in key biological processes

Modulation of cAMP/cGMP signaling as prevention of congenital heart defects in Pde2A deficient embryos: a matter of oxidative stress

Abstract Phosphodiesterase 2A (Pde2A) is a dual-specific PDE that breaks down both cAMP and cGMP cyclic nucleotides. We recently highlighted a direct relationship between Pde2A impairment, a consequent increase of cAMP, and the appearance of mouse congenital heart defects (CHDs). Here we aimed to characterize the pathways involved in the development of CHDs and in their prevention by pharmacological approaches targeting cAMP and cGMP signaling. Transcriptome analysis revealed a modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, transcription, and oxidative stress in hearts from Pde2A −/− embryos. Metoprolol and H89 pharmacological administration prevented heart dilatation and hypertabeculation in Pde2A −/− embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Amelioration of cardiac defects was also observed by using the antioxidant NAC, indicating oxidative stress as one of the molecular mechanisms underpinning the CHDs. In addition, Sildenafil treatment recovered cardiac defects suggesting the requirement of cAMP/cGMP nucleotides balance for the correct heart development

Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.

<p>Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.</p

Analysis of the <i>EGFR</i> gene in the RA2 ERL-resistant cell line.

<p>A) Analysis of <i>EGFR</i> exon 19 nucleotides sequence. The pherogram of the parental cell line with peaks corresponding to the <i>EGFR</i> mutated sequence (ΔE746-A750) and the pherogram of the RA2 resistant cells with peaks corresponding to the mutated and wild type (WT) EGFR nucleotides sequence are shown. B) qPCR analysis. Relative <i>EGFR</i> gene copy number (GCN) in genomic DNA, normalized to the <i>Rnase P</i> gene, is expressed relative to the levels in parental cell lines (P) set as 1 (mean ± SD of triplicate determinations). Results are representative of those obtained from 2 independent analysis.</p