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The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

While PI3K/AKT/mTOR pathway is altered in a variety of cancers including non small cell lung cancer, abnormalities in this pathway are more common in squamous cell lung carcinoma than in adenocarcinoma of the lung. Moreover, aberrant activation of PI3K/AKT/mTOR pathway is one of the mechanisms of acquired resistance to EGFR-TK inhibitors in patients with adenocarcinoma carrying EGFR activating mutations. Several inhibitors of the PI3K pathway are undergoing evaluation in preclinical and clinical studies. These include pan and selective inhibitors of PI3K, AKT inhibitors, rapamycin and rapalogs for mTOR inhibition, dual mTORC1-mTORC2 inhibitors and dual PI3K-mTOR inhibitors. This review focuses on recent preclinical and clinical data on the efficacy of PI3K pathway inhibitors in NSCLC either as monotherapy approach or in combination with chemotherapy or with drugs that target other signaling transduction pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: HER-2 represents a relatively new therapeutic target for non small cell lung cancer (NSCLC) patients. The incidence for reported HER-2 overexpression/amplification/mutations ranges from 2 to 20% in NSCLC. Moreover, HER-2 amplification is a potential mechanism of resistance to tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) (about 10% of cases). T-DM1, trastuzumab emtansine is an antibody-drug conjugate composed by the monoclonal antibody trastuzumab and the microtubule polymerization inhibitor DM1. The activity of T-DM1 has been studied in breast cancer but the role of T-DM1 in lung cancer remains unexplored., Methods: Antiproliferative and proapoptotic effects of T-DM1 have been investigated in different NSCLC cell lines by MTT, crystal violet staining, morphological study and Western blotting. HER-2 expression and cell cycle were evaluated by flow cytometry and Western blotting. Antibody dependent cell cytotoxicity (ADCC) was measured with a CytoTox assay. Xenografted mice model has been generated using a NSCLC cell line to evaluate the effect of T-DM1 on tumor growth. Moreover, a morphometric and immunohistochemical analysis of tumor xenografts was conducted., Results: In this study we investigated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation and in gefitinib resistant HER-2 overexpressing PC9/HER2cl1 cell clone. T-DM1 efficiently inhibited proliferation with arrest in G2-M phase and induced cell death by apoptosis in cells with a significant level of surface expression of HER-2. Antibody-dependent cytotoxicity assay documented that T-DM1 maintained the same activity of trastuzumab. Our data also suggest that targeting HER-2 with T-DM1 potentially overcomes gefitinib resistance. In addition a correlation between cell density/tumor size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model., Conclusions: Our results indicate that targeting HER-2 with T-DM1 may offer a new therapeutic approach in HER-2 over-expressing lung cancers including those resistant to EGFR TKIs.

Background: [18F]fluorodeoxyglucose (FDG)-PET is being evaluated as a tool for the early detection of response to various targeted agents in solid tumors. The aim of this study was to evaluate the predictive value of PET response after 2 days of erlotinib in unselected pretreated patients with stage IV NSCLC., Patients and Methods: FDG-PET/CT scans were conducted at baseline and after 2 days of erlotinib, with a CT evaluation performed at baseline and after 45-60 days of therapy. PET responses were evaluated by quantitative changes on SUVmax tumor/non-tumor ratio and classified according to EORTC criteria. PET responses were compared with RECIST responses and related to progression-free (PFS) and overall (OS) survival. Erlotinib effects on glucose uptake were also studied in a panel of NSCLC cell lines., Results: Fifty-three patients were enrolled. At 2 days of erlotinib, 20 (38 %) patients showed partial metabolic response (PMR), 25 (47 %) had stable metabolic disease (SMD) and 8 (15 %) had progressive metabolic disease (PMD). All patients with PMD had confirmed RECIST progression at 45-60 days. Patients with early PMR and SMD had significantly longer PFS (p < 0.001 and p = 0.001, respectively) and OS (p = 0.001 for both) than PMD patients., Conclusions: FDG-PET assessment after 2 days of erlotinib could be useful to identify early resistant patients and to predict survival in unselected NSCLC pretreated population.

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.

Background: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC., Results: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone., Conclusion: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation., (2010 Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor (EGFR) is a validated target for therapy in non-small cell lung cancer (NSCLC). Most patients, however, either do not benefit or develop resistance to specific inhibitors of the EGFR tyrosine kinase activity, such as gefitinib or erlotinib. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation and survival pathways and has been associated with resistance to EGFR tyrosine kinase inhibitors. In this study, we assessed the effects of combining the mTOR inhibitor everolimus (RAD001) with gefitinib on a panel of NSCLC cell lines characterized by gefitinib resistance and able to maintain S6K phosphorylation after gefitinib treatment. Everolimus plus gefitinib induced a significant decrease in the activation of MAPK and mTOR signaling pathways downstream of EGFR and resulted in a growth-inhibitory effect rather than in an enhancement of cell death. A synergistic effect was observed in those cell lines characterized by high proliferative index and low doubling time. These data suggest that treatment with everolimus and gefitinib might be of value in the treatment of selected NSCLC patients that exhibit high tumor proliferative activity.

Introduction: Epidermal growth factor receptor (EGFR) gene intron 1 contains a polymorphic single sequence dinucleotide repeat (CA)n whose length has been found to inversely correlate with transcriptional activity. This study was designed to assess the role of (CA)n polymorphism in predicting the outcome of gefitinib treatment in advanced non-small cell lung cancer (NSCLC)., Methods: Blood and tumor tissue from 58 patients with advanced NSCLC submitted to gefitinib were collected. EGFR intron 1 gene polymorphism, along with EGFR gene mutation, gene copy number and immunohistochemistry expression were determined. Moreover, a panel of lung cancer cell lines characterized for EGFR intron 1 polymorphism was also studied., Results: EGFR intron 1 polymorphism showed a statistically significant correlation with the gefitinib response (response rate 25 versus 0%, for patients with a (CA)16 and with a (CA)else genotype, respectively; p = 0.044). Patients with a (CA)16 genotype had a longer survival compared with those with a (CA)else genotype (11.4 versus 4.8 months, respectively; p = 0.037). In addition, cell lines lacking the (CA)16 allele showed a statistically significant higher IC50 compared with cell lines bearing at least one (CA)16 allele (p = 0.003)., Conclusions: This study supports a potential role of EGFR intron 1 polymorphism in predicting the outcome of gefitinib treatment in advanced NSCLC.

In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non-small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21(WAF1) proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 mumol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors.