Francesca Saccani

Gefitinib Inhibits Invasive Phenotype and Epithelial-Mesenchymal Transition in Drug-Resistant NSCLC Cells with MET Amplification

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.

Trastuzumab emtansine is active on HER-2 overexpressing NSCLC cell lines and overcomes gefitinib resistance.

BackgroundHER-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.MethodsAntiproliferative 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.ResultsIn 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.ConclusionsOur 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.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

BackgroundThe 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.ResultsIn 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.ConclusionOur 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.

Dual-Acting Drugs: an in vitro Study of Nonimidazole Histamine H3 Receptor Antagonists Combining Anticholinesterase Activity

Dual‐acting compounds that combine H3 antagonism with anticholinesterase properties are currently emerging as a novel and promising therapeutic approach in the treatment of multifactorial disorders primarily characterized by cholinergic deficits such as Alzheimers disease. A series of novel nonimidazole H3 ligands was developed from the chemical manipulation of 1,1′‐octa‐, ‐nona‐, and ‐decamethylene‐bis‐piperidines—H3 antagonists that had been the subject of previous investigations. These compounds were evaluated for in vitro binding affinity, antagonistic potency, and selectivity at rodent and human histamine H3 receptors, inhibitory activity at rat brain cholinesterase, and in vivo CNS access and cholinomimetic effects. Within the present series, the tetrahydroaminoacridine hybrid 18 stands out as one of the most attractive molecules, synergistically combining nanomolar and selective H3 antagonism with remarkable anticholinesterase activity. From this original starting point, it is hoped that future investigations will lead to dual‐acting compounds that can selectively enhance central cholinergic neurotransmission and thus facilitate the treatment of cognitive disorders.

Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

Inhibition of PI3K Pathway Reduces Invasiveness and Epithelial-to-Mesenchymal Transition in Squamous Lung Cancer Cell Lines Harboring PIK3CA Gene Alterations

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA–mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients. Mol Cancer Ther; 14(8); 1916–27. ©2015 AACR.

Suppression of inflammatory events associated to intestinal ischemia–reperfusion by 5-HT1A blockade in mice

Intestinal ischemia and reperfusion (I/R) is a potentially life-threatening disease, ensuing from various clinical conditions. Experimentally, either protective or detrimental roles have been attributed to 5-HT in the functional and morphological injury caused by mesenteric I/R. Recently, we proved the involvement of 5-HT2A receptors in the intestinal dysmotility and leukocyte recruitment induced by 45min occlusion of the superior mesenteric artery (SMA) followed by 24h reperfusion in mice. Starting from these premises, the aim of our present work was to investigate the role played by endogenous 5-HT in the same experimental model where 45min SMA clamping was followed by 5h reflow. To this end, we first observed that ischemic preconditioning before I/R injury (IPC+I/R) reverted the increase in 5-HT tissue content and in inflammatory parameters induced by I/R in mice. Second, the effects produced by intravenous administration of 5-HT1A ligands (partial agonist buspirone 10mgkg(-1), antagonist WAY100135 0.5-5mgkg(-1)), 5-HT2A antagonist sarpogrelate (10mgkg(-1)), 5-HT3 antagonist alosetron (0.1mgkg(-1)), 5-HT4 antagonist GR125487 (5mgkg(-1)) and 5-HT re-uptake inhibitor fluoxetine (10mgkg(-1)) on I/R-induced inflammatory response were investigated in I/R mice and compared to those obtained in sham-operated animals (S). Our results confirmed the significant role played by 5-HT2A receptors not only in the late but also in the early I/R-induced microcirculatory dysfunction and showed that blockade of 5-HT1A receptors protected against the intestinal leukocyte recruitment, plasma extravasation and reactive oxygen species formation triggered by SMA occlusion and reflow. The ability of α7 nicotinic receptor (α7nAchR) antagonist methyllycaconitine (5mgkg(-1)) to counteract the beneficial action provided by buspirone on I/R-induced neutrophil infiltration suggests that the anti-inflammatory effect produced by 5-HT1A receptor antagonism could be partly ascribed to the indirect activation of α7nAch receptors.

Lung mesenchymal cells function as an inductive microenvironment for human lung cancer propagating cells

OBJECTIVES The aim of the present study was to characterize the biological properties and in vivo tumourigenic potential of mesenchymal cells (MCs) obtained from non-small-cell lung cancer (NSCLC) samples. METHODS NSCLC samples (53 adenocarcinomas and 24 squamous-cell carcinomas) surgically removed from 46 males and 31 females were processed to identify mesenchymal cells from human lung cancer (hLc-MCs). hLc-MCs were separated from neoplastic epithelial cells, expanded and extensively characterized in vitro. Subsequently, female BALB/c nude mice were subcutaneously injected with either 10(6) or 2.5 × 10(6) Calu-3 (human adenocarcinoma cell line able to reproducibly induce xenografted tumours) alone or in combination with equal doses of hLc-MCs. Control animals were injected with the two doses of hLc-MCs only. RESULTS Primary cultures of hLc-MCs were obtained from >80% of NSCLC specimens. The typical MCs immunophenotype was documented by the expression of CD90, CD105, CD73, CD13 and CD44 at fluorescence-activated cell sorting analysis. CD45, CD14, CD34 and epithelial antigens were negative while CD117 (c-kit) and CD133 (prominin) were partially expressed. Interestingly, nuclear transcription factors octamer-binding transcription factor 3/4 and sex determining region Y-box 2 involved in stemness, thyroid transcription factor 1 in bronchoalveolar commitment, and ETS1 in carcinogenesis, were expressed in hLc-MCs isolated from NSCLC. Specific conditioned media and cocultures confirmed the supportive role of hLc-MCs for cancer cells. In vivo experiments showed that at both doses Calu-3 xenografts doubled in size when hLc-MCs were coinjected. Cell tracking in xenografted tumours, by immunofluorescence combined with fluorescence in situ hybridization analysis, documented hX-chromosome-labelled, Calu-3-derived cytokeratin-positive adenocarcinoma structures surrounded by hLc-MCs. CONCLUSIONS Tumour-propagating cells require the inductive interaction of resident mesenchymal cells to foster lung cancer development.

Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted and antiplatelet/antiphlogistic activities evaluation.

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug.

Dual Role of Endogenous Serotonin in 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis

Background and Aims: Changes in gut serotonin (5-HT) content have been described in Inflammatory Bowel Disease (IBD) and in different experimental models of colitis: the critical role of this monoamine in the pathogenesis of chronic gastrointestinal inflammation is gradually emerging. Aim of the present study was to evaluate the contribution of endogenous 5-HT through the activation of its specific receptor subtypes to the local and systemic inflammatory responses in an experimental model of IBD. Materials and Methods: Colitis was induced by intrarectal 2,4,6-TriNitroBenzene Sulfonic acid in mice subacutely treated with selective antagonists of 5-HT1A (WAY100135), 5-HT2A (Ketanserin), 5-HT3 (Ondansetron), 5-HT4 (GR125487), 5-HT7 (SB269970) receptors and with 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin. Results: Blockade of 5-HT1A receptors worsened TNBS-induced local and systemic neutrophil recruitment while 5-HT1A agonist delayed and mitigated the severity of colitis, counteracting the increase in colonic 5-HT content. On the contrary, blockade of 5-HT2A receptors improved global health conditions, reduced colonic morphological alterations, down-regulated neutrophil recruitment, inflammatory cytokines levels and colonic apoptosis. Antagonism of 5-HT3, 5-HT4, and 5-HT7 receptor sites did not remarkably affect the progression and outcome of the pathology or only slightly improved it. Conclusion: The prevailing deleterious contribution given by endogenous 5-HT to inflammation in TNBS-induced colitis is seemingly mediated by 5-HT2A and, to a lesser extent, by 5-HT4 receptors and coexists with the weak beneficial effect elicited by 5-HT1A stimulation. These findings suggest how only a selective interference with 5-HT pro-inflammatory actions may represent an additional potential therapeutic option for intestinal inflammatory disorders.