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B. Solomon

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    MS27 - Mechanisms of Acquired Resistance to Targeted Therapy (ID 44)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Medical Oncology
    • Presentations: 4
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      MS27.1 - Resistance to EGFR TKIs (ID 589)

      10:30 - 12:00  |  Author(s): S. Yano

      • Abstract
      • Presentation
      • Slides

      Abstract
      Dramatic response has been achieved by EGFR inhibitors in lung cancer expressing EGFR activating mutations. However, cancer cells show either intrinsic or acquire resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib, and cause disease progression. Known major mechanisms for acquired resistance to EGFR-TKI include T790M gatekeeper mutation in the EGFR gene and activation of bypass survival signal via receptors other than EGFR. The latter mechanism can involve Met gene amplification and ligand-triggered receptor activation as well. For example, HGF, the ligand of a tyrosine kinase receptor Met, activates Met and the downstream PI3K/Akt pathway and triggers resistance to EGFR inhibitors in EGFR mutant cancer cells. Moreover, common polymorphism in BIM gene was recently reported to be found specifically in East Asian and confer intrinsic resistance to EGFR-TKI. These accumulating evidences suggest that mechanisms of EGFR-TKI resistance are complicated and heterogenous even in one individual. In this session, the resistance mechanisms will be introduced and therapeutic strategies will be discussed.

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      MS27.2 - Resistance to ALK Inhibitors (ID 590)

      10:30 - 12:00  |  Author(s): R.C. Doebele

      • Abstract
      • Presentation
      • Slides

      Abstract
      ALK gene rearrangements occur in approximately 5% of lung adenocarcinomas and less frequently in other histologic subtypes. Crizotinib is currently the standard of care for ALK+ NSCLC (1). Treatment with crizotinib leads to remarkable objective response rates, durable progression free-survival and superiority over standard second line chemotherapy. Unfortunately, patients eventually experience disease progression on crizotinib. Disease progression may occur primarily in the central nervous system (CNS) alone, likely because of poor penetration of crizotinib into this space, or simultaneously with systemic progression outside of the CNS (2). Systemic disease progression likely occurs via cellular resistance that occurs by multiple mechanisms, which have been observed in vitro or in patient tumor samples derived following progression on crizotinib. Mutations in the kinase domain of ALK currently account for approximately 25% of observed drug resistance (3-5). Resistance mutations occur at the ‘gatekeeper’ position, L1196M, but multiple other ALK kinase domain mutations have been observed in patient samples or in cell lines with induced drug resistance. Indeed, in the first case of published crizotinib resistance, the tumor harbored two separate ALK mutations (6). This pattern stands in contrast to EGFR mutant lung cancer, where the observed rate of resistance mutations is 50-60% and the majority of resistance mutations occur at the gatekeeper position, T790M (7). Also in contrast to EGFR mutant lung cancer, resistance mutations in ALK do not appear to confer a fitness disadvantage to the tumor cell (3). Mutations induce resistance by allowing persistent ALK signaling despite the presence of crizotinib. Copy number gain of the ALK fusion has also been observed in both cell line models and in patient tumor samples following crizotinib resistance (3, 4). It is hypothesized that that a fraction of ALK fusion proteins are not inhibited by clinically achievable doses of crizotinib and that increased expression may allow sufficient downstream signaling for tumor cell survival. Collectively, we have termed resistance mutations and copy number gain as ‘ALK-dominant’ mechanisms of resistance, because the tumor cells are still predicted to be ‘addicted’ to ALK signaling. Multiple mechanisms of ‘bypass’ signaling have been observed in both cell line models and post-progression tumor biopsies. These include activating mutations in EGFR and KRAS, and ligand dependent activation of EGFR or KIT (3, 4). In some cases the ALK gene rearrangement is no longer observed in post-crizotinib biopsy, also suggestive of an alternate or bypass signaling pathway. We have termed these bypass signaling mechanisms as ‘ALK non-dominant’ resistance as the tumor cells may no longer be dependent on ALK signaling. Approximately 50% of patients have been shown to harbor each class (dominant vs. non-dominant) resistance and this may have implications for post-progression therapy in these patients (1). Next generation ALK inhibitors such as LDK378, AP26113, and CH/RO5424802 which potently inhibit the ALK kinase and have activity against many of the resistance mutations in vitro, may be the favored post-progression therapy for patients with ALK dominant resistance. All of these drugs have also demonstrated anecdotal evidence of activity in the CNS. Although not ALK-specific, HSP90 inhibitors, such as ganetespib, IPI-504, and AUY-922, can inhibit ALK signaling by decreasing proper folding of the chimeric ALK fusion proteins and may also overcome ALK dominant resistance (8). ALK non-dominant resistance may require dual inhibition of ALK and a bypass signaling pathway to overcome resistance and the selection of drugs would be dependent on the alternate signaling pathway. Currently, no post-crizotinib therapies are approved in ALK+ lung cancer, but systemic chemotherapy remains as useful treatment strategy and some evidence suggests that pemetrexed-based regimens may be an optimal initial choice in the absence of a clinical trial (9). References 1. Camidge DR, Doebele RC. Treating ALK-positive lung cancer--early successes and future challenges. Nat Rev Clin Oncol. 2012;9:268-77. 2. Costa DB, Kobayashi S, Pandya SS, Yeo WL, Shen Z, Tan W, et al. CSF concentration of the anaplastic lymphoma kinase inhibitor crizotinib. J Clin Oncol. 2011;29:e443-5. 3. Doebele RC, Pilling AB, Aisner DL, Kutateladze TG, Le AT, Weickhardt AJ, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res. 2012;18:1472-82. 4. Katayama R, Shaw AT, Khan TM, Mino-Kenudson M, Solomon BJ, Halmos B, et al. Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers. Sci Transl Med. 2012;4:120ra17. 5. Huang D, Kim DW, Kotsakis A, Deng S, Lira P, Ho SN, et al. Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment. Genomics. 2013. 6. Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T, et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med. 2010;363:1734-9. 7. Yu HA, Arcila ME, Rekhtman N, Sima CS, Zakowski MF, Pao W, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19:2240-7. 8. Normant E, Paez G, West KA, Lim AR, Slocum KL, Tunkey C, et al. The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models. Oncogene. 2011;30:2581-6. 9. Camidge DR, Kono SA, Lu X, Okuyama S, Baron AE, Oton AB, et al. Anaplastic lymphoma kinase gene rearrangements in non-small cell lung cancer are associated with prolonged progression-free survival on pemetrexed. J Thorac Oncol. 2011;6:774-80.

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      MS27.3 - Clinical Definition of TKI Resistance and How to Overcome It (ID 591)

      10:30 - 12:00  |  Author(s): P.A. Jänne

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      MS27.4 - Trial Design as it Applies to Resistance (ID 592)

      10:30 - 12:00  |  Author(s): O. Mir, J. Soria

      • Abstract
      • Presentation
      • Slides

      Abstract
      Lung cancer therapy in the metastatic setting is invariably characterized by the emergence of resistance. This applies both to conventional chemotherapy and molecular targeted agents. This presentation will focus on the biological background and subsequent trial designs as it applies to resistance regarding molecular targeted agents (MTA). In the clinical setting when a patient develops progressive disease while exposed to a MTA some key basic considerations should be taken into account before considering acquired biological resistance. Compliance of the patient to the prescribed therapy is an obvious one. Indeed chronic exposure to tyrosine kinase inhibitors (TKI) exposes the patient to chronic toxicities. Some of these toxicities can become intolerable for the patient, thus leading to patient-driven therapeutic breaks that are frequently not specified to the clinician. This is notably true for week-end breaks as well holiday breaks. Honest dialogue with the patient is necessary to identify compliance issues and pharmacological (PK) dosage in the blood of the relevant TKI is an approach to be discussed. Further pharmacokinetic interactions represent a real issue with TKI: many TKI are metabolized by the liver and can have CYP liabilities (ie erlotinib, gefitinib are susbtrated of CYP3A4). Inter-individual PK variability due to genotypic background (polymorphisms in genes encoding drug metabolizing enzymes), inflammatory and nutritional status, may result in suboptimal drug concentrations and decreased efficacy. Significant decrease in drug exposure over time have also been described for and may result in secondary progression despite preserved sensitivity to these agents, with a potential role for subsequent dose escalation. From a biological perspective, 3 main mechanisms have been described to explain resistance to TKI: a) mutation/amplification in the target (ie T790M for EGFR or ALK L1196M), bypass mechanism (ie Met amplification, PI3K mutation, HER 3 activation,) and growth survival/apoptosis resistance (ie loss of BIM, SCLC, EMT). Stronger kinase inhibitors and use of combinations appear as potential solutions to deal with these resistance mechanisms. Some of the potential designs to address acquired resistance include: a) upfront combinations, b) use of third generation inhibitors or mutant-specific inhibitors, c) rolling trials (ie the use of sequential TKI therapies), c) alternating therapeutic approaches (TKI followed by chemotherapy or TKI followed by immunotherapy).

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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 8
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      O12.01 - Prospective isolation of human lung stem/progenitor cells and their role in the initiation of lung cancer (ID 1716)

      10:30 - 12:00  |  Author(s): C.E. Weeden, K. Viitaniemi, G. Ramm, J. Ciciulla, I. Bertoncello, M. Asselin-Labat

      • Abstract
      • Presentation
      • Slides

      Background
      Cells of origin of cancers acquire the first genetic aberration(s) that lead to tumourigenesis. An understanding of the cell of origin in different subtypes of lung cancer could allow earlier detection of malignancies and more effective treatment. Stem or progenitor cells are likely tumour initiating cells due to both their longevity, allowing for accumulation of genetic lesions, and their capacity for renewal. This study aims to isolate human lung progenitor subpopulations based on their differential expression of cell surface markers to evaluate their role as the cell of origin of the different subtypes of lung cancer.

      Methods
      Single cell suspensions were generated from adjacent normal tissue of patients undergoing lung cancer resection. Epithelial cells were immediately separated based on their expression of cell surface markers by fluorescence activated cell sorting (FACS). The progenitor cell capacity of epithelial cell subsets was then assessed using an in vitro colony forming assay. Subsets with progenitor activity were analysed for their expression of differentiated lung cell markers both before and after colony formation.

      Results
      We have identified a sort strategy that allows for enrichment of basal cells, Clara cells, type I and II pneumocytes from fresh human lung tissue as shown by qPCR and electron microscopy data. The basal cell and type II pneumocyte subpopulations consistently formed colonies in vitro - cell types that have previously shown progenitor activity in the mouse lung. The Clara cell and type I cell compartment did not consistently form colonies. Basal and type II cells formed phenotypically distinct colonies when cultured in a three-dimension matrix that expressed different lung specific markers (p63, SP-C…) as shown by RT-PCR analysis and immunofluorescence staining.

      Conclusion
      This study has demonstrated the prospective isolation of four epithelial cell subsets from fresh human lung tissue for the first time and confirmed the progenitor activity of basal cells and type II cells in the human lung. We are currently comparing the genetic profile of these human lung progenitor cells with the genetic profile of molecular subtypes of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using next generation sequencing. Future studies aim to transform lung progenitor cells with genetic alterations common in NSCLC and SCLC to further establish their role as cells of origin of these cancers.

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      O12.02 - Pathway Analysis of Gene Expression Profiles that Distinguish Persistent from Regressive Bronchial Dysplasia Indicate Synergistic Role for Polo-Like Kinase 1 (PLK1) and Epoxide Hydrolase 3 (EPHX3) in Malignant Progression. (ID 3334)

      10:30 - 12:00  |  Author(s): D.T. Merrick, M.G. Edwards, W. Franklin, M. Sugita, Y. Miller, M. Friedman, L. Dwyer-Nield, M. Tennis, K. Choo, G. Hickey, T. Kennedy, A. Van Bokhoven, L. Heasley, F.R. Hirsch, P.A. Bunn, M. Geraci, R.L. Keith, R. Nemenoff

      • Abstract
      • Presentation
      • Slides

      Background
      160,000 Americans die from lung cancer annually and the prognosis for invasive lung cancer is poor. Prevention of cancer represents an approach with high potential for significant reduction in mortality. Bronchial dysplasia (BD) is a precursor lesion of squamous cell carcinoma (SCC) of the lung, and persistent BDs represent a high risk subset of these lesions. Genomic instability is an important process underlying malignant progression. Gene expression microarray analyses were used to identify potential mediators of genomic instability in persistent BD and study their activity in these high risk lesions. Two genes, PLK1, which abrogates G2-M checkpoint DNA damage repair, and EPHX3, which converts tobacco smoke derived pro-carcinogens to mutagens, were selected for further analysis.

      Methods
      Sixty-three frozen baseline biopsies were classified into persistent/progressive BD, regressive BD , progressive non-dysplasia and stable non-dysplasia groups according to the presence or absence of BD on follow-up biopsies. H&E staining was performed on frozen sections to confirm histology, and RNA was harvested for global gene expression microarray analysis. Intergroup comparisons employed ANOVA statistical analysis with a false discovery rate of 10% to identify differentially expressed genes associated with persistence and gene expression alterations related to baseline histology used Spearman correlation coefficient cutoff of r= +/- 0.5. A pathway analysis (Ingenuity) using the persistence related genelist was performed to identify active pathways associated with persistence of BD. Validational studies were performed by quantitative RT-PCR in cell lines established from persistent and regressive bronchial sites. Inhibitors of persistence associated enzymes were used in tissue culture based assays of cellular proliferation.

      Results
      Gene expression analyses support the unique biological nature of persistent BD. Intergroup comparisons showed significant numbers of differentially expressed genes only in the comparisons of persistent BD with regressive BD (318 genes) or stable non-dysplasia (6254 genes). 831 genes showed differential expression associated with increasing baseline dysplastic grade regardless of outcome. While approximately half of these genes also differentiated persistent from regressive BD, the presence of numerous persistence related genes that are independent of histology further substantiates the unique high risk nature of persistent BD. A pathway analysis revealed “mitotic roles of PLKs” as having the most significant association with persistence. Quantitative RT-PCR using cultures of 8 persistent BD and 6 regressive BD validated increased expression in persistent BD of PLK1 (2.77X, p=0.002) and EPHX3 (2.36X, p=0.081). Using a classification of dysplastic specimens as high or low expressers of PLK1 and/or EPHX3 (high > mean), we found a significant direct relationship with increased level of outcome diagnosis score: low expression of both genes (2.58); high expression of only one gene (3.60); and high expression of both (5.06). The baseline diagnosis did not differ between groups. Culture of the SCC cell line H2009 with EPHX inhibitor revealed a non-significant trend toward decreased proliferation (80.4% vs untreated).

      Conclusion
      Gene expression data confirms the biologically distinct nature of persistent BD. PLK1 and EPHX3 overexpression demonstrate a cooperative effect in respect to increased outcome histology suggesting a potential role for these enzymes in persistence/progression of BD via promotion of genomic instability.

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      O12.03 - Pseudogenes as miRNA sponges in non-small cell lung cancer (ID 3455)

      10:30 - 12:00  |  Author(s): G.L. Stewart, K.S. Enfield, S. Lam, W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer is the most common cause of cancer death worldwide, with a five-year survival of less than 15%. This poor therapeutic outcome is largely due to complex molecular backgrounds as well as typically late stage at diagnosis, with most patients presenting with unresectable local tumours or metastatic disease. While mutations of driver genes is a well known mechanism of tumorigenesis, approximately half of all non-small cell lung cancer (NSCLC) tumours harbour no known actionable oncogenic drivers, emphasizing the need to explore alternative mechanisms. New sequencing technologies have allowed investigation of previously unexplored areas of the genome and revealed that several classes of non coding RNAs (ncRNAs), those with no protein product, are involved in tumourigenesis, emphasizing the need for further exploration and study. MicroRNAs (miRNAs) have emerged as major players in lung carcinogenesis, displaying both oncogenic and tumor suppressive functions through translational inhibition of genes containing miRNA target sequences. Pseudogenes are non-coding relatives of protein-coding genes that contain a high degree of sequence similarity with their parent genes, thus sharing many of the same miRNA target sequences. As a result, when overexpressed, a pseudogene can function as a miRNA "decoy" protecting its parent gene from miRNA-mediated translational inhibition. DNA copy number (CN) alterations (gain of oncogenes/loss of tumour suppressors), is a major molecular mechanism driving cancer. Like protein coding genes, CN alterations can influence ncRNA expression levels, and several pseudogenes have been reported to be deregulated at the CN level in other cancer types. We hypothesize that pseudogenes of lung cancer-related genes are deregulated at the CN level in NSCLC.

      Methods
      Global CN profiles for 83 lung adenocarcinomas, and 12 squamous cell carcinomas, as well as paired adjacent non-malignant tissues were generated on the Affymetrix SNP 6.0 array. Frequencies of DNA CN alterations were assessed at candidate pseudogene loci (gain>2.3 copies, loss<1.7 copies). Candidate pseudogenes (1) have a parent gene that has been previously reported to play a role in cancer biology, (2) are expressed in human tissue, and (3) share at least one conserved miRNA binding site with its parent gene.

      Results
      Several pseudogenes for OCT4 (octamer-binding transcription factor 4), an early embryonic transcription factor, were found to be frequently gained (46.9-34.9%), and could protect OCT4 from miRNA-mediated translational inhibition. Additionally, pseudogenes for E2F3 (E2F Transcription Factor 3), a potent cell cycle regulator, as well as those for the well known lung cancer oncogene BRAF, were found to have high frequencies of CN alteration (36.1%, and 19.2%, respectively). These high frequencies of alteration suggest that these pseudogenes play an important role in NSCLC.

      Conclusion
      These results suggest that pseudogenes are clonally selected for at the DNA level, and pseudogene-mediated protection of oncogenic transcripts from miRNA-mediated translational inhibition may represent a novel mechanism of oncogenicity in NSCLC. Analyses of pseudogene expression and corresponding parent gene protein level in cell models will yield insight into how this class of ncRNA affects tumourigenesis, potentially leading to improvements in early detection, diagnosis, and treatment.

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      O12.04 - DISCUSSANT (ID 3896)

      10:30 - 12:00  |  Author(s): K. Sutherland

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      O12.05 - Defining the role of ZEB1 in the pathogenesis of non-small cell lung cancer (NSCLC) using immortalized human bronchial epithelial cells (HBECs) (ID 1139)

      10:30 - 12:00  |  Author(s): J.E. Larsen, J.K. Osborne, A. Augustyn, J.P. Sullivan, M. Sato, L. Girard, A.F. Gazdar, J.D. Minna

      • Abstract
      • Presentation
      • Slides

      Background
      To study the role of common lung cancer mutations in transforming lung epithelial cells in an appropriate cellular context we used cdk4/hTERT-immortalized normal HBECs. We developed an isogenic series of HBECs by introducing genetic manipulations representing common lung cancer mutations (such as p53, KRAS[V12], cMYC, and LKB1). This defined in vitro system allows characterization of specific tumorigenic contributions as well as identification of acquired changes, likely representing tumor acquired vulnerabilities and novel therapeutic targets (Mol Cancer Res 2013). One acquired change observed with oncogenic transformation of HBECs is a spontaneous epithelial-to-mesenchymal transition (EMT), an important biologic process in cancer. This study sought to characterize the role of EMT in driving tumorigenesis in HBECs and, in turn, lung cancer to identify novel therapeutic targets.

      Methods
      Genetic manipulations were introduced into cell lines using siRNA/shRNA or over-expression constructs. Tumorigenicity was measured using in vitro (anchorage-dependent and -independent colony formation, proliferation, migration and transwell Matrigel invasion assays) and in vivo (subcutaneous or intravenous injection into NOD/SCID mice) methods. Genome-wide mRNA expression data from five independent datasets was obtained either in-house using Illumina HumanHT-12v4 BeadChips or from publicly available databases.

      Results
      Analysis of EMT-promoting transcription factors in our isogenic series of oncogenically-manipulated HBECs found ZEB1 expression highly correlated with mesenchymal-like HBECs. Functional studies confirmed ZEB1 was a significant driver of tumorigenic phenotypes in both oncogenic HBECs and human lung cancer cell lines where loss of ZEB1 resulted in decreased colony formation, migration and invasion in vitro and subcutaneous tumor growth and intravenous colonization in vivo. A set of ZEB1-associated genes was identified from analyzing five independent mRNA microarray datasets comprising both cell lines and lung adenocarcinomas. From this gene set we found ZEB1 directly represses ESRP1 by binding to its promoter, which leads to increased mesenchymal splicing of the ESRP1 target CD44. The mesenchymal isoform of CD44, CD44s, conferred a CD44[hi] flow cytometry profile which, in turn, could be used to select for a highly tumorigenic subpopulation in partially transformed HBECs. To identify candidate ZEB1-activated targets we screened ZEB1-upregulated genes in a siRNA invasion assay. Several genes including PMP22 and CD70 could phenocopy ZEB1 where siRNA-mediated loss of expression led to decreased invasiveness in multiple NSCLC cell lines. CD70 (also called TNFSF7, tumor necrosis factor ligand superfamily member 7) may represent a prime therapeutic target for anti-metastatic growth in lung cancer. The ligand for CD27, it is involved in immune regulation, upregulated in some cancers and is being studied as a potential target for antibody therapeutics. Importantly, an anti-CD70 monoclonal antibody inhibited invasion of NSCLC cell lines comparably to siCD70 and siZEB1.

      Conclusion
      We demonstrate in vitro models of defined oncogenic HBEC transformation provide an invaluable tool to study lung cancer progression where EMT is an important mediator. ZEB1 is spontaneously expressed with malignant transformation of HBECs and is a significant driver of oncogenic progression in both HBECs and NSCLC cells. Identification of CD70 and PMP22 as downstream targets of ZEB1 may represent novel therapeutic targets for lung cancer.

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      O12.06 - Hedgehog/Gli Promotes Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer (ID 2091)

      10:30 - 12:00  |  Author(s): H. Li, D. Yue, T.H. Tseng, D.M. Jablons, T. Cheng, B. He, M.J. Mann

      • Abstract
      • Presentation
      • Slides

      Background
      A majority of non-small cell lung cancer (NSCLC) patients are diagnosed with metastatic phenotypes. Epithelial-to-Mesenchymal Transition (EMT), characterized by loss of epithelial markers, such as E-cadherin, is suggested to be involved in the metastatic process. In addition, aberrant activation of the Hedgehog-Gli(Hh/Gli) signaling pathway is implicated in various cancers, including NSCLC. We hypothesize that the Hh/Gli signaling pathway may regulate EMT in NSCLC, and inhibition of Hh/Gli pathway may provide a novel strategy to treat NSCLC and prevent metastasis.

      Methods
      Tumor tissues of 324 NSCLC patients were analyzed by immunohistochemistry for Gli and E-cadherin expression. Mechanistic studies were carried out in four NSCLC cell lines, A549, H1666, H2170 and H1703. Our lab has developed a novel small molecule Gli inhibitor (Gli-I )that effectively suppresses lung cancer in vitro and in vivo. Gli-I and a Smoothened inhibitor vismodegib were applied to suppress Hh/Gli signaling, while Hh protein was utilized to stimulate the pathway. Upon different treatments, EMT phenotypes were evaluated by wound healing assays and 3D cell invasion assays. Expression of EMT markers was measured by immunofluorescent staining and western blot at protein levels, as well as quantitative RT-PCR at mRNA levels.

      Results
      Our results demonstrated elevated Gli expression in 78% of NSCLC patient tissues. Gli expression was reversely correlated with E-Cadherin in patient tissues and culture cell lines. Inhibition of Hh signaling reduced cell migration and invasion, while stimulation of Hh signaling promoted EMT phenotypes. Specifically, Gli-I significantly suppressed cell proliferation, migration and invasion more effectively than vismodegib. Furthermore, mechanistic studied showed Hh/Gli signaling may regulate EMT through suppressing E-Cadherin.

      Conclusion
      Our results suggested that SHh/Gli signaling promotes cell proliferation and EMT, leading to NSCLC cell invasion and metastasis. Inhibition of Hh/Gli signaling by a novel Gli inhibitor Gli-I suppresses cell proliferation and invasion. Our novel Gli inhibitor holds the promise to provide an effective therapeutics to treat NSCLC and prevent metastasis.

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      O12.07 - Pulmonary fibroblasts increase EMT signaling and decrease sensitivity to chemotherapy in non-small cell lung cancer cells via TGF-beta and IL-6 signaling (ID 265)

      10:30 - 12:00  |  Author(s): Y. Shintani, A. Abulaiti, T. Kimura, S. Funaki, T. Nakagiri, M. Inoue, N. Sawabata, M. Minami, E. Morii, M. Okumura

      • Abstract
      • Presentation
      • Slides

      Background
      The tumor microenvironment is a key factor in tumor progression. A specific subset of stromal cells, termed cancer associated fibroblasts (CAFs), modulate the behavior of adjacent cancer cells by secreting various growth factors and cytokines. The purpose of this study was to clarify the roles of transforming growth factor (TGF)-β and interleukin (IL)-6 in the communication between CAFs and non-small-cell lung cancer (NSCLC) cells.

      Methods
      Fibroblasts obtained during surgical exploration were co-cultured with human lung adenocarcinoma cell lines. We defined fibroblasts obtained from tumors as CAFs and those from normal lung tissue as lung normal fibroblasts (LNFs). Immunohistochemistry was used to examine the fibroblast distribution, as well as TGF-β and IL-6 expression in 60 tumor specimens obtained from patients with NSCLC after undergoing induction chemotherapy or chemoradiotherapy (ITx).

      Results
      The expression levels of myofibroblast markers were higher in CAFs than LNFs after 5 passages in the absence of continuing interaction with carcinoma cells, and we used at least 2 pairs of those CAFs and LNFs in the following experiments. Conditioned medium (CM) from both types of fibroblasts induced epithelial mesenchymal transition (EMT) and acquisition of cancer stemness in lung cancer cells (A549 and NCI-H358), indicating it to be biologically active. Phenotypic changes of cancer cells by CM from CAFs were greater than those induced by CM from LNFs. These CAF-induced changes were inhibited by addition of the TGF-β inhibitor SB431542 or IL-6 receptor neutralizing antibody (IL6-R-Ab). The concentrations of TGF-β1 and IL-6 were higher in CM from CAFs as compared to that from LNFs. Subcutaneous co-injection of lung cancer cells and CAFs in mice enhanced tumor growth when compared with cancer cells alone, which was attenuated by administration of SB431542 or IL-6R-Ab. These findings suggested that CAFs may be more activated in our experimental system as compared to LNFs, and stimulate tumor progression via TGF-β and IL-6 signaling. In addition, decreased expression of epithelial markers and upregulation of mesenchymal markers were detected in surgically resected specimens after ITx as compared with biopsy specimens obtained before treatment. The disease-free survival rate of patients with EMT marker-positive tumors was significantly lower than that of those with EMT marker-negative tumors, indicating that EMT changes are associated with insensitivity to ITx. Furthermore, an increased diffuse distribution pattern of SMA-positive activated fibroblasts was significantly correlated with the expression of EMT markers. Also, though SMA-stained fibroblasts expressed IL-6 in the surgical specimens, TGF-β was expressed in cancer cells as well as CAFs after ITx. Together, our results suggest that tumor stromatic tissues including CAFs increase in response to ITx, while CAFs secrete TGF-β and IL-6, inducing EMT in cancer cells.

      Conclusion
      The TGF-β and IL-6 axis induces EMT and stimulates tumor progression, while TGF-β and IL-6 may play roles to contribute to communication between CAFs and NSCLC cells for tumor progression. Targeting CAFs as a therapeutic strategy against cancer is an intriguing concept that would benefit from further study.

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      O12.08 - DISCUSSANT (ID 3897)

      10:30 - 12:00  |  Author(s): J. Minna

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-002 - RLIP76 expression is prognostic and predictive of chemotherapy benefit in resected NSCLC (ID 247)

      09:30 - 16:30  |  Author(s): B. Solomon

      • Abstract

      Background
      Despite adjuvant chemotherapy improving overall survival of resected Stage II and III non-small cell lung cancer (NSCLC), acute and late toxicities of chemotherapy have highlighted the need to better predict which patients will benefit from treatment. RLIP76 is a ubiquitously expressed multi-functional transporter that is associated with cisplatin and vinorelbine resistance. Our aim was to analyse the prognostic and predictive value of RLIP76 expression in NSCLC.

      Methods
      We identified 367 NSCLC patients resected between 1996 and 2009. A tissue microarray was created and immunohistochemistry (IHC) performed with an anti-human RLIP76 rabbit polycloncal antibody (Millipore, Temecula, CA). The intensity (0-3) and proportion of tumour cells (0-100) with staining was scored. The product of RLIP76 intensity and proportion of tumour cells staining was calculated (range 0-300) and divided into high (>100) and no/low expression (≤100). RLIP76 expression was correlated with clinical features and patient outcome.

      Results
      IHC was available for 285 patients, 173(60.7%) of which were male. Number of patients according to stage I, II, III and IV was 150(52.6%), 83(29%), 44(15.4%) and 8(3%), respectively. RLIP76 was overexpressed in 117(41%) specimens. There was no relationship between RLIP76 expression and stage, histology, sex or age. High RLIP76 expression was associated with an improved prognosis on univariate (HR 0.62, CI 9.44-0.90, p=0.012,Figure 1) and multivariate analysis (HR 0.57, CI 0.39-0.83, p=0.003). Adjuvant chemotherapy was also associated with an improved survival on multivariate analysis (HR 0.52, CI 0.33-0.82, p=0.005). When stratifying by RLIP76 expression, the benefit of chemotherapy was limited to patients with no/low expression (HR =0.44, CI 0.24-0.8, p=0.008)(Figure 2). No benefit of chemotherapy was seen in patients with high RLIP76 expression (HR=0.75, CI 0.34-1.63, p=0.5). Figure 1 Figure 2

      Conclusion
      High RLIP76 expression is associated with an improved prognosis in resected NSCLC.Interestingly no/low RLIP76 expression may predict for benefit of adjuvant chemotherapy. Further studies are needed to confirm these results.

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    PL03 - Presidential Symposium Including Top Rated Abstracts (ID 85)

    • Event: WCLC 2013
    • Type: Plenary Session
    • Track:
    • Presentations: 1
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      PL03.08 - DISCUSSANT (ID 3887)

      08:15 - 09:45  |  Author(s): B. Solomon

      • Abstract
      • Slides

      Abstract not provided

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