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A. Baird



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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.01-011 - Targeting the Urokinase Plasminogen Activator (uPA) System to overcome cisplatin resistance in NSCLC (ID 3347)

      11:50 - 12:04  |  Author(s): A. Baird

      • Abstract

      Background
      The urokinase plasminogen activator (uPA) system (uPAS) has been shown to play a significant multifunctional role in tumour progression including angiogenesis, adhesion and migration. Increased levels of urokinase plasminogen activator (uPA) and its receptor uPAR (CD87) strongly correlate with poor prognosis and a poor clinical outcome. It has been shown previously that a subpopulation of uPAR-positive cells in Small Cell Lung Cancer (SCLC) cell lines demonstrate significant drug resistance to traditional chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU) and etoposide. The uPAS is regulated by NF-κB which has been shown to be constitutively activated in several cancer types including non-small cell lung cancer (NSCLC). Furthermore, we have shown NF-κB to be involved in the development of resistance to cisplatin in NSCLC. This project focuses on determining the role of the uPA system in the invasive phenotype of cisplatin resistant NSCLC cells.

      Methods
      Expression of NF-κB (p65) in parent and resistant NSCLC cell lines was quantified by qPCR, western blot and high content screening (HCS). The expression profiles of NFκB target genes were quantified using a Roche custom NFκB RTPCR array. Gene “hits” with a fold change >2 between parent and cisplatin resistant cells were validated by qPCR analysis. The upregulation of the urokinase-type plasminogen activator (uPA) in cisplatin resistant cells was determined by western blot. The effect of uPA inhibition on cell migration and invasion, using the monoclonal anti-uPAR antibody ATN-658, is being determined using the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform.

      Results
      Gene expression data, from the NFκB target gene array identified a panel of genes including; PLAU (gene for uPA), RIPK and NLRP12 amongst others that were over-expressed in H460 cisplatin resistant cell lines compared to the isogenic parent cell line. uPA overexpression at the protein level was confirmed in a panel of cisplatin resistant cells compared to parent cell lines. The effect of ATN-658 on the inhibition of cell migration and invasion in cisplatin sensitive and resistant cell lines will be presented.

      Conclusion
      Overexpression of uPA across a panel of cisplatin resistant NSCLC cell lines highlights its significance as a marker of resistance. Targeting the uPA system may be exploited in cisplatin resistant NSCLC to inhibit cell migration and invasion.

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      P1.01-016 - Targeting NF-κB regulated pathways to overcome cisplatin resistance in non small cell lung cancer (ID 3270)

      13:00 - 13:14  |  Author(s): A. Baird

      • Abstract

      Background
      Cisplatin based doublet chemotherapy is the mainstay of non small cell lung cancer (NSCLC) treatment with an initial objective response rate of approximately 40-50%. However, intrinsic and acquired resistance to cisplatin constitutes a major clinical obstacle in lung cancer management and has yet to be fully understood. Inflammatory mediators may play an important role in the development of cisplatin resistance, such as those regulated by NF-κB. We have previously demonstrated that levels of NF-κB are increased in cisplatin resistant cells compared with sensitive Parent cells. We are currently assessing a number of NF-κB regulated targets in cisplatin resistant cell line models, using DHMEQ, a specific NF-κB inhibitor. DHMEQ treatment results in greater cell death in the cisplatin resistant cells compared with Parent. This study will elucidate the efficacy of DHMEQ to overcome cisplatin resistance and identify novel targets within the NF-κB pathway that may improve therapeutic strategies for NSCLC patients.

      Methods
      NF-κB downstream targets and signalling mediators were examined using NF-κB signalling and target pathway qPCR arrays (168 genes) in the H460 CisR and Parent cell line model. Targets identified are currently undergoing validation using qPCR and western blot. Biological and functional relevance of these targets in the development of cisplatin resistance will be examined further using DHMEQ and siRNA knockdown strategies. In addition, a xenograft murine model will be utilised to assess the effect of DHMEQ alone and in combination with cisplatin on tumour growth in vivo.

      Results
      Data from qPCR arrays have demonstrated that a number of genes are differentially regulated between the CisR and Parent cell lines. These include genes which activate the NF-κB signalling cascade (TLR3, TLR4), regulators of the pathway (BIRC3, CASP1), transcription factors (Myc) and NF-κB responsive genes (TNF, CXCL8). A number of these genes will be modulated to determine their involvement in cisplatin resistance. In addition, DHMEQ is being used in combination studies to determine, whether it can re-sensitise cells to cisplatin therapy. At present a dosing study is ongoing to establish the effect of DHMEQ on xenograft tumours derived from Parent and CisR cells. The results of which will be presented.

      Conclusion
      Preliminary data indicates that NF-κB and a number of its downstream targets are deregulated in cisplatin resistant cells. This project aims to validate the role of these NF-κB regulated genes in cisplatin resistant NSCLC. It will also determine whether DHMEQ may be a novel targeted agent for the treatment of NSCLC. The data obtained in this study will ultimately benefit patients by providing insights into novel druggable targets and new clinical strategies to re-sensitise patients to cisplatin therapy.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-055 - The RON (MST1R)/MSP pathway is a potential therapeutic target in malignant plural mesothelioma (ID 3250)

      09:30 - 09:30  |  Author(s): A. Baird

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer. Treatment options are limited and drug resistance is common. Thus, there is a need to identify novel therapeutic targets in this disease in order to improve treatment options and survival times. Macrophage stimulating protein (MSP) is the only ligand recognised to bind to the RON receptor (MST1R). RON is a member of the MET proto-oncogene family. The MSP-RON signalling pathway has been implicated in a variety of cellular functions such as macrophage morphogenesis and phagocytosis. De-regulation of this pathway has been linked to tumour progression and metastasis in a number of cancers. We have previously identified RON as frequently activated in MPM and high positivity for RON by IHC was an independent predictor of favourable prognosis.

      Methods
      A panel of mesothelioma cell lines were screened for the expression of MSP and RON at the mRNA (RT-PCR) and protein (Western blot) level. The effect of MSP, IMC-RON8 (a humanised IgG1 monoclonal antibody), LCRF004 (a small molecule inhibitor) and NRWHE (a small peptide) was examined in the H226 cell line using proliferation (BrdU ELISA), apoptosis (Multi-parameter apoptosis assay) and migration assays (xCELLigence). A phospho-kinase proteome profiler array was utilised to detect the downstream signalling pathways activated upon MSP stimulation. The expression of MSP and the macrophage marker, CD-68, was examined by IHC using MPM TMAs. Studies are ongoing to determine the effect of the LCRF004 compound in vivo using a xenograft murine model with the H226 cells.

      Results
      The mRNA and protein levels of RON and MSP were differentially expressed in a panel of MPM cell lines. Treatment with LCRF004 resulted in significantly decreased proliferation and increased apoptosis in the H226 cells. MSP was unable to rescue the cells from the effects of LCRF004. NRWHE and RON8 had little effect on either proliferation or apoptosis. All of the compounds examined inhibited the migration capacity of the H226 cells. The combination of LCRF004 and MSP produced a synergistic effect, showing greater inhibition of migration than either compound alone. However, MSP treatment resulted in the up-regulation of a number of phosphor-kinases including Akt, ERK and the Src family. Currently, a number of proteins identified in the array studies are undergoing validation. Results of an in vivo H226 murine model using the LCRF004 compound will be presented at the meeting.

      Conclusion
      From previous work performed in this laboratory, we have determined that high expression of RON in MPM is an independent predictor of favourable prognosis. IHC was performed on a TMA of MPM patient samples and high expression levels of MSP correlated with better survival. There was no association between CD68 staining and MSP, nor correlation of CD68 expression with survival. Targeting the RTK domain of the RON receptor with a small molecule inhibitor is an effective interventional strategy in MPM. The seemingly counter intuitive results obtained from the MPM TMA studies and the in vitro experimental data, may be RON isoform dependant. Additional studies are ongoing to further delineate the RON-MSP axis in MPM.

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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 3
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      P2.01-010 - A model of chronic inflammation affects the invasive capacity and induces several hallmarks of cancer in a normal bronchial epithelial cell line (ID 3260)

      09:30 - 09:30  |  Author(s): A. Baird

      • Abstract

      Background
      Acute inflammation is usually a rapid and self-limiting process, however it does not always resolve. This leads to the establishment of a chronic inflammatory state and provides the perfect environment for the transformation process. It has been estimated that approximately 25% of all malignancies are initiated or exacerbated by inflammation caused by infectious agents. Furthermore, inflammation is linked to all of the six hallmarks of cancer (evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis, increase in survival factors and invasion and metastasis). It is thought that inflammation may play a critical role in lung carcinogenesis given that individuals with inflammatory lung conditions have an increased risk of lung cancer development. This study focuses on inflammation as a contributory factor in non small cell lung cancer (NSCLC), concentrating primarily on the pathological involvement of the pro-inflammatory cytokines, TNF-α, IL-1β, and hypoxia.

      Methods
      A normal bronchial epithelial cell line (HBEC4) was modified to stably and functionally over-express TNF-α and IL-1β (alone or in combination) and were continuously cultured for three months under normoxic or hypoxic (Invivo~2~ Hypoxia Workstation - 0.5% oxygen) conditions. Subsequently a range of experimental assays were carried out to assess functional cell change. These included: transformation assay (soft agar), proliferation (BrdU ELISA), invasion (Cell Invasion assay), migration (Scratch assay) and angiogenesis (Endothelial tube formation). Gene expression changes were also assessed using qPCR Cancer PathwayFinder arrays.

      Results
      Although transformation was not evident by soft agar, the adhesion potential (ICAM and VCAM levels) of normoxic and hypoxic clones has amplified and the growth rate has increased over time. Under normoxia the IL-1β and the TNF-α/IL-1β clones displayed an increased invasive capacity compared with the empty vector control (p<0.05). Differences were also detected in the gene expression profile implicated in the pathways involved in the hallmarks of cancer - cell signalling (FOS, JUN), apoptosis (BAD, BAX, BCL2), angiogenesis (CXCL8), adhesion (ITGB3), invasion (S100A4, TIMP1) and cell cycle regulation (p53, c-myc). A number of these targets are currently undergoing validation.

      Conclusion
      This study provides a valuable isogenic cell line model in which to study the effect of prolonged chronic inflammation. Although the cells have not developed anchorage independent growth as assessed by soft agar, there are distinct indications that phenotypic changes occurred within the three month time frame. As pro-longed chronic exposure to inflammation is a pre-requisite for many disease states, these results warrant extended growth studies to further delineate the complex roles of TNF-α, IL-1β and hypoxia in the process of carcinogenesis. This will assist in the development of novel cancer target therapeutics and chemo-preventive agents in lung cancer.

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      P2.01-011 - The IL-23 family may be a novel therapeutic target in non small cell lung cancer (ID 3304)

      09:30 - 09:30  |  Author(s): A. Baird

      • Abstract

      Background
      IL-23A is a member of the IL-6 super-family and is composed of covalently bound IL-12p40 and IL-23p19 (IL-23A) subunits. The functional IL-23R is composed of the IL-23R and IL-12Rβ1 subunits and once activated can promote the STAT3 signalling pathway. IL-23A plays a key role in chronic inflammation through the promotion of IL-17 production by T helper 17 cells (Th 17). Stimulation of IL-23A associated pathways produces pro and anti oncogenic effects which are cell type dependant. We have previously identified IL-23A axis as deregulated in NSCLC and sought to examine the family in greater detail.

      Methods
      The expression of IL-23A/IL-23R was examined in a series of resected fresh frozen NSCLC tumours and in a panel of normal (HBEC) and NSCLC cell lines. Epigenetic regulation of IL-23A/IL-23R was determined in NSCLC cell lines using histone deacetylase (HDi) and DNA methyltransferase (DNMTi) inhibitors. A ChIP assay was performed to investigate the direct effect of Trichostatin A (TSA) on the IL-23A/IL-23R promoter regions. Additionally, the effect of Gemcitabine on IL-23A/IL-23R expression was examined. Finally, the effect of recombinant IL-23 treatment and Apilimod (STA 5326) (a small molecule which blocks the expression of IL-23 and IL-12) on NSCLC cell line proliferation was examined. IL-23 expression was also studied in a panel of NSCLC cisplatin sensitive and resistant cell lines. Furthermore due to its role in immune regulation, we are currently studying the effect of IL-23 on innate immune cell function.

      Results
      IL-23A expression was significantly elevated in a cohort of NSCLC patient tumour samples (p<0.05). Differential IL-23R expression was evident in a panel of NSCLC normal/tumour matched pairs (n=20), with no expression in 25%, reduced levels in 20% and increased levels in 55% of tumour samples compared with matched normal. In addition, IL-23A/IL-23R was found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation in the A549 cell line (p<0.05), with associated chromatin remodelling at both promoters. Gemcitabine, also induced IL-23A/IL-23R expression in this cell line (p<0.05). Furthermore, treatment with recombinant IL-23 resulted in increased cellular proliferation in the A549 cell line, while Apilimod reduced cellular proliferation. Preliminary results indicate that IL-23 can potentially affect the function of innate γδ T cells. In a panel of cisplatin resistant and sensitive cell lines, IL-23A was up-regulated in 3/5 resistant cell lines. Work is ongoing to assess the IL-23R expression in CisR and Parent cells lines and the effect of co-treating cells with HDi and Apilimod and to determine whether these compounds can re-sensitise cells to cisplatin therapy.

      Conclusion
      Based on our results the IL-23A/IL-23R axis is dysregulated in NSCLC. The IL-23 family is subject to epigenetic regulation through HDAC modifications and CpG island methylation. Gemcitabine treatment also affected the expression of this family. Recombinant IL-23 was pro-proliferative in NSCLC and blocking IL-23 with Apilimod reduced the proliferative capacity of the cells. Levels of IL-23A are differentially expressed between cisplatin sensitive and resistant cell lines. Apilimod may be a novel therapeutic drug in the treatment of NSCLC.

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      P2.01-016 - Targeting the PI3K-mTOR-NFκB pathway to overcome cisplatin resistance in NSCLC. (ID 2788)

      09:30 - 09:30  |  Author(s): A. Baird

      • Abstract

      Background
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality in the Western world with a poor overall 5 year survival of <15%. The most effective systemic chemotherapy for NSCLC is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem and understanding the mechanisms involved is critical to the development of new therapeutic intervention strategies. The PI3K pathway plays an important role in NSCLC and we and others have shown increased PI3K signaling to be associated with a more aggressive disease with poor prognosis. Several proteins in this pathway have been indicated as potential mediators of cisplatin resistance in other cancers, and our group has previously identified the PI3K-activated transcription factor NFκB as a key player in this setting. In this study, targeted inhibition of three strategic points of the PI3K pathway was carried out with the aim of overcoming acquired resistance to cisplatin in these cell lines.

      Methods
      A panel of cisplatin resistant cell lines was previously generated in our laboratory through prolonged exposure to the drug. Expression of PI3K pathway related genes was compared between H460 parent (H460PT) and H460 cisplatin resistant (H460CR) cells using a PI3K pathway SABiosciences RTPCR array. Identified genes of interested were further investigated via PCR and Western blot in these cells as well as A549 parent (A549PT) and A549 cisplatin resistant (A549CR) cells. Three strategic points of the pathway were inhibited using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFkB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors on the parent & cisplatin resistant cell lines both with and without cisplatin were assessed by BrdU proliferation assay and multiparameter apoptosis assay (High Content Analysis).

      Results
      One of the most notable targets to emerge from the PI3K pathway RTPCR array screen was NFKBIA; the gene which codes for NFκB inhibitor IκBα. This gene was shown to be 12 fold overexpressed in H460CR compared to H460PT. This finding was validated at both the RNA and protein level by PCR and Western blot. NFκB was also found to be overexpressed in cisplatin resistant cells compared to their respective parent cells. Inhibition of NFκB by DHMEQ led to significantly improved inhibition of proliferation and induction of apoptosis in cisplatin resistant cells compared to parent cells. Preliminary data indicates that inhibition of PI3K and mTOR by GDC-0980 did not offer as significant a benefit as inhibition of NFκB in the cisplatin resistance setting, though further data from combination studies will be presented.

      Conclusion
      We conclude that the PI3K pathway plays an important role in resistance to cisplatin in NSCLC, particularly when signaling proceeds through the transcription factor NFκB. Targeting this pathway may be of benefit in re-sensitizing cisplatin resistant tumours to the drug.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-021 - Targeting HDACs to overcome cisplatin resistance in malignant plural mesothelioma (ID 3207)

      09:30 - 09:30  |  Author(s): A. Baird

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer of the mesothelial cells and is becoming a worldwide health burden. Progress in the treatment of MPM remains difficult, underscored by the fact that no single treatment option has proven particularly effective and chemo-resistance is a common problem. However, epigenetic modifiers, such as histone deacetylase inhibitors (HDi) have emerged as a novel class of anti-cancer agent and are currently undergoing clinical trials in numerous cancers. There is also evidence to suggest that HDAC expression may play a role in the development of chemo-resistance. We investigated the levels of HDACs in MPM cell lines, patient samples and determined the effect of HDi on MPM cisplatin sensitive and resistant cell lines.

      Methods
      A panel of MPM cell lines (n=16) was screened for the expression of HDAC11, Class I (HDAC1/2/3/8) and Class II (HDAC4/5/6/7/9/10) HDACs at the mRNA level (RT-PCR) and at the protein level (Western Blot). The HDAC expression profile was determined in an isogenic cell line model consisting of a cisplatin sensitive (Parent) and resistant (CisR) MPM cell line, P31. In addition, HDAC expression was examined in panel of twenty patient samples (benign/biphasic/sarcomatoid/epithelial). Also, MPM TMAs were stained by imummo-histochemistry for HDAC5 expression. Furthermore proliferation assays (BrdU ELISA) were performed to determine the effect of two HDi on the p31 cell lines. The HDi used were Vorinostat (pan HDAC inhibitor) and 19i (selective HDAC5 inhibitor).

      Results
      HDAC11, Class I and II HDACs were detected to varying degrees at the mRNA and protein level within our cell line panel. In the P31 isogenic parent/cisplatin resistant, HDAC protein expression was decreased (HDAC2/3/4/5/7) in the CisR compared with the Parent. HDAC5 was significantly reduced at both the protein and the mRNA level (p<0.05). The expression of HDACs 1/2/3/5 were increased in the MPM patient samples (n=15) compared with benign (n=5) (HDAC2/3/5, p<0.05). HDAC5 staining in a cohort of MPM samples, demonstrated no significant association between survival and a low HDAC5 score. However females had a greater median survival than their male counterparts. Vorinostat and 19i significantly reduced the proliferative capacity of the p31 Parent and CisR. SAHA was more effective in the Parent cells compared with CisR. The effect of 19i was similar in both cell lines.

      Conclusion
      Altered HDAC expression was observed in an isogenic Parent and CisR MPM cell model. Work ongoing in non-small cell lung cancer (NSCLC) has also demonstrated significantly reduced HDAC5 levels in CisR compared with Parent. This may suggest that a reduction in HDAC expression is involved in cisplatin resistance. Reduced levels of HDAC5, in an MPM TMA, were not associated with survival. It should be noted, however that patient numbers were small and biphasic and sarcomatoid sub-types had the lowest expression levels of HDAC5. We are currently increasing our patient numbers for an additional IHC study. HDi significantly reduced the proliferative capacity of CisR and Parent MPM cells. HDAC levels may represent an important biomarker in stratifying patients for future epigenetic targeted therapies in MPM.