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S. Gray



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    MO09 - Mesothelioma I (ID 120)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track:
    • Presentations: 1
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      MO09.08 - NF-kB in cisplatin resistance and as a prognostic marker in Malignant pleural mesothelioma (ID 3338)

      16:55 - 17:00  |  Author(s): S. Gray

      • Abstract
      • Presentation
      • Slides

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Currently rates of MPM are rising and estimates indicate that the incidence of MPM will peak in western world within the next 10-15 years. Untreated, MPM has a median survival time of 6 months, with poor survival rates for most patients after 24 months of diagnosis. Nuclear Factor kappa B (NF-kB) is a pro-inflammatory transcription factor which is activated in many cancer types, including MPM. The NF-kB pathway regulates important cellular processes including survival and proliferation signals, which are often found to be dysregulated in cancer. Furthermore, we and others have shown that increased NF-kB activation is linked to development of cisplatin resistance. We aim to outline the potential role of NF-kB as a mediator of cisplatin resistance in MPM and determine its value as a potential candidate for therapeutic intervention.

      Methods
      NF-kB expression was examined in a cohort of MPM patients (n=200) by IHC, and correlated with clinicopathological variables and survival. NF-kB expression was examined in both a panel of MPM cell lines and isogenic parent/cisplatin resistant cell lines by Western blot analysis. The effect of NF-kB inhibition on cellular proliferation was measured by BrdU assay, in a panel of MPM and isogenic parent/cisplatin resistant cell lines, using the novel NF-kB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ). In addition, the effect of DHMEQ on nuclear translocation of NF-kB was examined by high content screening (HCS).

      Results
      Cytoplasmic or membranous immunostaining was seen in the majority of tumour samples (96.5%), but nuclear localisation of NF-kB was seen in only 11% cases. Kaplan-Meier survival analysis showed that nuclear NF-kB expression correlated with reduced survival (p=0.05). There was no significant correlation between the level of expression of NF-kB and standard clinicopathological parameters. NF-kB was expressed in all MPM cell lines tested to a varying extent (n=20), with no associations to histology. NF-kB levels were shown to be elevated in cisplatin resistant cell lines when compared to the isogenic parent from which they were derived. DHMEQ was shown to reduce nuclear translocation of NF-kB, inhibiting cell proliferation in all cell lines but to a lesser extent in NCI 2596 cells which have low NFkB expression.

      Conclusion
      Nuclear NFkB expression is a poor prognostic factor in MPM. DHMEQ, which inhibits nuclear translocation of NF-kB, inhibits cell proliferation in MPM cell lines. Furthermore, increased NF-kB expression in resistant cells suggests this pathway may play a role in development of cisplatin resistance in MPM. Inhibition of NF-kB may therefore prove to be of potential therapeutic benefit in MPM treatment and re-sensitisation of resistant MPM to cisplatin.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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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): S. Gray

      • 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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      P1.06-056 - Isolation & enumeration of Circulating Tumor Cells in Non-small Cell Lung Cancer, using Screencell & VitaAssay techniques. (ID 3318)

      09:30 - 09:30  |  Author(s): S. Gray

      • Abstract

      Background
      Circulating Tumour Cells (CTCs) have been the subject of much interest as a potential biomarker however methods for isolating CTCs are still in their infancy. A promising method of CTC detection is ScreenCell. This technique uses polycarbonate filtration membranes containing multiple tiny pores. When blood is made to flow across the membrane, tumour cells are captured due to their greater size. Another such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype.

      Methods
      Peripheral blood samples were obtained from patients with advanced NSCLC using both Screencell & VitaAssay. In addition healthy blood samples spiked with NSCLC cells were also analysed. ScreenCell: Peripheral blood is diluted with specified buffer and drawn across the Screencell filter using a vacuum tube. The filters with captured fixed cells are then stained with H&E and/or immunocytochemistry. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12-18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs due to their invasive phenotype. Captured cells are fixed and stained using immunocytochemistry.

      Results
      Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. CTCs were detected in 70% of patient samples with. (n=10) Numbers of CTCs detected ranged from 6-82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli (CTM) were detected in 50% of patient samples. (An example of CTM is illustrated in Fig. 1) Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 & H2228 cells, approximately 20% (range 9%-26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified. (range 14-52, n = 6) (An example of CTCs detected by immunocytochemistry is illustrated in Figs. 2 & 3) Figure 1

      Conclusion
      ScreenCell & VitaAssay techniques both appear to be viable methods of isolating & enumerating CTCs, in both model cell-spiking experiments and in NSCLC patient samples, as determined by morphology and antigen expression detected with immunocytochemistry. Of particular interest many of the CTCs isolated using Screencell, were detected as clusters or microemboli. Additional samples are being taken to compare CTC & CTM numbers with clinical outcomes.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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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): S. Gray

      • 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): S. Gray

      • 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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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-017 - Long non coding RNAs (lncRNAs) are dysregulated in malignant pleural mesothelioma (MPM) (ID 1524)

      09:30 - 09:30  |  Author(s): S. Gray

      • Abstract

      Background
      Malignant Pleural Mesothelioma (MPM) is an aggressive disease, often diagnosed at an advanced stage. It is characterized by a long latency period and prior asbestos exposure. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates rarely exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) plays an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. The aims of this study were to characterize the expression and function of these lncRNAs in MPM.

      Methods
      To identify novel lncRNAs involved in MPM, microarray profiling was performed on five cell lines - the immortalized normal mesothelial cell line (MeT-5A) and four MPM lines (two epithelioid H28 and H226 and two biphasic MM05 and MSTO) using Invitrogen’s NCode lncRNA microarrays. These allow simultaneous assessment of mRNA and lncRNA content. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological (>3-fold difference) significance. Expression of high priority candidates were technically validated using RT-qPCR, and biologically validated in three independent test sets. Pathway analyses were performed to interrogate the relationship between lncRNA and mRNA expression. Cell proliferation and colony formation assays were used to investigate lncRNA function.

      Results
      Microarray profiling and real-time qPCR validation identified 9 lncRNA candidates with significant differential expression in MPM compared with normal mesothelial cells Validation in three independent test sets by RT-qPCR analysis demonstrated consistent up-regulation of four of these lncRNAs. Receiver Operating Curve analysis showed that two of these candidates were able to separate benign pleura and MPM with high sensitivity and specificity. In addition, high expression of AK054908 was associated with nodal metastases with lower levels of AK130275 and AF268386 observed in patients receiving induction chemotherapy. Cases with higher EF177379 levels also demonstrated a trend to improved survival. The majority of mRNAs co-expressed with candidate lncRNAs were associated with cellular and metabolic processes including cell cycle, cell death and apoptosis. In functional studies, siRNA knockdown of AK130275 showed suppression of cell growth and colony formation in MPM cells with moderate changes observed following knockdown of EF177379.

      Conclusion
      To our knowledge this is the first systematic study of lncRNA expression profiles in MPM. We have found that lncRNA expression profiles can distinguish malignant mesothelium from normal pleural tissue, and that some lncRNAs are associated with nodal metastasis and long term survival. We also demonstrate that lncRNAs have potential prognostic and diagnostic utility with functional roles in regulating cell growth. Further work is required to evaluate whether these lncRNAs are capable of differentiating mesothelioma from lung cancer and benign asbestos-related diseases, and to reveal their specific functions in MPM pathogenesis.

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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): S. Gray

      • 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.