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D. Richard



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    P2.01 - Poster Session with Presenters Present (ID 461)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 2
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      P2.01-031 - CCL Chemokines May Play an Important Role in Cisplatin Resistance (ID 4861)

      14:30 - 15:45  |  Author(s): D. Richard

      • Abstract

      Background:
      In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumour heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC.

      Methods:
      A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilised to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterise inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin.

      Results:
      A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite affects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterisation is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumour growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumour samples appeared more necrotic with DHMEQ and are currently being characterised using IHC for necrosis and proliferation.

      Conclusion:
      Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitising patients to chemotherapy.

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      P2.01-081 - CDCA3 is a Novel Prognostic Cell Cycle Protein and Target for Therapy in Non-Small Cell Lung Cancer (ID 5823)

      14:30 - 15:45  |  Author(s): D. Richard

      • Abstract
      • Slides

      Background:
      Lung cancer is the leading cause of cancer-related mortality worldwide with a 5 year survival rate of 15%. Non-small cell lung cancer (NSCLC) is the most commonly diagnosed form of lung cancer. Cisplatin-based regimens are currently the most effective chemotherapy for NSCLC, however, chemoresistance poses a major therapeutic problem. New and reliable strategies are required to avoid drug resistance in NSCLC. Cell division cycle associated 3 (CDCA3) is a key regulator of the cell cycle. CDCA3 modulates this process by enabling cell entry into mitosis through degradation of the mitosis-inhibitory factor WEE1. CDCA3 itself is also degraded in G1 yet re-expressed in G2/M phase, to allow successful progression through the cell cycle. Herein, we describe CDCA3 as a novel prognostic factor in NSCLC and target to delay or prevent cisplatin resistance in NSCLC.

      Methods:
      CDCA3 expression was investigated in squamous and non-squamous NSCLC using several approaches including bioinformatic analysis of publicly available datasets, immunohistochemistry of a tissue microarray and western blot analysis of matched tumour and normal tissue and NSCLC cell lines. CDCA3 function in NSCLC was determined using several in vitro assays by siRNA depleting CDCA3 in a panel of three immortalized bronchial epithelial cell lines (HBEC) and seven NSCLC cell lines.

      Results:
      CDCA3 transcripts and protein levels are elevated in NSCLC patient tissue and highly expressed in tumour cells relative to proximal normal cells. High mRNA levels are associated with poor survival in resected NSCLC. Depletion of CDCA3 in vitro markedly impairs proliferation in seven NSCLC cell lines by inducing a mitotic cell cycle arrest, ultimately resulting in p21-dependent cellular senescence. Importantly, silencing of CDCA3 also greatly sensitises NSCLC cell lines to cisplatin. In line with these in vitro data, NSCLC patients that have elevated levels of CDCA3 and are treated with cisplatin have a poorer outcome than patients with reduced levels of the protein. To improve patient response to cisplatin, we are exploring novel strategies to suppress CDCA3 expression in tumour cells.

      Conclusion:
      Our data highlight CDCA3 as a novel factor in mediating NSCLC. We propose that evaluating novel strategies to target CDCA3 may prove a useful strategy is enhancing the anti-tumour activity of platinum-based chemotherapy and may ultimately benefit patient outcomes by preventing cisplatin resistance.

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    P2.03b - Poster Session with Presenters Present (ID 465)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P2.03b-096 - Utilisation of a Novel 3D Culture Technology for the Assessment of Chemo-Resistance in Non-Small Cell Lung Cancer (ID 4954)

      14:30 - 15:45  |  Author(s): D. Richard

      • Abstract
      • Slides

      Background:
      Elucidation of the key mechanisms underlying resistance to chemotherapy is an on-going and complex process. Owing to its suggested increased biological relevance, many are now transitioning from two-dimensional (2D) to three-dimensional (3D) cellular-based assay systems. These systems permit the formation of 3D multicellular structures (MCS). The internal micro-environment of these structures mimics closely those found in vivo. In addition they are considered to provide a more biologically relevant model of chemo-resistance. This study focuses on the utilisation of a novel 3D culture technology to compare chemo-resistant models of non-small cell lung cancer (NSCLC) in 3D culture with those cultured in 2D monolayers. Critically, this will provide a valuable tool to determine the biological discrepancies which exist between the two culture methods with the aim to identify novel mechanisms of chemo-resistance in NSCLC.

      Methods:
      Isogenic NSCLC models of cisplatin resistance, which encompassed sensitive parent (PT) and matched cisplatin resistant (CisR) cell lines, were used for this study. The 3D MCS were cultured in Happy Cell Advanced Suspension Medium™ and 2D monolayers as standard. Both 2D and 3D cultures were exposed to a range of cisplatin concentrations (0 – 100 µM) for a period of 72 h. Subsequently, cellular viability, hypoxia, proteomic and morphological assays were conducted in order to compare the response of both PT and CisR cells in 2D and 3D.

      Results:
      High content imaging has identified a central necrotic core within the 3D MCS, which is a feature of the asymmetric growth patterns observed in vivo; that being a decrease in viable cells as you move inwards from the periphery of the MCS. Preliminary data suggests that at equivalent cisplatin concentrations, H460 3D MCS exhibit increased resistance to cisplatin compared with 2D monolayers in both PT and CisR cell lines. Proteomic analysis has also identified diverse pathway modifications in 3D compared with 2D culture, with a number of proteins expressed exclusively in each. Additional cellular characterisation and further bioinformatic analysis is on-going.

      Conclusion:
      Chemotherapeutic intervention is most frequently employed in the treatment of NSCLC, however many patients exhibit intrinsic and acquired resistance to common chemotherapy drugs, such as cisplatin and targeted agents. As it has been argued that 3D models and their micro-environment are more reflective of the in vivo situation, 3D culture may provide a more accurate in vitro model to elucidate mechanisms of chemo-resistance and possibly aid in the identification of novel targets to re-sensitise and stratify patients for therapy.

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    P3.01 - Poster Session with Presenters Present (ID 469)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 2
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      P3.01-042 - Lung Cancer Cells Can Stimulate Functional and Genotypic Modifications in Normal Bronchial Epithelial Cells (ID 4852)

      14:30 - 15:45  |  Author(s): D. Richard

      • Abstract

      Background:
      Normal lung epithelium cells may act in concert with tumour cells, given that bystander effects may exist between the two. This interaction may lead to inappropriate activation of pro-oncogenic signalling pathways, which may result in high mutational load and tumour heterogeneity. The aim of this project is to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalised normal bronchial epithelial cell line.

      Methods:
      A normal bronchial epithelial cell line (HBEC4) was exposed to A549 (adenocarcinoma), H460 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) NSCLC cell lines in a trans-well co-culture system. Cellular characteristics were examined using a Cytell Cell Imaging System (cell number, viability, apoptosis, cell cycle). The gene expression profile was also determined in terms of inflammatory mediators, stem cell markers (RT-PCR) and miRNA profiling (Nanostring). The proliferative effect of NSCLC cancer exosomes was also examined (BrdU ELISA) on the HBEC4 cell line.

      Results:
      A number of functional and gene modifications were observed in the HBEC4 cell line after seven days of co-culture. While patterns were similar amongst all NSCLC subtypes, SK-MES-1 elicited the most significant effects in terms of cell number, viability, cell cycle progression and proliferative potential of isolated cancer exosome fraction. Promotion of both inflammatory mediators and stem cell marker expression was evident at the mRNA level. There was no apparent consensus between NSCLC subtypes and miRNA expression, as exposure to each cell line resulted in distinct profiles of miRNAs in HBEC4 cells. Bioinformatic analysis of miRNA target genes, demonstrated that pathways such as p53, MAPK, VEGF, TLR and Wnt were amongst those altered.

      Conclusion:
      Cancer cells may promote significant genotypic and phenotypic alterations within the normal lung epithelium though multiple mechanisms. These modifications may, in part, contribute to the heterogeneity of lung cancer tumours and influence response to both chemotherapeutics and targeted agents.

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      P3.01-064 - The Overexpression and Cleavage of SASH1 by Caspase-3 Stimulates Cell Death in Lung Cancer Cells (ID 5811)

      14:30 - 15:45  |  Author(s): D. Richard

      • Abstract
      • Slides

      Background:
      SASH1 (SAM and SH3 domain-containing protein 1) is a recently identified gene with tumour suppressor properties and has a role in induction of apoptosis. Previous work has shown that 90 % of lung cancer cell lines have a decrease in SASH1 mRNA levels (Zeller et al., 2003), however little characterisation of SASH1 function in lung cancer has been undertaken.

      Methods:
      We evaluated SASH1 expression in transformed normal and malignant non-small cell lung cancer cell lines. We also utilised cell based assays to study the effects of altered SASH1 levels on cell survival and proliferation. Identification of a novel SASH1 targeting drug was performed through connectivity mapping.

      Results:
      SASH1 protein expression was down regulated in two of the five lung cancer cell lines compared to immortalized normal bronchial epithelial cells. Prognoscan assessment identified decreased SASH1 mRNA expression reduced patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated the Chloropyramine inhibited lung cancer proliferation in vitro. We also explored the role of SASH1 in apoptosis. Following ultraviolet light exposure SASH1 is cleaved by Caspase-3. The C-terminal fragment of SASH1 then translocates from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild type SASH1 or cleaved SASH1 amino acids 231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-κB to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a co-dependence between SASH1 and NF-κB for this process.

      Conclusion:
      We have shown that SASH1 contributes to apoptosis via a NF-κB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.

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