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M.P. Barr



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    MO04 - Lung Cancer Biology I (ID 86)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO04.03 - Chromosomal and mutational analysis of the cisplatin resistant phenotype in NSCLC cells (ID 3313)

      16:15 - 17:45  |  Author(s): M.P. Barr

      • Abstract
      • Presentation
      • Slides

      Background
      Primary and acquired resistance to platinum agents such as cisplatin have become a major obstacle in the management of lung cancer patients, in particular non-small cell lung cancer (NSCLC). The availability of comprehensive genomic data on DNA copy number changes in cisplatin resistant NSCLC is limited, and little is known about the genes driving this chemoresistant phenotype. Detailed molecular portraits through high density genomic DNA arrays and genome wide mutation profiles will aid in understanding the molecular basis of individual responses to new molecular therapies.

      Methods
      A panel of cisplatin resistant (CisR) NSCLC cell lines were recently generated and characterised in our laboratory. In this study, high resolution array-based comparative genome hybridization (aCGH) was performed on a panel of five CisR NSCLC cell lines to examine DNA copy number gains, losses and amplifications. Cellular DNA (500ng) and control DNA was differentially labelled with Cy3 and Cy5, respectively. Labelled test (4µg) and reference DNA were hybridised to a 12-plex 135,000 probe array (Roche NimbleGen) for 18 hours in a MAUI hybridisation station (BioMicro Systems) at 42°C. Fluorescent intensities were extracted and log 2 ratios calculated and normalized using NimbleScan Software (version 2.4). Chromosomal aberrations were identified using the CGH-segMNT algorithm (NimbleScan 2.4). A significance log 2 ratio threshold of <−0.25 for loss and >0.25 for gain was used to identify DNA copy number imbalances. For mutational analysis, Sequenom®, a mass-spectrometry-based SNP genotyping technology, was used to identify mutations in our panel of resistant cell lines. Using a literature search and the Catalogue of Somatic Mutations in Cancer (COSMIC) database, a mutation panel was identified for the detection of 547 frequently occurring and potentially clinically relevant mutations in 49 cancer-related genes. Some of these include KRAS, NRAS, BRAF, PIK3CA, MET, CTNNB1, STK11, AKT, and EGFR. Matrix chips were analysed on a Sequenom® MassArray MALDI-TOF system. Visual inspection and Sequenom® typer software were used to perform genotyping based on mass spectra.

      Results
      Using aCGH arrays, a number of gains, losses and amplifications of various chromosomes were found across a panel of CisR cell lines, relative to corresponding PT cells. The most frequently occurring of these chromosomal imbalances included gains, losses and homozygous deletions on chromosome 3 (MOR, A549, H1299), deletions and amplifications on chromosome 7 (H460, A549, H1299) and deletions and gains on chromosome 15 (MOR, A549, H1299) and chromosome X (MOR, H460, SKMES-1). Deletions on chromosomes 4, 6, 11, 12, 14, and amplification of chromosome 5, were also identified among the different CisR cell lines. The collation and analysis of data arising from mutation analysis of CisR cells using the Sequenom® platform are currently being completed.

      Conclusion
      High-resolution mapping of chromosomal imbalances may offer potential in the identification of genes, including oncogenes and tumour suppressor genes, affected by these imbalances. These findings may further contribute to the delineation of the genomic profile of cisplatin resistant lung cancer, and offer perspectives for the identification of genes contributing to this disease phenotype and in assessing the response to new molecular treatments.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.01-001 - VEGF is an autocrine survival factor in non-small cell lung cancer, mediating its effects via the Neuropilin-1 receptor. (ID 3344)

      09:30 - 16:30  |  Author(s): M.P. Barr

      • Abstract

      Background
      The VEGF pathway has become an important therapeutic target in lung cancer. In Phase III trials, blocking VEGF using Bevacizumab (Avastin[®]) has yielded improved progression-free and overall survival in NSCLC patients when combined with standard chemotherapy, demonstrating the use of VEGF as a target for therapy. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC.

      Methods
      NSCLC cells (H460, H647, A549 and SKMES-1) were screened for expression of VEGF and its receptors at the mRNA and protein levels. The effect of recombinant VEGF (100ng/ml) and its blockade using neutralising antibodies (1µg/ml) on lung tumour cell proliferation (BrdU) and cell cycle (FACS) was examined. Phosphorylation of Akt and Erk1/2 proteins was examined by High Content Analysis (HCA) and confocal microscopy. The effects of silencing VEGF (100nM siVEGF)) on cell proliferation and survival signalling were assessed using BrdU and Western blot analysis, respectively. The role of NP1 in cell proliferation (BrdU), apoptosis (HCA) and cell survival signalling was also examined. A NP1 stable transfected H460 cell line (NP1-negative) was generated and NP1 overexpression verified at the mRNA (RT-PCR) and protein (Western Blot) levels relative to empty vector controls (EVC). Cell proliferation, pAkt and pErk1/2 levels were assessed in response to recombinant VEGF and VEGF antibodies. Tumour growth studies were carried out in female Balb/c nude mice following subcutaneous injection of NP1-overexpressing cells (n=8) and EVC control cells (n=8). The role of epigenetic modifications in the regulation of VEGF and its receptors was also examined using the histone deacetylase (HDAC) inhibitors, Trichostatin-A (TSA) and Virinostat (SAHA).

      Results
      VEGF increased proliferation of NP1-expressing NSCLC cells (H647, A549 and SKMES-1). Blocking VEGF inhibited VEGF-mediated proliferation and induced growth arrest in the G0/G1 phase of the cell cycle. Phosphorylation of Akt and Erk1/2 proteins was significantly upregulated in response to VEGF, while antibodies to VEGF inhibited this effect. VEGF siRNA significantly inhibited lung tumour cell proliferation and decreased phosphorylation of pAkt and pErk1/2. NP1 blockade significantly inhibited proliferation and apoptosis of NP1-expressing NSCLC cells, with no effect on the NP1-negative cell line, H460. Cell proliferation was significantly decreased in response to NP1 siRNA. Stable transfection of H460 cells with NP1 pcDNA significantly increased proliferation relative to EVC cells. This effect was further increased in NP1 stable transfectants upon the addition of VEGF, while neutralising antibodies to VEGF inhibited this effect. Expression levels of pAkt protein was significantly increased following treatment of cells with VEGF, with little or no effect on the pErk1/2 pathway. Tumour growth was significantly increased in mice injected with NP1-overexpressing cells (p=0.0052). HDAC inhibitors increased VEGFR1 and VEGFR2 and downregulated NP1 and NP2 expression. Significant inhibition in proliferation of NP1-positive lung cancer cells was associated with a decrease in the NP1 receptor at the mRNA and protein levels.

      Conclusion
      We demonstrate that VEGF is an autocrine survival factor for NSCLC cells, mediating its effects through the Neuropilin-1 receptor. Combining anti-VEGF therapies with HDAC inhibitors may offer potential as a promising avenue for future clinical research in the treatment of NSCLC.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-024 - PARP inhibition increases sensitivity of NSCLC cells to cisplatin (ID 3300)

      09:30 - 16:30  |  Author(s): M.P. Barr

      • Abstract

      Background
      Primary and acquired resistance to platinum agents is a serious clinical problem in lung cancer. Its mechanisms are probably multifactorial and remain poorly understood. Enhanced DNA repair can lead to increased cell viability in the face of DNA damage and has been proposed to be important in mediating platinum resistance. PARPs (poly(ADP-ribose) polymerases) are a family of nuclear enzymes that regulate the repair of DNA single-strand breaks (SSBs). Cisplatin sensitivity and DNA repair mechanisms following treatment with the PARP inhibitor, PJ34, was investigated in this study.

      Methods
      A panel of isogenic cisplatin resistant (CisR) NSCLC cells lines (MOR, SKMES-1, H1299) previously generated in our laboratory were used. The cisplatin resistant phenotype was initially assessed by treating CisR and parental (PT) cells with increasing doses of cisplatin (0-80uM) for 72h, after which time, cell proliferation was measured (BrdU). The effects of PJ34 on cell survival were also examined in a similar dose-response study. IC~25~ concentrations were calculated for each cell line using GraphPad statistical software. Cells were treated with PJ34 (IC~25~) alone, or in combination with cisplatin and cell survival/proliferation measured after 72h. Under similar experimental conditions, RNA was isolated from cells from which cDNA was reverse transcribed. All cell lines were screened for PARP1, PARP2, BRCA1, BRCA2 and ERCC1 mRNA at basal levels, and in response to treatment (RT-PCR). To investigate DNA double strand break (DSB) repair capacity in our panel of cell lines in response to PARP inhibition and cisplatin, phosphorylated γH2AX foci was examined by High Content Analysis (HCA) following treatment of cell lines for 24h. Cisplatin-DNA adduct formation (Pt-GpG) was studied following treatment of cells for 24h. Cells (1x10[6]/ml) were spotted on Superfrost® Gold glass slides. Immunofluorescence staining of specific DNA platination products, and quantification of adducts, was performed using an antibody that specifically recognises cisplatin-GpG DNA adducts.

      Results
      MOR and H1299 CisR cells were significantly more resistant to cisplatin (10µM and 20µM) compared to PT cells. SKMES-1 CisR cells were also significantly more resistant at 10µM, 20µM and 40µM cisplatin. While PJ34 had no effect on NSCLC cells when treated as a single agent, cell proliferation was significantly inhibited in MOR and H1299 cells when used in combination with cisplatin. No effect however was observed in our panel of CisR cell lines. While baseline expression levels of PARP1/2, BRCA1/2 and ERCC1 mRNA levels were similar in PT and CisR cell lines, BRAC1/2 mRNA expression was increased in cells treated with cisplatin alone, and in combination with PJ34 in PT cells but not in CisR cells. The formation of γH2AX foci and measurement of cisplatin-GpG DNA adducts in response to PARP inhibition and cisplatin are currently being investigated.

      Conclusion
      Data from this study show that inhibition of NSCLC cells with the PARP inhibitor, PJ34, sensitises lung cancer cells to the cytotoxic effects of the platinum drug, cisplatin. Further studies are warranted to investigate the role of PARP inhibitors in cisplatin resistant NSCLC cells.