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K.A. Gately

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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): K.A. Gately

      • Abstract

      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.

      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).

      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.

      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.