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VEGF and the epigenetic regulation of its receptors in non-small cell lung cancer
Lung Cancer 63 Suppl. 1 (2009) S1–S38
Posters, 7th Annual BTOG Meeting, 2009 Basic Science 1 VEGF and the epigenetic regulation of its receptors in non-small cell lung cancer M. Barr1 , S.G. Gray1 , K. Gately1 , N. Al-Sarraf1 , G.P. Pidgeon2 , K.J. O’Byrne1 . 1 Thoracic Oncology, St. James’s Hospital, Dublin 8, Ireland, 2 Clinical Surgery, St. James’s Hospital, Dublin 8, Ireland Introduction: VEGF the most potent angiogenic cytokine and is overexpressed in non-small cell lung cancer (NSCLC). In this study, we examined the role of VEGF as a survival factor in non-small cell lung cancer cells and the epigenetic mechanisms regulating VEGF receptor expression. Methods: A549 and SKMES1 NSCLC cells were screened for the VEGF receptors. The effect of VEGF on proliferation and apoptosis was examined using the BrdU assay and FACS analysis, respectively. The effect of VEGF blockade on the cell cycle was assessed by propidium iodide staining. Phosphorylation of the downstream signalling proteins, Akt/PKB and Erk1/2, was examined by confocal microscopy. The effect of TSA (Trichostatin A), on VEGF receptor expression in addition to proliferation and apoptosis, in the presence or absence of VEGF, was examined. NSCLC cells were treated with TSA and VEGF levels were measured in the conditioned media by ELISA. The ChIP assay was used to study the association between histone proteins and DNA while the effect of TSA on acetylation of histones H3 and H4 was examined by Western blotting. Results: VEGF increased proliferation and decreased apoptosis of Neuropilin-1-expressing NSCLC cells while VEGF blockade induced growth arrest in the G0/G1 phase of the cell cycle. VEGF induced phosphorylation of the cell signal proteins Akt and Erk1/2. TSA upregulated the expression of VEGFR1 and VEGFR2 and downregulated NP1 and NP2. VEGF was unable to rescue cells from TSA-induced cell death. VEGF secretion by NSCLC cells was decreased in response to TSA. De novo protein synthesis was required for downregulation of the Neuropilin receptors by TSA but not for VEGFR1 and VEGFR2. Increased acetylation of histones H3 and H4 by TSA was observed in both cell lines. Conclusion: This study demonstrates that the inhibitory effects of TSA on lung cancer cell survival are associated with its regulation of the VEGF receptors. Although TSA upregulated VEGFR1 and VEGFR2 and downregulated the Neuropilin receptors, in particular NP1, VEGF was unable to rescue cells from TSA-induced cell death, indicating NP1 as a critical survival factor for NSCLC cells. 2 Metabolic targeting, HDAC inhibitors, and non small cell lung cancer? S.G. Gray, K.J. O’Byrne, S. Ahmad. Department of Clinical Medicine, St. James’s Hospital, Dublin, Ireland Introduction: Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP, known as the Warburg effect to provide for their energy needs. At the same time, overexpression of the 0169-5002/$
see front matter © 2009 Elsevier Ireland Ltd.
insulin receptor is associated with increased risk of metastasis and decreased survival in NSCLC. Uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT4 is essential for insulin-stimulated glucose uptake. We suggest that epigenetic therapies which reactivate GLUT4 in non-small cell lung cancer (NSCLC) may be useful for (a) NSCLC diagnosis and (b) to metabolically targeting strategies. Methods: Normal and NSCLC cancer cell lines were screened for expression of GLUT4, and responses to inhibitors to histone deacetylases (HDi) and DNA methyltransferase (DNMT) were examined by RT-PCR.. Chromatin immunoprecipitation (ChIP) was used to examine changes to the GLUT4 promoter in response to HDi. Results: GLUT4 expression can be strongly induced in NSCLC cell lines following treatment with agents which target HDACs or DNMTs, with minimal responses in transformed normal human bronchial epithelial cells. Bioinformatic analysis of the promoter for GLUT4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT4 is dynamically remodelled in response to HDi. Conclusions: These results may have value within the clinical setting. (1) It may be possible to use this to enhance FDG-PET imaging sensitivity. (2) Alternatively, it may be possible to target NSCLC through the use of HDi and insulin mediated uptake of the metabolic targeting drug 2-deoxyglucose (2-DG). (3) Finally, both 2-DG and HDi have been shown to enhance or sensitize NSCLC to chemotherapy. These represent novel avenues for further study, which we are currently pursuing. 3 Evaluating the roles of TNF, IL-1 and hypoxia in lung carcinogenesis A.M. Baird, S.G. Gray, K.J. O’Byrne. Department of Clinical Medicine, TCD/St. James’s Hospital, Dublin, Ireland Introduction: Hypoxia and chronic inflammation are key triggers in the transformation process with at least 20% of all malignancies initiated or exacerbated by inflammation. The aim of this project is to examine inflammation in the process of lung carcinogenesis with a particular emphasis on TNF, IL-1 and hypoxia. This will provide a cell line model in which to examine their effects on normal bronchial epithelial cells with the ultimate aim to modify the cells’ phenotype from normal to malignant. Methods: A normal bronchial epithelial cell line (HBEC4) was modified to functionally over-express TNF and IL-1 (alone or in combination). The cells were grown for three months under normoxia or hypoxia (0.5% oxygen) after which time a range of experimental assays were carried out to assess cell change. These included: proliferation, invasion, transformation, migration, angiogenesis, sequencing and the use of a Cancer PathwayFinder PCR Array. Results: Although transformation was not detected as assayed by soft agar, the expression levels of c-Myc and p53 have changed.
Posters, 7th Annual BTOG Meeting, 2009 Basic Science 1 VEGF and the epigenetic regulation of its receptors in non-small cell lung cancer M. Barr1 , S.G. Gray1 , K. Gately1 , N. Al-Sarraf1 , G.P. Pidgeon2 , K.J. O’Byrne1 . 1 Thoracic Oncology, St. James’s Hospital, Dublin 8, Ireland, 2 Clinical Surgery, St. James’s Hospital, Dublin 8, Ireland Introduction: VEGF the most potent angiogenic cytokine and is overexpressed in non-small cell lung cancer (NSCLC). In this study, we examined the role of VEGF as a survival factor in non-small cell lung cancer cells and the epigenetic mechanisms regulating VEGF receptor expression. Methods: A549 and SKMES1 NSCLC cells were screened for the VEGF receptors. The effect of VEGF on proliferation and apoptosis was examined using the BrdU assay and FACS analysis, respectively. The effect of VEGF blockade on the cell cycle was assessed by propidium iodide staining. Phosphorylation of the downstream signalling proteins, Akt/PKB and Erk1/2, was examined by confocal microscopy. The effect of TSA (Trichostatin A), on VEGF receptor expression in addition to proliferation and apoptosis, in the presence or absence of VEGF, was examined. NSCLC cells were treated with TSA and VEGF levels were measured in the conditioned media by ELISA. The ChIP assay was used to study the association between histone proteins and DNA while the effect of TSA on acetylation of histones H3 and H4 was examined by Western blotting. Results: VEGF increased proliferation and decreased apoptosis of Neuropilin-1-expressing NSCLC cells while VEGF blockade induced growth arrest in the G0/G1 phase of the cell cycle. VEGF induced phosphorylation of the cell signal proteins Akt and Erk1/2. TSA upregulated the expression of VEGFR1 and VEGFR2 and downregulated NP1 and NP2. VEGF was unable to rescue cells from TSA-induced cell death. VEGF secretion by NSCLC cells was decreased in response to TSA. De novo protein synthesis was required for downregulation of the Neuropilin receptors by TSA but not for VEGFR1 and VEGFR2. Increased acetylation of histones H3 and H4 by TSA was observed in both cell lines. Conclusion: This study demonstrates that the inhibitory effects of TSA on lung cancer cell survival are associated with its regulation of the VEGF receptors. Although TSA upregulated VEGFR1 and VEGFR2 and downregulated the Neuropilin receptors, in particular NP1, VEGF was unable to rescue cells from TSA-induced cell death, indicating NP1 as a critical survival factor for NSCLC cells. 2 Metabolic targeting, HDAC inhibitors, and non small cell lung cancer? S.G. Gray, K.J. O’Byrne, S. Ahmad. Department of Clinical Medicine, St. James’s Hospital, Dublin, Ireland Introduction: Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP, known as the Warburg effect to provide for their energy needs. At the same time, overexpression of the 0169-5002/$
see front matter © 2009 Elsevier Ireland Ltd.
insulin receptor is associated with increased risk of metastasis and decreased survival in NSCLC. Uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT4 is essential for insulin-stimulated glucose uptake. We suggest that epigenetic therapies which reactivate GLUT4 in non-small cell lung cancer (NSCLC) may be useful for (a) NSCLC diagnosis and (b) to metabolically targeting strategies. Methods: Normal and NSCLC cancer cell lines were screened for expression of GLUT4, and responses to inhibitors to histone deacetylases (HDi) and DNA methyltransferase (DNMT) were examined by RT-PCR.. Chromatin immunoprecipitation (ChIP) was used to examine changes to the GLUT4 promoter in response to HDi. Results: GLUT4 expression can be strongly induced in NSCLC cell lines following treatment with agents which target HDACs or DNMTs, with minimal responses in transformed normal human bronchial epithelial cells. Bioinformatic analysis of the promoter for GLUT4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT4 is dynamically remodelled in response to HDi. Conclusions: These results may have value within the clinical setting. (1) It may be possible to use this to enhance FDG-PET imaging sensitivity. (2) Alternatively, it may be possible to target NSCLC through the use of HDi and insulin mediated uptake of the metabolic targeting drug 2-deoxyglucose (2-DG). (3) Finally, both 2-DG and HDi have been shown to enhance or sensitize NSCLC to chemotherapy. These represent novel avenues for further study, which we are currently pursuing. 3 Evaluating the roles of TNF, IL-1 and hypoxia in lung carcinogenesis A.M. Baird, S.G. Gray, K.J. O’Byrne. Department of Clinical Medicine, TCD/St. James’s Hospital, Dublin, Ireland Introduction: Hypoxia and chronic inflammation are key triggers in the transformation process with at least 20% of all malignancies initiated or exacerbated by inflammation. The aim of this project is to examine inflammation in the process of lung carcinogenesis with a particular emphasis on TNF, IL-1 and hypoxia. This will provide a cell line model in which to examine their effects on normal bronchial epithelial cells with the ultimate aim to modify the cells’ phenotype from normal to malignant. Methods: A normal bronchial epithelial cell line (HBEC4) was modified to functionally over-express TNF and IL-1 (alone or in combination). The cells were grown for three months under normoxia or hypoxia (0.5% oxygen) after which time a range of experimental assays were carried out to assess cell change. These included: proliferation, invasion, transformation, migration, angiogenesis, sequencing and the use of a Cancer PathwayFinder PCR Array. Results: Although transformation was not detected as assayed by soft agar, the expression levels of c-Myc and p53 have changed.