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S. Finn
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MINI 08 - Prognostic/Predictive Biomarkers (ID 106)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.E. Stinchcombe, N. Pavlakis
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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MINI08.08 - VEGF-Mediated Cell Survival in NSCLC: Implications for Epigenetic Targeting of VEGF Receptors as a Therapeutic Approach (ID 2721)
16:45 - 18:15 | Author(s): S. Finn
- Abstract
- Presentation
Background:
We have recently shown that VEGF, at least in part, is an autocrine growth factor for NSCLC cells, mediating its survival effects via VEGFR2 (KDR) in addition to the more novel receptor, Neuropilin-1 (Barr et al., Mol Cancer, 2015). In this study, we evaluated the potential therapeutic utility of histone deacetylase (HDAC) inhibitors in targeting the VEGF-VEGFR signalling axis in non-small cell lung cancer (NSCLC) cells.
Methods:
The effect of the HDAC inhibitor, Trichostatin-A (TSA) on modulating the expression of the VEGF receptors, VEGFR1, VEGFR2, NP1 and NP2, in A549 and SKMES-1 cells was examined and validated at the mRNA level and protein levels using RT-PCR and Western blot analysis. Gene expression was further validated by quantitative real-time PCR. To investigate the effect of TSA on the viability of NSCLC cells, these were treated with increasing concentrations of TSA (2.5 ng/ml-250 ng/ml) for 24h. Cell proliferation and apoptosis was measured by BrdU and Annexin V/PI (FACS), respectively. VEGF protein secretion in response to TSA was assessed in conditioned media from lung tumour cells by ELISA. To determine if the effects of TSA on VEGFR receptors were mediated through immediate to early responses, cells were pre-treated with cycloheximide (10 µg/ml) for 2 h followed by treatment with TSA (250 ng/ml) for 24 h. To confirm whether the observed effects of HDAC inhibition by TSA were due to increased histone hyperacetylation at the VEGFR1 and VEGFR2 gene promoters, chromatin immunoprecipitation (ChIP) analysis was carried out following treatment with TSA.
Results:
NP1 and NP2 mRNA levels were decreased in both A549 and SKMES-1 lung cancer cells in response to TSA and induced the expression of VEGFR1 and VEGFR2 at higher concentrations. TSA however, had no effect on VEGF mRNA expression. Critically, the effect of TSA was more marked at the protein level, with complete loss of Neuropilin-1 protein. HDAC inhibition resulted in a significant decrease in the viability of A549 and SKMES-1 cells in a dose-dependent fashion. While TSA induced significant apoptosis of both lung tumour cell lines, VEGF was unable to rescue cells from TSA-induced cell death. VEGF secretion was significantly decreased in both cell lines. Treatment with cycloheximide was unable to abrogate the TSA-mediated increase in the VEGF receptors examined, indicating that de novo protein synthesis is not required for these observed effects, but may be due to direct effects at the promoter level. Direct histone acetylation of histones H3 and H4 was observed, indicating an increase in histone hyperacetylation of VEGFR1 and VEGR2 promoters. A significant trend in the modulation of the VEGF receptors similar to that seen in response to TSA was shown when treated with Vorinostat (SAHA).
Conclusion:
Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for NSCLC patients in the clinical setting. The possibility of novel targeted agents decreasing the levels, or function, of tumour VEGF receptors, in particular NP1, may lead to more successful treatments and prolonged overall survival in these patients.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-099 - Differential Regulation of DNA Repair Genes in Cisplatin Resistant Non-Small Cell Lung Cancer Cells (ID 2746)
09:30 - 17:00 | Author(s): S. Finn
- Abstract
Background:
In the absence of specific treatable mutations, cisplatin-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to this drug. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. DNA repair is therefore a vital target to improving cancer therapy and overcoming resistance of tumour cells to DNA damaging agents currently used in the treatment of NSCLC patients.
Methods:
DNA Repair Pathway RT[2 ]Profiler Arrays were used to elucidate the key DNA repair genes implicated in cisplatin resistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) H460 NSCLC cells previously established in our laboratory. The regulation of the trans-activation of p53 in response to DNA damage was studied by examining protein accumulation, post-translational modifications (p53[Ser15]) and whether depletion of the novel DNA repair protein, hSSB1, affects the regulation of p53 in response to cisplatin. The repair of cisplatin-induced double strand breaks (DSBs) was examined by immunofluorescence imaging of γH2AX foci. Expression of p53[Ser15] (phosphorylated & total) in addition to hSSB1 was also assessed by HCA and Western blot analysis.
Results:
We identified a number of critical DNA repair genes that were differentially regulated between parental and cisplatin resistant NSCLC cells, some of which are known to be implicated in the nucleotide and mismatch repair pathways. H2AX was shown to be a reliable and specific marker of DNA double strand DNA breaks induced by platinum agents such as cisplatin. Cisplatin induced the translocation of p53 from the cytoplasmic compartment of H460 PT cells to the nuclear compartment, while significant levels of p53 were retained within the cytoplasmic compartment of CisR cells. Using both HCS and Western blot analysis, hSSB1 protein was undetectable.
Conclusion:
To date, despite reports that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.
