NSCLC Cancer Cells Research Articles

Abstract 1892: Identification of differentially methylated genes associated with cetuximab and erlotinib resistance

Abstract Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. Two classes of EGFR-directed therapeutic inhibitors are currently in clinical use: anti-EGFR monoclonal antibodies, such as cetuximab, and small-molecule tyrosine kinase inhibitors (TKIs) of EGFR, such as erlotinib. Although both classes of drugs are capable of inducing an initial therapeutic response, primary as well as acquired drug resistance is frequently observed. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of cytotoxic chemotherapeutic drug resistance. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we employed two parental NSCLC and HNSCC cell lines and their resistant derivatives to either erlotinib or cetuximab. DNA from each pair was used for methylation-specific array containing a panel of 64 genes that are commonly known to be regulated through promoter methylation. We found that death-associated protein kinase 1 (DAPK1) was hypermethylated in drug resistant cells generated from both parental cell lines. Restoration of DAPK1 into the resistant cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK1 induced resistance in the parental sensitive cells. These results demonstrate that DAPK1 plays an important role in both cetuximab and erlotinib resistance in NSCLC and HNSCC cancer cells, and that promoter methylation of its gene frequently occurs during the chronic treatment of anti-EGFR agents. In conclusion, DAPK1 could be a novel target for the improvement of therapeutic benefit of anti-EGFR therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1892. doi:1538-7445.AM2012-1892

Abstract A73: The Bcl-2/BclXL inhibitor, ABT263, enhances paclitaxel-induced apoptosis in NSCLC models

Abstract The anti-apoptotic protein, BclXL, is thought to be an important chemo-resistance factor (Amundsen et al, 2000), and the combination of a BclXL inhibitor and paclitaxel exhibits increased NSCLC cancer cell killing in culture and in xenograft models (Shoemaker et al, 2006). ABT263 is a small molecule inhibitor of Bcl-2, BclXL and Bcl-w, that is currently in clinical trials (Tse et al, 2008). We set out to address the following questions 1) is ABT-263 broadly synergistic with paclitaxel in NSCLC cells, 2) what is the mechanism of the synergistic interaction and 3) can we identify biomarkers to guide the selection of patients that are likely to respond to the combination of ABT263 and taxane based chemotherapy regimens? We screened a panel of 26 NSCLC cell lines for synergy with ABT263. All 26 cell lines exhibited a synergistic response to the combination, as evaluated by Bliss additivity, and 20 cell lines had Bliss sum values in excess of 100, considered to be a robust synergy response in this assay. In order to address the mechanism of synergy, we performed time-lapse microscopy on GFP-H2b expressing A549 cells. We find that the addition of ABT263 alters the timing and fate of cells that enter mitosis in the presence of paclitaxel. Paclitaxel alone caused prolonged mitotic arrest usually followed by mitotic slippage. ABT263 as a single agent had little effect on mitosis, but caused cells to die rapidly from a mitotic arrest state when added to paclitaxel. This resulted in an increase in the rate and percentage of cell death compared to either single agent. To identify biomarkers that might influence synergy between ABT263 and paclitaxel, we identified genes whose mRNA levels had significant correlations with the Bliss score. Together these data suggest that the combination of ABT263 and paclitaxel has the potential to have improved efficacy in cancers that are refractory to paclitaxel. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A73.

Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines

Glucose transporters (Gluts) facilitate glucose uptake and tumors frequently over express the Gluts, especially Glut-1. Breast cancer and lung cancer (NSCLC) cell lines were incubated with anti-Glut-1 antibodies alone or with cisplatin, paclitaxel or gefitinib. Inhibition of proliferation and apoptosis was assessed. Antibodies to Glut-1 inhibited proliferation by 50% and 75% in the tested NSCLC and breast cancer cell lines, respectively. They also potentiate the anti-proliferative effects of cisplatin, paclitaxel and gefitinib. Our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated cell lines and provide preliminary evidence of their anti-tumor activity.

The TGF-beta1 antisense oligonucleotide AP 11014 for the treatment of non-small cell lung, colorectal and prostate cancer: Preclinical studies

3132 Background: Tumor derived transforming growth factor beta (TGF-β) is a pivotal factor for malignant progression by inducing metastasis, angiogenesis and tumor cell proliferation and by mediating the tumors' escape from immunosurveillance. In colorectal, non-small cell lung and prostate cancer in particular expression of the TGF-β1 isoform has been correlated with tumor progression and poor clinical prognosis. Significantly elevated TGF-β1 plasma levels to more than 3fold in colon cancer (median control: 6,200 pg/ml, range: 1,977 - 17,515 pg/ml; median patients: 19,948 pg/ml, range: 5,014 - 38,900 pg/ml) and NSCLC (median control: 3,482 pg/ml, range: 747 - 9,984 pg/ml; median patients: 11,572 pg/ml, range: 4,952 - 30,985 pg/ml) and to more than 4fold in prostate cancer (median control: 2,152 pg/ml, range: 1,275 - 3,958 pg/ml; median patients: 10,066 pg/ml, range: 3,435 - 41,574 pg/ml) further support the role of TGF-β1 as a key tumor promoter. Methods: The in vitro effects of AP 11014, a TGF-β1 specific phosphorothioate antisense oligonucleotide, on TGF-β1 secretion, proliferation, migration of and immunosuppression by various cancer cell lines were determined. Results: AP 11014 significantly reduced TGF-β1 secretion by 43 - 100% in different NSCLC (A549, NCI-H661, SW 900), colon cancer (HCT-116) and prostate cancer (DU-145, PC-3) cell lines. Tumor cell proliferation was inhibited in a dose-dependent manner in all cell lines. In a scratch assay AP 11014 reduced migration of a NSCLC (SW 900) and prostate cancer (PC-3) cell line by max. 65% after 24h. Additionally, AP 11014 reversed immunosuppression mediated by NSCLC (NCI-H661, A-549) and colon cancer (HCT-116) cell derived TGF-β1 by increasing LAK cell cytotoxicity up to 368% of the untreated control (effector/target ratio 5:1). Conclusion: These preclinical data clearly indicate antisense mediated suppression of TGF-β1 by AP 11014 as a highly promising approach for the therapy of non-small cell lung, colorectal and prostate cancer in humans. Based on these data a clinical trial with AP 11014 in TGF-β1 overexpressing tumors is planned. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antisense Pharma GmbH Antisense Pharma GmbH

The TGF-beta1 antisense oligonucleotide AP 11014 for the treatment of non-small cell lung, colorectal and prostate cancer: Preclinical studies

3132 Background: Tumor derived transforming growth factor beta (TGF-β) is a pivotal factor for malignant progression by inducing metastasis, angiogenesis and tumor cell proliferation and by mediating the tumors' escape from immunosurveillance. In colorectal, non-small cell lung and prostate cancer in particular expression of the TGF-β1 isoform has been correlated with tumor progression and poor clinical prognosis. Significantly elevated TGF-β1 plasma levels to more than 3fold in colon cancer (median control: 6,200 pg/ml, range: 1,977 - 17,515 pg/ml; median patients: 19,948 pg/ml, range: 5,014 - 38,900 pg/ml) and NSCLC (median control: 3,482 pg/ml, range: 747 - 9,984 pg/ml; median patients: 11,572 pg/ml, range: 4,952 - 30,985 pg/ml) and to more than 4fold in prostate cancer (median control: 2,152 pg/ml, range: 1,275 - 3,958 pg/ml; median patients: 10,066 pg/ml, range: 3,435 - 41,574 pg/ml) further support the role of TGF-β1 as a key tumor promoter. Methods: The in vitro effects of AP 11014, a TGF-β1 specific phosphorothioate antisense oligonucleotide, on TGF-β1 secretion, proliferation, migration of and immunosuppression by various cancer cell lines were determined. Results: AP 11014 significantly reduced TGF-β1 secretion by 43 - 100% in different NSCLC (A549, NCI-H661, SW 900), colon cancer (HCT-116) and prostate cancer (DU-145, PC-3) cell lines. Tumor cell proliferation was inhibited in a dose-dependent manner in all cell lines. In a scratch assay AP 11014 reduced migration of a NSCLC (SW 900) and prostate cancer (PC-3) cell line by max. 65% after 24h. Additionally, AP 11014 reversed immunosuppression mediated by NSCLC (NCI-H661, A-549) and colon cancer (HCT-116) cell derived TGF-β1 by increasing LAK cell cytotoxicity up to 368% of the untreated control (effector/target ratio 5:1). Conclusion: These preclinical data clearly indicate antisense mediated suppression of TGF-β1 by AP 11014 as a highly promising approach for the therapy of non-small cell lung, colorectal and prostate cancer in humans. Based on these data a clinical trial with AP 11014 in TGF-β1 overexpressing tumors is planned. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antisense Pharma GmbH Antisense Pharma GmbH