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Targeting RET G691S single nucleotide polymorphism and oncogene addiction in pancreatic cancer cells
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Surgical Forum Abstracts
quantitative chromatin immunoprecipitation (ChIP) was performed using mouse KH20 and KH95 E2F antibodies. RESULTS: ELISA showed 43% of normal BMPR1A protein levels compared to controls in the JP patient with the E2F mutation. Cotransfection of BMPR1A wt promoter with the E2F expression vector resulted in an 11.3 fold increase in Luciferase activity as compared to completely abolished promoter activity in the E2F mutant construct. Semi-quantitative ChIP confirmed binding of E2F to the regulatory region of the BMPR1A promoter. CONCLUSIONS: We have shown that E2F is a key factor in the regulation of BMPR1A transcription, and this study provides evidence that germline mutation in the E2F RBS of the BMPR1A promoter causes JP.
Molecular network, pathway and functional analysis of gene signatures associated with colon cancer stem cells Dana Haddad MD, Susanne G Carpenter MD, Nicholas D Socci PhD, Yuman Fong MD, FACS Memorial Sloan-Kettering Cancer Center, New York, NY INTRODUCTION: Cancer stem cells (CSCs) are a subpopulation of cancer cells believed capable of tumor initiation and resistance to adjuvant therapy. This study aimed to determine changes in gene expression patterns associated with putative colon CSCs compared to regular colon cancer cells. METHODS: HCT-8 colon cancer cells were cultured in serum-free medium. CSCs were identified by tumorsphere formation and nonadherent growth, and tumorigenicity confirmed in vivo. HG-U133A cDNA microarray chips were used to compare gene expression patterns of CSCs to regular HCT-8 cells. Data was analyzed using Ingenuity Pathways Analysis software in the context of gene function, networks, and pathways. RESULTS: CSC tumorsphere formation was confirmed in vitro and tumorigenicity confirmed in vivo, with CSCs showing enhanced tumor growth versus regular HCT-8 cells with as little as 1E3 cells. Microarray revealed changes in gene expression, with gene networks involving functions such as cell death, cellular assembly, cell cycle, and DNA repair (all p⬍0.01). Gene changes involved pathways such as BRCA1 in DNA damage response (downregulated, p⬍0.01), altered cell cycle checkpoint control (p⬍0.01), and ERK5 signaling (upregulated, p⬍0.05); as well as observed involvement of CXCR4 signaling, Oct4, AKT/PI3K and Wnt pathways. Preliminary work confirms some of these changes on a protein level, including upregulation of AKT1 in CSCs on western blot. CONCLUSIONS: Our study confirms changes in gene expression patterns in colon CSCs as compared to regular colon cancer cells in vitro. This suggests that molecular assays may be useful to identify potential treatment targets to overcome progression of, and eradicate, this cancer cell subpopulation.
J Am Coll Surg
Targeting RET G691S single nucleotide polymorphism and oncogene addiction in pancreatic cancer cells Christina Prendergast DO, Jianming Lu PhD, Xiaoming Shen PhD, Desmond Jackson PhD, Avo Artinyan MD, MS, Joseph Kim MD, FACS City of Hope, Duarte, CA INTRODUCTION: We previously demonstrated RET G691S in pancreatic cancer, which amplifies receptor tyrosine kinase signaling resulting in increased pancreatic cancer cell invasiveness. We hypothesized that pancreatic cancer cells harboring RET G691S become dependent on RET signaling, rendering these cells more susceptible to targeting with the small molecule RET inhibitor ZD6474. METHODS: Human pancreatic cancer cells were exposed to the RET ligand, glial-derived neurotropic factor (GDNF). We assessed the activation of ERK1/2 by Western blot assay and beta-catenin by immunofluorescence. Changes in cell proliferation and apoptosis were measured by tetrazolium-based and Annexin-V assays, respectively. Cells were exposed to a range of concentrations of ZD6474 for durations of 24-72 h. Cytotoxicity was measured with an acid phosphatase activity assay. RESULTS: RET G691S was detected in pancreatic cancer cells Capan-2 and MIAPaca-2, but not PANC-1 and xPA-1 cells. First, we observed that exposure of cells to GDNF increased the phosphorylation of ERK1/2 in all cells, but only increased cytoplasmic localization of beta-catenin in RET G691S-positive cells. GDNF also induced proliferation but did not alter apoptosis in all cells. We observed lower half maximal inhibitory concentrations (IC50) for Capan-2 and MIAPaCa-2 cells (8 and 10 M, respectively) in comparison to xPA-1 and PANC-1 cells (50 and 25 M, respectively). CONCLUSIONS: Targeting RET G691S with ZD6474 results in enhanced cell cytotoxicity potentially via beta-catenin signaling, suggesting that these cells may require RET signaling for survival. The high incidence of RET G691S indicates that this may be a potential novel target in patients with pancreatic cancer.
Pterostilbene inhibition of pancreatic cancer growth: A genomic profile Patrick W Mannal MD, Debbie McDonald BS, David W McFadden MD, MBA, FACS Fletcher Allen Health Care, Burlington, VT INTRODUCTION: Pterostilbene, a naturally occurring phenolic compound found in grapes and blueberries, has been found to have inhibitory effects on neoplastic cells. To date, Pterostilbene’s precise mechanism of action has been elusive, but experimentally linked to mitochondrially-derived apoptosis in pancreatic cancer cells. Herein, we hope to identify significant genomic alterations in human pancreatic cancer cells after exposure to Pterostilbene. METHODS: MIA-PACA cells were grown in culture and exposed to 50M Pterostilbene in DMSO for 3, 6, 9, and 12 hour increments. The culture media from the 6 and 12 hour time points was harvested and RNA extracted. The RNA was amplified, fragmented, and la-
Surgical Forum Abstracts
quantitative chromatin immunoprecipitation (ChIP) was performed using mouse KH20 and KH95 E2F antibodies. RESULTS: ELISA showed 43% of normal BMPR1A protein levels compared to controls in the JP patient with the E2F mutation. Cotransfection of BMPR1A wt promoter with the E2F expression vector resulted in an 11.3 fold increase in Luciferase activity as compared to completely abolished promoter activity in the E2F mutant construct. Semi-quantitative ChIP confirmed binding of E2F to the regulatory region of the BMPR1A promoter. CONCLUSIONS: We have shown that E2F is a key factor in the regulation of BMPR1A transcription, and this study provides evidence that germline mutation in the E2F RBS of the BMPR1A promoter causes JP.
Molecular network, pathway and functional analysis of gene signatures associated with colon cancer stem cells Dana Haddad MD, Susanne G Carpenter MD, Nicholas D Socci PhD, Yuman Fong MD, FACS Memorial Sloan-Kettering Cancer Center, New York, NY INTRODUCTION: Cancer stem cells (CSCs) are a subpopulation of cancer cells believed capable of tumor initiation and resistance to adjuvant therapy. This study aimed to determine changes in gene expression patterns associated with putative colon CSCs compared to regular colon cancer cells. METHODS: HCT-8 colon cancer cells were cultured in serum-free medium. CSCs were identified by tumorsphere formation and nonadherent growth, and tumorigenicity confirmed in vivo. HG-U133A cDNA microarray chips were used to compare gene expression patterns of CSCs to regular HCT-8 cells. Data was analyzed using Ingenuity Pathways Analysis software in the context of gene function, networks, and pathways. RESULTS: CSC tumorsphere formation was confirmed in vitro and tumorigenicity confirmed in vivo, with CSCs showing enhanced tumor growth versus regular HCT-8 cells with as little as 1E3 cells. Microarray revealed changes in gene expression, with gene networks involving functions such as cell death, cellular assembly, cell cycle, and DNA repair (all p⬍0.01). Gene changes involved pathways such as BRCA1 in DNA damage response (downregulated, p⬍0.01), altered cell cycle checkpoint control (p⬍0.01), and ERK5 signaling (upregulated, p⬍0.05); as well as observed involvement of CXCR4 signaling, Oct4, AKT/PI3K and Wnt pathways. Preliminary work confirms some of these changes on a protein level, including upregulation of AKT1 in CSCs on western blot. CONCLUSIONS: Our study confirms changes in gene expression patterns in colon CSCs as compared to regular colon cancer cells in vitro. This suggests that molecular assays may be useful to identify potential treatment targets to overcome progression of, and eradicate, this cancer cell subpopulation.
J Am Coll Surg
Targeting RET G691S single nucleotide polymorphism and oncogene addiction in pancreatic cancer cells Christina Prendergast DO, Jianming Lu PhD, Xiaoming Shen PhD, Desmond Jackson PhD, Avo Artinyan MD, MS, Joseph Kim MD, FACS City of Hope, Duarte, CA INTRODUCTION: We previously demonstrated RET G691S in pancreatic cancer, which amplifies receptor tyrosine kinase signaling resulting in increased pancreatic cancer cell invasiveness. We hypothesized that pancreatic cancer cells harboring RET G691S become dependent on RET signaling, rendering these cells more susceptible to targeting with the small molecule RET inhibitor ZD6474. METHODS: Human pancreatic cancer cells were exposed to the RET ligand, glial-derived neurotropic factor (GDNF). We assessed the activation of ERK1/2 by Western blot assay and beta-catenin by immunofluorescence. Changes in cell proliferation and apoptosis were measured by tetrazolium-based and Annexin-V assays, respectively. Cells were exposed to a range of concentrations of ZD6474 for durations of 24-72 h. Cytotoxicity was measured with an acid phosphatase activity assay. RESULTS: RET G691S was detected in pancreatic cancer cells Capan-2 and MIAPaca-2, but not PANC-1 and xPA-1 cells. First, we observed that exposure of cells to GDNF increased the phosphorylation of ERK1/2 in all cells, but only increased cytoplasmic localization of beta-catenin in RET G691S-positive cells. GDNF also induced proliferation but did not alter apoptosis in all cells. We observed lower half maximal inhibitory concentrations (IC50) for Capan-2 and MIAPaCa-2 cells (8 and 10 M, respectively) in comparison to xPA-1 and PANC-1 cells (50 and 25 M, respectively). CONCLUSIONS: Targeting RET G691S with ZD6474 results in enhanced cell cytotoxicity potentially via beta-catenin signaling, suggesting that these cells may require RET signaling for survival. The high incidence of RET G691S indicates that this may be a potential novel target in patients with pancreatic cancer.
Pterostilbene inhibition of pancreatic cancer growth: A genomic profile Patrick W Mannal MD, Debbie McDonald BS, David W McFadden MD, MBA, FACS Fletcher Allen Health Care, Burlington, VT INTRODUCTION: Pterostilbene, a naturally occurring phenolic compound found in grapes and blueberries, has been found to have inhibitory effects on neoplastic cells. To date, Pterostilbene’s precise mechanism of action has been elusive, but experimentally linked to mitochondrially-derived apoptosis in pancreatic cancer cells. Herein, we hope to identify significant genomic alterations in human pancreatic cancer cells after exposure to Pterostilbene. METHODS: MIA-PACA cells were grown in culture and exposed to 50M Pterostilbene in DMSO for 3, 6, 9, and 12 hour increments. The culture media from the 6 and 12 hour time points was harvested and RNA extracted. The RNA was amplified, fragmented, and la-