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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
SNU-449, and SNU-475. ELF expression was lost in one human HCC cell line (SNU-398), and decreased in SNU-182 and SNU475. TBRII expression was lost in three cell lines (SNU-398, SNU-182, and SNU-475). 2. Immunohistochemical analysis of cell cycle protein expression revealed increased expression of cyclin D1 and Cdk4. 3. Restoration of ELF protein in human HCC cell line results in 2-fold reduction in cyclin D1 expression in SNU-182 and 3.5-fold reduction in SNU 398. 4. The decrease in cyclin D1 is accompanied by decreased expression of hyperphosphorylated-Rb in these two cell lines. 5. Further analysis of the role of ELF in human HCC confirms markedly reduced nuclear expression of ELF in 9 out of 10 human HCC samples by immunohistochemistry (p⬍0.01). Conclusions: Diminished or absent ELF expression in mouse and human HCC as well as cell cycle deregulation are due to disruption of TGF-beta signaling. Loss of ELF can serve as a primary event in progression towards a fully transformed phenotype. Exploration of the mechanisms behind inactivation of the TGF-beta signaling pathway and its restoration holds promise for new therapeutic approaches in human hepatocellular cancer.
P72. MODULATING AKT ACTIVITY ALTERS CELLULAR EFFECTS OF CHEMOTHERAPY IN PANCREATIC CANCER. C. M. Parsons, T. L. Bowles, S. Virudachalam, R. J. Bold; U.C. Davis Medical Center, Sacramento, CA Background: Apoptotic resistance to current chemotherapeutic agents is a significant barrier to effective therapy in pancreatic cancer. The PI3K/Akt signaling pathway is constitutively activated in pancreatic cancer and blocks induction of apoptosis. Studies in various tumors demonstrate improved efficacy of chemotherapy with inhibition of the PI3K/Akt pathway. We hypothesized that the level of Akt activity correlates with the apoptotic resistance to chemotherapy observed in pancreatic cancer. Methods: Mia-PaCa-2 and PANC-1 human pancreatic cells were used for all studies. Akt activity was increased by transfection of a constitutively active myr-Akt plasmid, while knockdown was achieved using Akt siRNA or a novel small molecule inhibitor (A-443654). Western blot analysis and Akt kinase assays were used to evaluate the change in Akt activity. Chemotherapies examined include paclitaxel and gemcitabine. Effects on cell cycle distribution and apoptosis were assessed with fluorescence activated cell sorting (FACS). Induction of apoptosis was also quantitated using an ELISA for the active form of caspase-3. Results: Mia-PaCa-2 demonstrated greater Akt activity than PANC-1; Akt activity could be successfully increased by transfecting the myr-Akt plasmid, while inhibition was achieved using either Akt siRNA or A-443654. Inhibition of Akt induced apoptosis in both cell lines to similar degrees (Figure 1A). In both cell lines, paclitaxel induced a G2/M cell cycle arrest while gemcitabine induced a G0 cell cycle arrest with subsequent induction of apoptosis. Unexpectedly, increasing Akt activity potentiated the G2/M cell cycle arrest following paclitaxel treatment while Akt inhibition reduced the G2/M cell cycle arrest and paclitaxel-induced apoptosis (Figure 1B, data shown for Mia-PaCa-2). Conversely, Akt inhibition increased the induction of gemcitabine-induced apoptosis. Paclitaxel treatment increased caspase-3 activity; however, neither increasing nor decreasing Akt activity significantly altered the ability of paclitaxel to activate caspase-3. Conclusions: Inhibition of Akt permits the induction of apoptosis independent of basal Akt activity, suggesting a role for Akt in the generalized promotion of cell survival in pancreatic cancer cells. However, Akt appears to mediate the induction of G2/M arrest by paclitaxel, so that inhibiting Akt reduced the efficacy of paclitaxel; these effects were not observed with gemcitabine. Therefore, the cellular effect of combining inhibition of Akt with conventional chemotherapy is dependent on the specific chemotherapy selected. In conclusion, detailed elucidation of the cellular events regulated by Akt is essential before combining Akt-directed therapy with conventional chemotherapy.
P73. TGFBETA SIGNALING AND GATA6 COOPERATIVELY REGULATE THE UROKINASE PLASMINOGEN ACTIVATOR (UPA). N. S. Belaguli, M. Zhang, M. Aftab, M. Rigi, D. H. Berger; Baylor College of Medicine, Houston, TX Introduction: uPA is a serine protease associated with tumor cell invasion and metastasis. TGF, normally an inhibitor of epithelial growth and a promoter of epithelial differentiation, is also a promoter of tumor cell invasion and metastasis in late stage colon cancer. This tumor promoting effect of TGF is mediated, in part, through upregulation of uPA. Recently, we have demonstrated that uPA gene expression is activated by GATA6, a member of GATA family of zinc finger proteins expressed in human colon cancers and cancer-derived cell lines. We hypothesized that the effect of the TGF signaling pathway on the activation of uPA gene expression, in colon cancer, is mediated through association with GATA6. Materials and Methods: uPA promoter activity was analyzed by transfecting wild type and deletion mutants of uPA promoter-luciferase vector into HCT116 cells along with GATA6 expression vector. Cooperative activation of uPA promoter was assessed by cotransfecting GATA6 and Smad2/3/4 expression vectors along with the uPA promoter-luciferase reporter. Physical interaction between GATA6 and Smad2/3/4 was analyzed by coimmunoprecipitation and GST fusion protein pull-down experiments. Results: GATA6 strongly activated uPA promoter activity and this activation required at least 520 bp of the uPA promoter region. GATA6 mediated activation of the uPA promoter was further enhanced by coexpression of Smad2/ 3/4. In vitro GST fusion protein pull-down experiments demonstrated physical association between GATA6 and Smad2/3/4. These results were confirmed by coimmunoprecipitation experiments performed in HCT116 cells with HA epitope tagged GATA6 and FLAG epitope tagged Smad2/3/4. Conclusion: Physical interaction between GATA6, which is expressed strongly in colon cancer cells, and TGF signaling factors such as, Smad2/3/4, activate uPA gene expression cooperatively. P74. TOLL-LIKE RECEPTOR-4 SIGNALING DOES NOT INCREASE RATE OF METASTATIC TUMOR ENGRAFTMENT IN A MURINE MODEL OF COLORECTAL CARCINOMA. T. M. Earl, I. B. Nicoud, J. Pierce, J. P. Wright, C. M. Jones, R. S. Chari; Vanderbilt University Medical Center, Nashville, TN Introduction: Obesity and newer chemotherapeutic agents used to treat metastatic colorectal carcinoma are associated with hepatic steatosis and Toll-like receptor-4 (TLR4) is increased in fatty liver. TLR4 is known to have pro-neoplastic effects in primary malignancies; however, the effect of hepatic TLR4 on metastatic liver tumor
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
SNU-449, and SNU-475. ELF expression was lost in one human HCC cell line (SNU-398), and decreased in SNU-182 and SNU475. TBRII expression was lost in three cell lines (SNU-398, SNU-182, and SNU-475). 2. Immunohistochemical analysis of cell cycle protein expression revealed increased expression of cyclin D1 and Cdk4. 3. Restoration of ELF protein in human HCC cell line results in 2-fold reduction in cyclin D1 expression in SNU-182 and 3.5-fold reduction in SNU 398. 4. The decrease in cyclin D1 is accompanied by decreased expression of hyperphosphorylated-Rb in these two cell lines. 5. Further analysis of the role of ELF in human HCC confirms markedly reduced nuclear expression of ELF in 9 out of 10 human HCC samples by immunohistochemistry (p⬍0.01). Conclusions: Diminished or absent ELF expression in mouse and human HCC as well as cell cycle deregulation are due to disruption of TGF-beta signaling. Loss of ELF can serve as a primary event in progression towards a fully transformed phenotype. Exploration of the mechanisms behind inactivation of the TGF-beta signaling pathway and its restoration holds promise for new therapeutic approaches in human hepatocellular cancer.
P72. MODULATING AKT ACTIVITY ALTERS CELLULAR EFFECTS OF CHEMOTHERAPY IN PANCREATIC CANCER. C. M. Parsons, T. L. Bowles, S. Virudachalam, R. J. Bold; U.C. Davis Medical Center, Sacramento, CA Background: Apoptotic resistance to current chemotherapeutic agents is a significant barrier to effective therapy in pancreatic cancer. The PI3K/Akt signaling pathway is constitutively activated in pancreatic cancer and blocks induction of apoptosis. Studies in various tumors demonstrate improved efficacy of chemotherapy with inhibition of the PI3K/Akt pathway. We hypothesized that the level of Akt activity correlates with the apoptotic resistance to chemotherapy observed in pancreatic cancer. Methods: Mia-PaCa-2 and PANC-1 human pancreatic cells were used for all studies. Akt activity was increased by transfection of a constitutively active myr-Akt plasmid, while knockdown was achieved using Akt siRNA or a novel small molecule inhibitor (A-443654). Western blot analysis and Akt kinase assays were used to evaluate the change in Akt activity. Chemotherapies examined include paclitaxel and gemcitabine. Effects on cell cycle distribution and apoptosis were assessed with fluorescence activated cell sorting (FACS). Induction of apoptosis was also quantitated using an ELISA for the active form of caspase-3. Results: Mia-PaCa-2 demonstrated greater Akt activity than PANC-1; Akt activity could be successfully increased by transfecting the myr-Akt plasmid, while inhibition was achieved using either Akt siRNA or A-443654. Inhibition of Akt induced apoptosis in both cell lines to similar degrees (Figure 1A). In both cell lines, paclitaxel induced a G2/M cell cycle arrest while gemcitabine induced a G0 cell cycle arrest with subsequent induction of apoptosis. Unexpectedly, increasing Akt activity potentiated the G2/M cell cycle arrest following paclitaxel treatment while Akt inhibition reduced the G2/M cell cycle arrest and paclitaxel-induced apoptosis (Figure 1B, data shown for Mia-PaCa-2). Conversely, Akt inhibition increased the induction of gemcitabine-induced apoptosis. Paclitaxel treatment increased caspase-3 activity; however, neither increasing nor decreasing Akt activity significantly altered the ability of paclitaxel to activate caspase-3. Conclusions: Inhibition of Akt permits the induction of apoptosis independent of basal Akt activity, suggesting a role for Akt in the generalized promotion of cell survival in pancreatic cancer cells. However, Akt appears to mediate the induction of G2/M arrest by paclitaxel, so that inhibiting Akt reduced the efficacy of paclitaxel; these effects were not observed with gemcitabine. Therefore, the cellular effect of combining inhibition of Akt with conventional chemotherapy is dependent on the specific chemotherapy selected. In conclusion, detailed elucidation of the cellular events regulated by Akt is essential before combining Akt-directed therapy with conventional chemotherapy.
P73. TGFBETA SIGNALING AND GATA6 COOPERATIVELY REGULATE THE UROKINASE PLASMINOGEN ACTIVATOR (UPA). N. S. Belaguli, M. Zhang, M. Aftab, M. Rigi, D. H. Berger; Baylor College of Medicine, Houston, TX Introduction: uPA is a serine protease associated with tumor cell invasion and metastasis. TGF, normally an inhibitor of epithelial growth and a promoter of epithelial differentiation, is also a promoter of tumor cell invasion and metastasis in late stage colon cancer. This tumor promoting effect of TGF is mediated, in part, through upregulation of uPA. Recently, we have demonstrated that uPA gene expression is activated by GATA6, a member of GATA family of zinc finger proteins expressed in human colon cancers and cancer-derived cell lines. We hypothesized that the effect of the TGF signaling pathway on the activation of uPA gene expression, in colon cancer, is mediated through association with GATA6. Materials and Methods: uPA promoter activity was analyzed by transfecting wild type and deletion mutants of uPA promoter-luciferase vector into HCT116 cells along with GATA6 expression vector. Cooperative activation of uPA promoter was assessed by cotransfecting GATA6 and Smad2/3/4 expression vectors along with the uPA promoter-luciferase reporter. Physical interaction between GATA6 and Smad2/3/4 was analyzed by coimmunoprecipitation and GST fusion protein pull-down experiments. Results: GATA6 strongly activated uPA promoter activity and this activation required at least 520 bp of the uPA promoter region. GATA6 mediated activation of the uPA promoter was further enhanced by coexpression of Smad2/ 3/4. In vitro GST fusion protein pull-down experiments demonstrated physical association between GATA6 and Smad2/3/4. These results were confirmed by coimmunoprecipitation experiments performed in HCT116 cells with HA epitope tagged GATA6 and FLAG epitope tagged Smad2/3/4. Conclusion: Physical interaction between GATA6, which is expressed strongly in colon cancer cells, and TGF signaling factors such as, Smad2/3/4, activate uPA gene expression cooperatively. P74. TOLL-LIKE RECEPTOR-4 SIGNALING DOES NOT INCREASE RATE OF METASTATIC TUMOR ENGRAFTMENT IN A MURINE MODEL OF COLORECTAL CARCINOMA. T. M. Earl, I. B. Nicoud, J. Pierce, J. P. Wright, C. M. Jones, R. S. Chari; Vanderbilt University Medical Center, Nashville, TN Introduction: Obesity and newer chemotherapeutic agents used to treat metastatic colorectal carcinoma are associated with hepatic steatosis and Toll-like receptor-4 (TLR4) is increased in fatty liver. TLR4 is known to have pro-neoplastic effects in primary malignancies; however, the effect of hepatic TLR4 on metastatic liver tumor