- Email: [email protected]
Su1832 Cetuximab and Panitumumab Do Not Inhibit Reg4-CD44-Mediated Activation of EGFR in Colorectal Cancer Cells
Su1833
BACKGROUND & AIMS: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase expressed in epithelial cells of the normal gastrointestinal tract, where its expression is associated with growth inhibition, differentiation, and DNA-damage induced apoptosis. However, PTK6 also promotes the development of colon cancer; disruption of the Ptk6 gene in the mouse leads to resistance to azoxymethane-induced colon carcinogenesis. The aim of our study was to identify novel PTK6 substrates and interacting proteins in intestinal cells to gain a better understanding of the signaling pathways regulated by PTK6. METHODS: Ectopic expression of active PTK6 in the SW620 human colon carcinoma cell line led to robust tyrosine phosphorylation of proteins at 120 kDa, 80 kDa and 60 kDa. These tyrosine phosphorylated proteins were immunoprecipitated from SW620 cells using anti-phosphotyrosine antibodies, subjected to SDS-PAGE and excised for mass spectrometry sequencing. Several interesting and potentially novel PTK6 substrates and interacting proteins along with previously confirmed substrates of PTK6 were identified from the mass spectrometry analysis. RESULTS: Known substrates of PTK6 such as the RNA-binding proteins Sam68 and PSF (PTB-associated splicing factor), as well as Paxillin were identified by mass spectrometry. In addition, several proteins not previously linked to PTK6 signaling were identified, including p130 Crk-associated substrate (p130CAS) and focal adhesion kinase (FAK). We demonstrate that p130CAS and FAK are novel direct substrates of PTK6. Several tyrosine residues are targeted by PTK6 on both proteins and have been identified using mass spectrometry and phospho-specific antibodies. Both proteins associate with PTK6. Using Src/Yes/Fyn deficient mouse embryonic fibroblasts, we show that PTK6 phosphorylates p130Cas and FAK in a Src kinase-independent manner. CONCLUSIONS: p130CAS and FAK are novel direct substrates of PTK6 that are involved in cell spreading, adhesion, and migration. The functional significance of PTK6 mediated phosphorylation and association with p130CAS and FAK in intestinal epithelial cells is currently under investigation.
OR, odds ratio; CI, confidence interval; * defined as hip, vertebral or Colles' fractures Su1831 Variation in Association of CRC in Prostate Cancer Patients Destined for Radiation Therapy Across Different Ethnic Groups Martin Tobi, Yu-Xiao Yang, Keith Davies, Bradley Irwin, Steven Marcus The association between external beam radiation and later development of colorectal cancer (CRC) remains controversial. We reported (DDW 2011) the incidence of CRC for each fiscal year (FY) before diagnosis, from diagnosis to 5 years of follow-up, and after follow-up in irradiated and non-irradiated prostate cancer patients from the VHA National Database (VHAND) to determine a basis for suggested early screening for CRC within 5 years of irradiation. We confirmed a significant increase of CRC in irradiated patients. The overall proportion of CRC in the irradiation group (1,635/24,706) was greater (15,846/442,238) than the non-irradiation group (RR1.76;CI1.68-1.85;p<0.0001). In the current study, we aimed to determine whether this association varies across different racial groups. Methods: We interrogated the VHAND using relevant ICD-9 and CPT codes to identify all patients with a diagnosis of prostate cancer who either were destined to receive irradiation (i.e., irradiation group) or never received irradiation (i.e., no irradiation group) for the period of FY 1999 to FY 2006. We then calculated the relative risk of developing CRC between the irradiation group and the no irradiation group stratified by racial groups. Results The relative risk of developing colorectal cancer between the irradiation group and the no irradiation group within each racial group is shown in the following table: Conclusions: The increase in CRC risk associated with being destined to receive irradiation is much more pronounced among African Americans and Hispanics compared to Caucasians and other racial groups. Future studies should be conducted to define the underlying reasons to explain this phenomenon
Su1834 Reversal of ABCG2-Mediated Multidrug Resistance by the c-JUN NH2Terminal Kinase Signaling Pathway in Colon Cancer Ming Ming Zhu, Jin L. Tong, Qi Xu, Xi T. Xu, Fang Nie, Zhi H. Ran Background and Aim: Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy of colon cancer. Apart from well-studied P-glycoprotein and MRP1, ABCG2 as another member of the ABC transporters, is known to play a crucial role in the development of MDR. However, the molecular mechanism of controlling ABCG2 expression in drug resistance of colon cancer is unclear and scarcely reported. In the hydroxycamptothecin (HCPT) resistant cell line SW1116/HCPT that is from human colon cancer cell line SW1116, ABCG2 is the major transporter inducing drug resistance, but not P-glycoprotein or MRP1. In the present study, we systematically investigate the potential role of the JNK/c-jun signal pathway in ABCG2-induced MDR in colon cancer. Methods: The activation levels of JNK and downstream transcription factor c-Jun were detected in SW1116 and SW1116/HCPT cells by western blot analysis. The specific inhibitor SP600125 and siRNA targeting the JNK1/2 gene were used to block the JNK/c-jun pathway. Then the mRNA and protein expression of ABCG2 were measured by Taqman real-time PCR and Western blot. The transport functions of ABCG2 and cell apoptosis were demonstrated by flow cytometry. Cell viabilities were determined by MTT at different endpoints. Results: The activation level of JNK/c-jun in SW1116/HCPT cells was higher than that in SW1116 cells. SP600125 could inhibit the activation of JNK and downstream transcription factor c-Jun. After treatment with SP600125 for 48 hours, the mRNA and protein expression of ABCG2 were down regulated 67.83±3.10% and 54.29±5.19% in SW1116/HCPT cells, respectively. The accumulation of drug in cells was increased 4.76±0.29 times. Notably, the experiments of siRNA directed against JNK1/2 indicated that partial silence of JNK1 gene increased the dephosphorylation of transcription factor c-Jun, suppressed ABCG2 protein expression 54.36±3.66%, raised the intracellular concentration of ABCG2 substrates 2.61±0.15 times, and increased the sensitivity of the MDR cells to HCPT by > 7-fold, while no corresponding function was observed after silence of JNK2 gene. Moreover, inhibition of JNK pathway induced the apoptosis of SW1116/HCPT cells by promoting the cleavage of PARP and suppressing the anti-apoptotic protein survivin and bcl-2. Conclusion: Taken together, our work indicates that JNK1/c-jun signaling pathway is involved in ABCG2-mediated MDR in colon cancer cells. Definitely, inhibition of the JNK1/c-jun pathway may be useful for reversing ABCG2-related resistance in HCPT-resistant colon cancer cells.
* no irradiation group as the reference group; Cl-Confidence Intervals Su1832 Cetuximab and Panitumumab Do Not Inhibit Reg4-CD44-Mediated Activation of EGFR in Colorectal Cancer Cells Kumar S. Bishnupuri, Satheesh K. Sainathan, Wu Jin, Brian K. Dieckgraefe Colorectal cancer (CRC) is a leading cause of cancer death. We identified Regenerating gene 4 (Reg4) as an important regulator of CRC growth. We previously reported Reg4-mediated EGFR activation to be involved in the induction of CRC cell proliferation and observed an increase of CRC cells' susceptibility to irradiation-induced apoptosis following an antagonism of Reg4-signaling using Reg4-specific monoclonal antibodies (mAbs) and siRNAs. Cetuximab and panitumumab are FDA-approved mAbs used for the treatment of CRC, which block EGF-mediated EGFR activation and associated CRC growth. However, it is not known if these mAbs block Reg4-mediated EGFR activation. This study tests the hypothesis that cetuximab and panitumumab will not block Reg4-mediated EGFR activation and associated CRC growth. By western blot analysis, we observed an EGF and Reg4-mediated activation of EGFR phosphorylation at Tyr1068 residues. Addition of cetuximab and panitumumab to CRC cell cultures prior to EGF treatment inhibited EGF-mediated EGFR phosphorylation; however, similar treatments did not block Reg4-mediated EGFR phosphorylation at Tyr1068 residues. In addition, these treatments didn't inhibit Reg4-mediated induction of Akt activity, a key component of Reg4-EGFR signaling pathway. In order to define the mechanism of Reg4-mediated activation of EGFR, we observed a direct binding of Reg4 protein to CD44, a glycoprotein whose expression on the cell surface changes profoundly during tumor metastasis, particularly during the progression of various carcinomas. Previous studies have shown that CD44 can transactivate the EGFR. In this study, we demonstrated the involvement of CD44 receptor in Reg4-mediated activation of EGFR. These results suggested that EGFR is not only activated by its direct binding to EGF family ligands, but also by the binding of Reg4 to CD44 receptor, which transactivates EGFR to induce downstream signaling pathways associated with CRC growth. Therefore, interruptions of Reg4-CD44 binding will likely exhibit synergy with existing anti-EGFR biologic agents, and provide a basis for more effective treatment of colorectal cancer and other gastrointestinal malignancies.
Su1835 Regulation of Epithelial Mesenchymal Transition Through Protein Kinase CK2 in Helicobacter pylori Infected Gastric Cancer Cells Yeo Song Lee, Ki Taek Nam, SangHun Lee, Dayeon Yu, Go Choi, Sung Kwan Shin, Yong Chan Lee Background/Aims; Helicobacter pylori (H. pylori) has been identified as the major etiological agent in the development of gastric carcinoma. H. pylori harbor a pathogenicity island encoding oncoprotein, CagA which is injected into gastric epithelial cells through T4SS. The CagA-host protein interactions disrupt tight and adherent junctions, leading to a loss of cell polarity and may evolve into invasive phenotype. Protein kinase CK2 (PKCK2) plays a crucial role in many cellular processes and regulates several important growth/survival related signal pathways such as WNT/ β-Catenin, NF-kB and DNA damage responsible pathways. Elevated PKCK2 has pro-survival and anti-apoptotic consequences and it has been showed recently that elevated CK2 is crucial for cancer growth and metastasis. The aim of this study was to evaluate the role of CK2 in epithelial mesenchymal transition (EMT) through α/β-catenin phosphorylation in H. pylori stimulated gastric epithelial cells. Methods; H. pylori strains 60190 (cagPAI+), 8822 (cagPAI-), and ΔcagA (an isogenic mutant of 60190) were used. H. pylori were cultured on blood agar plates containing 10% horse serum at
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AGA Abstracts
AGA Abstracts
Identification and Characterization of Novel Protein Tyrosine Kinase 6 Substrates and Associated Proteins in Intestinal Cells Jessica Gierut, Yu Zheng, Angela L. Tyner
BACKGROUND & AIMS: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase expressed in epithelial cells of the normal gastrointestinal tract, where its expression is associated with growth inhibition, differentiation, and DNA-damage induced apoptosis. However, PTK6 also promotes the development of colon cancer; disruption of the Ptk6 gene in the mouse leads to resistance to azoxymethane-induced colon carcinogenesis. The aim of our study was to identify novel PTK6 substrates and interacting proteins in intestinal cells to gain a better understanding of the signaling pathways regulated by PTK6. METHODS: Ectopic expression of active PTK6 in the SW620 human colon carcinoma cell line led to robust tyrosine phosphorylation of proteins at 120 kDa, 80 kDa and 60 kDa. These tyrosine phosphorylated proteins were immunoprecipitated from SW620 cells using anti-phosphotyrosine antibodies, subjected to SDS-PAGE and excised for mass spectrometry sequencing. Several interesting and potentially novel PTK6 substrates and interacting proteins along with previously confirmed substrates of PTK6 were identified from the mass spectrometry analysis. RESULTS: Known substrates of PTK6 such as the RNA-binding proteins Sam68 and PSF (PTB-associated splicing factor), as well as Paxillin were identified by mass spectrometry. In addition, several proteins not previously linked to PTK6 signaling were identified, including p130 Crk-associated substrate (p130CAS) and focal adhesion kinase (FAK). We demonstrate that p130CAS and FAK are novel direct substrates of PTK6. Several tyrosine residues are targeted by PTK6 on both proteins and have been identified using mass spectrometry and phospho-specific antibodies. Both proteins associate with PTK6. Using Src/Yes/Fyn deficient mouse embryonic fibroblasts, we show that PTK6 phosphorylates p130Cas and FAK in a Src kinase-independent manner. CONCLUSIONS: p130CAS and FAK are novel direct substrates of PTK6 that are involved in cell spreading, adhesion, and migration. The functional significance of PTK6 mediated phosphorylation and association with p130CAS and FAK in intestinal epithelial cells is currently under investigation.
OR, odds ratio; CI, confidence interval; * defined as hip, vertebral or Colles' fractures Su1831 Variation in Association of CRC in Prostate Cancer Patients Destined for Radiation Therapy Across Different Ethnic Groups Martin Tobi, Yu-Xiao Yang, Keith Davies, Bradley Irwin, Steven Marcus The association between external beam radiation and later development of colorectal cancer (CRC) remains controversial. We reported (DDW 2011) the incidence of CRC for each fiscal year (FY) before diagnosis, from diagnosis to 5 years of follow-up, and after follow-up in irradiated and non-irradiated prostate cancer patients from the VHA National Database (VHAND) to determine a basis for suggested early screening for CRC within 5 years of irradiation. We confirmed a significant increase of CRC in irradiated patients. The overall proportion of CRC in the irradiation group (1,635/24,706) was greater (15,846/442,238) than the non-irradiation group (RR1.76;CI1.68-1.85;p<0.0001). In the current study, we aimed to determine whether this association varies across different racial groups. Methods: We interrogated the VHAND using relevant ICD-9 and CPT codes to identify all patients with a diagnosis of prostate cancer who either were destined to receive irradiation (i.e., irradiation group) or never received irradiation (i.e., no irradiation group) for the period of FY 1999 to FY 2006. We then calculated the relative risk of developing CRC between the irradiation group and the no irradiation group stratified by racial groups. Results The relative risk of developing colorectal cancer between the irradiation group and the no irradiation group within each racial group is shown in the following table: Conclusions: The increase in CRC risk associated with being destined to receive irradiation is much more pronounced among African Americans and Hispanics compared to Caucasians and other racial groups. Future studies should be conducted to define the underlying reasons to explain this phenomenon
Su1834 Reversal of ABCG2-Mediated Multidrug Resistance by the c-JUN NH2Terminal Kinase Signaling Pathway in Colon Cancer Ming Ming Zhu, Jin L. Tong, Qi Xu, Xi T. Xu, Fang Nie, Zhi H. Ran Background and Aim: Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy of colon cancer. Apart from well-studied P-glycoprotein and MRP1, ABCG2 as another member of the ABC transporters, is known to play a crucial role in the development of MDR. However, the molecular mechanism of controlling ABCG2 expression in drug resistance of colon cancer is unclear and scarcely reported. In the hydroxycamptothecin (HCPT) resistant cell line SW1116/HCPT that is from human colon cancer cell line SW1116, ABCG2 is the major transporter inducing drug resistance, but not P-glycoprotein or MRP1. In the present study, we systematically investigate the potential role of the JNK/c-jun signal pathway in ABCG2-induced MDR in colon cancer. Methods: The activation levels of JNK and downstream transcription factor c-Jun were detected in SW1116 and SW1116/HCPT cells by western blot analysis. The specific inhibitor SP600125 and siRNA targeting the JNK1/2 gene were used to block the JNK/c-jun pathway. Then the mRNA and protein expression of ABCG2 were measured by Taqman real-time PCR and Western blot. The transport functions of ABCG2 and cell apoptosis were demonstrated by flow cytometry. Cell viabilities were determined by MTT at different endpoints. Results: The activation level of JNK/c-jun in SW1116/HCPT cells was higher than that in SW1116 cells. SP600125 could inhibit the activation of JNK and downstream transcription factor c-Jun. After treatment with SP600125 for 48 hours, the mRNA and protein expression of ABCG2 were down regulated 67.83±3.10% and 54.29±5.19% in SW1116/HCPT cells, respectively. The accumulation of drug in cells was increased 4.76±0.29 times. Notably, the experiments of siRNA directed against JNK1/2 indicated that partial silence of JNK1 gene increased the dephosphorylation of transcription factor c-Jun, suppressed ABCG2 protein expression 54.36±3.66%, raised the intracellular concentration of ABCG2 substrates 2.61±0.15 times, and increased the sensitivity of the MDR cells to HCPT by > 7-fold, while no corresponding function was observed after silence of JNK2 gene. Moreover, inhibition of JNK pathway induced the apoptosis of SW1116/HCPT cells by promoting the cleavage of PARP and suppressing the anti-apoptotic protein survivin and bcl-2. Conclusion: Taken together, our work indicates that JNK1/c-jun signaling pathway is involved in ABCG2-mediated MDR in colon cancer cells. Definitely, inhibition of the JNK1/c-jun pathway may be useful for reversing ABCG2-related resistance in HCPT-resistant colon cancer cells.
* no irradiation group as the reference group; Cl-Confidence Intervals Su1832 Cetuximab and Panitumumab Do Not Inhibit Reg4-CD44-Mediated Activation of EGFR in Colorectal Cancer Cells Kumar S. Bishnupuri, Satheesh K. Sainathan, Wu Jin, Brian K. Dieckgraefe Colorectal cancer (CRC) is a leading cause of cancer death. We identified Regenerating gene 4 (Reg4) as an important regulator of CRC growth. We previously reported Reg4-mediated EGFR activation to be involved in the induction of CRC cell proliferation and observed an increase of CRC cells' susceptibility to irradiation-induced apoptosis following an antagonism of Reg4-signaling using Reg4-specific monoclonal antibodies (mAbs) and siRNAs. Cetuximab and panitumumab are FDA-approved mAbs used for the treatment of CRC, which block EGF-mediated EGFR activation and associated CRC growth. However, it is not known if these mAbs block Reg4-mediated EGFR activation. This study tests the hypothesis that cetuximab and panitumumab will not block Reg4-mediated EGFR activation and associated CRC growth. By western blot analysis, we observed an EGF and Reg4-mediated activation of EGFR phosphorylation at Tyr1068 residues. Addition of cetuximab and panitumumab to CRC cell cultures prior to EGF treatment inhibited EGF-mediated EGFR phosphorylation; however, similar treatments did not block Reg4-mediated EGFR phosphorylation at Tyr1068 residues. In addition, these treatments didn't inhibit Reg4-mediated induction of Akt activity, a key component of Reg4-EGFR signaling pathway. In order to define the mechanism of Reg4-mediated activation of EGFR, we observed a direct binding of Reg4 protein to CD44, a glycoprotein whose expression on the cell surface changes profoundly during tumor metastasis, particularly during the progression of various carcinomas. Previous studies have shown that CD44 can transactivate the EGFR. In this study, we demonstrated the involvement of CD44 receptor in Reg4-mediated activation of EGFR. These results suggested that EGFR is not only activated by its direct binding to EGF family ligands, but also by the binding of Reg4 to CD44 receptor, which transactivates EGFR to induce downstream signaling pathways associated with CRC growth. Therefore, interruptions of Reg4-CD44 binding will likely exhibit synergy with existing anti-EGFR biologic agents, and provide a basis for more effective treatment of colorectal cancer and other gastrointestinal malignancies.
Su1835 Regulation of Epithelial Mesenchymal Transition Through Protein Kinase CK2 in Helicobacter pylori Infected Gastric Cancer Cells Yeo Song Lee, Ki Taek Nam, SangHun Lee, Dayeon Yu, Go Choi, Sung Kwan Shin, Yong Chan Lee Background/Aims; Helicobacter pylori (H. pylori) has been identified as the major etiological agent in the development of gastric carcinoma. H. pylori harbor a pathogenicity island encoding oncoprotein, CagA which is injected into gastric epithelial cells through T4SS. The CagA-host protein interactions disrupt tight and adherent junctions, leading to a loss of cell polarity and may evolve into invasive phenotype. Protein kinase CK2 (PKCK2) plays a crucial role in many cellular processes and regulates several important growth/survival related signal pathways such as WNT/ β-Catenin, NF-kB and DNA damage responsible pathways. Elevated PKCK2 has pro-survival and anti-apoptotic consequences and it has been showed recently that elevated CK2 is crucial for cancer growth and metastasis. The aim of this study was to evaluate the role of CK2 in epithelial mesenchymal transition (EMT) through α/β-catenin phosphorylation in H. pylori stimulated gastric epithelial cells. Methods; H. pylori strains 60190 (cagPAI+), 8822 (cagPAI-), and ΔcagA (an isogenic mutant of 60190) were used. H. pylori were cultured on blood agar plates containing 10% horse serum at
S-515
AGA Abstracts
AGA Abstracts
Identification and Characterization of Novel Protein Tyrosine Kinase 6 Substrates and Associated Proteins in Intestinal Cells Jessica Gierut, Yu Zheng, Angela L. Tyner