- Email: [email protected]
S1991 The PI3K-AKT Pathway Is a Downstream Effector of CD24
towards reducing co-carcinogenic effects of PG peptides. These studies were supported by NIH grants # CA097959 and CA 114264 to PS.
promoter in the cytosol. Here, we dissected ligand-specific effects on p21waf1 expression, modulation and its functional consequences. METHODS: ACVR2/TGFBR2 (WT)/SMAD4 (WT) FET and ACVR2/TGFBR2 (WT)/SMAD4 (null) SW480 colon cancer cells were assessed for p21waf1 expression after ligand treatment and subcellular localization using Western blots. p21waf1 and SMAD4-specific siRNA were used to establish functional consequences of p21waf1 loss and SMAD4 dependency. Cellular viability, apoptosis, and migration were determined using MTT assay, ADP/ATP apoptosis assay, and Boyden chambers. p21-specific transactivation was determined using p21-luciferase, and p21waf1 transcription using quantitative PCR. Expression of activin/TGFβ pathway members was correlated to p21waf1 subcellular localization in primary colon cancers via immunohistochemistry. RESULTS: Activin was a more potent inducer of apoptosis, while TGFβ was more growth suppressive, and both functions were SMAD4-dependent. TGFβ-induced growth suppression coincided with increased nuclear p21waf1 protein through increased p21waf1 transactivation, transcription and protein stabilization. However, activin decreased nuclear p21waf1 protein with a nuclear to cytosolic shift. Induction of p21waf1 by TGFβ was SMAD4-dependent, but decrease of nuclear p21waf1 by activin was SMAD4-independent. Knock down of p21waf1 reversed growth suppressive effects, enhanced survival, and increased the migratory capacity of both ligands. Increased cytosolic p21waf1 in primary colon cancers correlated with intact ACVR2. CONCLUSIONS: Despite identical downstream SMAD signaling, activin and TGFβ have opposing effects on the cdk2 inhibitor p21waf1, resulting in differing effects on growth suppression, migration, and survival in colon cancer. Activin-specific modulation of p21waf1 functions through non-canonical, SMAD-independent pathways and may represent a potential target for therapy of metastatic disease.
AGA Abstracts
S1988 The Absence of the Proto-Oncogene Fer Increases Colon Cancer Development Maitham A. Khajah, Stefan J. Urbanski, Donna-Marie McCafferty Background: The group IV non-receptor protein tyrosine kinase Fer kinase has been described as a proto-oncogene Fer and is implicated in signal transduction pathways regulated by growth factors. Fer is highly expressed in different malignant cell lines and In Vitro downregulation of Fer reduces the proliferation of prostate carcinoma cells and attenuates the development of prostate cancer. The Aim of this study was to determine the role of Fer kinase in an In Vivo model of colitis-associated adenocarcinoma: the interleukin 10 (IL-10\) model. Methods: WT (129SvEv), IL-10-\-, and IL-10-\-FerDR/DR double knockout mice were used at 3 months of age. Macroscopic and histological scoring of inflammation and dysplasia were performed on the colon (n= 13-25 mice). Myeloperoxidase (MPO) activity was determined as an indication of neutrophil recruitment to the colonic tissues. Bone marrow-derived neutrophils chemotaxis toward different chemoattractants (WKYMVm; fMLP peptide, keratinocyte derived cytokine; KC, macrophage inflammatory protein; MIP-2, and granulocyte macrophage colony stimulating factor; GM-CSF) was performed using the under agarose assay In Vitro. Results: A significant increase in inflammatory parameters (macroscopic and histological score, and MPO activity) was noted in IL-10-\- compared to WT mice at 3 months of age. IL-10-\- mice had a 34 % incidence of mucosal hyperplasia (polyps) with no evidence of neoplastic changes, with the exception of 1 (of 14) mouse with low grade dysplasia. A moderate but significant increase in inflammation was observed in IL-10-\-FerDR/ DR double knockout compared to IL-10-\- mice (p<0.05). Surprisingly, in the absence of Fer kinase, IL-10-\- mice had a 52% incidence of polyps with a 61% incidence of adenocarcinoma. Multiple adenocarcinomas were noted in some mice (2 mice). Using bone marrow derived neutrophils we demonstrated a significant increase in chemotaxis of neutrophils from IL10-\- mice compared with wild type (116±12 vs. 48.8±8.3 toward WKYMVm, 45.3±6.1 vs. 21.6±5.4 toward KC, 55.8±8 vs. 23.6±5.3 toward MIP-2, and 101±4.2 vs. 42±4.1 toward GM-CSF respectively). Neutrophil chemotaxis was further enhanced in the absence of Fer kinase (166±8.9) toward WKYMVm only. Conclusions: These data demonstrate for the first time an important role for Fer kinase in regulating the development adenocarcinoma in a chronic model inflammation In Vivo. This may be associated with enhanced leukocyte recruitment to the colon in the absence of Fer kinase contributing to the inflammationdysplasia-adenocarcinoma sequence.
S1991 The PI3K-AKT Pathway Is a Downstream Effector of CD24 Inna Naumov, Sarah Kraus, Diana Kazanov, Shiran Shapira, Eyal Sagiv, Nadir Arber Background: CD24 is a cell-surface molecule, thought to be involved in adhesion and signaling processes. However, a signaling cascade for CD24 has not been identified. CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of gastrointestinal tumors at a fairly early stage in the multistep process of carcinogenesis (Gastroenterology, Vol 131(2) 630639, 2006). Anti-CD24 mAb induced a significant growth inhibition in CD24-expressing colorectal and pancreatic cancer cells (Clin Can Res, Vol 13(22), p. 6807-6815, 2007). Xenograft models showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA) (Can Res, Vol 68(8), 2803-2812, 2008). The PI3K-AKT pathway has been implicated in the molecular pathogenesis of a variety of cancers and its overexpression was recently demonstrated to be an early event in colorectal carcinogenesis. Aim: To examine the role of PI3K-AKT in the signaling cascade leading to growth inhibition induced by anti-CD24 mAb treatment. Methods: Pancreatic cancer cells, Colo357, were treated with anti-CD24 mAb for various time-points (24, 48, 72hrs) and AKT activation was examined by Western blotting with anti-phospho-AKT (Ser473). Cell proliferation was determined by methylene blue assay following exposure to mAb alone or combined with wortmannin (PI3K inhibitor). Cell cycle and apoptosis was evaluated by FACS analysis following PI staining. Results: Cell growth was significantly inhibited in a time- and dosedependent manner. The level of apoptosis was 25% and 49% after 48 and 72hrs respectively, without a change in cell cycle. Combined mAb treatment with wortmannin increased apoptosis by 11%. Surprisingly, mAb treatment resulted in the down-regulation of AKT activation with a significant AKT degradation. Conclusions: Here we report, for the first time, that AKT is a downstream effector of CD24. CD24 inhibits AKT activity. The combined targeting of CD24 and AKT might serve as a potent anticancer treatment. Understanding the functional aspects of CD24-induced signaling will pave the way for successful cancer therapy using anti-CD24 antibodies and selective promotion of apoptosis.
S1989 Identification of a Functional p53 Responsive Element Within the Promoter of XAF1 Gene in Gastrointestinal Cancer Cells Qing Gu, Wenjing Zhang, Jide Wang, Juan Ma, Zesong Li, Hui Y. Lan, Benjamin C.Y. Wong BACKGROUND: XAF1 was first identified as a XIAP-interacting protein and functioned as a tumor suppressor to sensitize cells to apoptosis. XAF1 expression in gastric cancer tissues was negative correlated with p53 status with cells harboring wildtype p53 expressed low level of XAF1. The purpose of this study was to clarify the regulatory mechanism of p53 on XAF1 expression. METHODS: XAF1 expressions and promoter activities in several gastrointestinal (GI) cancer cell lines with different p53 status were detected by western blot and dual luciferase assay. The effects of ectopic over-expressed wildtype and mutant p53 on XAF1 expression were evaluated after transient transfection. A 107bp of XAF1 core promoter were separated into five probes to examine their binding capacity to the recombinant p53 protein. Site-directed mutation of the putative p53 binding sequence and p53 knockdown by siRNA were performed to confirm the interaction between p53 and XAF1 promoter segments. RESULTS: The protein expression and the promoter activities of XAF1 in cell lines with null p53 were higher than that in cell lines with wildtype and mutant p53. Ectopic over-expression of wildtype p53 suppressed XAF1 protein expression and transcriptional activities. A halfsite (-95nt~-86nt, translation starting codon was defined as +1) and a quartersite of p53 responsive element (-4nt~+1nt) were found within XAF1 promoter. Both sequences bound to recombinant p53 effectively determined by electrophoresis mobility shift assay (EMSA) and the binding could be blocked specifically by non-labelled consensus p53 oligonucleotide. Site-mutation of the putative p53 responsive sequences abrogated the binding between probes and p53 protein. However, only the mutation of halfsite but not the quartersite sequence increased XAF1 promoter activities. Suppression of p53 by siRNA could not only decrease the binding capacity of p53 responsive halfsite but also increase XAF1 expression and its promoter activity. CONCLUSIONS: p53 could suppress the transcription of XAF1 through interaction with a high affinity responsive element (-95nt~-86nt) within XAF1 promoter. The significance of this exclusive interaction between these two tumor suppressors remains to be elucidated.
S1992 The Interaction of β-Catenin and Telomerase and Its Role During Carcinogenesis Falk Mancke, Angela Queisser, Heike Kunert, Steffen Heeg, Nina Hirt, Frank Götschel, Andreas Hecht, Steve Artandi, Oliver G. Opitz Introduction: Maintenance of telomere length is one major determinant of immortalization and thus malignant transformation. Most human tumor cells maintain their telomere length through reactivation of telomerase. Recently, several lines of evidence demonstrated that telomerase possesses additional functions during carcinogenesis and hair follicle stem cell biology, independent of its ability to elongate telomeres. Studies investigating the molecular mechanisms of this telomere elongating independent function of telomerase analyzed the genome wide transcriptional response to acute changes of TERT levels, the protein component of telomerase, in mouse skin. Further statistical comparison to other micro array gene sets revealed that TERT exerts its function on hair follicle stem cells through transcriptional control of WNT related genes. Taking these findings in consideration our aim was to further elucidate the telomere elongating independent functions of telomerase during carcinogenesis, investigating the potential interaction between telomerase and WNT signaling components. Methods: We analyzed distinct cell types representing different steps in the malignant transformation of squamous cell carcinoma and adenocarcinoma of the esophagus. We performed transient transfection assays determining the transcriptional activity of the canonical WNT pathway, co-immunoprecipitation experiments looking for protein-protein interactions and immunoprecipitation (IP) experiments with telomerase repeat amplification protocol (TRAP). This allowed us to study interactions of the catalytically active enzyme. To knock down TERT expression we carried out siRNA transfection experiments. Results: IPImmunoblot experiments demonstrate a interaction of β-catenin and TERT in 293T cells. IP-TRAP experiments identified β-catenin and catalytically active telomerase as interaction partners in 293T cells and fully malignant adenocarcinoma cells. Using siRNA mediated knock down of TERT we observed a TERT dependent reduction in transcriptional activity of canonical WNT signaling, suggesting a functional relevance of the described interaction. Despite robust telomerase activity squamous cell carcinoma cells show neither transcriptional
S1990 Opposing P21waf1 Regulation By TGFβ/SMAD4 and Activin/SMAD4 in Colon Cancer Cells Barbara H. Jung, Jennifer Cabral, Przemyslaw K. Slowik, Jessica Gomez, Stayce E. Beck, John M. Carethers BACKGROUND: Activin and transforming growth factor β (TGFβ) are growth suppressive TGFβ family ligands that signal through a primary receptor type 2 receptor (activin receptor 2, ACVR2, and TGFβ receptor 2, TGFBR2, respectively) to activate specific type 1 receptors, ACVR1 and TGFBR1. This is followed by phosphorylation of cytosolic SMAD2/3 that pairs with SMAD4 to translocate to the nucleus as a transcription factor. Despite the utilization of identical SMADs, microsatellite unstable colon cancers inactivate both activin and TGFβ signaling simultaneously. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21waf1, which has growth suppressive properties in the nucleus, but may act as a tumor
AGA Abstracts
A-308
promoter in the cytosol. Here, we dissected ligand-specific effects on p21waf1 expression, modulation and its functional consequences. METHODS: ACVR2/TGFBR2 (WT)/SMAD4 (WT) FET and ACVR2/TGFBR2 (WT)/SMAD4 (null) SW480 colon cancer cells were assessed for p21waf1 expression after ligand treatment and subcellular localization using Western blots. p21waf1 and SMAD4-specific siRNA were used to establish functional consequences of p21waf1 loss and SMAD4 dependency. Cellular viability, apoptosis, and migration were determined using MTT assay, ADP/ATP apoptosis assay, and Boyden chambers. p21-specific transactivation was determined using p21-luciferase, and p21waf1 transcription using quantitative PCR. Expression of activin/TGFβ pathway members was correlated to p21waf1 subcellular localization in primary colon cancers via immunohistochemistry. RESULTS: Activin was a more potent inducer of apoptosis, while TGFβ was more growth suppressive, and both functions were SMAD4-dependent. TGFβ-induced growth suppression coincided with increased nuclear p21waf1 protein through increased p21waf1 transactivation, transcription and protein stabilization. However, activin decreased nuclear p21waf1 protein with a nuclear to cytosolic shift. Induction of p21waf1 by TGFβ was SMAD4-dependent, but decrease of nuclear p21waf1 by activin was SMAD4-independent. Knock down of p21waf1 reversed growth suppressive effects, enhanced survival, and increased the migratory capacity of both ligands. Increased cytosolic p21waf1 in primary colon cancers correlated with intact ACVR2. CONCLUSIONS: Despite identical downstream SMAD signaling, activin and TGFβ have opposing effects on the cdk2 inhibitor p21waf1, resulting in differing effects on growth suppression, migration, and survival in colon cancer. Activin-specific modulation of p21waf1 functions through non-canonical, SMAD-independent pathways and may represent a potential target for therapy of metastatic disease.
AGA Abstracts
S1988 The Absence of the Proto-Oncogene Fer Increases Colon Cancer Development Maitham A. Khajah, Stefan J. Urbanski, Donna-Marie McCafferty Background: The group IV non-receptor protein tyrosine kinase Fer kinase has been described as a proto-oncogene Fer and is implicated in signal transduction pathways regulated by growth factors. Fer is highly expressed in different malignant cell lines and In Vitro downregulation of Fer reduces the proliferation of prostate carcinoma cells and attenuates the development of prostate cancer. The Aim of this study was to determine the role of Fer kinase in an In Vivo model of colitis-associated adenocarcinoma: the interleukin 10 (IL-10\) model. Methods: WT (129SvEv), IL-10-\-, and IL-10-\-FerDR/DR double knockout mice were used at 3 months of age. Macroscopic and histological scoring of inflammation and dysplasia were performed on the colon (n= 13-25 mice). Myeloperoxidase (MPO) activity was determined as an indication of neutrophil recruitment to the colonic tissues. Bone marrow-derived neutrophils chemotaxis toward different chemoattractants (WKYMVm; fMLP peptide, keratinocyte derived cytokine; KC, macrophage inflammatory protein; MIP-2, and granulocyte macrophage colony stimulating factor; GM-CSF) was performed using the under agarose assay In Vitro. Results: A significant increase in inflammatory parameters (macroscopic and histological score, and MPO activity) was noted in IL-10-\- compared to WT mice at 3 months of age. IL-10-\- mice had a 34 % incidence of mucosal hyperplasia (polyps) with no evidence of neoplastic changes, with the exception of 1 (of 14) mouse with low grade dysplasia. A moderate but significant increase in inflammation was observed in IL-10-\-FerDR/ DR double knockout compared to IL-10-\- mice (p<0.05). Surprisingly, in the absence of Fer kinase, IL-10-\- mice had a 52% incidence of polyps with a 61% incidence of adenocarcinoma. Multiple adenocarcinomas were noted in some mice (2 mice). Using bone marrow derived neutrophils we demonstrated a significant increase in chemotaxis of neutrophils from IL10-\- mice compared with wild type (116±12 vs. 48.8±8.3 toward WKYMVm, 45.3±6.1 vs. 21.6±5.4 toward KC, 55.8±8 vs. 23.6±5.3 toward MIP-2, and 101±4.2 vs. 42±4.1 toward GM-CSF respectively). Neutrophil chemotaxis was further enhanced in the absence of Fer kinase (166±8.9) toward WKYMVm only. Conclusions: These data demonstrate for the first time an important role for Fer kinase in regulating the development adenocarcinoma in a chronic model inflammation In Vivo. This may be associated with enhanced leukocyte recruitment to the colon in the absence of Fer kinase contributing to the inflammationdysplasia-adenocarcinoma sequence.
S1991 The PI3K-AKT Pathway Is a Downstream Effector of CD24 Inna Naumov, Sarah Kraus, Diana Kazanov, Shiran Shapira, Eyal Sagiv, Nadir Arber Background: CD24 is a cell-surface molecule, thought to be involved in adhesion and signaling processes. However, a signaling cascade for CD24 has not been identified. CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of gastrointestinal tumors at a fairly early stage in the multistep process of carcinogenesis (Gastroenterology, Vol 131(2) 630639, 2006). Anti-CD24 mAb induced a significant growth inhibition in CD24-expressing colorectal and pancreatic cancer cells (Clin Can Res, Vol 13(22), p. 6807-6815, 2007). Xenograft models showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA) (Can Res, Vol 68(8), 2803-2812, 2008). The PI3K-AKT pathway has been implicated in the molecular pathogenesis of a variety of cancers and its overexpression was recently demonstrated to be an early event in colorectal carcinogenesis. Aim: To examine the role of PI3K-AKT in the signaling cascade leading to growth inhibition induced by anti-CD24 mAb treatment. Methods: Pancreatic cancer cells, Colo357, were treated with anti-CD24 mAb for various time-points (24, 48, 72hrs) and AKT activation was examined by Western blotting with anti-phospho-AKT (Ser473). Cell proliferation was determined by methylene blue assay following exposure to mAb alone or combined with wortmannin (PI3K inhibitor). Cell cycle and apoptosis was evaluated by FACS analysis following PI staining. Results: Cell growth was significantly inhibited in a time- and dosedependent manner. The level of apoptosis was 25% and 49% after 48 and 72hrs respectively, without a change in cell cycle. Combined mAb treatment with wortmannin increased apoptosis by 11%. Surprisingly, mAb treatment resulted in the down-regulation of AKT activation with a significant AKT degradation. Conclusions: Here we report, for the first time, that AKT is a downstream effector of CD24. CD24 inhibits AKT activity. The combined targeting of CD24 and AKT might serve as a potent anticancer treatment. Understanding the functional aspects of CD24-induced signaling will pave the way for successful cancer therapy using anti-CD24 antibodies and selective promotion of apoptosis.
S1989 Identification of a Functional p53 Responsive Element Within the Promoter of XAF1 Gene in Gastrointestinal Cancer Cells Qing Gu, Wenjing Zhang, Jide Wang, Juan Ma, Zesong Li, Hui Y. Lan, Benjamin C.Y. Wong BACKGROUND: XAF1 was first identified as a XIAP-interacting protein and functioned as a tumor suppressor to sensitize cells to apoptosis. XAF1 expression in gastric cancer tissues was negative correlated with p53 status with cells harboring wildtype p53 expressed low level of XAF1. The purpose of this study was to clarify the regulatory mechanism of p53 on XAF1 expression. METHODS: XAF1 expressions and promoter activities in several gastrointestinal (GI) cancer cell lines with different p53 status were detected by western blot and dual luciferase assay. The effects of ectopic over-expressed wildtype and mutant p53 on XAF1 expression were evaluated after transient transfection. A 107bp of XAF1 core promoter were separated into five probes to examine their binding capacity to the recombinant p53 protein. Site-directed mutation of the putative p53 binding sequence and p53 knockdown by siRNA were performed to confirm the interaction between p53 and XAF1 promoter segments. RESULTS: The protein expression and the promoter activities of XAF1 in cell lines with null p53 were higher than that in cell lines with wildtype and mutant p53. Ectopic over-expression of wildtype p53 suppressed XAF1 protein expression and transcriptional activities. A halfsite (-95nt~-86nt, translation starting codon was defined as +1) and a quartersite of p53 responsive element (-4nt~+1nt) were found within XAF1 promoter. Both sequences bound to recombinant p53 effectively determined by electrophoresis mobility shift assay (EMSA) and the binding could be blocked specifically by non-labelled consensus p53 oligonucleotide. Site-mutation of the putative p53 responsive sequences abrogated the binding between probes and p53 protein. However, only the mutation of halfsite but not the quartersite sequence increased XAF1 promoter activities. Suppression of p53 by siRNA could not only decrease the binding capacity of p53 responsive halfsite but also increase XAF1 expression and its promoter activity. CONCLUSIONS: p53 could suppress the transcription of XAF1 through interaction with a high affinity responsive element (-95nt~-86nt) within XAF1 promoter. The significance of this exclusive interaction between these two tumor suppressors remains to be elucidated.
S1992 The Interaction of β-Catenin and Telomerase and Its Role During Carcinogenesis Falk Mancke, Angela Queisser, Heike Kunert, Steffen Heeg, Nina Hirt, Frank Götschel, Andreas Hecht, Steve Artandi, Oliver G. Opitz Introduction: Maintenance of telomere length is one major determinant of immortalization and thus malignant transformation. Most human tumor cells maintain their telomere length through reactivation of telomerase. Recently, several lines of evidence demonstrated that telomerase possesses additional functions during carcinogenesis and hair follicle stem cell biology, independent of its ability to elongate telomeres. Studies investigating the molecular mechanisms of this telomere elongating independent function of telomerase analyzed the genome wide transcriptional response to acute changes of TERT levels, the protein component of telomerase, in mouse skin. Further statistical comparison to other micro array gene sets revealed that TERT exerts its function on hair follicle stem cells through transcriptional control of WNT related genes. Taking these findings in consideration our aim was to further elucidate the telomere elongating independent functions of telomerase during carcinogenesis, investigating the potential interaction between telomerase and WNT signaling components. Methods: We analyzed distinct cell types representing different steps in the malignant transformation of squamous cell carcinoma and adenocarcinoma of the esophagus. We performed transient transfection assays determining the transcriptional activity of the canonical WNT pathway, co-immunoprecipitation experiments looking for protein-protein interactions and immunoprecipitation (IP) experiments with telomerase repeat amplification protocol (TRAP). This allowed us to study interactions of the catalytically active enzyme. To knock down TERT expression we carried out siRNA transfection experiments. Results: IPImmunoblot experiments demonstrate a interaction of β-catenin and TERT in 293T cells. IP-TRAP experiments identified β-catenin and catalytically active telomerase as interaction partners in 293T cells and fully malignant adenocarcinoma cells. Using siRNA mediated knock down of TERT we observed a TERT dependent reduction in transcriptional activity of canonical WNT signaling, suggesting a functional relevance of the described interaction. Despite robust telomerase activity squamous cell carcinoma cells show neither transcriptional
S1990 Opposing P21waf1 Regulation By TGFβ/SMAD4 and Activin/SMAD4 in Colon Cancer Cells Barbara H. Jung, Jennifer Cabral, Przemyslaw K. Slowik, Jessica Gomez, Stayce E. Beck, John M. Carethers BACKGROUND: Activin and transforming growth factor β (TGFβ) are growth suppressive TGFβ family ligands that signal through a primary receptor type 2 receptor (activin receptor 2, ACVR2, and TGFβ receptor 2, TGFBR2, respectively) to activate specific type 1 receptors, ACVR1 and TGFBR1. This is followed by phosphorylation of cytosolic SMAD2/3 that pairs with SMAD4 to translocate to the nucleus as a transcription factor. Despite the utilization of identical SMADs, microsatellite unstable colon cancers inactivate both activin and TGFβ signaling simultaneously. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21waf1, which has growth suppressive properties in the nucleus, but may act as a tumor
AGA Abstracts
A-308