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Xu Cao - One of the best experts on this subject based on the ideXlab platform.
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acute myelogenous leukemia derived Smad4 mutations target the Protein to ubiquitin proteasome degradation
Human Mutation, 2006Co-Authors: Lei Yang, Yi Tang, Xu Cao, Ning Wang, Mei WanAbstract:Disruption of transforming growth factor-β (TGFB1/TGF-β) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-β signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with Protein instability; however, the precise mechanism by which mutated Smad4 Proteins undergo rapid degradation remains to be elucidated. A missense mutation of the Smad4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Δ483–552) have been identified in acute myelogenous leukemia. It is not known whether Protein instability of these Smad4 mutants is one of the contributors to TGF-β signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia–derived Smad4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated Proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that β-transducin-repeat-containing Protein 1 (β-TrCP1), an F-box Protein in the ubiquitin E3 ligase Skp1-Cullin-F-box Protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated Smad4 Proteins. In addition, small interference RNA (siRNA)-triggered endogenous β-TrCP1 suppression increased the Protein expression level of both overexpressed Smad4 mutants and endogenous mutated Smad4 Protein in acute myelogenous leukemia cells. These data suggest that mutated Smad4 Proteins undergo rapid degradation in acute myelogenous leukemia cells via SCFβ-TrCP1 E3 ligase-mediated Protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation. Hum Mutat 27(9), 897–905 2006. Published 2006 Wiley-Liss, Inc.
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scfβ trcp1 controls Smad4 Protein stability in pancreatic cancer cells
American Journal of Pathology, 2005Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Selwyn M Vickers, Lei Yang, Jin Huang, Nirag Jhala, Ning Wang, Xu CaoAbstract:Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 Protein degradation in pancreatic cancer cells. We found that F-box Protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 Protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 Proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated Protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 Protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.
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Smad4 Protein stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 stability.
Mei Wan - One of the best experts on this subject based on the ideXlab platform.
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acute myelogenous leukemia derived Smad4 mutations target the Protein to ubiquitin proteasome degradation
Human Mutation, 2006Co-Authors: Lei Yang, Yi Tang, Xu Cao, Ning Wang, Mei WanAbstract:Disruption of transforming growth factor-β (TGFB1/TGF-β) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-β signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with Protein instability; however, the precise mechanism by which mutated Smad4 Proteins undergo rapid degradation remains to be elucidated. A missense mutation of the Smad4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Δ483–552) have been identified in acute myelogenous leukemia. It is not known whether Protein instability of these Smad4 mutants is one of the contributors to TGF-β signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia–derived Smad4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated Proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that β-transducin-repeat-containing Protein 1 (β-TrCP1), an F-box Protein in the ubiquitin E3 ligase Skp1-Cullin-F-box Protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated Smad4 Proteins. In addition, small interference RNA (siRNA)-triggered endogenous β-TrCP1 suppression increased the Protein expression level of both overexpressed Smad4 mutants and endogenous mutated Smad4 Protein in acute myelogenous leukemia cells. These data suggest that mutated Smad4 Proteins undergo rapid degradation in acute myelogenous leukemia cells via SCFβ-TrCP1 E3 ligase-mediated Protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation. Hum Mutat 27(9), 897–905 2006. Published 2006 Wiley-Liss, Inc.
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scfβ trcp1 controls Smad4 Protein stability in pancreatic cancer cells
American Journal of Pathology, 2005Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Selwyn M Vickers, Lei Yang, Jin Huang, Nirag Jhala, Ning Wang, Xu CaoAbstract:Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 Protein degradation in pancreatic cancer cells. We found that F-box Protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 Protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 Proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated Protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 Protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.
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Smad4 Protein stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 stability.
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Smad4 Protein stability is regulated by ubiquitin ligase scf
2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming ShiAbstract:Smad4 is a key intracellular mediator for the transforming growth factor(TGF) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCF , a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein -TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The -TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing -TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous -TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF -TrCP1 abrogates TGFfunction in vivo by decreasing Smad4 stability.
Lei Yang - One of the best experts on this subject based on the ideXlab platform.
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acute myelogenous leukemia derived Smad4 mutations target the Protein to ubiquitin proteasome degradation
Human Mutation, 2006Co-Authors: Lei Yang, Yi Tang, Xu Cao, Ning Wang, Mei WanAbstract:Disruption of transforming growth factor-β (TGFB1/TGF-β) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-β signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with Protein instability; however, the precise mechanism by which mutated Smad4 Proteins undergo rapid degradation remains to be elucidated. A missense mutation of the Smad4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Δ483–552) have been identified in acute myelogenous leukemia. It is not known whether Protein instability of these Smad4 mutants is one of the contributors to TGF-β signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia–derived Smad4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated Proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that β-transducin-repeat-containing Protein 1 (β-TrCP1), an F-box Protein in the ubiquitin E3 ligase Skp1-Cullin-F-box Protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated Smad4 Proteins. In addition, small interference RNA (siRNA)-triggered endogenous β-TrCP1 suppression increased the Protein expression level of both overexpressed Smad4 mutants and endogenous mutated Smad4 Protein in acute myelogenous leukemia cells. These data suggest that mutated Smad4 Proteins undergo rapid degradation in acute myelogenous leukemia cells via SCFβ-TrCP1 E3 ligase-mediated Protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation. Hum Mutat 27(9), 897–905 2006. Published 2006 Wiley-Liss, Inc.
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scfβ trcp1 controls Smad4 Protein stability in pancreatic cancer cells
American Journal of Pathology, 2005Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Selwyn M Vickers, Lei Yang, Jin Huang, Nirag Jhala, Ning Wang, Xu CaoAbstract:Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 Protein degradation in pancreatic cancer cells. We found that F-box Protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 Protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 Proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated Protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 Protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.
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Smad4 Protein stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 stability.
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Smad4 Protein stability is regulated by ubiquitin ligase scf
2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming ShiAbstract:Smad4 is a key intracellular mediator for the transforming growth factor(TGF) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCF , a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein -TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The -TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing -TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous -TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF -TrCP1 abrogates TGFfunction in vivo by decreasing Smad4 stability.
Hirokazu Nagawa - One of the best experts on this subject based on the ideXlab platform.
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chromosome 18q deletion as a novel molecular predictor for colorectal cancer with simultaneous hepatic metastasis
Diagnostic Molecular Pathology, 2009Co-Authors: Toshiaki Tanaka, Toshiaki Watanabe, Joji Kitayama, Takamitsu Kanazawa, Yoshihiro Kazama, Junichiro Tanaka, Shinsuke Kazama, Hirokazu NagawaAbstract:Currently, surgical treatment for colorectal hepatic metastasis is performed with low mortality and morbidity rates. However, there is no definitive marker that predicts patient outcome. The aim of this study is to identify the molecular predictor of survival along with its clinical properties. Fifty-six patients were surgically treated for colorectal cancer and synchronous hepatic metastasis from January 1994 to December 2004. Clinicopathologic and molecular factors were reviewed in association with overall survival (OS) and disease-free survival (DFS). Chromosome 18q deletion in the primary tumor was a molecular predictor that affected OS (P=0.021). Decreased expression of the Smad4 Protein tended to affect the outcome; however, no statistical significance was observed (P=0.29:OS, P=0.45:DFS). Preoperative carcinoembryonic antigen (P=0.013) and carbohydrate antigen 19-9 (CA19-9) (P<0.0001) levels were poor clinical predictors of OS. The number of primary lymph nodes was the only pathologic factor that affected DFS (P=0.0052). The number and diameter of hepatic metastasis had no influence on survival. In conclusion, we demonstrated that chromosome 18q deletion, in conjunction with high carcinoembryonic antigen and CA19-9 levels, is an unfavorable prognostic factor. This novel molecular predictor is helpful in identifying patients who would benefit from surgical resection.
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loss of Smad4 Protein expression and 18qloh as molecular markers indicating lymph node metastasis in colorectal cancer a study matched for tumor depth and pathology
Journal of Surgical Oncology, 2008Co-Authors: Toshiaki Tanaka, Toshiaki Watanabe, Takamitsu Kanazawa, Yoshihiro Kazama, Junichiro Tanaka, Shinsuke Kazama, Hirokazu NagawaAbstract:Purpose Chromosome 18q21 deletion and Smad4 Protein inactivation have been reported as molecular markers predicting unfavorable outcome in colorectal cancers and, in a previous report, we recently revealed that these molecules are closely associated with distant metastasis, which is one of the clinical factors affecting postoperative survival. However, there has been no discussion as to how these molecules influence another clinical factor, namely, lymph node metastasis. In this report, we studied the significance of chromosome 18q deletion and loss of Smad4 Protein expression in association with lymph node metastasis. Method Forty pairs of colorectal cancer specimens were studied; one group was positive for lymph node metastasis while the other was negative. We examined Smad4 Protein expression level and chromosome 18q deletion in the two groups. Results Immunohistochemical staining revealed that more cases showed a weaker stain for Smad4 Protein in the lymph node positive group compared with the negative group (P = 0.00075). Furthermore, a higher ratio of 18q21 deletion was observed in the lymph node positive group (P = 0.029). Conclusion We revealed that chromosome 18q deletion and Smad4 Protein inactivation are the essential molecular events in the process of lymph node metastasis. J. Surg. Oncol. 2008;97:69–73. © 2007 Wiley-Liss, Inc.
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Chromosome 18q deletion and Smad4 Protein inactivation correlate with liver metastasis: A study matched for T- and N- classification.
British journal of cancer, 2006Co-Authors: Toshiaki Tanaka, Toshiaki Watanabe, Takamitsu Kanazawa, Yoshihiro Kazama, Junichiro Tanaka, Shinsuke Kazama, Hirokazu NagawaAbstract:Smad4 Protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 Protein, we focused on the specific impact of Smad4 Protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 Protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P=0.024) in the level of Smad4 Protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 Protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.
Yi Tang - One of the best experts on this subject based on the ideXlab platform.
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acute myelogenous leukemia derived Smad4 mutations target the Protein to ubiquitin proteasome degradation
Human Mutation, 2006Co-Authors: Lei Yang, Yi Tang, Xu Cao, Ning Wang, Mei WanAbstract:Disruption of transforming growth factor-β (TGFB1/TGF-β) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-β signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with Protein instability; however, the precise mechanism by which mutated Smad4 Proteins undergo rapid degradation remains to be elucidated. A missense mutation of the Smad4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Δ483–552) have been identified in acute myelogenous leukemia. It is not known whether Protein instability of these Smad4 mutants is one of the contributors to TGF-β signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia–derived Smad4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated Proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that β-transducin-repeat-containing Protein 1 (β-TrCP1), an F-box Protein in the ubiquitin E3 ligase Skp1-Cullin-F-box Protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated Smad4 Proteins. In addition, small interference RNA (siRNA)-triggered endogenous β-TrCP1 suppression increased the Protein expression level of both overexpressed Smad4 mutants and endogenous mutated Smad4 Protein in acute myelogenous leukemia cells. These data suggest that mutated Smad4 Proteins undergo rapid degradation in acute myelogenous leukemia cells via SCFβ-TrCP1 E3 ligase-mediated Protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation. Hum Mutat 27(9), 897–905 2006. Published 2006 Wiley-Liss, Inc.
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scfβ trcp1 controls Smad4 Protein stability in pancreatic cancer cells
American Journal of Pathology, 2005Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Selwyn M Vickers, Lei Yang, Jin Huang, Nirag Jhala, Ning Wang, Xu CaoAbstract:Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 Protein degradation in pancreatic cancer cells. We found that F-box Protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 Protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 Proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated Protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 Protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.
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Smad4 Protein stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 stability.
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Smad4 Protein stability is regulated by ubiquitin ligase scf
2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming ShiAbstract:Smad4 is a key intracellular mediator for the transforming growth factor(TGF) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein stability is unclear. We have identified SCF , a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein -TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The -TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing -TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous -TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF -TrCP1 abrogates TGFfunction in vivo by decreasing Smad4 stability.