Background: Ankyrin repeat and SOCS box protein 3(ASB3) is a member of ASB family and contains ankyrin repeat sequence and SOCS box domain. Previous studies indicated that it mediates the ubiquitination and degradatio...Background: Ankyrin repeat and SOCS box protein 3(ASB3) is a member of ASB family and contains ankyrin repeat sequence and SOCS box domain. Previous studies indicated that it mediates the ubiquitination and degradation of tumor necrosis factor receptor 2 and is likely involved in inflammatory responses. However, its effects on oncogenesis are unclear. This study aimed to investigate the effects of ASB3 on the growth and metastasis of colorectal cancer(CRC).Methods: We used next?generation sequencing or Sanger sequencing to detect ASB3 mutations in CRC specimens or cell lines, and used real?time quantitative polymerase chain reaction, Western blotting, and immunohistochemical or immunofluorescence assay to determine gene expression. We evaluated cell proliferation by MTT and colony for?mation assays, tested cell cycle distribution by flow cytometry, and assessed cell migration and invasion by transwell and wound healing assays. We also performed nude mouse experiments to evaluate tumorigenicity and hepatic metastasis potential of tumor cells.Results: We found that ASB3 gene was frequently mutated(5.3%) and down?regulated(70.4%) in CRC cases. Knock?down of endogenous ASB3 expression promoted CRC cell proliferation, migration, and invasion in vitro and facilitated tumorigenicity and hepatic metastasis in vivo. Conversely, the ectopic overexpression of wild?type ASB3, but not that of ASB3 mutants that occurred in clinical CRC tissues, inhibited tumor growth and metastasis. Further analysis showed that ASB3 inhibited CRC metastasis likely by retarding epithelial?mesenchymal transition, which was characterized by the up?regulation of β?catenin and E?cadherin and the down?regulation of transcription factor 8, N?cadherin, and vimentin.Conclusion: ASB3 dysfunction resulted from gene mutations or down?regulated expression frequently exists in CRC and likely plays a key role in the pathogenesis and progression of CRC.展开更多
背景与目的锚蛋白重复序列和SOCS盒蛋白3(ankyrin repeat and SOCS box protein 3, ASB3)是ASB家族成员,包含锚蛋白重复序列和SOCS盒结构域。以往研究表明,它介导肿瘤坏死因子受体2的泛素化和降解,并可能参与炎症反应。然而它对肿瘤发...背景与目的锚蛋白重复序列和SOCS盒蛋白3(ankyrin repeat and SOCS box protein 3, ASB3)是ASB家族成员,包含锚蛋白重复序列和SOCS盒结构域。以往研究表明,它介导肿瘤坏死因子受体2的泛素化和降解,并可能参与炎症反应。然而它对肿瘤发生的作用尚不清楚。本研究旨在探讨ASB3对结直肠癌(colorectal cancer, CRC)的生长与转移的影响。方法使用新一代测序或Sanger测序法检测结直肠癌标本或细胞系中的ASB3突变,并用实时定量PCR、蛋白免疫印迹、免疫组化或免疫荧光法来测定基因的表达。通过MTT和集落形成实验检测细胞的增殖,用流式细胞仪检测细胞周期分布,并用Transwell和划痕实验检测细胞迁移和侵袭。应用裸鼠实验来检测肿瘤细胞的成瘤性和肝转移。结果在结直肠癌病例中ASB3基因经常发生突变(5.3%)和下调(70.4%)。沉默内源性ABS3的表达在体外可促进结直肠癌细胞的增殖、迁移和侵袭,在体内可促进成瘤和肝转移。相反,过表达野生型ASB3可抑制肿瘤的生长和转移,而过表达ASB3突变体不具有这种抑制效应。进一步分析表明,ASB3通过上调β?catenin、E?cadherin和下调转录因子8、N?cadherin和vimentin阻止上皮?间充质转换来抑制结直肠癌转移。结论在结直肠癌中常存在ASB3基因突变或表达下调,在结直肠癌的发生和进展中起到重要作用。展开更多
Apoptosis and autophagy are distinct cellular processes that can be highly interconnected. The cross talk between the two processes is indispensable in determining the overall cell fate. Although the apoptosis-promoti...Apoptosis and autophagy are distinct cellular processes that can be highly interconnected. The cross talk between the two processes is indispensable in determining the overall cell fate. Although the apoptosis-promoting effect of caspases has been demonstrated, the roles of autophagy-related proteins and even autophagy itself in regulating apoptosis remain poorly understood. In our present study, we found that downregulation of ubiquitin E3 ligase ASB3 led to enhanced mitochondrial apoptosis as well as autophagy, which synergistically promoted cell death in hepatocellular carcinoma(HCC). We observed the activation of caspase-8 and decrease of autophagy protein Beclin1 in apoptotic cells that were depleted of ASB3. Beclin1 was mainly cleaved by activated caspase-8 and active Beclin1 initiated mitochondrial apoptosis via locating its C-terminal fragment to mitochondria. In addition, knocking down of Beclin1 markedly blocked the apoptosis, indicating its essential role in the process.Notably, our study indicated that enhanced autophagy level might be involved in the activation of caspase-8 and promote the apoptosis. Taken together, our results demonstrated that ASB3 can regulate mitochondrial pathway of apoptosis by controlling caspase-8 mediated cleavage of Beclin1 in HCC. Therefore, ASB3 may potentially serve as a novel target for HCC therapy,especially when combined with autophagy agonist.展开更多
基金supported by the National Natural Science Foundation of China (No. 81472256, 81272638)the Guangdong Provincial Science and Technology Project (No. 2016A020215081, 2016A020217007)the National High Technology Research and Development Program of China (863 Program, No. 2012AA02A204)
文摘Background: Ankyrin repeat and SOCS box protein 3(ASB3) is a member of ASB family and contains ankyrin repeat sequence and SOCS box domain. Previous studies indicated that it mediates the ubiquitination and degradation of tumor necrosis factor receptor 2 and is likely involved in inflammatory responses. However, its effects on oncogenesis are unclear. This study aimed to investigate the effects of ASB3 on the growth and metastasis of colorectal cancer(CRC).Methods: We used next?generation sequencing or Sanger sequencing to detect ASB3 mutations in CRC specimens or cell lines, and used real?time quantitative polymerase chain reaction, Western blotting, and immunohistochemical or immunofluorescence assay to determine gene expression. We evaluated cell proliferation by MTT and colony for?mation assays, tested cell cycle distribution by flow cytometry, and assessed cell migration and invasion by transwell and wound healing assays. We also performed nude mouse experiments to evaluate tumorigenicity and hepatic metastasis potential of tumor cells.Results: We found that ASB3 gene was frequently mutated(5.3%) and down?regulated(70.4%) in CRC cases. Knock?down of endogenous ASB3 expression promoted CRC cell proliferation, migration, and invasion in vitro and facilitated tumorigenicity and hepatic metastasis in vivo. Conversely, the ectopic overexpression of wild?type ASB3, but not that of ASB3 mutants that occurred in clinical CRC tissues, inhibited tumor growth and metastasis. Further analysis showed that ASB3 inhibited CRC metastasis likely by retarding epithelial?mesenchymal transition, which was characterized by the up?regulation of β?catenin and E?cadherin and the down?regulation of transcription factor 8, N?cadherin, and vimentin.Conclusion: ASB3 dysfunction resulted from gene mutations or down?regulated expression frequently exists in CRC and likely plays a key role in the pathogenesis and progression of CRC.
文摘背景与目的锚蛋白重复序列和SOCS盒蛋白3(ankyrin repeat and SOCS box protein 3, ASB3)是ASB家族成员,包含锚蛋白重复序列和SOCS盒结构域。以往研究表明,它介导肿瘤坏死因子受体2的泛素化和降解,并可能参与炎症反应。然而它对肿瘤发生的作用尚不清楚。本研究旨在探讨ASB3对结直肠癌(colorectal cancer, CRC)的生长与转移的影响。方法使用新一代测序或Sanger测序法检测结直肠癌标本或细胞系中的ASB3突变,并用实时定量PCR、蛋白免疫印迹、免疫组化或免疫荧光法来测定基因的表达。通过MTT和集落形成实验检测细胞的增殖,用流式细胞仪检测细胞周期分布,并用Transwell和划痕实验检测细胞迁移和侵袭。应用裸鼠实验来检测肿瘤细胞的成瘤性和肝转移。结果在结直肠癌病例中ASB3基因经常发生突变(5.3%)和下调(70.4%)。沉默内源性ABS3的表达在体外可促进结直肠癌细胞的增殖、迁移和侵袭,在体内可促进成瘤和肝转移。相反,过表达野生型ASB3可抑制肿瘤的生长和转移,而过表达ASB3突变体不具有这种抑制效应。进一步分析表明,ASB3通过上调β?catenin、E?cadherin和下调转录因子8、N?cadherin和vimentin阻止上皮?间充质转换来抑制结直肠癌转移。结论在结直肠癌中常存在ASB3基因突变或表达下调,在结直肠癌的发生和进展中起到重要作用。
基金supported by the National Natural Science Foundation of China (81372652, 81772529 and 81770579)the Development Fund for Shanghai Talents (201660).
文摘Apoptosis and autophagy are distinct cellular processes that can be highly interconnected. The cross talk between the two processes is indispensable in determining the overall cell fate. Although the apoptosis-promoting effect of caspases has been demonstrated, the roles of autophagy-related proteins and even autophagy itself in regulating apoptosis remain poorly understood. In our present study, we found that downregulation of ubiquitin E3 ligase ASB3 led to enhanced mitochondrial apoptosis as well as autophagy, which synergistically promoted cell death in hepatocellular carcinoma(HCC). We observed the activation of caspase-8 and decrease of autophagy protein Beclin1 in apoptotic cells that were depleted of ASB3. Beclin1 was mainly cleaved by activated caspase-8 and active Beclin1 initiated mitochondrial apoptosis via locating its C-terminal fragment to mitochondria. In addition, knocking down of Beclin1 markedly blocked the apoptosis, indicating its essential role in the process.Notably, our study indicated that enhanced autophagy level might be involved in the activation of caspase-8 and promote the apoptosis. Taken together, our results demonstrated that ASB3 can regulate mitochondrial pathway of apoptosis by controlling caspase-8 mediated cleavage of Beclin1 in HCC. Therefore, ASB3 may potentially serve as a novel target for HCC therapy,especially when combined with autophagy agonist.
