Objective:Partitioning defective protein 3(Par3)has recently been found to have important roles in cancer progression.Interestingly,Par3's functions vary among cancers:both Par3 elevation(in the prostate or liver)...Objective:Partitioning defective protein 3(Par3)has recently been found to have important roles in cancer progression.Interestingly,Par3's functions vary among cancers:both Par3 elevation(in the prostate or liver)and loss(in the breast or lung)have been implicated in cancer metastasis.Although Par3 overexpression has been correlated with diminished survival in renal cell carcinoma(RCC),data indicating the role of Par3 in RCC metastasis are lacking.Given reports of interactions between Par3 and oncoproteins such as Yesassociated protein(YAP)/WW domain-containing transcription regulator 1(TAZ),we investigated whether Par3-mediated RCC metastasis might be due to activation of the Hippo pathway components YAP and TAZ.Methods:Par3 levels were analyzed in RCC cell lines and human RCC patient tissues by western blotting and immunohistochemical(IHC)staining,as appropriate.Co-immunoprecipitation(co-IP)and immunofluorescence studies were conducted to examine the interaction between Par3 and YAP.Quantitative PCR and luciferase assays were used to investigate the effects of Par3 on YAP target gene expression and co-transcriptional regulation.PDZ domain deletion mutants of Par3 were generated to elucidate the structural basis of the interaction between Par3 and YAP.Results:Higher Par3 levels were found in distant-organ-RCC-metastasis-derived ACHN sublines than wild type ACHN cell lines.Par3 levels were also higher in the patient tissue obtained from metastatic sites than in normal kidney and primary RCC tumor tissues.Co-IP and IHC experiments demonstrated that Par3 directly interacted and co-localized with YAP/TAZ proteins.Moreover,Par3 upregulated the transcription of YAP/TAZ downstream target genes and increased the luciferase activity of YAP/TAZ responsive elements.PDZ domain 3 in the PARD3 gene was demonstrated to be particularly important in the interactions between Par3 and YAP.Furthermore,Par3 was found to upregulate intracellular levels of YAP/TAZ molecules and promote nuclear translocation of YAP.Conclusions:Together,these results indicate the role of Par3 in RCC metastasis,via driving metastatic RCC progression by promoting the YAP/TAZ pathway.展开更多
目的探讨极性蛋白Par3(partitioning defective protein 3,Par3)和磷酸化的信号传导子和转录激活子3(p-signal transducers and activators of transcription 3,p-STAT3)在宫颈病变组织中的表达及临床意义。方法采用免疫组织化学染色法...目的探讨极性蛋白Par3(partitioning defective protein 3,Par3)和磷酸化的信号传导子和转录激活子3(p-signal transducers and activators of transcription 3,p-STAT3)在宫颈病变组织中的表达及临床意义。方法采用免疫组织化学染色法检测Par3和p-STAT3蛋白在慢性宫颈炎、宫颈上皮内瘤变Ⅱ-Ⅲ(cervical intraepithelial neoplasia Ⅱ-Ⅲ,CIN Ⅱ-Ⅲ)以及宫颈鳞状细胞癌(cervical squamous carcinoma,CSCC)和淋巴结转移灶中的表达情况,分析其与临床病理参数之间的关系。结果Par3蛋白在慢性宫颈炎、CIN Ⅱ-Ⅲ、CSCC和淋巴结转移灶中的蛋白表达量呈现逐渐降低趋势(P<0.001);上述4种组织中p-STAT3蛋白的表达量是随着宫颈病变加重而逐渐增高(P=0.0062)。比较二者与临床病理参数的关系发现,宫颈鳞癌中Par3表达与肿瘤淋巴结转移(P=0.003)以及组织学分化程度(P=0.017)有关,宫颈鳞癌组织p-STAT3阳性表达与肿瘤临床分期、组织学分化程度有关(P=0.001)。CINⅡ-Ⅲ组织中极性蛋白Par3与p-STAT3蛋白水平呈负相关(r=-5.553,P<0.05)。结论宫颈鳞癌中极性蛋白Par3低表达,p-STAT3蛋白高表达,二者表达差异与宫颈鳞癌发生、组织学分化、肿瘤淋巴结转移以及临床分期有关。展开更多
The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting ...The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase （DNA-PK）, which plays an essential role in repairing double-strand DNA breaks （DSBs）. We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by γ-irradiation （IR）, a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.展开更多
基金supported by grants from the American Urology Association(AUA)Urology Care Foundation Research Scholar AwardKidney Cancer Research Alliance Research Grant+3 种基金Simmons Comprehensive Cancer Center(SCCC)Early-Stage Clinical Investigator AwardDedman Scholar Awardsupported by the Simmons Cancer Center’s Tissue Management Shared Resourcethe National Cancer Institute of the National Institutes of Health(grant No.P30 CA142543)。
文摘Objective:Partitioning defective protein 3(Par3)has recently been found to have important roles in cancer progression.Interestingly,Par3's functions vary among cancers:both Par3 elevation(in the prostate or liver)and loss(in the breast or lung)have been implicated in cancer metastasis.Although Par3 overexpression has been correlated with diminished survival in renal cell carcinoma(RCC),data indicating the role of Par3 in RCC metastasis are lacking.Given reports of interactions between Par3 and oncoproteins such as Yesassociated protein(YAP)/WW domain-containing transcription regulator 1(TAZ),we investigated whether Par3-mediated RCC metastasis might be due to activation of the Hippo pathway components YAP and TAZ.Methods:Par3 levels were analyzed in RCC cell lines and human RCC patient tissues by western blotting and immunohistochemical(IHC)staining,as appropriate.Co-immunoprecipitation(co-IP)and immunofluorescence studies were conducted to examine the interaction between Par3 and YAP.Quantitative PCR and luciferase assays were used to investigate the effects of Par3 on YAP target gene expression and co-transcriptional regulation.PDZ domain deletion mutants of Par3 were generated to elucidate the structural basis of the interaction between Par3 and YAP.Results:Higher Par3 levels were found in distant-organ-RCC-metastasis-derived ACHN sublines than wild type ACHN cell lines.Par3 levels were also higher in the patient tissue obtained from metastatic sites than in normal kidney and primary RCC tumor tissues.Co-IP and IHC experiments demonstrated that Par3 directly interacted and co-localized with YAP/TAZ proteins.Moreover,Par3 upregulated the transcription of YAP/TAZ downstream target genes and increased the luciferase activity of YAP/TAZ responsive elements.PDZ domain 3 in the PARD3 gene was demonstrated to be particularly important in the interactions between Par3 and YAP.Furthermore,Par3 was found to upregulate intracellular levels of YAP/TAZ molecules and promote nuclear translocation of YAP.Conclusions:Together,these results indicate the role of Par3 in RCC metastasis,via driving metastatic RCC progression by promoting the YAP/TAZ pathway.
基金This work was supported by the grants from National Natural Science Foundation of China(Nos.30170208,30623003 and 30170208)from the Ministry of Science and Technology,China(No.2001AA233031 and No.2001CB510205)from US NIH(CA50519)(to DJ Chen).
文摘The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase （DNA-PK）, which plays an essential role in repairing double-strand DNA breaks （DSBs）. We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by γ-irradiation （IR）, a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.
