Ccdc6 knock-in mice develop thyroid hyperplasia associated to an enhanced CREB1 activity (original) (raw)
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PLoS ONE, 2012
CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424) which are highly conserved in vertebrates. We demonstrate that the posttranslational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.
CCDC6 represses CREB1 activity by recruiting histone deacetylase 1 and protein phosphatase 1
Oncogene, 2010
RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation.
The thyroid hormone receptor antagonizes CREB-mediated transcription
The EMBO Journal, 2003
Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein±protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein±protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences.
Tumor Suppressor Role of theCL2/DRO1/CCDC80Gene in Thyroid Carcinogenesis
The Journal of Clinical Endocrinology & Metabolism, 2013
Context: Thyroid carcinoma is one of the most common malignancies of the endocrine system, and, despite the high frequency of oncogene activation in thyroid neoplastic lesions, the tumor suppressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicated a link between the CL2/CCDC80 gene and thyroid cancer. Objective: The objective of the study was to examine the expression of the CL2/CCDC80 gene in human thyroid carcinomas in the attempt to determine whether it plays a role in thyroid carcinogenesis. Design: We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissue samples differing in degree of malignancy. We also investigated the effects of its restoration in 2 human thyroid carcinoma cell lines characterized by very low levels of CL2/CCDC80 expression. Results: CL2/CCDC80 expression was much lower in almost all the thyroid carcinomas analyzed than in normal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozygosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restoration of CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a higher susceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression positively regulated the expression of E-cadherin, thereby halting cancer progression. Conclusions: These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroid carcinogenesis.
The cl2/dro1/ccdc80 null mice develop thyroid and ovarian neoplasias
Cancer Letters, 2015
We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knockout mice. We found that embryonic fibroblasts from cl2/ccdc80 −/− mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80 −/− mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knockout mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis.
Cloning of TC-1 (C8orf4), a Novel Gene Found to Be Overexpressed in Thyroid Cancer
Genomics, 2000
A novel gene highly expressed in thyroid cancer, designated TC-1 (thyroid cancer-1), was cloned from suppression subtractive hybridization between papillary thyroid carcinoma and its surrounding normal thyroid tissue. Overexpression of TC-1 in thyroid cancer was confirmed in 15/16 paired samples by RT-PCR and Northern analysis. Ubiquitously expressed in human tissues, the TC-1 sequence showed no homology to any known gene, but matched a cluster of ESTs. After alignment of our sequence with the ESTs, the missing transcription start site was obtained by 5-RACE and verified by primer extension analysis. The full-length mRNA sequence of 1327 bp has an open reading frame of 321 bp, which encodes a highly conserved protein. Three regulatory motifs were identified at the expected positions within 1 kb of the 5 flanking sequence obtained by genome walking. Using fluorescence in situ hybridization, TC-1 was localized to chromosome 8p11.2. The overexpression of TC-1 in papillary carcinoma suggests that it may have an important role in thyroid carcinogenesis.
Journal of Biological Chemistry, 2010
Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser 136 in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP⅐CRTC⅐CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2. Pin1 was initially cloned as a NIMA kinase-interacting protein (1). Since its discovery, numerous proteins have been identified as Pin1 substrates, including p53, cyclin D1, and Tau (2-5). Pin1 interacts with a number of target proteins through recognition of phospho-Ser/Pro motifs, and the proline conformational change induced by Pin1 modifies the structures and functions, such as stabilization, phosphorylation, and translocation, of target proteins (4-7). Pin1 possesses the WW and PPIase 3 domains in its N-terminal (amino acids 1-38) and C-terminal (amino acids 39-163) regions, respectively. To date, many reports have supported an important role for Pin1 in diseases such as cancer and Alzheimer disease (4, 5). In this study, we demonstrated that Pin1 is also involved in metabolic disease via regulation of CRTC2 (CREB-regulated transcriptional co-activator 2; also known as TORC). The cAMP-responsive element (CRE)-binding protein (CREB) stimulates transcriptional activity through recruitment of the histone acetylase CBP and through an association with CRTC, leading to formation of the CREB⅐CBP⅐CTRC complex on a CRE site (8-16). Thus, multiple molecular mechanisms affect the CREB⅐CBP⅐CTRC complex, resulting in the regulation of CRE transcriptional activity. They include the phosphorylations of CREB at Ser 133 , CBP at Ser 436 , and CRTC2 at Ser 171 (16, 17). The phosphorylation of CRTC2 at Ser 171 reportedly leads to an association with 14-3-3 protein and thereby to its nuclear exclusion and degradation (16). The CRTC family consists of three members, CRTC1, CRTC2, and CRTC3 (16, 18). CRTC1 is highly expressed in the brain, whereas the other two are ubiquitously expressed (19). In the liver, insulin induces the phosphorylation of CRTC2 at Ser 171 , and this phosphorylation leads to the aforementioned * This work was supported in part by Grant-in-aid for Young Scientists 20790648 (to Y. N.) from the Ministry of Education, Science, Sports, and Culture, Japan.