Oncogene (2010) 29, 4341–4351 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE CCDC6 represses CREB1 activity by recruiting histone deacetylase 1 and phosphatase 1

V Leone1,2, G Mansueto1,2, GM Pierantoni1, M Tornincasa1, F Merolla1, A Cerrato1, M Santoro1, M Grieco1, A Scaloni3, A Celetti1 and A Fusco1,2

1Istituto di Endocrinologia ed Oncologia Sperimentale del CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta` di Medicina e Chirurgia di Napoli, Universita` degli Studi di Napoli ‘Federico II’, Naples, Italy; 2NOGEC (Naples Oncogenomic Center)-CEINGE, Biotecnologie Avanzate-Napoli, & SEMM—European School of Molecular Medicine—Naples Site, Naples, Italy and 3Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples, Italy

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is Santoro et al., 1994, 2006). In RET/PTC1, the fusion frequently activated in human PTCs. It is characterized occurs with the CCDC6 (Grieco et al., 1990) after a by the fusion of the intracellular kinase-encoding domain chromosomal inversion [inv (10) (q11.2q21)] (Pierotti of RET to the first 101 amino acids of CCDC6. The aim et al., 1992). of our work is to characterize the function of the CCDC6 All the fused to RET are ubiquitously expressed protein to better understand the function of its truncation, and, therefore, are able to drive the expression of that results in the loss of the expression of one allele, in truncated forms of RET in thyroid follicular cells, which the process of thyroid carcinogenesis. Here, we report that normally do not express it. Certainly, the inappropriate CCDC6 interacts with CREB1 and represses its tran- expression of RET/PTC in the thyroid follicular cells, scriptional activity by recruiting histone deacetylase 1 and the mislocalization of the RET-tyrosine kinase from the protein phosphatase 1 at the CRE site of the membrane to the cytoplasm, the absence of the CREB1 target genes. Finally, we show an increased extracellular regulatory region of RET, and the presence CREB1 phosphorylation and activity in PTCs carrying of coiled-coil domains in the RET-partner coding the RET/PTC1 oncogene. Consistently, an increased sequences, that favors the dimerization process, account expression of two known CREB1 target genes, AREG for the oncogenic activity of the chimeric RET/PTC and cyclin A, was observed in this subgroup of thyroid genes. The function of RET/PTC oncogene in thyroid papillary carcinomas. Therefore, the repression of carcinogenesis has been validated by in vitro and CREB1 activity by CCDC6 has a critical function in in vivo experiments. Indeed, PCCl 3 cells expressing the development of human thyroid papillary carcinomas the RET/PTC1 oncogene lose the differentiated pheno- carrying RET/PTC1 activation. type, but are not tumorigenic. Equally, transgenic mice Oncogene (2010) 29, 4341–4351; doi:10.1038/onc.2010.179; with thyroid-targeted expression of PTC1 developed published online 24 May 2010 thyroid carcinomas, indicating that the fusion has a primary function in the pathogenesis of the disease with Keywords: CREB1; CCDC6; HDAC1; RET/PTC1; which it is associated (Santoro et al., 1996). RET/PTC papillary thyroid carcinomas rearrangements are specific for thyroid carcinomas of papillary histotype and are considered early events in the tumorigenesis process, as they are frequently found in clinically silent small PTCs (Viglietto et al., Introduction 1995). As the fusion of CCDC6 with RET results in the loss The RET/papillary thyroid carcinoma (PTC) oncogene of the function of one allele, and in some cases, also of is activated in about 20% of PTC (Kondo et al., 2006). the normal unrearranged CCDC6 allele, it is reasonable RET/PTCs are chimeric genes generated by the fusion to hypothesize that this loss might result in growth of the RET tyrosine kinase domain with the 50 terminal advantage during tumor progression. Therefore, it region of other genes. There are at least 15 types of becomes important to elucidate the function of the RET/PTC rearrangements involving RET and 10 CCDC6 gene product also in consideration that the different genes. RET/PTC1 and RET/PTC3 are the CCDC6 gene has been reported to be fused to genes most prevalent RET/PTC variants (Grieco et al., 1990; other than RET in thyroid and non-thyroid human neoplastic diseases (Kulkarni et al., 2000; Schwaller Correspondence: Professor A Fusco, Dipartimento di Biologia e et al., 2001; Puxeddu et al., 2005; Drechsler et al., 2007). Patologia Cellulare e Molecolare, Istituto di Endocrinologia ed Earlier work has shown that CCDC6 gene product is Oncologia Sperimentale, del Consiglio Nazionale delle Ricerche, via an ubiquitously expressed 65 kDa nuclear and cytosolic Pansini 5, Naples 80131, Italy. E-mails: [email protected] or [email protected] protein, is phosphorylated by extracellular signal- Received 21 December 2009; revised 29 March 2010; accepted 19 April regulated after serum stimulation (Grieco 2010; published online 24 May 2010 et al., 1994; Celetti et al., 2004); it has also been proposed CCDC6 represses CREB1 activity V Leone et al 4342 as a proapoptotic factor (Celetti et al., 2004). More and direct physical interaction of CCDC6 with CREB1 recently, it has been shown that CCDC6 is involved in and ATF2, but not with other CREB family proteins, at ATM-mediated cellular response to DNA damage, least in these experimental conditions. supporting the idea that the impairment of CCDC6 To verify that the CCDC6/CREB1 interaction occurs gene function might have a function in thyroid also in cultured cells, protein lysates from B-CPAP cells, carcinogenesis (Merolla et al., 2007). The aim of our which express the endogenous CCDC6 protein, were work has been to further characterize the function of the immunoprecipitated with anti-CREB1 antibodies and CCDC6 protein first by searching for the interacting immunoblotted with anti-CCDC6 antibodies. As shown proteins. By a functional proteomic approach, we in Figure 1b, specific bands were detected, indicating identified several proteins among which we selected the that CCDC6 and CREB1 form complexes in cultured CREB1 protein because of its relevance in the regulation cells. The reciprocal experiment confirmed the interac- of thyroid cell proliferation (Kimura et al., 2001). tion (Figure 1b). To investigate whether the portion First, we have confirmed the interaction between of CCDC6 included in the RET/PTC1 oncoprotein CCDC6 and CREB1 and then, we showed that CCDC6 is crucial for the binding of CREB1, we performed is responsible for the transcriptional repression of co-immunoprecipitation experiments in B-CPAP cells CREB1 target genes, promoting HDAC1 activity. transiently transfected with the CCDC6 (1–101) aa Finally, we report that the repression of the CREB1 deleted mutant. Protein lysates were first immunopreci- activity is achieved by CCDC6 activating the PP1 pitated with anti- antibody, and then the filters were phosphatase that is able to dephosphorylate CREB1 immunoblotted with anti-Myc and anti-CREB1 anti- at Ser133. bodies. As shown in Figure 1c, left panel, CCDC6wt and CREB1 form complexes in cultured cells, confirm- ing the results obtained with the endogenous proteins. The same results were obtained immunoprecipitating Results with anti-CREB1 antibodies and immunoblotting with anti-Myc antibodies (Figure 1c, lower panel). The CCDC6 protein binds CREB1 mutant CCDC6 (1–101) was not able to interact with To identify CCDC6-interacting proteins, a proteomic CREB1 (Figure 1c, right panel), indicating that this approach was used. To achieve this, we transiently domain is not sufficient for the binding to CREB1. transfected B-CPAP cells with a V5-tagged CCDC6- expression vector. Nuclear protein lysates were immu- CCDC6 inhibits the transcriptional activity of CREB1 noprecipitated with anti-V5 antibodies, fractionated by To understand the functional consequences of the SDS–PAGE, and visualized by ammoniacal silver interaction of CCDC6 with CREB1, we investigated staining (Supplementary Figure 1). Single components whether CCDC6 can be directly involved in the of the immunoprecipitated complexes were digested transactivating activity of CREB1. Therefore, we with trypsin and identified by mass spectrometry transfected B-CPAP cells with a CRE-luc reporter gene (Talamo et al., 2003). In Supplementary Table 1, we (De Angelis et al., 2003) in which tandem three CRE report some representative CCDC6-interacting proteins. universal sites were fused upstream to a luciferase Among them, we focused our attention on the CREB1 cDNA together with increasing amounts of CCDC6 in protein because of its relevance in thyroid cell prolifera- the presence of CREB1. As shown in Figure 2a, the tion. Indeed, thyroid cells are dependent for growth and over-expression of CREB1 activated the reporter more differentiation on thyrotropin that through the activa- than sixfold, whereas cotransfection of CCDC6 resulted tion of its stimulates the c-AMP pathway, in a decrease of CREB-mediated transcriptional activity eventually leading to CREB1 phosphorylation and in a dose-dependent manner. Indeed, a decrease of 20 activation of its transcriptional activity (Kimura et al., and 70% was obtained when 1 and 5 mg of CCDC6 were 2001). co-expressed with CREB1, respectively, whereas The following step of our work was to validate the CCDC6 alone slightly modified the CRE-luc reporter CCDC6–CREB1 interaction by a pull-down assay. For basal activity. As expected, the CCDC6 (1–101) mutant this purpose, the GST-CCDC6 fusion protein was tested does not inhibit the CREB activity (Figure 2a). for its ability to bind CREB1 and the other members of the CREB protein family in vitro. Protein lysates from B-CPAP cells, a thyroid papillary cancer line, were CCDC6 reduces CREB binding to CRE element incubated with GST-CCDC6 or GST alone, and the To unveil the biochemical mechanisms underlying the bound proteins were then analyzed by western blot negative regulation of CREB1 target gene using antibodies against the different CREB family by CCDC6, we investigated by electrophoretic mobility proteins. As shown in Figure 1a, specific bands were shift assay the effect of CCDC6 on the binding of CREB observed in the GST-CCDC6 lane when the blots were to the CRE element. Nuclear extracts from B-CPAP incubated with antibodies versus CREB1 and ATF2. cells over-expressing CCDC6 or from control cells were Conversely, no specific bands were observed with incubated with a CRE radiolabelled oligonucleotide. As antibodies versus the other members of the CREB shown in Figure 2b, nuclear proteins were capable of family, and when TCEs were incubated with GST binding the CRE oligonucleotide (lane 1). The expres- protein used as control. These data indicate a specific sion of CCDC6 reduced the binding of CREB to CRE

Oncogene CCDC6 represses CREB1 activity V Leone et al 4343

Figure 1 Characterization of the CCDC6/CREB1 interaction. (a) GST pull-down assays were performed between total cellular extracts (TCEs) from B-CPAP cells and the GST or GST-CCDC6 fusion proteins. The bound complexes and TCEs were separated on SDS–PAGE and analyzed by western blotting with the indicated antibodies. (b) The co-immunoprecipitation was performed on the endogenous CCDC6 and CREB1 proteins (TCE from parental B-CPAP cells). (c) B-CPAP cells were transfected with CCDC6 or the CCDC6 (1–101) deleted mutant constructs. TCEs were prepared and equal amounts of proteins were immunoprecipitated with anti- Myc or anti-CREB1. Then, the immunocomplexes were analyzed by western blotting using the indicated antibodies. IgG indicates negative control of immunoprecipitation using an unrelated antibody. element (lanes 2 and 3) in a dose-dependent manner. CCDC6 allele, was transfected with CCDC6, and the The binding specificity was shown by competition chromatin was immunoprecipitated with anti-CCDC6 experiments showing loss of binding with the addition or anti-IgG antibodies used as control. The immuno- of a 200-fold molar excess of unlabelled CRE oligonu- precipitated DNA-chromatin was subsequently ana- cleotide (lanes 4 and 5). This result shows that CCDC6 lyzed by semiquantitative polymerase chain reaction reduces the binding of CREB to CRE element. (PCR) (Figure 2c, upper left panel) and quantitative To confirm whether the binding is really due to real-time PCR (Figure 2c, upper right panel) CREB1 protein, supershift experiment was performed using specific primers spanning the region using specific antibodies directed against CREB1 (À274/À267). The anti-CCDC6 antibodies precipitated protein. As shown in Figure 2b, the complex was the AREG promoter region from CCDC6-expressing specifically supershifted by the CREB1 antibodies, TPC1 cells (Figure 2c, upper panel) and from B-CPAP indicating that the complex consisted mainly of CREB1 cells, which express endogenous CCDC6, too (Figure 2c, protein. To verify these data in cells, we investigated lower panel). Moreover, we investigated the effect of by chromatin immunoprecipitation (ChIP) whether CCDC6 over-expression on the binding of CREB1 to CCDC6 protein binds a CREB1 target gene promoter, the CRE of AREG promoter. The anti-CREB1 anti- such as AREG, a member of an EGF-like growth factor bodies precipitated AREG promoter both in the parental (Berasain et al., 2007). Therefore, the thyroid papillary and in expressing-CCDC6 TPC1 cells (Figure 2d). cancer cell line TPC1, which harbors the RET/PTC1 However, the AREG promoter amplification was rearrangement and has lost the normal unrearranged lower (about 50%) in the CCDC6-transfected cells in

Oncogene CCDC6 represses CREB1 activity V Leone et al 4344

Figure 2 CCDC6 reduces CREB1 transcriptional activity. (a) Luciferase assay was performed using the indicated amounts of CCDC6 or mutant CCDC6 (1–101)-expression constructs on the CRE-luciferase-reporter vector in B-CPAP cells. All transfections were performed in duplicate; data are mean±s.d. of three independent experiments, Po0.01. (b) Electrophoretic mobility shift assay (EMSA) assay was performed with a radiolabelled oligonucleotide containing the CRE element incubated with nuclear extracts from B-CPAP cells transfected with increased amount of CCDC6 vector. Nuclear extracts were incubated with a 200-fold excess of unlabelled oligonucleotide used as competitor. Supershift analysis was performed using anti-CREB1 antibodies. (c) ChIP was performed in CCDC6-expressing TPC1 cells (upper panel) or in B-CPAP cells, which express endogenous CCDC6 protein (lower panel). The cells were crosslinked and immunoprecipitated with anti-CCDC6. The precipitated DNA was subjected to PCR (left panel) or qRT–PCR (right panel) with specific primers, which amplify CRE element of human AREG promoter or primers for the GAPDH promoter as control. IgG were used as an immunoprecipitation control. (d) CCDC6-expressing TPC1 cells were crosslinked and immunoprecipitated with anti-CREB1 antibodies. The precipitated DNA was subjected to PCR (upper panel) or qRT–PCR (lower panel) with specific primers, which amplify CRE element of human AREG promoter or primers for the GAPDH promoter as control. IgG were used as an immunoprecipitation control. (e) AREG by qRT–PCR from TPC1 cells untransfected and transfected with CCDC6-expressing vector. Relative expression indicates the change in expression levels in comparison with parental cells. Data are mean±s.d. of three independent experiments.

comparison with the control cells, indicating that the Subsequently, we evaluated the expression of AREG binding of CREB1 to the AREG promoter is reduced by by real-time PCR in parental and expressing-CCDC6 CCDC6 expression. Conversely, no amplification was TPC1 cells. In CCDC6-expressing cells, the AREG observed when primers for the control promoter expression was reduced about 40% in comparison with GAPDH were used. These results indicate that CCDC6 parental cells, confirming that CCDC6 reduces the reduces CREB1 binding to AREG promoter. expression of the AREG gene (Figure 2e).

Oncogene CCDC6 represses CREB1 activity V Leone et al 4345 CCDC6 reduces the phosphorylation of CREB1 at Ser133 cells. Conversely, the use of antibodies versus the It is known that CREB1 activity is also dependent on its CREB1 protein revealed equal amount of CREB1 in phosphorylation status (Gonzalez and Montminy, both the cells. These data indicate that the expression of 1989). In particular, phosphorylation at Ser133 repre- CCDC6 reduces the phosphorylation status of CREB1, sents a critical modification for CREB1 activity. There- regulating its transcriptional activity. fore, we investigated whether CCDC6 could reduce the To further show that CCDC6 expression is able to CREB1 activity modifying the phosphorylation status reduce the phosphorylation at Ser133 of CREB1 of Ser133. To achieve this, TPC1 cells were transfected protein, the endogenous CCDC6 gene expression was with CCDC6 and the proteins were analyzed by western transiently silenced transfecting B-CPAP cells with blot with antibodies against pSer133 CREB1 and small-interfering RNAs (CCDC6i-345). As shown in against total CREB1. As shown in Figure 3a (left Figure 3a (right panel), silencing of CCDC6 gene panel), the amount of phosphorylated CREB1 is lower expression increased the phosphorylation status of in CCDC6-expressing cells in comparison with control CREB1, in comparison with the same cells treated with

Figure 3 CCDC6 promotes the dephosphorylation of CREB1 at Ser 133. (a) Western blot analysis of the indicated proteins in TCEs from TPC1 cells transfected with CCDC6 vector (left panel) and from B-CPAP cells transiently transfected with small-interfering RNAs (siRNAs) for CCDC6 expression (CCDC6i-345 and scrambled oligo) (right panel). a-tubulin expression was used to normalize the amount of loaded proteins. (b) B-CPAP cells were transfected with CCDC6-expression vector. TCEs were prepared, and equal amounts of proteins were immunoprecipitated with anti-Myc. The immunocomplexes were analyzed by western blotting using the anti-Myc, anti-PP1, and anti-CREB1 antibodies. The relative inputs are TCEs derived from empty vector or the CCDC6-expression vector-B-CPAP-transfected cells. IgG indicates the negative control of immunoprecipitation. (c) TPC1 cells were transfected with CCDC6-expression vector, crosslinked, and immunoprecipitated with anti-PP1. The precipitated DNA was subjected to PCR and qRT–PCR with specific primers for the CRE element of human AREG promoter or primers for the GAPDH promoter, used as control. IgG were used as an immunoprecipitation control. (d) ChIP and Re-ChIP experiments in expressing CCDC6-TPC1 cells (upper panel) or in B-CPAP cells (lower panel). Soluble chromatin immunoprecipitated with anti-CCDC6 antibodies was eluted and reimmunoprecipitated with anti-PP1 and anti-CREB1. The precipitated DNA was subjected to PCR as above.

Oncogene CCDC6 represses CREB1 activity V Leone et al 4346 the scrambled oligo. Western blot with anti-CCDC6 HDAC8 proteins (Gao et al., 2009). HDACs catalyze protein confirmed silencing of CCDC6 gene expression. the removal of acetyl groups from core histones (Marks CCDC6 mRNA levels in transiently silenced B-CPAP et al., 2001), inducing local condensation of chromatin. cells was analyzed by RT–PCR and is shown in Therefore, HDACs are generally considered repressors Supplementary Figure 2. of transcription (Marks et al., 2001). Our data strongly suggested an interaction between CCDC6 and the proteins of HDAC family. To test this CCDC6 physically interacts with PP1 on the AREG hypothesis, protein lysates from B-CPAP cells were promoter incubated with GST-CCDC6 or GST alone for 2 h, and PP1, PP2A, and PP2B, belonging to the PPP family of then a pull-down assay was performed. As shown in Ser/Thr protein phosphatases, participate in regulating Figure 4a, HDAC1 was able to interact with GST- many important physiological processes, such as cell CCDC6, and not with GST alone. Then, we confirmed cycle control and regulation of cell growth and division the CCDC6–HDAC1 interaction also by co-immuno- regulation (Klumpp and Krieglstein, 2002; Terrak et al., precipitation experiment between the endogenous pro- 2004; Bennett, 2005; Trinkle-Mulcahy and Lamond, teins in B-CPAP cells (Figure 4b). 2006; Han et al., 2007; Wang et al., 2008; Virshup and Shenolikar, 2009). It has also been reported that CREB1 CCDC6 interacts with HDAC1 on the AREG promoter is inactivated by PP1 through dephosphorylation at To determine whether HDAC1 binds the AREG Ser133 (Hagiwara et al., 1992). To understand the promoter, ChIP experiments were performed in wt and mechanism by which CCDC6 is able to modify in CCDC6-expressing TPC1 cells. As expected, CCDC6 the phosphorylation status of CREB1, therefore, we binds AREG promoter (Figure 4c). HDAC1 is also able hypothesized that a possible interaction of CCDC6 with to bind this promoter, and its occupancy increases after PP1 could account for this CCDC6-mediated effect. We CCDC6 expression (Figure 4c). first investigated whether CCDC6 was able to physically Subsequently, to determine whether CCDC6 occupies interact with PP1. To this aim, B-CPAP cells were the AREG promoter together with HDAC1, ChIP, and transiently transfected with the CCDC6-expressing Re-ChIP experiments were performed in TPC1 cells wt vector, and protein lysates were immunoprecipitated and transfected with the CCDC6-expressing vector. The with the indicated antibodies. As shown in Figure 3b, anti-CCDC6 were released, reimmunoprecipitated with PP1 and CREB1 co-immunoprecipitated indicating that anti-HDAC1 (Re-ChIP), and then analyzed by semi- PP1 forms a complex with CCDC6 and CREB1. quantitative PCR and qRT–PCR. The results, shown in To investigate whether the physical interaction Figure 4c lower panel, reveal that the antibodies against between CCDC6, PP1, and CREB1 takes place on the HDAC1 precipitate the AREG promoter in TPC1- human AREG promoter, we first evaluated whether PP1 CCDC6 cells and not in the control ones after their protein binds the AREG promoter in cultured cells and release from anti-CCDC6, indicating that CCDC6 then whether the binding is influenced by CCDC6 occupies this region with HDAC1 (Figure 4c). The expression, by ChIP assay using antibodies raised versus same results were obtained in B-CPAP cells expressing the PP1 protein. PP1 precipitated AREG promoter the endogenous CCDC6 protein (data not shown). region in CCDC6-expressing TPC1 cells (Figure 3c) and Taken together, these results indicate that CCDC6 in B-CPAP cells (data not shown). Indeed, the binds the human AREG promoter and that participates immunoprecipitated DNA-chromatins were analyzed in the same DNA-bound complex that contains by semiquantitative PCR and quantitative reverse HDAC1. transcription PCR (qRT–PCR) using specific primers spanning the AREG promoter region containing CRE CCDC6 expression is correlated with AREG promoter elements. The results shown in Figure 3c indicate that deacetylation PP1 protein binds the AREG promoter region and that The fact that CCDC6 and HDAC1 are present on the this binding was increased after CCDC6 expression. same CRE region of the AREG promoter suggests that To determine whether CCDC6, PP1, and CREB1 HDAC1 participates in the CCDC6-mediated repres- occupy this AREG promoter region all together, sion of CREB1 target genes. Therefore, we analyzed the ReChIP experiments were performed in CCDC6-expres- acetylation status of histones H3 at the AREG promoter sing TPC1 cells with the indicated antibodies. The by ChIP assays. Parental and CCDC6-expressing TPC1 results showed that PP1, CREB1, and CCDC6 partici- cells were immunoprecipitated with antibodies against pate to the same DNA-bound complex on AREG acetylated-histones H3. The immunoprecipitated DNA- promoter (Figure 3d, upper panel). It is noteworthy chromatin was subsequently analyzed by semiquantita- that the same results were obtained in B-CPAP cells tive PCR and qRT–PCR using primers specific for the expressing the endogenous CCDC6 protein (Figure 3d, AREG promoter. A lower amplification was observed in lower panel). the CCDC6-expressing cells in comparison with the control cells (Figure 4d), indicating that CCDC6 CCDC6 physically interacts with HDAC1 expression is associated with a strong decrease in Recently, it has been shown that PP1 can be targeted to the levels of acetylated-histones H3 at the AREG CREB1, for efficient dephosphorylation, through bind- promoter. No amplification was observed with anti- ing of PP1 to HDAC1 (Canettieri et al., 2003) or IgG precipitates.

Oncogene CCDC6 represses CREB1 activity V Leone et al 4347

Figure 4 CCDC6 interacts with HDAC1. (a) GST pull-down assay was performed between total cellular extracts (TCEs) from B-CPAP cells and the GST or GST-CCDC6 fusion proteins. Bound complexes and TCEs were separated on SDS–PAGE and analyzed by western blotting with aHDAC1 antibodies. (b) TCEs from B-CPAP cells were prepared and proteins were immunoprecipitated with anti-CCDC6 antibodies. The immunocomplexes were analyzed by western blotting using antibodies for CCDC6 and HDAC1. IgG indicates the negative control of immunoprecipitation using an unrelated antibody. (c) ChIP and Re-ChIP experiments were performed with the soluble chromatin from CCDC6-expressing TPC1 cells immunoprecipitated with anti-CCDC6 and HDAC1 antibodies. Precipitated DNA was subjected to PCR and qRT–PCR with specific primers for CRE element of human AREG promoter or primers for the GAPDH promoter as control. IgG were used as an immunoprecipitation control. Soluble chromatin immunoprecipitated with anti-CCDC6 antibodies was re-immunoprecipitated with anti-HDAC1 and analyzed as above. (d) Soluble chromatin from TPC1 cells transfected as in (c) was immunoprecipitated with anti-acetyl-histone H3 (aAc-H3). The DNAs were amplified by PCR and qRT–PCR as in (c). IgG was used as an immunoprecipitation control.

CCDC6 requires HDAC1 activity to repress CREB1 activity in the same cells in the presence or absence of target gene transcription TSA (300 nM for 24 h), which specifically inhibits The data shown above prompted us to investigate HDACs (Figure 5b). As expected, in the presence of whether CCDC6 could mediate the repression of TSA, the HDAC activity was inhibited, in both the CREB1 target gene transcription by the recruitment of empty and CCDC6-expressing vector-transfected cells. HDACs. Therefore, we first analyzed HDAC activity in However, the inhibition of HDAC activity was higher in nuclear extracts from B-CPAP over-expressing and not the CCDC6 over-expressing cells with respect to the CCDC6. As shown in Figure 5a, CCDC6 expression control cells. increased the HDAC activity in a dose-dependent Finally, we analyzed the effects of TSA in the manner. To confirm these results, we analyzed HDAC CCDC6-mediated repression of CREB1 target genes

Oncogene CCDC6 represses CREB1 activity V Leone et al 4348 whereas it was significantly reduced by co-expression of CCDC6 as earlier shown. The treatment with TSA reduced the inhibitory effect of CCDC6 (Figure 5c).

CCDC6 inhibits CREB1 activity also in human PTCs To evaluate whether the relationship between CCDC6 over-expression and CREB1 activity defined in tumor cell lines can occur also in vivo, we analyzed the levels of the phosphorylated CREB1 protein at Ser133 and the consequently CREB1 transcriptional activity in normal thyroid, in several PTCs carrying either RET/PTC1 or BRAF mutations, and in PTCs in which no known genetic lesion was detected. As shown in Figure 6a, the levels of pCREB1 at Ser133 were higher in PTCs carrying the RET/PTC1 oncogene, which did not express CCDC6 in comparison with normal thyroid tissue and PTCs carrying other genetic lesions than RET/PTC1 and expressing CCDC6. Then, we analyzed the expression of CREB1 target genes, AREG and Cyclin A. Consistent with these results, AREG and Cyclin A gene expression, evaluated by qRT–PCR, essentially paralleled the level of phosphorylation of CREB1 (Figure 6b). Surprisingly, the levels of specific CREB1 mRNA were lower in PTCs compared with normal thyroid (Supplementary Figure 3). Analogous results have been described earlier (Luciani et al., 2003). Post-translational mechanisms, therefore, account for the increased CREB1 protein levels in PTCs. Therefore, these results clearly indicate that the reduced expression of CCDC6, that follows the RET/ PTC1 rearrangement (Sheils et al., 2000), results in an increased CREB1 phosphorylation and transcriptional activity also in vivo.

Discussion

We have investigated the physiological function of CCDC6 in thyroid cellular functions searching for the interacting proteins. Among the proteins binding to CCDC6 identified by a proteomic approach, we focused our attention on the CREB1 protein as CREB1 activation represents the final step of the thyrotropin Figure 5 CCDC6 requires HDAC1 activity to repress CREB1 pathway that is critical for thyroid cell growth and target gene transcription. (a) B-CPAP cells were transfected with differentiation. We first showed a physical interaction increasing amounts of CCDC6-expression vector. Then, nuclear between CCDC6 and CREB1, but not with the other extracts were assayed for HDAC activity after 48 h. (b) B-CPAP members of the CREB family. Then, we showed that cells were transfected with the CCDC6 or the backbone vectors, CCDC6 binds CRE element present in target genes and and the nuclear extracts were assayed for HDAC activity after 24 h from TSA treatment (300 nM). (c) CRE promoter activity was reduces CREB1 binding to these sequences. Subse- analyzed in B-CPAP cells in the presence of CREB1 and CCDC6- quently, in the attempt to verify the functional expression vectors. The cells were treated with TSA (300 nM)or consequences of this interaction, we showed that the with ethanol as control for 24 h after transfection. Luciferase expression of CCDC6 results in a decreased CREB1 activity were determined 24 h after treatment. All transfections were performed in duplicate; data are mean±s.d. of three activity. Consequently, our efforts were finalized to the independent experiments, Po0.01. comprehension of the mechanism by which CCDC6 is able to inhibit the CREB1 transcriptional activity. It is known that the phosphorylation is critical for through a luciferase assay on the CRE-luc reporter CREB1 activity, and that CREB1 is dephosphorylated construct in the presence of CCDC6 and CREB1 after by PP1. Our results show that CCDC6 is able to bind TSA treatment. As shown in Figure 5c, the CRE-luc PP1 leading to a reduced CREB1 phosphorylation at the basal activity was increased by CREB1 expression, critical Ser133. The reduced CREB1 phosphorylation is

Oncogene CCDC6 represses CREB1 activity V Leone et al 4349

Figure 6 Correlation of CREB phosphorylation at Ser 133 and AREG and Cyclin A expression in human PTCs. (a) Western blot analysis of pCREB at Ser133 in human normal thyroid and PTC tissues. CREB1 expression was used to normalize the amount of loaded proteins. (b) AREG and Cyclin A expression in a pool of five human normal thyroid and PTC tissues (11 samples) analyzed by qRT–PCR. Fold-change indicates the change in expression levels between PTCs with different genetic lesions and a pool of normal thyroids, assuming that the average value of normal samples is equal to 1. Data are mean±s.d. of three independent experiments. also associated to a reduced binding of CREB1 to CRE that results in a decreased acetylation status of histone elements. It is likely that CCDC6 protein exerts a critical H3 at the CREB1 responsive promoters. All these function in recruiting PP1 to CREB1. In fact, it is results, showing that CCDC6 is able to reduce CREB1 known that PP1 does not have good substrate specifi- activity, account for the inhibition of cell growth city, and that the PP1-interacting proteins not only induced by CCDC6, as already shown (Merolla et al., localize PP1 to different cellular compartments, but can 2007). also deliver PP1 to close proximity of potential We finally showed that the reduced CCDC6 expres- substrates, increasing PP1 substrate specificity and sion, as it occurs in human PTCs carrying the RET/ catalytic efficiency (Hu et al., 2006). Consistently, it PTC1 oncogene, leads to an increased CREB1 activity. has been recently reported that HDAC1 interacts with This could result in a higher proliferation rate that PP1 and is critical for the efficient action of PP1 on associated with other mitogenic stimuli, as that of the CREB1 (Canettieri et al., 2003). These data and the RET/PTC activity, may lead to thyroid cell malignant interaction between PP1 and CCDC6, showed in our transformation. experiments, suggested a possible interaction also Therefore, all the data reported here let us to propose between HDAC1 and CCDC6 that we have analyzed CCDC6 as a tumor suppressor in thyroid carcinogenesis and confirmed. Moreover, we have shown that this inhibiting the CREB1-dependent gene expression. interaction occurs at the level of the CREB1 responsive promoters, and that the CCDC6 expression increases HDAC1 activity first resulting in a reduced acetylation of the histone H3 at this region, and then, in a reduced Materials and methods transcription of the CREB1 responsive genes. Therefore, we hypothesize that PP1, CREB1, Cell cultures and transfection HDAC1, and CCDC6 form a unique complex in which B-CPAP is a cell line obtained from a differentiated PTC. Mutations of H-ras, PTC, and trk were not observed (Fabien the expression of CCDC6 works as a negative mod- et al., 1994). TPC1 cells are derived from PTC, which harbors ulator of the CREB1 activity by increasing the PP1 the RET/PTC1 rearrangement and have lost the expression of activity on CRE element that leads to a decreased the normal unrearranged CCDC6 allele. B-CPAP and TPC1 binding of CREB1 to the responsive regions. Moreover, cells were maintained as reported elsewhere (Dettori et al., CCDC6 inhibits CREB1 activity by activating HDAC1 2004; Merolla et al., 2007). B-CPAP cells were transfected by

Oncogene CCDC6 represses CREB1 activity V Leone et al 4350 Fugene reagent (Roche, Welwyn Garden City, UK) and TPC1 Electrophoretic mobility shift assay and supershift assay cells were transfected by Arrestin (OpenBiosystem, Huntsville, Electrophoretic mobility shift assay was performed as de- AL, USA) as suggested by the manufacturer. scribed earlier (Pierantoni et al., 2006). CREB consensus oligonucleotide probe was from Santa Cruz Biotechnology Inc Fresh human thyroid tissue samples (TransCruz Gel Shift Oligonucleotides). A 200-fold molar Neoplastic human thyroid tissues and normal adjacent tissue excess of unlabelled oligonucleotide was added as specific or the normal contralateral thyroid lobe were obtained as competitor. For supershift analyses, samples were preincu- reported earlier (Pallante et al., 2008). Thyroid tumors were bated, on ice for 2 h, with 2 mg of CREB1 antibodies. collected at the Laboratoire d’Histologie et de Cytologie, Centre Hospitalier (Lyon Sud, France) and the Laboratorie ChIP and Re-ChIP assays d’Anatomie Pathologique, Hospital de L’Antiquaille (Lyon, After transfection, chromatin samples were processed for ChIP France). and Re-ChIP experiments as reported elsewhere (Pierantoni et al., 2006). Samples were subjected to immunoprecipitation Expression constructs with the following specific antibodies: anti-CCDC6 (Celetti The expression CCDC6 and mutant CCDC6 (1–101) plasmids et al., 2004), anti-CREB1, anti-HDAC1(06720), anti-acetyl- and small-interfering RNAs (CCDC6i-345) have been de- Histone-H3, (Upstate Biotecnology); anti-PP1 (Santa Cruz scribed elsewhere (Celetti et al., 2004; Merolla et al., 2007). The Biotechnology Inc.). Detailed primer sequences are available expression plasmid pCMVSPORT6-CREB1 was from Invitro- as Supplementary Information. gen (Carlsbad, CA, USA). The GST-CCDC6 wt construct was obtained by amplifying the entire coding sequence (nt 1–1791) by PCR and then subcloned in the BamH1-EcoR1 restriction RNA extraction, cDNA preparation, semiquantitative, sites of pGEX2TK (Amersham, Rainham, UK). and qRT–PCR Total RNA isolation, RT—PCR, and qRT–PCR from human In vitro proteins translation and pull-down assay tissues or from cells were performed as described earlier (De GST pull-down experiments were carried out as reported Martino et al., 2009). Detailed primer sequences are available elsewhere (Pierantoni et al., 2007). as Supplementary Information.

Protein extraction, western blotting, and immunoprecipitation HDAC activity assays B-CPAP cells were transiently transfected with empty or Protein extraction, western blotting, and co-immunoprecipita- CCDC6 vectors. The total protein extracts (30 mg) were tion procedures were carried out as reported elsewhere incubated with [3H]-acetyl-Histone peptide to test the HDAC (Fedele et al., 2005). The Abs used for immunoprecipitation activity according to the manufacturer’s instructions of and western blotting were anti-CCDC6 (Celetti et al., 2004), Histone Deacetylase Assay kit (Upstate Biotechnology Inc.). anti-CREB1, and anti-HDAC1(06720) (Upstate Biotechno- logy Inc., Lake Placid, NY, USA); anti-Myc(9E10), anti- ATF1, anti-ATF2, anti-ATF3, anti-CREM1, anti-CREB2, anti-PP1, and anti-a-tubulin (Santa Cruz Biotechnology, Conflict of interest Santa Cruz, CA, USA); anti-pCREB Ser133 (Cell Signaling Technology, Inc., Danvers, MA, USA). The authors declare no conflict of interest.

Transactivation assay In the luciferase transactivation assay, B-CPAP cells were transiently transfected with the reporter construct (De Angelis Acknowledgements et al., 2003). Co-transfections were carried out in the presence of 200 ng of reporter construct and 500 ng of Renilla construct This work was supported by grants from the Associazione and of 5 mg of CCDC6 or 2 mg of pCMVSport6-CREB1 with Italiana Ricerca sul Cancro (AIRC) and from the Ministero or without the indicated amounts of CCDC6 wt or mutant dell’Istruzione, dell’Universita` e della Ricerca (MIUR) (MER- CCDC6 (1–101) constructs. Luciferase and Renilla activities IT and PRIN 2008 CCPKRP_002). We are grateful to were measured with the dual-luciferase reporter assay kit Konstantina Vergadou (Scientific Communication) for editing (Promega, Madison, WI, USA). the text and Mario Berardone for artwork.

References

Bennett D. (2005). Transcriptional control by -associated De Angelis R, Iezzi S, Bruno T, Corbi N, Di Padova M, Floridi A protein phosphatase-1. Biochem Soc Trans 33(Pt 6): 1444–1446. et al. (2003). Functional interaction of the subunit 3 of RNA Berasain C, Castillo J, Perugorrı´a MJ, Prieto J, Avila MA. (2007). polymerase II (RPB3) with -4 (ATF4). FEBS Amphiregulin: a new growth factor in hepatocarcinogenesis. Cancer Lett 547: 15–19. Lett 254: 30–41. De Martino I, Visone R, Wierinckx A, Palmieri D, Ferraro A, Canettieri G, Morantte I, Guzma´n E, Asahara H, Herzig S, Anderson Cappabianca P et al. (2009). HMGA proteins up-regulate CCNB2 SD et al. (2003). Attenuation of a phosphorylation-dependent gene in mouse and human pituitary adenomas. Cancer Res 69: activator by an HDAC-PP1 complex. Nat Struct Biol 10: 175–181. 1844–1850. Celetti A, Cerrato A, Merolla F, Vitagliano D, Vecchio G, Grieco M. Dettori T, Frau DV, Garcia JL, Pierantoni G, Lee C, Hernandez JM (2004). H4(D10S170), a gene frequently rearranged with RET in et al. (2004). Comprehensive conventional and molecular cytoge- papillary thyroid carcinomas: functional characterization. Oncogene netic characterization of B-CPAP, a human papillary thyroid 23: 109–121. carcinoma-derived cell line. Cancer Genet Cytogenet 151: 171–177.

Oncogene CCDC6 represses CREB1 activity V Leone et al 4351 Drechsler M, Hildebrandt B, Ku¨ndgen A, Germing U, Royer-Pokora Merolla F, Pentimalli F, Pacelli R, Vecchio G, Fusco A, Grieco M B. (2007). Fusion of H4/D10S170 to PDGFRbeta in a patient with et al. (2007). Involvement of H4(D10S170) protein in ATM- chronic myelomonocytic leukemia and long-term responsiveness to dependent response to DNA damage. Oncogene 26: 6167–6175. imatinib. Ann Hematol 86: 353–354. Pallante P, Federico A, Berlingieri MT, Bianco M, Ferraro A, Fabien N, Fusco A, Santoro M, Barbier Y, Dubois PM, Paulin C. Forzati F et al. (2008). Loss of the CBX7 gene expression correlates (1994). Description of a human papillary thyroid carcinoma cell with a highly malignant phenotype in thyroid cancer. Cancer Res 68: line. Morphologic study and expression of tumoral markers. Cancer 6770–6778. 73: 2206–2212. Pierantoni GM, Rinaldo C, Esposito F, Mottolese M, Soddu S, Fusco Fedele M, Pentimalli F, Baldassarre G, Battista S, Klein-Szanto AJ, A. (2006). High Mobility Group A1 (HMGA1) proteins interact Kenyon L et al. (2005). Transgenic mice over-expressing the wild- with and inhibit its apoptotic activity. Cell Death Differ 13: type form of the HMGA1 gene develop mixed growth / 1554–1563. prolactin cell pituitary adenomas and natural killer cell lymphomas. Pierantoni GM, Esposito F, Giraud S, Bienvenut WV, Diaz JJ, Fusco Oncogene 24: 3427–3435. A. (2007). Identification of new high mobility group A1 associated Gao J, Siddoway B, Huang Q, Xia H. (2009). Inactivation of CREB proteins. Proteomics 7: 3735–3742. mediated gene transcription by HDAC8 bound protein phospha- Pierotti MA, Santoro M, Jenkins RB, Sozzi G, Bongarzone I, Grieco M tase. Biochem Biophys Res Commun 379: 1–5. et al. (1992). Characterization of an inversion on the long arm of Gonzalez GA, Montminy MR. (1989). Cyclic AMP stimulates juxtaposing D10S170 and RET and creating the somatostatin gene transcription by phosphorylation of CREB at oncogenic sequence RET/PTC. Proc Natl Acad Sci USA 89: 1616–1620. serine 133. Cell 59: 675–680. Puxeddu E, Zhao G, Stringer JR, Medvedovic M, Moretti S, Fagin Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, JA. (2005). Characterization of novel non-clonal intrachromosomal Bongarzone I et al. (1990). PTC is a novel rearranged form of the rearrangements between the H4 and PTEN genes (H4/PTEN) in RET proto-oncogene and is frequently detected in vivo in human human thyroid cell lines and papillary thyroid cancer specimens. thyroid papillary carcinomas. Cell 60: 557–563. Mutat Res 570: 17–32. Grieco M, Cerrato A, Santoro M, Fusco A, Melillo RM, Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin C, Vecchio G. (1994). Cloning and characterization of H4(D10S170), Pierotti MA et al. (1994). Molecular characterization of RET/PTC a gene involved in RET rearrangements in vivo. Oncogene 9: 3: a novel rearranged version of the RET proto-oncogene in a 2531–2535. human thyroid papillary carcinoma. Oncogene 9: 509–516. Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T Santoro M, Chiappetta G, Cerrato A, Salvatore D, Zhang L, Manzo et al. (1992). Transcriptional attenuation following cAMP induction G et al. (1996). Development of thyroid papillary carcinomas requires PP-1-mediated dephosphorylation of CREB. Cell 70: secondary to tissue-specific expression of the RET/PTC1 oncogene 105–113. in transgenic mice. Oncogene 12: 1821–1826. Han Y, Haines CJ, Feng HL. (2007). Role(s) of the serine/threonine Santoro M, Melillo RM, Fusco A. (2006). RET/PTC activation in protein phosphatase 1 on mammalian sperm motility. Arch Androl papillary thyroid carcinoma. Eur J Endocrinol 155: 645–653. 53: 169–177. Schwaller J, Anastasiadou E, Cain D, Kutok J, Woyiski S, Willimas Hu XD, Huang Q, Roadcap DW, Shenolikar SS, Xia H. (2006). Actin- IR et al. (2001). CCDC6, a gene frequently rearranged in papillary associated neurabin-protein phosphatase-1 complex regulates hip- thyroid carcinomas, is fused to the platelet-derived growth factor pocampal plasticity. J Neurochem 98: 1841–1851. receptor b gene in atypical chronic myeloid leukaemia with Kimura T, Van Keymeulen A, Golstein J, Fusco A, Dumont JE, t(5;10)(q33;q22). Blood 97: 3910–3918. Roger PP. (2001). Regulation of thyroid cell proliferation by TSH Sheils OM, O0Leary JJ, Sweeney EC. (2000). Assessment of ret/PTC-1 and other factors: a critical evaluation of in vitro models. Endocr rearrangements in neoplastic thyroid tissue using TaqMan RT- Rev 22: 631–656. PCR. J Pathol 192: 32–36. Klumpp S, Krieglstein J. (2002). Serine/threonine protein phospha- Talamo F, D0Ambrosio C, Arena S, Del Vecchio P, Ledda L, tases in apoptosis. Curr Opin Pharmacol 2: 458–462. Zehender G et al. (2003). Proteins from bovine tissues and biological Kondo T, Ezzat S, Asa SL. (2006). Pathogenetic mechanisms in fluids: defining a reference electrophoresis map for liver, kidney, thyroid follicular-cell neoplasia. Nat Rev Cancer 6: 292–306. muscle, plasma and red blood cells. Proteomics 3: 440–460. Kulkarni S, Heath C, Parker S, Chase A, Iqbal A, Pocock F et al. TerrakM,KerffF,LangsetmoK,TaoT,DominguezR.(2004).Structure (2000). Fusion of H4/D10S170 to the platelet-derived growth factor basis of protein phosphatase1 regulation. Nature 429: 780–784. receptor b in BCR-ABL-negative myeloproliferative disorders with Trinkle-Mulcahy L, Lamond AI. (2006). Mitotic phosphatases: no a t(5;10)(q33;q21). Cancer Res 60: 3592–3598. longer silent partners. Curr Opin Cell Biol 18: 623–631. Luciani P, Buci L, Conforti B, Tonacchera M, Agretti P, Elisei R et al. Viglietto G, Chiappetta G, Martinez-Tello FJ, Fukunaga FH, Tallini (2003). Expression of cAMP response element-binding protein and G, Rigopoulou D et al. (1995). RET/PTC oncogene activation is an sodium iodide symporter in benign non-functioning and malignant early event in thyroid carcinogenesis. Oncogene 11: 1207–1210. thyroid tumours. Eur J Endocrinol 148: 579–586. Virshup DM, Shenolikar S. (2009). From promiscuity to precision: Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. protein phosphatases get a makeover. Mol Cell 33: 537–545. (2001). Histone deacetylases and cancer: causes and therapies. Nat Wang B, Zhang P, Wei Q. (2008). Recent progress on the structure of Rev Cancer 1: 194–202. Ser/Thr protein phosphatases. Sci China C Life Sci 51: 487–494.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene