Tumor Suppressor Role of the CL2/DRO1/CCDC80 Genein Thyroid Carcinogenesis
Angelo Ferraro, Filippo Schepis, Vincenza Leone, Antonella Federico,Eleonora Borbone, Pierlorenzo Pallante, Maria Teresa Berlingieri,Gennaro Chiappetta, Mario Monaco, Dario Palmieri, Lorenzo Chiariotti,Massimo Santoro, and Alfredo Fusco
Istituto per l’Endocrinologia e l’Oncologia Sperimentale “G. Salvatore” (A.Fer., V.L., A.Fed., E.B., P.P.,M.T.B., D.P., L.C., M.S., A.Fu.), Istituto per l=Endocrinologia e l=Oncologia Sperimentale, ConsiglioNazionale delle Ricerche, c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universitàdegli Studi di Napoli “Federico II”, and Istituto Nazionale dei Tumori (G.C., M.M.), Fondazione Pascale,80131 Napoli, Italy; Naples Oncogeomic Center (A.Fer., V.L., A.Fed., E.B., P.P., M.T.B., L.C., M.S., A.Fu.),Centro di Ingegneria Genetica-Biotecnologie Avanzate, 80145 Napoli, Italy; and Dipartimento diMedicina Sperimentale (F.S.), Facoltà di Medicina e Chirurgia di Catanzaro, Università degli Studi diCatanzaro “Magna Graecia”, Campus Universitario “Salvatore Venuta”, Viale Europa, LocalitàGermaneto, 88100 Catanzaro, Italy
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 sup-pressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicateda 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 inhuman thyroid carcinomas in the attempt to determine whether it plays a role in thyroidcarcinogenesis.
Design: We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissuesamples differing in degree of malignancy. We also investigated the effects of its restoration in 2human 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 innormal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozy-gosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restorationof CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a highersusceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression pos-itively regulated the expression of E-cadherin, thereby halting cancer progression.
Conclusions: These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroidcarcinogenesis. (J Clin Endocrinol Metab 98: 2834–2843, 2013)
Thyroid neoplasms are a good model with which tostudy the events involved in epithelial cell multistep
carcinogenesis. Indeed, they include a wide spectrum oflesions that differ in degree of malignancy, going from
benign follicular thyroid adenomas (FTAs), which are notinvasive and are well differentiated, to undifferentiatedanaplastic thyroid carcinomas (ATCs), which are very ag-gressive and are invariably fatal. Papillary (PTCs) and fol-
licular (FTCs) carcinomas are the most common forms ofthyroid cancer, and, being differentiated and having agood prognosis (1–5), they may represent intermediateforms of neoplasia.
Various oncogenes are involved in human thyroid car-cinomas, particularly in the papillary histotype (5). In fact,activation of the RET/PTC oncogene, which derives fromthe fusion of the tyrosine kinase domain of the RET proto-oncogene with ubiquitously expressed genes, is detected inabout 20% of PTCs (6–10). Rearrangements of TRK areless frequent (�5%) (11), and mutations of the BRAF genehave recently been detected in almost 50% of PTCs (12–14). Activating mutations of the RAS proto-oncogene arequite rare in classical PTC variants but rather frequent infollicular variants (�30%) and in FTCs (15–18) and arethe predominant molecular alterations in poorly differen-tiated carcinomas (19). Conversely, paired box 8 (PAX8)-peroxisome proliferator-activated receptor (PPAR) � re-arrangements are specific to FTCs (20, 21).
Alterations of PIK3CA (an effector of phosphoinosi-tide 3-kinase; PI3K) have also been found in thyroid car-cinomas. These alterations are generally observed in thelater stages of thyroid carcinogenesis and are more fre-quent in ATC than in PTC or FTC (22, 23). In contrast,mutations of MAPK effectors are preferentially associatedwith early stages of thyroid carcinogenesis. Mutations inthe AKT1 oncogene have been observed in poorly differ-entiated thyroid carcinoma (24). These mutations are gen-erally associated with BRAF mutations, and they do notoverlap with PIK3CA mutations in poorly differentiatedthyroid carcinoma (24). Despite the high frequency of on-cogene activation in neoplastic thyroid lesions, the tumorsuppressor genes involved in thyroid carcinogenesis re-main unknown.
Impairment of p53 tumor suppressor gene function is atypical feature of ATCs (25–27). Decreased levels of phos-phatase and tensin homolog deleted from chromosome 10(28, 29) and protein tyrosine phosphatase, receptor type,J (30, 31), a dual specific phosphatase and a receptor typetyrosine phosphatase, respectively, have been reported inthyroid malignancies. Both these proteins increase the sta-bility of p27(KIP1), which in turn can negatively regulatetransition through the cell cycle. These findings promptedthe search for other genes whose impaired function mightfavor thyroid cell transformation.
In an earlier study, we identified a gene, designated cl2(32), that is up-regulated in PC Cl3 cells after transfectionwith the adenovirus E1A gene (PC E1A). The rat cl2 tran-script is 4.4 kb long and encodes a 949-amino acid protein.Its expression was drastically down-regulated in humanthyroid neoplastic cell lines and tissues, although the num-ber of the samples analyzed was limited. Subsequently the
human ortholog of rat cl2 was identified, ie, DRO1(down-regulated by oncogenes 1) and reported to bedown-regulated in rat RK3E epithelial cells transformedby the �-catenin oncogene (33). Currently the NationalCenter for Biotechnology Information database recordsthe human gene, corresponding to DRO1 and CL2, ascoiled-coil domain containing 80 (CCDC80).
To verify whether the CL2/CCDC80 gene is involved inthyroid carcinogenesis, we measured its expression in alarge number of thyroid neoplastic samples and deter-mined the effects of the restoration of CL2/CCDC80 in 2human thyroid carcinoma cell lines that express very lowlevels of this gene. We found that CL2/CCDC80 geneexpression is drastically down-regulated in thyroid carci-nomas, particularly in the follicular variant of PTC. Res-toration of CL2/CCDC80 expression led to the inhibitionof the growth of thyroid carcinoma cell lines and impair-ment of their ability to grow in a semisolid medium and toinduce tumorigenesis in athymic mice. These results indi-cate that CL2/CCDC80 is a putative tumor suppressorgene in thyroid carcinogenesis.
Materials and Methods
Collection of neoplastic tissue specimens andextraction of nucleic acids
Human thyroid neoplastic and normal (contralateral normalthyroid lobe) specimens were obtained from surgical specimensand immediately frozen in liquid nitrogen. Total RNA was iso-lated from human tissues and cell lines with Trizol (Invitrogen,Carlsbad, California) according to the manufacturer’s instruc-tions. The integrity of the RNA was assessed by denaturing aga-rose gel electrophoresis. DNA was prepared for each samplefrom a portion of tissue pulverized with liquid nitrogen using theQIAamp DNA minikit (QIAGEN, Valencia, California) follow-ing the manufacturer’s instructions.
Quantitative real-time PCR
cDNA preparationOne microgram of total RNA of each sample was reverse
transcribed with the QuantiTect reverse transcription kit (QIA-GEN) using an optimized blend of oligo-deoxythymidine nucle-otides and random primers according to the manufacturer’sinstructions.
Selection of primers and probesPrimers and probes for quantitative real-time (qRT)-PCR
analyses are reported in Supplemental Materials and Methods,published on The Endocrine Society’s Journals Online web siteat http://jcem.endojournals.org.
Relative quantificationTaqMan qRT-PCR was performed on the 7900HD SDS ma-
chine (Applied Biosystems, Foster City, California) in 384-well
plates using a final volume of 20 �L. For PCR we used 8 �L of2.5� Real Master Mix probe ROX (Eppendorf AG, Hamburg,Germany), 200 nM of each primer, 100 nM probe, and cDNAgenerated from 50 ng of total RNA. The conditions used for PCRwere 2 minutes at 95°C and then 45 cycles of 20 seconds at 95°Cand 1 minute a 60°C. Each reaction was performed in duplicate.We used the 2-��CT method to calculate relative expression levels(34).
Immunohistochemical analysis, mutation analysis, and lossof heterozygosity analysis are described in Supplemental Mate-rials and Methods.
Thyroid cell lines and generation of CL2/CCDC80stable clones
In this study, we used the ATC-derived FRO and FB-1 celllines, and human embryonic kidney 293 (HEK293) cells. Theywere grown in DMEM (Life Technologies, Grand Island, NewYork) containing 10% fetal calf serum, glutamine, and ampicil-lin/streptomycin (all from Life Technologies) in a 5% CO2 at-mosphere. All cell lines were available in our laboratories andwere routinely tested according to standard required procedures.Thyroid carcinoma cell lines were transfected using Arrest-inreagent (Open Biosystems, Huntsville, Alabama) withp3XFLAG-CMV-10 plasmid (Sigma, St Louis, Missouri) carry-ing the human full-length coding sequence of the CL2/CCDC80gene (pCMV-10-CL2/CCDC80) or with empty vector. Trans-fected cells were selected in a medium containing 1250 �g/mLgeneticin (G418; Life Technologies) and several G418-resistantclones, and the mass cell population were isolated and expandedfor further analyses.
Proliferation, apoptosis, migration and transformation as-says are described in Supplemental Materials and Methods.
Protein extraction, Western blotting, andcoimmunoprecipitation
Protein extraction, Western blotting, and coimmunoprecipi-tation procedures were carried out as reported elsewhere (35)and are described in Supplemental Materials and Methods.
Chromatin immunoprecipitationChromatin samples, derived from transfected cells, were pro-
cessed for chromatin immunoprecipitation (ChIP) and re-ChIPexperiments as reported elsewhere (35). Samples were subjectedto immunoprecipitation with the specific antibodies anti-FLAG(F7425; Sigma) and anti-ZBRK1 (ab77085; Abcam, Cambridge,United Kingdom). The primers used for E-cadherin promoterChIP experiments are reported elsewhere (35). Input DNA val-ues were used to normalize the values from quantitative ChIPsamples. Data are reported as percent input and were calculatedwith the following formula: 2�Ct � 3, where � cycle threshold(Ct) is the difference between Ctinput and CtIP (35).
Transactivation assayHEK293 cells were transiently transfected with the reporter
construct containing the Luciferase gene under the control of afragment of the E-cadherin gene promoter (35) and with thepCMV-10-CL2/CCDC80 (see above) and ZBRK1 (SC320303;OriGene Technologies, Rockville, Maryland) expression vec-tors. The �-galactosidase construct was cotransfected for datanormalization. Protein lysates were prepared 36 hours after
transfection, and lysates were analyzed for luciferase activitiesusing the dual-light system (Life Technologies) and a luminom-eter (Lumat LB9507; Berthold Technologies, Bad Wildbad,Germany).
Statistical analysisA Student’s t test was used for the comparison between 2
groups of experiments. Statistical significant difference was con-sidered when P � .05. A Pearson correlation coefficient (R2)close to 1 was considered indicative of a significant direct cor-relation. All experiments were done in triplicate and the data aremean � SD of 3 independent experiments.
Study approvalHuman cancer samples, with paired normal adjacent tissue,
were obtained from the Service d’Anatomo-Pathologie, CentreHospitalier Lyon Sud (Pierre Benite, France) according to theguidelines of the local review board. Informed consent was ob-tained from all patients for the scientific use of biologicalmaterial.
Results
CL2/CCDC80 expression is drastically reduced inhuman thyroid carcinomas
In a previous analysis of a limited number of humanthyroid neoplastic samples, we found that CL2/CCDC80expression was lower in most of the PTC samples than inthe normal counterparts (32). This finding prompted us tomeasure CL2/CCDC80 mRNA expression levels in a va-riety of human thyroid neoplasias (10 FTAs, 36 classicPTCs, 33 follicular variants of PTC, 8 FTCs, and 8 ATCs)using a qRT-PCR assay. As shown in Figure 1A, CL2/CCDC80 expression was significantly down-regulated(with a fold change greater than 5) in 63.5% of carcinomaswith respect to normal thyroid tissue. The reduction inCL2/CCDC80 gene expression was particularly strikingin the follicular variant of PTC (average fold change�51.3). In 7 of the 33 cases of this histotype analyzed, thefold change exceeded �70, and in some cases CL2/CCDC80 expression was barely detectable. The reductionin CL2/CCDC80 gene expression was less pronounced inthe classic forms of PTC, FTC, and ATC (average foldchange �8.9, �33.2, and �22.4, respectively). CL2/CCDC80 gene expression was slightly down-regulated inFTAs (average fold change �2.7, Figure 1A).
We also screened the 69 PTCs analyzed in this study forthe genetic alterations known to be associated with thesetypes of carcinomas, ie, RET/PTC1 and RET/PTC3 acti-vation, BRAF mutations, TRK-T1 rearrangements, andmutations of the RAS gene family. Figure 1B shows theexpression of CL2/CCDC80 in the PTCs carrying RET orTRK-T1 activation or BRAF mutations. The reduction ofCL2/CCDC80 expression in PTCs mutated in BRAF was
2836 Ferraro et al CL2/CCDC80 Is a Potential Tumor Suppressor Gene J Clin Endocrinol Metab, July 2013, 98(7):2834–2843
comparable with that observed in thePTCswithoutmutations (average foldchange �9.4). Conversely, CL2/CCDC80 expression was moderatelydown-regulated (average fold change�4.9) in the PTCs carrying the RET/PTC1 rearrangement and drasticallydown-regulated in the only 2 PTCscarrying RET/PTC3 rearrangements(average fold change �68). RAS mu-tations were found in 8 cases of follic-ular PTC: 5 mutations in the Ha-RASgene and 3 in the N-RAS gene. Theaveragefoldchangewas�70,which issimilar to that found in the same his-totypes without RAS mutations (datanot shown).
Because cl2/ccdc80 gene expres-sion is down-regulated by �-catenin(33), we measured the expression of�-catenin in the thyroid tumor sam-ples. As shown in Supplemental Fig-ure 1, �-catenin-specific mRNA ex-pression was increased, albeit notsignificantly, in thyroid carcinomas(Supplemental Figure 1). Immuno-histochemical analysis of 10 PTCs ex-cludedthepresenceofthe�-cateninpro-tein at nuclear level (SupplementalFigure 2). However, activation of theWNT/�-catenin pathway is a constantfeature of human thyroid carcinomas,even in the absence of �-catenin up-reg-ulation or of mutation (36).
To evaluate CL2/CCDC80 pro-tein expression, we performed an im-munohistochemical analysis of 56paraffin-embedded thyroid tumorspecimens, including some of thesamples previously analyzed by RT-PCR. The results of the immunohis-tochemical study of these thyroidspecimens are reported Table 1. Pro-tein CL2/CCDC80 expression wasnot detected in a high percentage ofPTCs, FTCs, and ATCs, therebyconfirming the down-regulation ofCL2/CCDC80 gene expression.However, interestingly, when de-
Figure 1. CL2/CCDC80 mRNA levels are reduced in human thyroid carcinomas. A, CL2/CCDC80quantitative RT-PCR of thyroid tumors. Samples are divided according to histotype. FV-PTC,follicular variant of PTC. Values are expressed as fold change in a logarithmic scale vs a pool ofnormal samples, which were set equal to 1. The variability of CL2/CCDC80 expression in normalthyroid tissues is below 10%. B, Expression of CL2/CCDC80 mRNA in relation to oncogenemutations detected in thyroid carcinoma samples. The average fold change of CL2/CCDC80down-regulation in the BRAFV600E group was �9.4 and �4.9 in the RET/PTC1 group and �68.0in the RET/PTC3 group. The fold change in CL2/CCDC80 expression in the only sample with aTRK-T1 rearrangement was �4.4. Values are expressed as fold change values in a logarithmicscale vs a pool of normal samples, which were set equal to 1.
tectable, CL2/CCDC80 protein was mislocalized from thenucleus to cytoplasm. Surprisingly, no staining was ob-served in 8 of 10 ATCs, even though mRNA expressionwas reduced in some cases.
Some representative immunohistochemical results areshown in Figure 2. A positive nuclear signal was detectedin normal thyroid (Figure 2, A and B), goiter (Figure 2, Cand D), and FTA (Figure 2E), whereas no nuclear stainingwas observed in PTC (Figure 2F), FTC (Figure 2G), orATC (Figure 2H). The specificity of the reaction was con-firmed by the lack of tissue immunoreactivity after prein-cubation of the antibody with a molar excess of the CL2/CCDC80 synthetic peptide (data not shown) or byomitting the primary antibody (data not shown).
Loss of heterozygosity at the CL2/CCDC80 locus(3q13.2) in thyroid neoplastic samples
Loss of one allele often accounts for reduced gene ex-pression. Therefore, we examined the thyroid neoplasticsamples for loss of heterozygosity (LOH) at the CL2/CCDC80 locus. As shown in Supplemental Table 1 (45selected informative cases), LOH at the CL2/CCDC80locus was observed in 16 cases (35.5%), with the highestfrequency in the follicular PTC variants (10 of 20, 50%).A lower frequency was observed in the classic PTCs (3 of13, 23%). Among the 5 informative ATC samples, wefound LOH in 3 cases (3 of 5, 60%). Conversely, no LOHwas observed in the 7 FTAs analyzed. Interestingly, CL2/CCDC80 expression was much lower in the PTC samples(both variants) with LOH (average fold change �85.2)than in those without LOH (average fold change �12.4,Supplemental Figure 3).
Restoration of CL2/CCDC80 gene expressionreverts the malignant phenotype of thyroidcarcinoma cell lines
We assessed the tumor suppressor activity of CL2/CCDC80inthyroidcarcinogenesisbyperformingfunctional
studies. First, we evaluated the growth rate of 2 thyroid car-cinoma cell lines (FRO and FB-1), not expressing CL2/CCDC80, after restoration of CL2/CCDC80 expression. Tothis aim, we carried out a colony-forming assay in FRO andFB-1cellsafter transfectionwith thevectorcarrying theCL2/CCDC80geneor theemptybackbonevector (Figure3A).Asshown inFigure3B, cells transfectedwith theCL2/CCDC80gene (FRO-CL2 and FB-1-CL2) generated fewer colonies(less than 50% for FRO-CL2) than cells transfected with thebackbone vector (FRO-V and FB-1-V). A 2,3-bis-(2-me-thoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxani-lideassay,whichgivesameasureof thecellproliferationrate,yielded similar results. Indeed, as shown in Figure 3C, theFRO cells expressing the CL2/CCDC80 gene (FRO-CL2-Band FRO-CL2-E) grew at a significantly slower rate than theuntransfected (FRO) and backbone vector-transfected cells(FRO-V).
We next investigated the cell cycle phase distribution ofthe CL2/CCDC80-transfected FRO cells by flow cyto-metric analysis. As shown in Figure 3D, this analysis re-vealed a significant shift of the DNA profile to a sub G1position of the FRO-CL2 cells compared with the FRO-Vcells (from 8.85% of the FRO-V cells to 26.92% of theFRO-CL2 cells). Consistent with this finding, a terminaldeoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labeling (TUNEL) assay showedpositive staining for the FRO-CL2 cells but not for theFRO-V cells (Figure 3E). Similar results were obtainedwith the FB-1 cells (data not shown). These results impli-cate the CL2/CCDC80 gene in the process of apoptosis.
To evaluate whether CL2/CCDC80 was able to revertthe malignant phenotype of thyroid cancer cells, we ana-lyzed the ability of FRO-CL2 cells to grow in soft agar. Therestoration of CL2/CCDC80 expression drastically re-duced the ability of FRO cells to grow in agar (Figure 4A).Indeed, the number of colonies formed by FRO-CL2 cells
Table 1. Immunohistochemical Analysis of CL2/CCDC80 Protein Expression in Human Thyroid Normal andNeoplastic Samples
2838 Ferraro et al CL2/CCDC80 Is a Potential Tumor Suppressor Gene J Clin Endocrinol Metab, July 2013, 98(7):2834–2843
was much lower than that formed by FRO-V cells, and thesize of these colonies was much smaller (Figure 4B).
We also evaluated the tumorigenicity of FRO-CL2 andFRO-V cells by injecting 2 � 106 of them in athymic mice.Xenograft tumors did not develop in mice 6 weeks afterinjection of FRO-CL2 cells, whereas they did develop 3–4weeks after injection with FRO-V cells (Supplemental Ta-ble 2).
Because migration ability is a feature of malignant cells,we evaluated the ability of FRO-CL2 and FRO-V cells tomigrate. First, we performed a scratch wound healing assay.
Wound healing was pronouncedwithin 20 hours in control FRO-Vcells, whereas it was only marginal inFRO-CL2 cells at the same time point(Figure 4C). Lastly, we performed amigration assay in which cells wereseeded in serum-free medium on thetop chamber of a 2-chamber transwellcell culture plate. Colorimetric evalu-ation of the cells migrated to the lowerchamber revealed fewer migratedFRO-CL2 cells with respect to FRO-Vcells (Figure 4D), which indicates thatthe CL2/CCDC80 gene inhibits cellmigration.
Taken together, our results impli-cate loss of CL2/CCDC80 expressionin the progression of carcinogenesis.Because the lossofE-cadherin isahall-mark of the epithelial-mesenchymaltransition (37, 38), which is a criticalevent in cancer progression, we inves-tigated whether CL2/CCDC80 is in-volved in the regulation of E-cadherinexpression.
To this aim, we analyzed the ex-pression of the E-cadherin gene afterrestoration of CL2/CCDC80 expres-sion. As shown in Figure 5A, CL2/CCDC80 restoration resulted in theup-regulation of E-cadherin expres-sion in FRO cells, as assessed byqRT-PCR and Western blot analyses(Figure 5A). To investigate whetherCL2/CCDC80 was also able to bindthe E-cadherin promoter in vivo, weperformed a ChIP assay usingHEK293 cells with FLAG-taggedCL2/CCDC80 expression vector.
Anti-FLAG antibodies were able to precipitate in the E-cadherin promoter region (�300�40 of the region en-compassing trascription start site). No immunoprecipita-tion was observed with IgG precipitates or with primersfor the control promoter glyceraldehyde-3-phosphatedehydrogenase (data not shown), which indicates thatthe binding is specific for the E-cadherin promoter (Fig-ure 5B).
Finally, to evaluate the effects of CL2/CCDC80 expres-sion on E-cadherin transcription, we transiently cotrans-
Figure 2. CL2/CCDC80 protein levels are decreased in human thyroid carcinomas.Immunohistochemical analysis of CL2/CCDC80 protein expression was evaluated in humanbenign and malignant thyroid tissues. A and B, Normal thyroid with 2 different specimens (�200and �400, respectively), showing nuclear immunoreactivity. C and D, Goiter with 2 differentspecimens (�200 and �400, respectively), showing nuclear immunoreactivity. E, Thyroidadenoma (�200), showing nuclear positivity (inset: �400 magnification). F, Thyroid papillarycarcinoma (�200), negative for protein expression (inset: �400 magnification). G, Thyroidfollicular carcinoma (�200) without CL2/CCDC80 protein expression (inset: �400magnification). H, Anaplastic thyroid carcinoma (�200) without CL2/CCDC80 protein expression(inset: �400 magnification).
fected HEK293 cells with an expression vector encodingCL2/CCDC80 and with a reporter vector carrying theLuciferase gene under the control of the E-cadherin pro-moter. As shown in Figure 5C, CL2/CCDC80 dose de-pendently increased the transcriptional activity of the E-cadherin promoter. These results indicate that CL2/CCDC80 positively regulates E-cadherin transcription bydirect binding to its promoter.
CL2/CCDC80 interacts with zinc finger protein 350(ZBRK1/ZNF350) protein and counteracts itsnegative effect on the E-cadherin promoteractivity
To investigate the mechanisms by which CL2/CCDC80positively regulates E-cadherin expression, we carried outa yeast 2-hybrid screening to search for CL2/CCDC80interacting proteins. Among the interacting proteins iden-tified, we focused our attention on ZBRK1/ZNF350,whichhasbeen implicated in tumorbiology (39,40). Indeed,ZBRK1 is a direct mediator of a specific transcriptional
corepressor function for BRCA1(41), which is responsible for most hu-man familial breast and ovarian carci-nomas. We performed an immunopre-cipitation assay to verify the interactionbetween CL2/CCDC80 and ZBRK1.As shown in Figure 5D, we detected theassociation between transfected FLAG-tagged CL2/CCDC80 and the endoge-nous ZBRK1 protein in HEK293 cells,thereby confirming that CL2/CCDC80is able to form complexes with thisprotein.
We then carried out ChIP and re-ChIP analyses to determine whetherthese interactions take place on theE-cadherin promoter. As illustratedin Figure 5E, the antibody againstZBRK1 precipitated the E-cadherinpromoter after its release from anti-FLAG immunocomplexes, which in-dicates that CL2/CCDC80 occupiesthis promoter region together withthis interactor. The reciprocal exper-iment yielded similar results (datanot shown).
We hypothesized that CL2/CCDC80 interacts with ZBRK1 toregulate E-cadherin gene expression.To address this hypothesis, we trans-fected HEK293 cells with ZBRK1alone or with increasing amounts ofCL2/CCDC80 cDNA together with
the E-cadherin promoter construct. As shown in Figure5F, ZBRK1 negatively regulated the E-cadherin promoteractivity, whereas CL2/CCDC80 dose dependently coun-teracted this effect.
Discussion
To date, several oncogenes have been demonstrated to beinvolved in human thyroid carcinomas, particularly in thepapillary histotype, but little is known about tumor suppres-sor genes involved in thyroid carcinogenesis. Moreover,about 20%–25% of carcinomas affecting the thyroid glanddo not show mutations or changes in the expression of well-known oncogenes. Therefore, other mechanisms such as de-regulated expression of still unidentified genes may play animportant role in thyroid carcinogenesis.
We previously reported that the CL2 gene (also knownas CCDC80) was, down-regulated in a panel of thyroid
Figure 3. CL2/CCDC80 restoration reduces proliferation of thyroid carcinoma cell lines. A, CL2/CCDC80 expression in FB-1-V, FB-1-CL2, FRO-V, and FRO-CL2 cells evaluated by Western blotanalysis. B, Restoration of the CL2/CCDC80 gene in the FRO and FB-1 human thyroid carcinomacell lines reduces their ability to form colony in culture disk. C, Growth curve of FRO-CL2 stableclones (CL2-B and CL2-E), untransfected FRO cells, and FRO cells transfected with empty vector(FRO-V). Five days after plating, the number of cells expressing CL2/CCDC80 gene was more than50% lower than the control. D, Cell cycle analysis of FRO-V and FRO-CL2 cells showed thatreexpression of CL2/CCDC80 causes an increment of cells in the subG1 phase. E, TUNEL assay onFRO-V and FRO-CL2 cells. Virtual pictures of apoptotic nuclei were obtained by merging DAPI-stained nuclei (blue), with red fluorescence of DNA strand breaks labeled withtetramethylrhodamine-deoxyuridine 5-triphosphate. Results are expressed as percentage of redfluorescence stained nuclei over DAPI-stained nuclei. DAPI, 4�,6-diamidino-2-phenylindole.
2840 Ferraro et al CL2/CCDC80 Is a Potential Tumor Suppressor Gene J Clin Endocrinol Metab, July 2013, 98(7):2834–2843
carcinomas, which suggested that it exerts tumor suppres-sor activity. The results of the present study, conductedwith a large number of specimens, confirm CL2/CCDC80down-regulation in thyroid carcinomas. Moreover, theysupport the concept that CL2/CCDC80 plays a role inhuman thyroid carcinogenesis by mediating the control ofcell growth, invasion, and apoptosis.
The reduction of CL2/CCDC80 mRNA was greatest inthe follicular PTC variant with an average down-regula-tion of �51.3-fold. Moreover, CL2/CCDC80 mRNA wasalmost undetectable in some samples of this histotype.CL2/CCDC80 was also greatly reduced in the classic formof PTC and in FTC and ATC. Immunohistochemical dataconfirmed the qRT-PCR data. Interestingly, the CL2/CCDC80 protein was delocalized from the nucleus to thecytoplasm in those carcinoma samples that retained de-tectable levels of the protein. In partial contrast with RNAdata, the CL2/CCDC80 protein was undetectable in mostof the ATCs. This finding could be due to overexpressionin ATCs of microRNAs that specifically target CL2/CCDC80 mRNA, to mechanisms that impair CL2/CCDC80 mRNA translation in cancer cells or to contam-ination of normal cells in the tumor sample from which
RNA has been extracted. Moreover,given the finding that, upon apopto-tic stimuli, CL2/CCDC80 shuttlesfrom Golgi to the endoplasmic retic-ulum to exert its apoptotic function(42), it is feasible that the mislocal-ization of the protein could also in-terfere with apoptosis.
Our study also implicates LOH atthe CL2/CCDC80 locus in the re-duced expression of CL2/CCDC80in thyroid carcinomas. In fact, LOHat the CL2/CCDC80 locus was ob-served in 50% of the follicular PTCvariants and was well correlatedwith the down-regulation of theCL2/CCDC80 gene expression.
The results of our experiments onrestoration of CL2/CCDC80 geneexpression in thyroid carcinoma celllines indicate that loss of this geneexpression plays a critical role in thy-roid carcinogenesis. In fact, thegrowth rate of FRO cells transfectedwith a CL2/CCDC80 expressionvector was significantly reduced.Moreover, flow cytometric analysis,confirmed by the TUNEL assay,showed a shift of the DNA profile of
CL2/CCDC80 transfected cells to a subG1 position,which is consistent with the concept that CL2/CCDC80expression exerts an apoptotic effect. This finding is alsoin line with the recent finding that CL2/CCDC80 plays acrucial role in the apoptotic process (42). Finally, the on-cosuppressor function of the CL2/CCDC80 gene wasclearly indicated by the suppression of the neoplastic phe-notype by the cells reexpressing CL2/CCDC80. Indeed,these cells were no longer able to efficiently grow in semi-solid medium and did not induce tumors when injectedinto athymic mice.
Preliminary results of the characterization of the cl2/ccdc80 knockout mice, generated by our research group,seem to confirm that CL2/CCDC80 exerts a tumor sup-pressor function (Ferraro, A., and A. Fusco, manuscript inpreparation). Indeed, these mice develop thyroid adeno-mas and ovarian carcinomas with a significant frequency.Moreover, preliminary data indicate that mouse embry-onic fibroblasts heterozygous for cl2/ccdc80 grow fasterthan the wild-type counterparts and are less susceptible toapoptosis. These results indicate that even the expressionof only one allele is sufficient to exert a tumor-suppressiveeffect.
Figure 4. CL2/CCDC80 restoration reverts the malignant phenotype of thyroid carcinoma celllines. A and B, FRO cells transfected with the CL2/CCDC80 gene lose their ability to grow in asemisolid medium (detached from culture disk), as evaluated by soft agar assay. Histogramsrepresent the number of colonies detected by a microscopy observation. After 3 weeks thecolonies of both plates were photographed and counted. C, In vitro wound healing assayshowing the diverse ability of FRO-V and FRO-CL2 cells to migrate. Cells were photographedimmediately after scratch (0 hour) and then after 20 hours (20 h). FRO-CL2 cells show a lowermobility and therefore a less aggressive phenotype compared with FRO-V cells. D, FRO-CL2 cellshad a reduced ability to cross the transwell membrane compared with FRO-V cells asdemonstrated by the weaker staining of the transwells in which they were seeded. Opticalmicroscopy pictures show the transwells containing FRO-V cells and FRO-CL2 cells, respectively.
Regarding the mechanism by which the CL2/CCDC80gene affects cancer progression, it is feasible that CL2/CCDC80, by positively regulating E-cadherin expression,counteracts the negative regulation by ZBRK1 (39, 40).However, the mechanisms by which CL2/CCDC80 is in-volved in cell growth regulation and apoptosis need to bedefined.
In conclusion, the data reported herein are consistentwith the concept that loss of CL2/CCDC80 expression isinvolved in the process of thyroid carcinogenesis.
Acknowledgments
We thank Dr Yuri E. Nikiforov for theRAS gene mutations analysis. We aregrateful to Jean Ann Gilder (ScientificCommunication srl, Naples, Italy) forsubstantial editing of this article.
Address all correspondence and re-quests for reprints to: Angelo Ferraro,Signal Mediated Gene Expression Labo-ratory, Institute of Biology, MedicinalChemistry, and Biotechnology, NationalHellenic Research Foundation, 48 Vassi-leos Constantinou Avenue, 11635 Ath-ens Greece. E-mail: [email protected].
Thisworkwassupportedbygrants fromAssociazione Italiana per la Ricerca sul Can-cro (IG 5346) and the Ministerodell’Università e della Ricerca Scientifica eTecnologica, and Ministero dell’Istruzione,dell’Università e della Ricerca (PRIN 2008).
Disclosure Summary: The authorshave nothing to disclose.
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