Prohibitin Is Overexpressedin Papillary Thyroid CarcinomasBearing the BRAFV600E Mutation

Alessandra Franzoni,1 Mariavittoria Dima,1 Maria D’Agostino,2 Cinzia Puppin,1 Dora Fabbro,3

Carla Di Loreto,4 Maura Pandolfi,4 Efisio Puxeddu,5 Sonia Moretti,5 Marilena Celano,2

Rocco Bruno,6 Sebastiano Filetti,7 Diego Russo,2 and Giuseppe Damante1,3

Background: Prohibitin (PHB) is a multifunctional protein that is localized in different intracellular sites. PHB mayexert different roles in tumorigenesis, having either a permissive action on tumor growth or an oncosuppressorrole, depending on the cellular context. The objective of this study was to evaluate PHB expression in normalthyroid tissues, thyroid follicular adenomas (FAs), and papillary thyroid carcinomas (PTCs).Methods: PHB expression was analyzed by immunohistochemistry, Western blot, and quantitative reversetranscription polymerase chain reaction (RT-PCR). Transfections in the BCPAP and TPC-1 thyroid cancer cell lineswere used to evaluate the PHB promoter activity.Results: In terms of protein and mRNA levels, normal tissues from patients with serum thyrotropin (TSH) values>0.8 mU=L had PHB levels that were significantly reduced compared to specimens from patients with serum TSHvalues <0.5 mU=L, suggesting that TSH exerts an inhibitory effect on PHB expression. Consistent with this was thefinding that the presence of TSH was associated with low PHB levels in normal FRTL5 thyroid cells. Im-munohistochemical analysis showed relatively low and high PHB expression in FAs and PTCs, respectively. PHBmRNA and protein overexpression, as assessed by quantitative RT-PCR and Western blot, was noted only in PTCsbearing the BRAFV600E mutation. Notably, cell transfection experiments suggested that presence of the BRAFV600E

mutation may be associated to increase of the PHB promoter activity.Conclusions: PHB is overexpressed in PTCs bearing the BRAFV600E mutation. We postulate that the presence of theBRAFV600E mutation increases PHB promoter activity and therefore potentially mediates effects of this mutationon the behavior of BRAFV600E positive PTCs.

Introduction

Thyroid cancer tissues retain most of the biologicalproperties of normal thyroid cells, including thyroglob-

ulin production and radioiodine uptake. These characteristicsare used for postsurgical follow-up and treatment of persis-tent or recurrent disease, permitting an improved prognosisfor these patients (1). Decreased expression or function ofproteins that mediate the ability of thyrocytes to concentratethe iodine, however, imposes major limitations on the use ofradioactive isotopes in treating epithelial cell thyroid cancer

(2). Such a dedifferentiated phenotype is typical of a group ofthyroid cancers, with a more aggressive clinical behavior. Arelationship with particular genotypic alteration such as ac-tivation or inactivation of oncogenes and anti-oncogenes hasbeen hypothesized. Indeed, in papillary thyroid carcinomas(PTCs), the presence of the activation of the BRAF oncogenehas been correlated with a more aggressive phenotype (3–5).It may be related to the loss of differentiation markers such asgenes regulating iodine metabolism (6) as well as an increasein the angiogenetic markers (4,7) that have been detected inthese tumors.

1Dipartimento di Scienze e Tecnologie Biomediche, Universita di Udine, Udine, Italy.2Azienda Ospedaliero-Universitaria ‘‘S. Maria della Misericordia,’’ Udine, Italy.3Dipartimento di Ricerche Mediche e Morfologiche, Universita di Udine, Udine, Italy.4Dipartimento di Medicina Interna, Universita di Perugia, Perugia, Italy.5Dipartimento di Scienze Cliniche, Universita di Roma ‘‘la Sapienza,’’ Roma, Italy.6Dipartimento di Scienze Farmacobiologiche, Universita di Catanzaro, Catanzaro, Italy.7Ospedale di Tinchi-Pisticci, Matera, Italy.

THYROIDVolume 19, Number 3, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=thy.2008.0235

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Recently, the study of protein expression profiles has pro-vided useful information that has helped to characterize themalignant versus benign phenotype and characteristics as-sociated with more aggressive as opposed to less aggressivethyroid cancers (8–14). Preliminary studies, however, need tobe validated by studies with a large number of tumors andshould provide detail regarding the function, regulation, andexpression levels of potential markers of the aggressiveness oftumor behavior.

By using differential proteomic analysis of PTCs andmatched normal tissues, some of us and others previouslynoted (15,16) that prohibitin (PHB) expression was increasedin PTC compared to normal thyroid tissues.

Interestingly, the study by some members of our group wasexclusively of PTCs bearing the BRAFV600E mutation (16),while the others analyzed an unselected PTC cohort (15).

However, a different protein expression profiling study,also conducted in an unselected cohort, may have containedan inferior number of PTCs bearing the BRAFV600E mutationand could not detect differences in PHB expression betweencancer and normal tissue (17).

PHB is a multifunctional protein with a molecular weight ofabout 32 kDa. It is similar to prohibitin 2 (PHB2), which hasa molecular weight of 37 kDa (18). PHB is ubiquitously ex-pressed and localizes to different intracellular locations in-cluding mitochondria, nuclei, lipid rafts, and cytosol (19–22).In the mitochondria, PHB appears to exert a chaperone func-tion, protecting newly imported proteins from degradation(23). Moreover, it has been demonstrated that PHB inhibitspyruvate carboxylase (24), a mitochondrial enzyme involvedin the tricarboxylic acid cycle (25). Modification of the mito-chondrial PHB protein levels have been demonstrated duringcellular senescence in cultured human fibroblasts (26).

Accordingly, knockdown of PHB in endothelial cells in-creases mitochondrial production of reactive oxygen species,resulting in cellular senescence (27). In the nucleus PHB mod-ulates gene transcription (28,29). Structural and functionalinteractions with several transcription factors including p53,RB, E2F, and estrogen receptor have been described (20,30,31).Moreover, PHB interactions with chromatin-remodelling pro-teins have been shown (32).

There are conflicting experimental data regarding the in-volvement of PHB in tumorigenesis, showing a permissiveaction on tumor growth or, alternatively, an oncosuppressorrole depending on the cell context (33).

In this work we analyzed the expression levels of PHB in aseries of thyroid tumors, including follicular adenomas andpapillary thyroid carcinomas, the latter also characterized forBRAF mutational status. In addition, PHB expression and itsregulation by thyrotropin (TSH) was investigated in normalthyroid tissues and thyroid cell lines.

Materials and Methods

Thyroid tissues

Surgical specimens of 14 nonconsecutive sporadic thyroidfollicular adenomas (FAs) and 40 PTCs were analyzed. Thetissues were snap-frozen and stored at �808C until use. Pa-tients’ charts were reviewed to define the clinical features ofeach case. Normal tissues originating from the non-neoplastictissue of all FAs and all PTCs were also analyzed. All tumorswere histologically diagnosed and, when possible, staged

according to the criteria of the American Joint Committee onCancer (34). The study was approved by the local medicalethics committee. Before surgery, each study participant pro-vided written, informed consent to the collection of freshthyroid tissue for genetic studies.

We also analyzed 26 samples randomly selected from aseries of normal thyroid glands collected during a previousstudy from patients operated on for benign thyroid nodules(35). At the time of surgery, serum TSH levels of patients ofgroup 1 were within normal limits (1.0–4.0 mU=L) whereas inpatients of group 2 they were below 0.5 mU=L, as a result ofsuppressive levothyroxine (LT4) therapy. All sample aliquotswere immersed in liquid nitrogen and stored at �708C im-mediately after surgery.

Cell lines and transfection

BCPAP and TPC-1 are PTC-derived cell lines. The formerbears a BRAFV600E mutation, while the latter harbors aRET=PTC1 rearrangement (36). FRTL5 cells are a normal ratthyroid cell line. BCPAP and TPC-1 cells were cultured inDulbecco’s modified Eagle’s medium (DMEM) with 10% fetalcalf serum (Gibco, Milano, Italy). FRTL5 cells were main-tained in F12 Coon’s modified medium with 5% calf serumand hormone mixture as previously described (37). The cal-cium phosphate coprecipitation method was used for trans-fections, as described elsewhere (38). Twenty hours prior totransfection, BCPAP and TPC1 cells were plated at 6�105

cells=100-mm culture dish. FRTL5 cells were plated at1.5�106 cells=100-mm culture dish 48 hours prior to trans-fection, and 3 hours prior to the addition of the DNA-calciumphosphate precipitates, the medium was changed to DMEMcontaining 5% calf serum and growth factors. The prohibitinpromoter construct, Prohibitin-Luc, was kindly donated byDr. Theiss (39). The BRAFV600E mutant allele is cloned in theexpression vector pEFP, in which transcription is driven bythe EF-1a promoter. The transfection efficiency was normal-ized by cotransfecting the RSV-CAT plasmid, which con-tains the Rous sarcoma virus (RSV) promoter, linked to thechloramphenicolacetyltransferase (CAT) gene. Cells wereharvested 48 hours after transfection and cell extracts wereprepared by a standard freeze and thaw procedure. CATprotein levels were measured by enzyme-linked immuno-sorbent assay (Amersham-Pharmacia Biotech, Milano, Italy).Luciferase activity was measured by a standard chemilumi-nescence procedure (38).

Immunohistochemistry and Western blot

Dewaxed tissue sections of a different series of thyroid tu-mors, including 10 FAs and 10 PTCs as well as 10 normalthyroid tissues, were analyzed by immunohistochemistryto search for expression of the PHB protein. Immunohisto-chemistry was performed using a rabbit polyclonal antibody(Novus Biologicals, Littleton, CO) with a final dilution to1:100 and a polymer-based immunohistochemical detectionsystem (Super SensitiveTM Polymer-HRP IHC Detection Sys-tem, BioGenex, San Ramon, CA). Specificity of the immuno-histochemical signal was controlled by omitting the primaryantibody.

Total proteins were extracted from thyroid tissues aspreviously described (40). Briefly, frozen tissue or cell pelletwas thawed and homogenized in 1 mL of buffer containing

248 FRANZONI ET AL.

250 mM sucrose, 10 mM HEPES-KOH (pH 7.5), 1 mM EDTA,1 mM PMSF, 10 mg=mL leupeptin, and 10 mg=mL aprotinin(Sigma Aldrich srl, Milano, Italy). The homogenate was cen-trifuged at 14,000 g (48C for 15 minutes) and the supernatant(which contained the whole cell lysate) was quantified spec-trophotometrically using the Bradford method. Thirty mi-crograms of protein was loaded on a 9% sodium dodecylsulfate (SDS) polyacrylamide gel and subjected to electro-phoresis at a constant voltage (120 V). Electroblotting toa Hybond-P ECL nitrocellulose membrane (Amersham-Pharmacia Biotech) was performed for 2 hours at 225 mAusing a Mini Trans blot electroblotting system (Bio-Rad,Milano, Italy). Blocking was done using TTBS=milk (Tris-buffered saline [TBS], 1% Tween 20, and 5% nonfat dry milk)for 2 hours at room temperature. The membrane was thenincubated with a 1:500 dilution of affinity purified rabbit anti-PHB (Abcam, Cambridge, UK) polyclonal antibody or a 1:5000dilution of mouse monoclonal anti-human beta-actin anti-body (Sigma Aldrich srl), overnight at 48C in TTBS=milk.After one 15-minute and two 5-minute washes in TTBS, themembrane was incubated with a 1:20,000 dilution of a horseradish peroxidase–conjugated anti-rabbit or anti-mouse anti-body (Transduction Laboratories, Lexington TX) in TTBS=milk.After one 15-minute and two 5-minute washes in TTBS, theprotein was visualized by an enhanced chemiluminescenceWestern blot detection system (ECL plus, Amersham-Pharmacia Biotech). Quantification was achieved by densito-metric scanning.

PHB mRNA quantification and detectionof the BRAFV600E mutation

PHB mRNA levels were analyzed by quantitative reversetranscription polymerase chain reaction (RT-PCR). Total RNAfrom thyroid tissues and cell lines was extracted with RNeasyprotect mini kit (Qiagen, Milano, Italy). One microgram oftotal RNA was reverse-transcribed to single strand cDNAusing random exaprimers and 200 U of MMLV reverse tran-scriptase (Invitrogen, Milano, Italy) in a final volume of 20 mL,at 428C for 50 minutes followed by heating at 708C for 15minutes. Real-time PCR was performed using the ABI Prism

7300 Sequence Detection System (Applied Biosystems, FosterCity, CA).

Oligonucleotide primers and the probe for human PHBwere purchased from Applied Biosystems, designed by Pri-mer Express program. The sequences of the primers and theprobe are the following: PHB forward, 50-TCACACTGCGCATCCTCTTC-30; PHB reverse 50-CAAAGCGAGCCACCACTGA-30; PHB probe 50-TCGCCAGCCAGCTTCCTCGC-30.Oligonucleotide primers and probe for the human endoge-nous control beta-glucuronidase is described by Beillard et al.(41). To measure PHB mRNA levels in FRTL5 cells, b-actin

FIG. 1. Prohibitin expression in normal thyroid tissue. (A)Immunohistochemical detection. (B) Western blot analysisunder reduced conditions of protein extracts from normalthyroid tissues, using a polyclonal anti-prohibitin antibody(see Materials and Methods). A representative image of twosamples is shown.

FIG. 2. Levels of prohibitin (PHB) protein and mRNAin normal thyroid tissues with low (<0.5 mU=L) or high(>0.8 mU=L) thyrotropin (TSH) levels and in FRTL5 cells inthe presence or absence of TSH. (A) Western blot analyseswere carried out with protein extracts from thyroid tissuesusing a polyclonal anti-prohibitin antibody or a monoclonalanti-beta actin antibody (see Materials and Methods). Valuesrepresent the PHB=beta-actin protein ratio expressed inarbitrary units as measured by laser densitometry. (B) Quan-titative reverse transcription polymerase chain reaction (RT-PCR) was performed as described in Materials and Methods.Both panels show results from 14 and 12 subjects with lowand high TSH levels, respectively. (C) Western blot of FRTL5cells in the absence or presence of TSH. (D) PHB mRNAlevels in FRTL5 cells in absence or presence of TSH. Statis-tical analysis was performed by Student’s t test.

PROHIBITIN EXPRESSION IN PTCS 249

FIG. 3. Immunohistochemicaldetection of PHB in normal,thyroid follicular adenoma (FA),and papillary thyroid carcinoma(PTC) specimens; (A) normaltissue, (B) FA, and (C) PTC. Ineach panel, a representativeimmunohistochemical image ofPHB expression is shownon the left; a quantitation ofPHB-positive cells is shown onthe right, expressed as a per-centage of 1000 evaluated cells:� no expression, þ weak ex-pression, þþ moderate expres-sion, þþþ high expression.

FIG. 4. Levels of PHB mRNA in(A) thyroid FA and (B) PTCtissues. Expression levels of PHBgene were measured by quanti-tative RT-PCR (see Materials andMethods) in tumor tissues andcorresponding normal tissues(NT) of 14 FAs and 40 PTCs.Statistical analysis was per-formed by paired Student’s t test.

250 FRANZONI ET AL.

was used as the endogenous control; oligonucleotide primersand probes for rat PHB and b-actin were purchased fromApplied Biosystems as Assays-on-Demand Gene ExpressionProducts. A 25-mL reaction mixture containing 5mL of cDNAtemplate, 12.5 mL of TaqMan Universal PCR master mix(Applied Biosystems), and 1.25 mL of primer probe mixturewas amplified using the following thermal cycler parameters:incubation at 508C for 2 minutes and denaturation at 958C for10 minutes, then 40 cycles of the amplification step (dena-turation at 958C for 15 seconds and annealing=extension at608C for 1 minute). All amplification reactions were per-formed in triplicate. The DCT method, by means of the SDSsoftware (Applied Biosystem), was used to calculate themRNA levels. A pool of six human normal thyroid specimentotal RNAs was used as calibrator. Thus, PHB mRNA levelsare always expressed as multiple or fraction of the expressionlevels in this pool, considered arbitrarily as 1.

BRAF mutations were identified by single-stranded con-formation polymorphism screening of products obtained byRT-PCR amplification of exon 15, and results were confirmedby means of sequence analysis (42).

Results

PHB is localized in different cell districts (19–22) and, de-pending on the cell type, the relative amounts may change.Thus, by using immunohistochemistry, we first investigatedthe localization of PHB in normal thyroid tissues. As shownin Fig. 1A, PHB was mainly detected as a weak diffuse stain-ing in cytoplasm in about 20% of thyrocytes. A Western blot

analysis performed on total protein extracts from normal tis-sues indicated that the antibody detects a single protein spe-cies of approximately 30 kDa, which is the reported molecularweight of human PHB (Fig. 1B).

Since no data are reported on PHB regulation in normalthyrocytes, we next investigated PHB protein levels usingboth in vivo and in vitro experimental models. Normal humanthyroid tissue was harvested from the extra-nodular portionsof thyroids that were resected for benign thyroid nodulardisease. Some of these patients had received LT4 prior tosurgery and others had not. This therefore was an ‘‘in vivo’’model (35) to evaluate the effect of TSH on some aspects ofthyroid-specific gene expression. PHB protein levels wereevaluated by Western blot, using b-actin to normalize proteininput. As shown in Fig. 2A, normal tissues from patients withserum TSH values >0.8 mU=mL displayed PHB protein levelssignificantly reduced with respect to specimens from patientswith serum TSH values <0.5 mU=mL. Similar data were ob-tained when PHB transcripts were measured by quantitativeRT-PCR. In fact, as shown in Fig. 2B, PHB mRNA levelswere significantly lower in tissues from patients with serumTSH values >0.8 mU=L with respect to tissues from pa-tients with serum TSH values <0.5 mU=L. These data sug-gest that ‘‘in vivo’’ TSH may exert an inhibitory effect on PHBexpression. In order to corroborate the notion that TSH con-trols PHB expression an ‘‘in vitro’’ model was also investi-gated. We analyzed the rat thyroid cell line FRTL5 kept for1 week in presence or absence of TSH (10 mU=mL). As shownin Fig. 2C and 2D, both protein and mRNA PHB levels werereduced in the presence of TSH.

FIG. 5. Levels of PHBmRNA and protein levels inPTCs with or without theBRAFV600E mutation. Expres-sion levels of prohibitin genewere measured by quantita-tive RT-PCR (see Materialsand Methods) in tumor tissuesand (A) corresponding normaltissues (N) of 20 PTCs with thewild-type BRAF gene (PTCBRAF�) and (B) 20 PTCs withthe BRAFV600E mutation (PTCBRAFþ). Expression of PHBprotein in PTC tissues with theBRAFV600E mutation was ana-lyzed by Western blot. Valuesare calculated as in Fig. 2. Datafrom tissues of 14 patientsare shown. Statistical analysiswas performed by pairedStudent’s t test. (D) A Westernblot with three representativecases of patients having PTCwith the BRAFV600E mutationis shown. N, normal tissue; T,tumoral tissue.

PROHIBITIN EXPRESSION IN PTCS 251

PHB expression in thyroid FAs and PTCs was then inves-tigated and compared to normal tissues. We first evaluatedPHB protein expression by immunohistochemistry. Ten caseseach of normal thyroid tissue, FA, and PTC were investigatedand results are shown in Fig. 3. As noted above, normalthyroids were weakly stained in about 20% of thyrocytes. InFAs both the fraction of stained cells and the intensity ofstaining were higher than in normal tissues. However, themost intense PHB expression was detected in PTCs, in whichboth cell positivity rate and staining intensity were higherthan in the previous tissue types.

PHB mRNA levels were also investigated in a differentseries of thyroid tissues, constituted by 14 FAs and 40 PTCs. Ineach case, the normal tissue of the same patient was used ascontrol. As shown in Fig. 4A, FAs displayed a slight increasein PHB mRNA levels, but the difference was not statisticallysignificant. In contrast, PTCs showed PHB mRNA levels sig-nificantly higher than corresponding normal tissues, thoughthe mean differences were not dramatically different (Fig. 4B).

A previous study by some of us showed an increase ofPHB protein levels in PTCs bearing the BRAFV600E mutationcompared to normal tissues (16).

Therefore, we divided our PTC cohort based on BRAFmutational status and compared the expression of PHB inthe mutation positive and negative cases. As shown in Fig.5A and 5B, PTCs not bearing the BRAFV600E mutation showedPHB mRNA levels very similar to the corresponding normal

Table 1. Correlation Between Prohibitin Gene

Expression and Several Clinical and Biological

Features of Papillary Thyroid Carcinomas

PHB mRNA ratio (T=N)a

�1.0 >1.0 p value

BRAFþ 10=25 (40%) 6=7 (85.7%) 0.03Age at diagnosis 46.8� 15.2 53� 19.7 0.48Tumor size 1.957� 0.844 1.914� 0.823 0.91

SexMale 7 2 0.81Female 14 5

MulticenterYes 9 5 0.19No 12 2

Extrathyroidal invasionYes 5 1 0.59No 16 6

Lymph node metastasisYes 7 3 0.64No 14 4

Distant metastasisYes 2 0 0.68No 17 6

StageIþII 15 3 0.16IIIþIV 4 3

Classic papillary histotypeYes 13 4 0.82No 8 3

Follicular variant histotypeYes 5 3 0.33No 16 4

Other histotypeYes 3 0 0.28No 18 7

aPTCs were divided in two groups (�1 and >1), depending on theratio of the PHB mRNA levels between neoplastic (T) and normal (N).

FIG. 6. PHB mRNA expression and PHB gene promoteractivity in BCPAP and TPC-1 cells. (A) PHB mRNA levels,measured as described in Materials and Methods by quanti-tative RT-PCR. (B) PHB promoter activity in BCPAP and TPC-1 cells. Results obtained with 1 and 2 mg of PHB promotervector are shown. (C) PHB promoter activity in TPC-1 cellstransfected with an expression vector of the BRAFV600E

mutant oncogene (BRAFV600E) or with the empty vector(pEFP). In panels (B) and (C), results are expressed as ratiobetween the PHB promoter and the RSV promoter, which wasused to normalize for efficiency of transfection.

252 FRANZONI ET AL.

tissues; in contrast PTCs bearing the BRAFV600E mutationshowed significantly higher levels of PHB mRNA levels. Acorrespondence between increased PHB mRNA levels andprotein levels (evaluated by Western blot) could be observedin tumors bearing the BRAFV600E mutation, as shown in Fig.5C,D.

We then evaluated whether PHB expression in PTCs wasrelated to some clinical and biological features of these tu-mors. PTCs were divided into two different groups, depend-ing on the PHB mRNA ratio between the neoplastic andthe normal tissue of each patient (�1 and >1). As shown inTable 1, only the presence of the BRAFV600E mutation showeda significant difference between the two groups of PTCs,suggesting that this mutation increases PHB expression. Inorder to provide an explanation for this phenomenon, weused two human thyroid cancer cell lines, both derived fromPTC: BCPAP and TPC-1. The BCPAP cell line bears theBRAFV600E mutation. This mutation is not present in the TPC1cells, which instead host the RET=PTC rearrangement (36). Inaccordance with data obtained in tumor specimens, we firstobserved that PHB mRNA levels were higher in the BCPAPcell line (Fig. 6A). Then, by using cell transfection in the samecell lines, we investigated the PHB promoter activity. A con-struct containing the human PHB promoter, linked to theLUC reporter gene was used (37). As shown in Fig. 6B, theactivity of the PHB promoter is higher in BCPAP cell line withrespect to the TPC-1 cell line. Thus, the TPC-1 cell line wastransfected with the PHB promoter construct and the ex-pression vector of the BRAF oncogene bearing the V600Emutation. Figure 6C shows the results of this experiment. Thepresence of the BRAFV600E oncogene is associated to a sig-nificant increase of PHB promoter activity. These resultssuggest that the activation of the BRAF oncogene may exert anactivating effect on the PHB promoter.

Discussion

Prohibitin is a regulator of the cell cycle that has been shownto be up-regulated in various tumors or transformed cells (43–45). In this study, we demonstrate that such a phenomenonoccurs in PTCs and that the increase of PHB gene expression isrelated to the presence of the BRAFV600E mutation. The presentdata, obtained in a large series of thyroid tumors, extend pre-vious studies using differential proteomic analysis, in which anincrease of PHB protein levels was detected in PTCs bearing theBRAFV600E mutation with respect to matched normal tissues(16). Moreover, data in human thyroid cancer cell lines confirmsuch results, suggesting an altered regulation of the PHB pro-moter activity. Accordingly, it has previously been shown thatexpression of the BRAFV600E mutant is able to modify the ac-tivity of various promoters (46,47).

Conflicting experimental data are reported concerning therole of PHB in tumorigenesis. PHB is required in the controlof the RAS-induced RAF-MEK=-ERK activation, which is acentral signaling pathway involved in cell growth and ma-lignant transformation in many cell types including thyrocytes(48,49). Thus, PHB expression should be permissive to tumorgrowth. Moreover, it has been recently demonstrated thatPHB maintains the angiogenic property of endothelial cells(50). In contrast, several reports indicate PHB as a tumor sup-pressor gene. For example, it has been shown that the 30 un-translated region of PHB mRNA exerts a tumor suppression

function in human breast cancer cells (51). Also the RNAiknockdown approach has provided conflicting results: in en-dothelial cells it induces cellular senescence and decreasedproliferation, but in MCF-7 cells PHB down-regulation pre-vents cellular senescence and increases cell growth (52). Thus,it appears that functions of PHB depend on the cell type.Nevertheless, recent data would suggest that the evaluationof PHB expression may have a diagnostic relevance. In fact, ithas been reported that immunohistochemical detection ofPHB in prostate biopsies may distinguish hyperplasia andcancer (43).

According to our present data and in agreement with pre-vious data using differential proteomic analysis (16), an in-crease of PHB protein levels is present in PTCs bearing theBRAFV600E mutation. It is well established that the BRAFV600E

mutation results in increasing kinase activity, leading to ex-cessive activation of the RAS=RAF=MEK=MAPK pathway andto uncontrolled proliferation of thyroid cancer cells (53,54).Simultaneously, it has been shown that PHB is indispensablefor the activation of the RAF-MEK-ERK pathway by RAS(48). The increase of PHB protein levels may therefore playa permissive role in thyroid tumorigenesis induced by BRAFactivation. Alternatively, activation of the RAS=RAF=MEK=MAPK pathway may play a causal role in the increase ofPHB gene expression, as suggested by our finding of increase ofPHB promoter activity in BRAFV600E expressing cells. More-over, it has been recently reported that the PHB promoter ac-tivity is controlled by STAT3 (37) and that RAF may induceSTAT3 transcriptional activation (55,56). Thus, PHB over-expression may represent a link between BRAF activation andthe loss of differentiated phenotype described in this subset ofthyroid tumors (6). In accordance with such a hypothesis, ourpresent data demonstrate an inverse correlation between bloodTSH levels and PHB expression in normal tissues. This inhibi-tory effect exerted by TSH, documented also in vitro, is re-pressed in thyroid cancer cells harboring BRAFV600E mutation,presumably owing to the impairment of cAMP-regulated geneexpression, driven primarily by the inhibition of TTF-1 tran-scriptional activity (57,58).

Acknowledgments

This work is funded by grants from MIUR to GD (PRIN N82007N8P32H_002) and DR and by grants from the Fonda-zione Umberto Di Mario and Banca d’Italia to SF. This workwas also funded by the FIRB grant no RBRN07BMCT. AF issupported by a grant from Fondazione Umberto Di Mario.CP is supported by a grant from FIRC (Federazione ItalianaRicerca Cancro). We thank Dr. Theiss for the gift of the plas-mid containing the PHB promoter.

Disclosure Statement

No competing financial interests exist.

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Address reprint requests to:Giuseppe Damante, M.D., Ph.D.

Dipartimento di Scienze e Tecnologie BiomedichePiazzale Kolbe 1

33100 UdineItaly

E-mail: gdamante@makek.dstb.uniud.it

PROHIBITIN EXPRESSION IN PTCS 255