CD44 Proteolysis Increases CREB Phosphorylation and ...cancerres.aacrjournals.org/content/canres/72/6/1449.full.pdf · Molecular and Cellular Pathobiology CD44 Proteolysis Increases

Molecular and Cellular Pathobiology

CD44 Proteolysis Increases CREB Phosphorylation andSustains Proliferation of Thyroid Cancer Cells

Valentina De Falco1, Anna Tamburrino1, Simona Ventre1, Maria Domenica Castellone1,Mouhannad Malek2, Serge N. Mani�e2, and Massimo Santoro1

AbstractCD44 is a marker of cancer stem-like cells and epithelial–mesenchymal transition that is overexpressed in

many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44undergoes sequentialmetalloprotease- and g-secretase–mediated proteolytic cleavage, releasing the intracellularprotein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we showthat CD44-ICDbinds to the transcription factor CREB, increasing S133 phosphorylation andCREB-mediated genetranscription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 tran-scription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenesexhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease,or g-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation wasobstructed by RNA interference–mediated knockdown of CD44 or inhibition of g-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathwaythat is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeuticintervention in thyroid carcinoma. Cancer Res; 72(6); 1449–58. �2012 AACR.

IntroductionCD44 is a glycosylated transmembrane glycoprotein that is

implicated in tumor growth andmetastasis (1–3). CD44 under-goes intramembrane proteolysis by metalloprotease thatcleaves the extracellular juxtamembrane stem domain, and,then, by g-secretase that cleaves the transmembrane domain(4–7). The first cleavage results in the shedding of the ecto-domain (ecto-CD44) and the release of the membrane-boundCD44 C-terminal fragment (CD44-CTF). The second cleavagecauses the release of the CD44 intracellular domain fragment(CD44-ICD; refs. 5, 6). Shedding of ecto-CD44 regulates cell–extracellular matrix interaction (8), whereas CD44-ICD trans-locates to the nucleus and activates gene transcription (7).Papillary thyroid carcinoma (PTC) often features RET/PTC

or BRAF oncoproteins, both of which signal through theextracellular signal–regulated kinase (ERK) pathway (9–11).RET/PTC result from the fusion of the tyrosine kinase domainof the RET receptor with the N-terminus of heterologous

proteins (9). RET tyrosine 1062 (Y1062) plays an importantrole in RET and RET/PTC signaling, acting as the binding sitefor several proteins, thus leading to ERK and PI3K (phosphoi-nositide 3-kinase)/AKT signaling (12–16).

CD44 cleavage produces CD44-ICD in cells transformed byoncogenic RET point mutants (17). CD44 is overexpressed inPTC and in cell lines harboring RET/PTC or BRAF oncogenes(18, 19). Here, we show that CD44-ICD triggers activation of theCREB transcription factor; in thyroid cancer cells, RET/PTC-BRAF signaling cascade uses such a CD44-ICD-CREB axis tosustain cell proliferation.

Materials and MethodsCell lines

TPC-1 and BCPAP cell lines were obtained, respectively, in1990 from M. Nagao (National Cancer Center Research Insti-tute, Tokyo, Japan) and in 1994 from N. Fabien (CNRS, Oullins,France). The anaplastic thyroid carcinoma cell lines ACT-1(NRAS Q61K), HTH7 (NRAS Q61R), HTH74 (negative forHRAS,KRAS, and NRAS mutations) and C643 (HRAS G13R) wereobtained in 2005 fromN. Onoda (Osaka University ofMedicine,Japan; ACT-1) and from N.E. Heldin (University Hospital,Uppsala, Sweden; HTH7, HTH74, and C643). These cell lineswere DNA profiled by short tandem repeat (STR) analysis in2009 (20) and shown to be unique and identical to thosereported in Schweppe and colleagues (21). In brief, 15 STRloci were tested by using the Applied Biosystems AmpFLSTRIdentifiler kit (ABI no. 4322288); DNA profiles were comparedmanually with the American Type Culture Collection (ATCC)database and to the DNA profiles reported by Schweppe andcolleagues (21). Human primary culture of thyroid cells (P5)

Authors' Affiliations: 1Dipartimento di Biologia e Patologia Cellulare eMolecolare, "L. Califano", Universit�a di Napoli "Federico II" c/o Istituto diEndocrinologia e Oncologia Sperimentale del CNR, Napoli, Italy; and2Centre Leon Berard, UMR-CNRS 5201, Lyon, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Massimo Santoro, Dipartimento di Biologia ePatologia Cellulare eMolecolare, "L. Califano", Universit�a di Napoli"Feder-ico II", via S. Pansini 5, 80131 Naples, Italy. Phone: 39-081-7463056; Fax:39-081-7463037; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3320

�2012 American Association for Cancer Research.

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were obtained from F. Curcio (University Udine, Italy) in 2003and passaged in our laboratory, as described (22), for fewerthan 2 months after resuscitation. They were tested in 2010 byproliferation rate and expression of thyroid differentiationmarkers (TG, TPO, and TSHR). Nthy-ori 3-1 (hereafter referredto as NTHY), a human thyroid follicular epithelial cell lineimmortalized by the SV40 large T gene, was obtained fromEuropean Collection of Cell Cultures (ECACC) in 2010. Theywere tested by ECACC for identity verification byDNAprofilingof STR sequences and were passaged in our laboratory forfewer than 3 months after resuscitation. PC Cl 3 (hereafterreferred to "PC") is a differentiated thyroid follicular cell linederived from 18-month-old Fischer rats in our laboratory in1987 (23). They were kept in culture for fewer than 3 monthsafter resuscitation and tested in 2010 by proliferation rate,dependence on 6 hormones for growth and expression ofthyroid differentiation markers (TG, TPO, and TSHR).HEK293T cells were purchased in 2006 from the ATCC andtested by ATCC for identity by DNA profiling of STR sequences.HEK293T were passaged in our laboratory for fewer than 3months after resuscitation. The PC ICD and BCPAP ICD celllines were obtained by a stable transfection with the CD44-ICDconstruct by using the Fugene HD reagent from Roche Diag-nostics. A pool of several cell clones was isolated by G418selection. Transient transfections were carried out with theFugene HD reagent. All these cell lines were grown in standardconditions as detailed in Supplementary Methods.

PlasmidsThe RET/PTC constructs used in this study were cloned in

pBABE or pcDNA3(Myc-His; Invitrogen) and described else-where (14). RET/PTC3(K�) is a kinase-dead mutant, carryingthe substitution of the catalytic lysine (residue 758 in full-length RET) with a methionine. In RET/PTC3(4YF) mutant the4 major autophosphorylation sites (Y826, Y1015, Y1029, andY1062 in full-length RET) are mutated to phenylalanine; Y1062has been added back in RET/PTC3(3YF). These mutants weregenerated by site-directed mutagenesis using the QuikChangemutagenesis kit (Stratagene). BRAFV600E and HRAS(V12)plasmids are described elsewhere (14). The full-length rat CD44was cloned into pDEST47 vector and tagged at the C-ter withGFP (17); CD44-ICD was cloned in pDEST47 (GFP tagged),pDEST40 (V5 tagged), and pCDNA 3.1 (myc tagged; ref. 17).

Antibodies, compounds, and proteinsThe antibody against the cytosolic portion of CD44

(CD44cyto) and anti-RET are described elsewhere (14, 17). Alist of commercial antibodies, compounds, and recombinantproteins used in this study is provided in SupplementaryMethods.

Cell growth and stainingFor growth curves, cells were seeded in triplicate and

counted at the indicated time points. Thyroid cancer cellswere maintained in Dulbecco's modified Eagle's medium(DMEM) 2.5% FBS and PC cells in medium supplemented with5% calf serum and without TSH. DNA synthesis was measuredby the 50-bromo-30-deoxyuridine (BrdUrd) Labeling and Detec-

tion Kit from Boehringer Mannheim, as indicated by manu-facturer (Supplementary Methods).

ELISA assayExtracellular shedding of the soluble ectodomain of human

standard CD44 (CD44st) was measured using the InstantELISA (Bender MedSystems) according to the manufacturer'sinstructions. Conditioned media from cell cultures were ana-lyzed in triplicate at 450 nmol/L with an microplate reader(Model 550; Bio-Rad).

Protein studiesImmunoblotting and immunoprecipitation experiments

were conducted according to standard procedures. For nuclearextraction, cells were lysed by shearing with 15 passagesthrough a 26-gauge needle mounted in a 1-mL syringe. Nucleiwere recovered by centrifugation at 3,000 � g for 10 minutes(Supplementary Methods).

Pull-down assayThe GST-CD44-ICD vector codes for a chimeric protein with

CD44-ICD fused to the glutathione S-transferase (GST). It wasgenerated by the Invitrogen Gateway technique using as donorthe pDEST47-CD44-ICD vector and as recipient the pDEST15vector. GST-CD44-ICD was purified from pDEST15-CD44-ICD–transformed bacterial lysates using glutathione Sephar-ose beads. Recombinant proteins containing the differentdomains of CREB fused to GST are described elsewhere(24). Pull-down assayswere carried out by standard procedures(Supplementary Methods).

Reporter assayAll the Firefly luciferase reporters were kindly provided by J.

S. Gutkind (NIH, Bethesda,MD; refs. 25–28). Twenty-four hoursafter seeding, the cells were transiently transfected in triplicatewith reporters togetherwith pRL-null, a plasmid expressing theenzymeRenilla luciferase, used as an internal control (PromegaCorporation). Luciferase assays were carried out according tostandard procedures (Supplementary Methods).

Chromatin immunoprecipitationChromatin was extracted from CD44-ICD or empty vec-

tor–transfected HEK293T cells, and chromatin immunopre-cipitation (ChIP) was done with the chromatin immunopre-cipitation assay kit (Upstate Biotechnology Inc.) accordingto manufacturer's instructions, as described in Supplemen-tary Methods.

RNA silencingThe small inhibitor duplex RNAs (siRNA) were from Dhar-

macon and were ON-target plus SMARTpool siCD44 human#L-009999-00, siCREB human: #L-003619-00-0005, and siCREBrat: #L-092995-00-0010. The siCONTROL Nontargeting Pool(#D-001206-13-05) was used as a negative control. Cells weretransfected with 100 nmol/L siRNAs using DharmaFECTreagent. The day before transfection, cells were plated in 35-mm dishes at 40% of confluence in DMEM supplemented with10% FBS and without antibiotics. The Sh29merRNA constructs

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against human CD44 were from OriGene Technologies(TR314080 IDs: TI356313, TI356314, and TI356316). Shorthairpin RNA (shRNA) pRS plasmid TR20003 was used as anegative control. Transfection was done in 100-mm dishesusing Fugene HD reagent (Roche) with 4 mg shRNA construct.Cells were harvested 48 hours after transfection.

In vitro PP2A dephosphorylation assayRecombinant HIS-CREB was in vitro phosphorylated by the

catalytic subunit of PKA in a buffer containing 1 mmol/L ATP,10mmol/LMgCl2, 50mmol/L KCl, 10mmol/LHEPES, and 10%glycerol for 1 hour at 30�C. Phosphorylated CREB was incu-bated in a phosphatase buffer (40mmol/L Tris HCl, 34mmol/LMgCl2, 4 mmol/L EDTA, 2 mmol/L DTT, and 0.05 mg/mLbovine serum albumin) for 1 hour at 37�C with 10 mU of PP2Acatalytic subunit. GST-CD44-ICD, or the GST backbone alone,were added to the samples. The reaction was terminated byadding SDS gel loading dye and samples were run on 10% SDS-polyacrylamide gel. Phospho-CREB signal was detected withanti-phospho(Ser/Thr) PKA substrate antibody and the

amount of CREB in each reaction was estimated with anti-HIS antibody.

Statistical analysisThe 2-tailed unpaired Student t test (normal distributions

and equal variances) was used for statistical analysis. Differ-ences were significant when P < 0.05. Statistical analysis wasdone using the Graph Pad InStat software program, version3.06.3.

ResultsCD44-ICD stimulates CRE-mediated transcription

We analyzed the capability of a GFP-tagged CD44-ICDconstruct to trans-activate a panel of promoter elements,including AP-1 (activating protein-1), SRF (serum responsefactor), TCF (ternary complex factor), Gli (Glioma-associatedoncogene homolog), NF-kB, and CRE (cAMP-responsiveelement) reporters (25–28). CD44-ICD strongly (about 10-fold,P < 0.01) activated the CRE reporter in HEK293T cells, but notthe other promoters (Fig. 1A). This was a specific feature of the

Figure 1. A, luciferase assays were carried out in HEK293T cells to measure effects of a GFP-tagged CD44-ICD (ICD) on the indicated reporters. B, CRE-LUCreporter assay in HEK293T cells transiently transfected with GFP-tagged full-length CD44 or CD44-ICD (ICD). C, HEK293T cells were transiently transfectedwithCycD1(CCND1)-LUCandCREBsiRNAor control scrambled siRNA (siCTR). CREBsilencing andCD44-ICD (ICD) expressionwas verifiedby immunoblot.In A–C, cotransfectedRenilla luciferase was used for normalization. Results are reported as fold changewith respect to the empty vector (�). Triplicates�SDare shown. P values were determined by the 2-tailed unpaired Student t test. CD44 or CD44-ICD (ICD) expression was verified by immunoblot with anti-GFPantibody (inset). D, immunoblot stained with cyclin D1 antibody of HEK293T cells cotransfected with CD44-ICD-GFP together with p300 or p300LYRR-dominant negative mutant. CD44-ICD expression was verified with anti-GFP. Anti-tubulin was used for normalization. E, HEK293T cells weretransiently transfected with CD44-ICD. Mock or anti-CREB immunoprecipitated chromatin was analyzed by semiquantitative PCRwith primers spanning theCRE site on the CCND1 promoter. Input DNA levels are shown for normalization. Data in D and E are representative of 3 independent experiments.

CD44 Cleavage Increases CREB Phosphorylation

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cleaved CD44-ICD, because full-length GFP-tagged CD44 didnot significantly (P > 0.05) stimulate the CRE reporter (Fig. 1B).

CD44-ICD sustains CREB/p300-mediated CCND1expression

CRE elements bind the CREB (cAMP-responsive elementbinding protein) family of transcription factors (29, 30). CyclinD1 (CCND1) is a prototypic CREB transcriptional target, con-taining a CRE element upstream of themRNA start site (at�58bp; refs. 25, 31).We studied the effect of CD44-ICDon aCCND1-luciferase reporter. HEK293T cells were keptwithout serum for24 hours after the transfection to reduce the growth factor–dependent CCND1 promoter activity. CD44-ICD stimulated(approximately 12-fold, P < 0.01) the CCND1 promoter (Fig. 1C,left).

We silenced endogenous CREB by transiently transfectingCREB siRNA (siCREB) in CD44-ICD–transfected HEK293Tcells; CREB knockdown (about 70% of reduction) was verifiedby immunoblot (Fig. 1C, right). CREB silencing, but not ascrambled control, obstructed CD44-ICD–mediated activationof CCND1-luciferase (Fig. 1C, left, P < 0.01).

The coactivator CBP/p300 binds to active S133-phosphor-ylated CREB and triggers CREB-mediated gene transcription(32). Previous data showed that CD44-ICD stimulates p300-mediated transactivation (33). Thus, we cotransfected CD44-ICD with p300 or a p300-dominant negative construct (LYRR;ref. 34). CD44-ICD upregulated (about 3-fold) endogenousCCND1 expression; the p300-dominant negative mutantblocked CD44-ICD–induced CCND1 expression, whereas wtp300 slightly increased it (Fig. 1D).

Phosphorylated CREB is recruited to CRE sites in DNA (35).We carried out a ChIP to measure CREB binding to the CREelement of the CCND1 promoter. CD44-ICD expressionincreased the binding of CREB to the CCND1 promoter byabout 5-fold (Fig. 1E).

Taken together, these results showed that CD44-ICD stimu-lates CREB recruitment to CCND1 promoter and CREB/p300-mediated transcription of CCND1.

CD44-ICD increases CREB phosphorylation on serine133

Nuclear extracts were prepared from HEK293T cells trans-fected with CD44-ICD and phosphoS133 (CREB principalactivation site) levels were measured by immunoblot. As apositive control, we treated cells with the adenylate cyclaseactivator forskolin (FSK). Besides CREB, the phospho-CREBantibody recognizes phosphorylated CREB family membersCREM (30 kDa) and ATF-1 (38 kDa). Expression of CD44-ICDinduced a robust increase of pS133 CREB as well as of phos-phorylated ATF-1 (Fig. 2A, left).

Activated pS133 CREB binds p300 and this results in atranscriptionally active complex. Thus, we analyzed CREB-p300 interaction by immunoprecipitating CREB from nuclearextracts of HEK293T cells cotransfected with CD44-ICD andp300, and staining the immunoblot with p300 antibody. CD44-ICD, as well as FSK treatment, increased (about 5-fold) theCREB-p300 interaction (Fig. 2A, right).

These results showed that CD44-ICD expression increaseslevels of S133-phosphorylated CREB and its binding to p300.

CD44-ICD forms a protein complex with CREBWe transfected HEK293T cells with V5-tagged CD44-ICD

and stained anti-CREB immunoprecipitates with V5 antibody.CD44-ICD formed a protein complex with CREB (Fig. 2B). To

Figure 2. A, left, nuclear extracts were obtained by HEK293T transientlytransfected with CD44-ICD (ICD) and pS133 CREB levels weredetermined by immunoblot. FSK (40 mmol/L 30 minutes) was used as apositive control. Anti-CREB was used for normalization. Right, nuclearextracts were obtained by HEK293T transiently transfected with CD44-ICD (ICD); anti-CREB immunocomplexes (1 mg) were immunoblottedwith p300. Lysis buffer alone was used as a negative control (buffer); FSKwas used as a positive control. Anti-CREB and anti-p300 were used fornormalization. B, HEK293T were transiently transfected with V5-taggedCD44-ICD (ICD) or empty vector (�). Proteins were immunoprecipitated(1 mg) with anti-CREB. CREB antibody incubated with no lysate (buffer)was used as a control. Immunocomplexes were blotted with V5; CREBantibody was used for normalization. C, CD44-ICD-GST (30 mg) wasincubatedwith 1mgof recombinantCREB (CREB-REC). Complexeswererecovered by glutathione beads and analyzed by immunoblot with anti-CREB. CREB input is reported for normalization. D, top, schematicrepresentation of different portions of CREB used in pull-down assay;bottom, the indicated portions of GST-CREB were used (30 mg) topull downmyc-taggedCD44-ICD transiently expressed inHEK293T cells(1 mg). GST alone was used as negative control. Data are representativeof 3 independent experiments.

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verify whether the interaction between CREB and CD44-ICDwas direct ormediated by other proteins, we carried out a pull-down assay using recombinant CREB and GST-CD44-ICDproteins. Figure 2C shows that CREB and CD44-ICD readilyinteracted in vitro.We used different domains of CREB expressed as recombi-

nant proteins fused to GST (24) to pull-down myc-taggedCD44-ICD expressed in HEK293T cells. As shown in Fig. 2D,CD44-ICD bound 2 contiguous domains of CREB: Q2 (aa 160–283), the constitutive glutamine-rich activation domain, andbZIP (aa 284–341), the DNA binding/dimerization domain.CD44-ICDdid not bind the isolatedDbZIP construct (aa 1–283)of CREB that contains Q2 but not bZIP. It is possible that, in theabsence of bZIP, the presence of the amino-terminal part of theprotein interferes with the interaction between Q2 and CD44-ICD. The fact that CD44-ICD binds to a CREB domain differentto that binding to p300 (KID domain; ref. 29) is consistent withthe possibility that a complex of 3 proteins, CREB, p300, andCD44-ICD is formed.

CD44-ICD reduces the rate of CREB dephosphorylationVarious serine/threonine kinases, that is, PKA, RSK, and

MSK, are able to phosphorylate CREB on S133 and to stimulateCREB binding to DNA (29, 35, 36). We evaluated the effects ofthe blockage of these kinases. To inhibit PKA, we either treatedcells for 2 hours with the H89 compound, a potent selective

inhibitor of PKA, or transfected cells with a plasmid encodingthe PKA-specific peptide inhibitor (PKi; ref. 37). RSK and MSKare activated by MEK (mitogen-activated protein/ERK kinase;ref. 36); thus, to inhibit RSK and MSK, we treated cells for 2hours with the MEK inhibitor U0126. Nuclear extracts wereanalyzed by Western blot with anti-pS133 CREB and normal-ized with anti-CREB. The inhibition of both PKA and MEKefficiently blocked CD44-ICD–induced CREB phosphorylation(Supplementary Fig. S1).

These results prompted us to hypothesize that, in thepresence of CD44-ICD, the increase of CREB phosphorylationlevels occurs because of a reduced rate of CREB dephosphor-ylation, rather than to activation of a specific CREB kinase. Toevaluate this possibility, we stimulated serum-starvedHEK293T cells for 40 minutes with the cAMP-analog N6-benzoyl-cAMP to induce S133 CREB phosphorylation, andthen measured CREB binding to CD44-ICD. GST-CD44-ICDpulled down a larger amount of CREB upon induction of CREBphosphorylation (Fig. 3A), indicating that CD44-ICD, althoughable to bind dephosphorylated CREB, binds preferentially topS133 CREB.

Then, we induced CREB phosphorylation with N6-benzoyl-cAMP in the presence or not of GFP-tagged CD44-ICD andchased pS133 CREB dephosphorylation by immunoblot. Figure3B shows that the pS133 CREB half-life was increased by CD44-ICD; CREB pS133 levels did not decrease up to 12 hours in cells

Figure 3. A,HEK293Twere treated for 1 hourwithN6-benzoyl-cAMP (100mmol/L). Protein lysates (2mg)werepulleddownwithGST-CD44-ICD (ICD-GST) andanalyzed by immunoblot with the indicated antibodies. Normalization was obtained by CREB blotting, and CREB phosphorylation was determined byphosphoCREB. B, GFP-tagged CD44-ICD (ICD-GFP)–transfected HEK293T cells, or cells transfected with the empty vector (�), were treated with N6-benzoyl-cAMP (100 mmol/L) and harvested at different time points. CREB and ATF-1 phosphorylation was determined by immunoblot. Anti-GFP and anti-CREBwere used for normalization. C, PKA-phosphorylated recombinant HIS-tagged CREB (0.05 mg) was incubated with 10 mU of PP2A catalytic subunit for1 hour at 37�C. Where indicated, GST-CD44-ICD (ICD-GST), or the control GST, was added. CREB phosphorylation was detected with an antibody reactingwith phospho(Ser/Thr) PKA substrates. Normalization was done with anti-HIS. Data are representative of 3 independent experiments.






expressing CD44-ICD, whereas the half-life of pS133 CREB wasabout 8 hours in mock-transfected cells (Fig. 3B).

Finally, we carried out an in vitro CREB dephosphorylationassay. Recombinant HIS-tagged CREB was phosphorylated invitro by PKA and then incubated with PP2A phosphatase in thepresence or the absence of GST-CD44-ICD. As shown in Fig. 3C,GST-CD44-ICD, but not the GST backbone, protected phos-phoCREB from PP2A-mediated dephosphorylation.

These findings showed that CD44-ICD reduces the rate ofCREB dephosphorylation on S133.

Expression of CD44-ICD-CREB complex in humanthyroid carcinoma cell lines

Weselected thyroid cancer cells as amodelwhereby to studyeffects of CD44-ICD. Initially, we looked for CD44-ICD, in 2 PTCcell lines, TPC-1 and BCPAP, that feature the RET/PTC1rearrangement and the BRAFV600E mutation, respectively; asa control, we used P5, a normal thyroid primary cell culture.Both PTC cell lines, but not nontransformed thyrocytes,expressed full-length CD44 as well as CD44-ICD (Supplemen-tary Fig. S2A). Moreover, pS133 CREB as well as phosphoATF-1levels were detected in BCPAP and TPC-1, whereas they wereonly barely detectable in P5 cells (Supplementary Fig. S2A).

GST-CD44-ICD, but not the GST backbone, was able to pulldownpS133CREB and phosphoATF-1 in both BCPAP andTPC-1 cell lysates (Supplementary Fig. S2B, left). Finally, in thyroidcancer cells, the pCREB-CD44-ICD interaction was shown byimmunoprecipitating pS133-phosphorylated CREB and stain-ing the blot with anti-CD44cyto antibody (SupplementaryFig. S2B, right).

These findings showed that thyroid cancer cells expressCD44-ICD and that, in these cells, CD44-ICD exists in acomplex with CREB.

RET/PTC and BRAFV600E oncogenes induce CD44cleavage

We treated TPC-1 and BCPAP cells for 48 hours with BB94,a broad-spectrum metalloprotease inhibitor; moreover, wetreated the RET/PTC1-positive TPC-1 cell line with ZD6474,a RET kinase inhibitor, and the BRAFV600E-positive BCPAPcell line with U0126, a MEK inhibitor. EctoCD44 (sCD44st,soluble standard CD44) shedding into the cell culture mediawas measured by ELISA. EctoCD44 release was detected inTPC-1 and BCPAP conditioned media (Supplementary Fig.S3A, left); treatment with BB94, ZD6474, or U0126 blockedectoCD44 shedding (P < 0.01), indicating that RET and BRAFsignaling stimulates CD44 cleavage (Supplementary Fig. S3A,left). In TPC-1 cells, treatment with BB94, ZD6474, andUO126 downregulated CD44-ICD (Supplementary Fig. S3A,middle). CD44 downregulation by siRNA proved that theprotein band with a relative molecular weight of about 10kDa was indeed CD44-ICD (Supplementary Fig. S3A, right).Finally, g-secretase blockade by COMP X or DAPT decreasedthe amount of CD44-ICD, whereas it increased the amountof a polypeptide of approximately 31 kDa, which corre-sponds to the molecular weight of CD44-CTF (Supplemen-tary Fig. S3A, middle). This effect was consistent with a blockof transmembrane conversion of CD44-CTF to CD44-ICD. In

contrast, CD44-CTF did not accumulate upon RET/PTC1 orMEK inhibition, which suggests that the RET/PTC1 andMEK block acts upstream CD44-CTF generation, possiblyat the level of metalloprotease-mediated CD44 cleavage(Supplementary Fig. S3A, middle).

We coexpressed a GFP-tagged full-length CD44 with the 2most prevalent RET/PTC oncogenes, RET/PTC1 and RET/PTC3. RET/PTC oncogenes stimulated the generation ofCD44-ICD and CD44-CTF (Supplementary Fig. S3B). That theband migrating above CD44-ICD corresponded to CD44-CTFwas suggested by the fact that when we used the metallopro-tease inhibitor BB94 in cells coexpressing CD44-GFP and RET/PTC1, this band was strongly reduced (Supplementary Fig.S4A). Instead, when we used a CD44 mutant lacking g-secre-tase cleavage site (D287–290), this band was not affectedwhereas CD44-ICD bandwas strongly attenuated (Supplemen-tary Fig. S4B). RET/PTC-mediated CD44 cleavage depended onRET/PTC kinase activity and on the integrity of tyrosine 1062,because CD44-ICDdid not accumulatewhen kinase-dead (K�)and (Y1062F) RET/PTC1mutants were expressed (Supplemen-tary Fig. S3C, left). CD44-ICD did not accumulate when theRET/PTC3 4YF mutant (in which the 4 major RET signaling

Figure 4. A, BCPAP and TPC-1 were transiently transfected with shCD44or the empty vector (�) with or without RNA interference–refractory V5-tagged rat CD44-ICD (ICD). Silencing was verified by immunoblot withanti-CD44cyto that does not react with rat CD44. Anti-V5 was used toverify the expression of rat CD44. PhosphoCREB and phosphoATF-1levels were measured by immunoblot; tubulin and CREB were used fornormalization. B, BCPAP and TPC-1 cells were treated with BB94 orDAPT. CREBphosphorylationwasmeasuredby immunoblot. Anti-CREBand anti-phosphoMAPK were used for normalization. CD44-ICD levelsare shown. C, immunoblot stainedwith cyclin D1 antibody of BCPAP andTPC-1 cells treated with siCD44, siCREB, or scrambled control (siCTR).The efficiency of RNA interference was verified by immunoblot with anti-CD44cyto and anti-CREB. Data are representative of 3 independentexperiments.

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tyrosines are mutated to phenylalanine) was expressed (Sup-plementary Fig. S3C, right), but CD44 cleavage was rescuedwhen Y1062 was added back (3YF; Supplementary Fig. S3C,right). We treated RET/PTC1-transfected HEK293T cells withU0126. Treatment with U0126 blocked CD44-ICD generation(Supplementary Fig. S3D). Transient expression of the myc-tagged constitutively active forms of RAS (RASV12) and BRAF(BRAFV600E) induced CD44-ICD formation (SupplementaryFig. S3D), which further supports the concept that the ERKpathway plays an important role in CD44 cleavage. Accord-ingly, also human thyroid carcinoma cell lines featuringmutant RAS alleles upregulated both CD44 and CD44-ICDwith respect to nontransformed thyrocytes (SupplementaryFig. S5).These findings showed that RET/PTC and BRAF signaling to

ERK stimulates CD44 processing.

CD44-ICD sustains CREB phosphorylation and CCND1expression in thyroid cancer cellsIn BCPAP and TPC1 thyroid cancer cells, CD44 silencing

by shCD44, but not the empty vector, attenuated CREBphosphorylation on S133 (Fig. 4A). In shCD44-transfectedcells, CREB phosphorylation was rescued by the transfectionof a V5-tagged rat CD44-ICD mRNA that is shCD44 resistantbecause it has several mismatches with human shCD44 (Fig.4A). BB94 and DAPT, able to block CD44 cleavage, reducedCREB pS133 (Fig. 4B). Downregulation of either CREB orCD44 by siRNA, but not a scrambled control, reduced (about2-fold) the expression of CCND1 in BCPAP and TPC-1 celllines (Fig. 4C).These findings showed that CD44-ICD sustains CREB-

dependent CCND1 expression in thyroid cancer cells.

CD44-ICD sustains proliferation of thyroid cellsWe silenced CD44 by transiently transfecting BCPAP and

TPC-1 cells with a human shCD44 plasmid. To exclude off-target effects, cells were cotransfected with the shCD44-resis-tant rat CD44-ICD construct. CD44 silencing reduced BrdUrdincorporation (P < 0.05), and this effect was rescued byadoptive expression of rat CD44-ICD (P < 0.05; Fig. 5A).CD44-ICD was not able to rescue the reduction of BrdUrdincorporation mediated by CREB silencing (P > 0.05) that isconsistent with CREB acting downstream CD44-ICD (Fig. 5A).We treated BCPAP cells with the g-secretase inhibitor COMPXand, to rescue the effect, we transfectedCD44-ICDor the emptyvector. As shown in Fig. 5B, COMPX inhibitedDNA synthesis inBCPAP, and CD44-ICD was able to revert this effect. We stablyexpressed CD44-ICD in BCPAP and selected one mass popu-lation by G418 treatment. Although treatment with COMP Xreduced the growth rate of parental cells (P < 0.01; Fig. 5C, left),it did not change significantly (P > 0.05) the proliferation ofBCPAP-ICD cells (Fig. 5C, right).

Nontransformed thyroid PC cells require a mixture of 6hormones (6H), including TSH, for proliferation (23). Inhormone-starved PC cells, transiently transfected CD44-ICDstimulated CRE-mediated transcription (about 9-fold;Fig. 6A; P < 0.01) and activated the CCND1 promoter (almost4-fold; Fig. 6B; P < 0.01). CREB silencing, but not a scrambledcontrol, obstructed CD44-ICD–mediated activation ofCCND1 luciferase (Fig. 6B; P < 0.01).

A mass population of CD44-ICD–transfected PC cells (PCICD pool) showed increased (about 8-fold) CRE-reporter lucif-erase activity with respect to empty vector–transfected cells(Fig. 6C; P < 0.01). Finally, we measured the rate of DNAsynthesis in the absence of TSH. BrdUrd incorporation in PC

Figure 5. A, BrdUrd-positive cells inBCPAPandTPC-1mock-transfected(�), or transfected with siCREB orshCD44 plasmid with or withoutshRNA-refractory rat CD44-ICD. B,BrdUrd-positive cells in BCPAPtreated for 48 hours with 10 mmol/LCOMP X and transfected with CD44-ICD or empty PCDNA vector. In Aand B, at least 100 cells werecounted in 5 fields and results areexpressed as percentage of BrdUrd-positive cells � SD. P values weredetermined by the 2-tailed unpairedStudent t test. C, one masspopulation of BCPAP transfectedwith CD44-ICD was selected byG418; growth curves are reported astriplicate determinations�SDwith orwithout 10 mmol/L COMP X. P valueswere determined by the 2-tailedunpaired Student t test.






ICD cells was higher (about 10-fold) than in empty vector–transfected cells (Fig. 6D; P< 0.01). Thus, CD44-ICD is sufficientto trigger proliferation of nontransformed thyrocytes.

DiscussionHerewe report a novel functional link betweenCD44 and the

CREB transcription factor. CREB is involved in neoplastictransformation and, being activated via PKA by cAMP, is alsoinvolved in the growth of normal thyroid follicular cells (38–41). Our data show that CD44-ICD binds CREB and increasespS133 CREB levels. CD44-ICD stimulated CREB-mediated genetranscription and recruitment of CREB to CCND1 gene pro-moter. We noted that CD44-ICD preferentially binds to phos-phorylatedCREB and thatCD44-ICD expression attenuates therate of CREB dephosphorylation, thus suggesting that inter-action with CD44-ICD impairs CREB dephosphorylation onS133. Accordingly, CD44-ICD protected pCREB from PP2A-mediated dephosphorylation in vitro.

Thyroid carcinoma overexpresses CD44 and such overex-pression is associated to the oncogenic conversion of the ERK

signaling pathway (18, 19). Moreover, as in other cell types (42),CD44 was expressed in prospectively identified thyroid cancerstem cells that induce tumors when injected orthotopicallyinto mouse thyroid (43). Thus, it is feasible that CD44 plays arole in thyroid cancer as well as in thyroid cancer stem cells.

Oncogenic RET point mutants induce CD44 cleavage (17).Here, we show that CD44-ICD is expressed in thyroid cancercell lines harboring RET/PTC or BRAFV600E oncogenes andthat RET/PTC and BRAF trigger CD44 cleavage. CD44-ICD isnecessary for the proliferation of thyroid cancer cells andsufficient to trigger proliferation of nontransformed thyro-cytes. These effects are mediated by CREB and by increasedrate of CCND1 transcription. The fact that extracellular shed-ding of ectoCD44 accompanies the generation of CD44-ICDand that RET and BRAF blockade impairs CD44-CTF accu-mulation indicates that metalloprotease-mediated cleavage isone level at which CD44-ICD generation is stimulated in cellsexpressing RET/PTC and BRAF. Accordingly, RET/PTC andBRAF upregulate the transcription of several metalloproteases(44). CD44 cleavage can be triggered by binding to differentextracellular ligands, including low molecular weight hyalur-onan acid (45). RET, RAS, and BRAF oncogenes stimulate theexpression of osteopontin, one extracellular CD44 ligand;whether this facilitates CD44 cleavage remains to bedetermined.

Ourfindings support amodel whereby CD44 cleavage acts asan amplifier of oncogenes signaling to CREB (Fig. 7). Accordingto this model, RET/PTC and BRAF promote CREB phosphor-ylation via ERK-mediated activation of CREB kinases andstabilize pS133-CREB through CD44-ICD. In turn, the CD44-ICD-CREB axis stimulates CCND1 transcription and thyroidcell proliferation. Should these results be validated in vivo, they

Figure6. A,CRE-LUCassay inPCcells transiently transfectedwithCD44-ICD. B, CycD1-LUC assay in PC cells transiently transfected with CREBsiRNA or scrambled siRNA (siCTR). In A and B, cells were harvested at 48hours, luciferase assay was carried out, and results are reported as foldchange with respect to the vector (�); cotransfected Renilla luciferasewas used for normalization. Triplicates � SD are shown. P values weredetermined by the 2-tailed unpaired Student t test. C, top, PC cells werestably transfected with GFP-tagged CD44-ICD (PC-ICD pool) or theempty GFP vector and mass populations were isolated by G418selection. Immunoblot with anti-GFP was done; equal loading wasascertained by anti-tubulin immunostain. Bottom, CRE-LUC assay in PCICD cell pool. Results are reported as fold change with respect to emptyvector–transfected cells. Cotransfected Renilla luciferase was used fornormalization. D, cells were seeded without TSH on glass coverslips,pulsed with BrdUrd, and BrdUrd incorporation measured by indirectimmunoflorescence. At least 100 cells were counted in 5 differentmicroscopic fields and results are expressed as percentage of BrdUrd-positive cells � SD. P values were determined by the 2-tailed unpairedStudent t test.

Figure 7. A model of functional interaction between oncogene signaling,CD44, and CREB in thyroid cancer cells.

De Falco et al.





may prompt the possibility of pharmacologic manipulation ofthe pathway.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank AstraZeneca for ZD6474, A. Feliciello for PKA inhibitors, J.

S. Gutkind for reporter vectors, M. Montminy for CREB recombinant proteins, G.Blandino for p300 vector, F. Curcio for P5 cells, M. Nagao for TPC1, N. Fabien forBCPAP, N. Onoda for ACT-1, and N.E. Heldin for HTH7, HTH74 and C643 cellsand also thank J.A. Gilder for text editing and A.M. Cirafici for technicalassistance.

Grant SupportThis study was supported by the grant no. 5880 of Associazione Italiana per la

Ricerca sul Cancro (AIRC), grant no. PIO-2 of Italian Ministero della Salute,grants no. E61J11000300001 and E61J10000210001 of Ministero dell'Universit�a edella Ricerca, grant no. FP6-36495 of the European UnionContract (GENRISK-T),and grant no. 4915 of ARC and LNCL-comit�e du Rhone.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received October 4, 2011; revised December 29, 2011; accepted January 13,2012; published OnlineFirst January 23, 2012.

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2012;72:1449-1458. Published OnlineFirst January 23, 2012.Cancer Res Valentina De Falco, Anna Tamburrino, Simona Ventre, et al. Proliferation of Thyroid Cancer CellsCD44 Proteolysis Increases CREB Phosphorylation and Sustains

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