Activating BRAF and PIK3CA Mutations Cooperate …...tumor suppressors. Using mice with conditional,...

Cell Cycle and Senescence

Activating BRAF and PIK3CA Mutations Cooperate toPromote Anaplastic Thyroid Carcinogenesis

Roch-Philippe Charles1,2, Jillian Silva1,2, Gioia Iezza3, Wayne A. Phillips4, and Martin McMahon1,2

AbstractThyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes,

anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion ofthyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms ofcancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature geneticabnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAFprotooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC.Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in thep110 catalytic subunit of PI30-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTENtumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAFV600E, we previouslydeveloped amodel of PTC.However, as in humans, BRAFV600E-inducedmouse PTC is indolent and does not leadto rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA todemonstrate that, although mutationally activated PIK3CAH1047R is unable to drive transformation on its own,when combined with BRAFV600E in thyrocytes, this leads to development of lethal ATC inmice. Combined, thesedata demonstrate that the BRAFV600E cooperates with either PIK3CAH1074R or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma.

Implications:This genetically relevantmousemodel of ATCwill be an invaluable platform for preclinical testing ofpathway-targeted therapies for the prevention and treatment of thyroid carcinoma.Mol Cancer Res; 12(7); 979–86.�2014 AACR.

IntroductionThyroid cancers derived from follicular epithelial cells are

histologically classified into papillary thyroid carcinoma(PTC), follicular or anaplastic thyroid carcinoma (ATC;ref. 1). Although PTC is indolent and readily managed bysurgical resection combined with radioiodine therapy, ATCis highly aggressive with more than 50% of ATC patientsdyingwithin 1 year after diagnosis (2). Althoughmetastasis isobserved in 10% to 20% of patients with ATC, mostpatients with ATC succumb to locally invasive, inoperable

disease that is largely refractory to conventional chemother-apy (3).Mutationally activated BRAF (commonly T1799!A in

exon 15) encoding BRAFV600E is detected in approximately40% of PTC and 25% of ATC (4). BRAFV600E is aconstitutively active protein kinase that activates theERK1/2 MAPK pathway (5). The importance of mutatedBRAF in thyroid cancer maintenance is suggested byresponses of patients with thyroid cancer to vemurafenib,a pharmacologic inhibitor of BRAFV600E (6). Moreover,conditional, thyrocyte-specific expression of BRAFV600E ingenetically engineered mouse (GEM)models results in PTC(7). However, as in humans, PTC in this model is indolentand does not routinely result in progressively lethal disease.Human ATC displays multiple cooperating mutational

events in tumor suppressors and oncogenes such as TP53(70%–80%), PTEN (10%–20%), BRAF (25%), H-, N- orKRAS (20%–30%), PIK3CA (15%–25%), and CTNNB1(60%–65%; ref. 8). Hence, by analogy to other cancer types,it is likely that progression tomore aggressive disease is due tocooperative interactions between these various geneticabnormalities. To test this, we generated mice with thyr-ocyte-specific expression of BRAFV600E in conjunction withexpression of mutationally activated PIK3CAH1047R, a con-stitutively activated form of the p110 catalytic subunit of

Authors' Affiliations: 1Helen Diller Family Comprehensive Cancer Center;Departments of 2Cell and Molecular Pharmacology, and 3Pathology, Uni-versity of California, San Francisco, California; and 4Surgical OncologyLaboratory, Sir Peter MacCallum Cancer Center, University of Melbourne,Victoria, Australia

Corresponding Authors: Roch-Philippe Charles, Institute f€ur Biochemieund Molekular Medizin, University of Bern, B€uhlstrasse 28, CH-3012 Bern,Switzerland. Phone: þ41-31-631-4344; Fax: þ41-31-631-3737; E-mail:[email protected]; and Martin McMahon, Helen DillerFamily Comprehensive Cancer Center Research Building, University ofCalifornia, 1450 Third Street, MC-0128, Room HD-365, San Francisco, CA94158, [email protected].

doi: 10.1158/1541-7786.MCR-14-0158-T

�2014 American Association for Cancer Research.

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PI30-kinase a (9). Expression of PIK3CAH1047R, which isdetected in many cancer types, is predicted to promoteelevated PI30-lipid production leading to activation of AKTprotein kinases and other PI30-lipid effectors in the cell (10).In brief, whereas adult-onset, thyrocyte-specific expressionof PIK3CAH1047R had no detectable effect on the thyroid, itcooperated dramatically with BRAFV600E such that micedeveloped rapidly lethal ATC. Similar observations were alsomade with thyrocyte-specific expression of BRAFV600E

combined with PTEN silencing. Using cultured humanthyroid cancer cell lines, we demonstrated that these path-ways cooperate to regulate the activity of mTOR and thephosphorylation of 4E-BP1. Hence, we propose that thisGEMmodel of ATC, which recapitulates key features of thehuman disease, will be useful in understanding thyroidcancer progression and modeling the effects of pathway-targeted therapy in the preclinical setting.

Materials and MethodsMouse breeding and manipulationBRaf CA, Pik3calat-1047R, Ptenlox, and Thyroglobulin::

CreERT2 mice were described previously (7, 11; 9, 12).BRafCA mice have been backcrossed in FVB/N in thelaboratory for more than 10 generations; all the others havebeen obtained in C57BL/6 F129–mixed background andcrossed in FVB/N since obtained. All the mice consideredhere are predominantly FVB/N. Thyrocyte-specific activa-tion of CreERT2 activity was achieved by intraperitonealinjection of 1mg of tamoxifen dissolved in peanut oil into 4-week-old mice.

Cell linesThe 8505c line was cultured as directed in RPMI com-

plemented with 10% FCS [and validated by short tandemrepeat (STR) profiling, performed by Microsynth]. Ocut-2in DMEM complemented with 10% FCS and nonessentialamino acids, STR profiles showed that this cell line was notpresenting mouse or human contamination and was offemale origin as expected from the literature. The STRprofile of Ocut-2 did not present any relevant similaritiesto any registered cell lines of the American Type CultureCollection.

Histology and immunofluorescence of mouse thyroidtissue sectionsAnimal experiments were carried out in accordance with

protocols approved by the University of California, SanFrancisco (San Francisco, CA) Institutional Animal Careand Use Committee (IACUC). Mice were anesthetized byintraperitoneal injection before tissue dissection, and theneuthanized by section of the abdominal aorta. Thyroids wereremoved, rinsed in ice-cold PBS and fixed for 4 hours in Z-Fix (Anatech). Four- to 5-mm sections of formalin-fixed,paraffin-embedded tissues were stained with hematoxylinand eosin or processed for immunofluorescence. Epitopeunmaskingwas performed by boiling slides for 10minutes ina buffer comprising 10mmol/LTris, 0.5mmol/LEGTApH9.0. Primary antibodies were obtained from the listed

commercial sources and diluted as follow: anti–TTF-1(1:200; Santa Cruz Biotechnology), anti-Ki67 (1:300;Abcam), anti-CK19 (1:300, TROMA-III, Hybridomabank, University of Iowa, Iowa, IA), Vimentin (Cell Sig-naling Technology; 1-200), and anti–Galectin-3 (Abcam;1:200). Antigen–antibody complexes were detected usingeither goat anti-rabbit Alexa-488 (1:500) or goat anti-ratAlexa-488 (1:500; Molecular Probes) with slides counter-stained with DAPI to visualize nuclear DNA.

Ultrasound imagingMice were anesthetized using 2%(v/v) isofluorane at

which time fur around the neck was removed using Veetdepilatory cream. Ultrasound images were collected weeklyusing the Vevo770 system (VisualSonics). Thyroid size wasassessed by counting pixels at the largest diameter of thethyroid and converting pixel count into area (mm2) usingscaling software internal to the device in combination withImageJ (NIH, Bethesda, Maryland, USA).

ResultsPik3calat-1047R (Pik3caLat hereafter) mice carry a condi-

tional allele that expresses normal PIK3CA before Cre-mediated recombination after which mutationally activatedPIK3CAH1047R is expressed from the normal chromosomallocus at physiologically relevant levels of expression (9). Totest the effects of thyrocyte-specific expression ofPIK3CAH1047R, we treated compound adult Thyro::CreER;Pik3caLat mice with tamoxifen and monitored prospectivelyfor palpable goiter. Even 1 year after tamoxifen administra-tion, mice with thyrocyte-specific expression ofPIK3CAH1047R displayed no obvious thyroid abnormalities.Hence, unlike BRAFV600E, mutationally activatedPIK3CAH1047R is insufficient to initiate thyroid hypertro-phy or tumorigenesis (7).To test whether PIK3CAH1047R would influence

BRAFV600E-induced PTC, we initiated tumorigenesis inadult Thyro::CreER; BRaf CA/þ; Pik3caLat/þ and monitoredthem prospectively for disease (Fig. 1A). At 6.5 months, 2 of14mice became emaciated, displayed labored breathing, andhad readily palpable thyroid tumors that were �1 cm3

requiring euthanasia. Over the next 2 months 71% of mice(10/14) presented developed similar end-stage diseaserequiring euthanasia. The experiment was stopped 12.5months after tumor initiation, a time at which 85% (12/14) of the Thyro::CreER; BRaf CA/þ; Pik3caLat/þ mice hadbeen retired. In contrast, only 37% (4/11) of an independentcohort of initiated Thyro::CreER; BRaf CA/þ mice reachedend-stage and required euthanasia (tumors �1 cm3).Kaplan–Meier survival analysis clearly demonstrated theshortened survival of mice bearing BRAFV600E/PIK3-CAH1047R versus BRAFV600E-expressing thyroid tumors(Fig. 1A, P < 0.01). To further evaluate BRAFV600E/PIK3-CAH1047R cooperation in thyroid tumorigenesis, tumorgrowth was measured in two additional cohorts of Thyro::CreER; BRaf CA/þ; Pik3caLat/þ or Thyro::CreER; BRaf CA/þ

mice using ultrasound imaging 2 months after initiation(Fig. 1B).Mice bearing BRAFV600E/PIK3CAH1047R thyroid

Charles et al.

Mol Cancer Res; 12(7) July 2014 Molecular Cancer Research980




tumors displayed an approximately 60% greater tumorburden compared with BRAFV600E alone tumors.To assess the effects of oncogene expression on tumor

histology, thyroid specimens were prepared from normalmice or mice with thyrocyte-specific expression ofPIK3CAH1047R, BRAFV600E, or combined BRAFV600E/PIK3CAH1047R (Fig. 1C and D). Both normal- andPIK3CAH1047R-expressing thyroid (Fig. 1C) tissue displayeda characteristic follicular architecture composed of a mono-layered cuboidal epithelium of thyrocytes. Indeed, even 12months after PIK3CAH1047R expression, no alteration inthyroid architecture was observed (data not shown).

BRAFV600E expression elicited PTC displaying the charac-teristic papillary thyroid architecture at 6 to 9 months afterinitiation as described previously (Fig. 1C, middle; ref. 7).In contrast, when BRAFV600E and PIK3CAH1047R werecoexpressed, we observed clear evidence of PTC within 3to 6 months that extended throughout the entire thyroid(Fig. 1D, left). Moreover, these PTC lesions displayedaggressive characteristics with evidence of invasion intosurrounding tissues, most notably the trachea (Fig. 1D,middle). Such invasion resulted in reduced airway diameterthat was observed in 70% of the animals represented in theKaplan–Meier survival curve (Fig. 1A). In addition, the

Figure 1. Combined thyrocyte-specific expression of BRAFV600E

and PIK3CAH1047R results in lethalthyroid carcinogenesis. A, Kaplan–Meier survival curves comparingmice survival of tamoxifen-treated Thyro::CreER; BRafCA/þ;Pik3caLat/þ and Thyro::CreER;BRafCA/þ mice (��, P � 0.01; log-rank test). B, thyroid tumor burdenin tamoxifen-treated Thyro::CreER;BRafCA/þ; Pik3caLat-HR/þ andThyro::CreER; BRafCA/þ micemeasured by ultrasonography(��,P�0.01; t test) 2.5months aftertumor induction. C, hematotoxylinand eosin (H&E) staining ofhistologic section of thyroid fromcontrol BRafCA/þ (WT, wild-type):Thyro::CreER; BRafCA/þ

(BRAFV600E) and Thyro::CreER;Pik3caLat/þ (PI3KCAH1047R)mice, at�40 (top row) and �200 (bottomrow) magnifications. D, H&Estaining of histologic section ofthyroid Thyro::CreER; BRafCA/þ;Pik3caLat/þ (BRAFV600E

PI3KCAH1047R) showing an area ofPTC (left), tracheal airway invasion(middle), and ATC area (right), at�40 (top row) and �200 (bottomrow) magnifications.

BRAF and PI3K Cooperate to Carry Thyroid Cancer Progression

www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 981




thyroid tumors in 80% of these animals displayed polygonalgiant cells and/or spindle-like cells (Fig. 1D, right), which is acharacteristic of human ATC. Such features (local invasionand aberrant tumor cytology) were not observed in PTCsinduced by expression of BRAFV600E alone (7).To further characterize BRAFV600E/PIK3CAH1047R col-

laboration in this model, we generated Thyro::CreER;BRaf CA mice that were either wild-type, heterozygous, orhomozygous for the conditionalPik3caLat allele. Of note, 2.5months after initiation, Thyro::CreER; BRafCA; Pik3caLat/þ

mice displayed the expected increase in tumor size comparedwith mice lacking the conditional Pik3caLat allele. Asexpected, Thyro::CreER; BRafCA; Pik3caLat/Lat mice alsodisplayed an increase in tumor size (�37%) compared withmice lacking the Pik3caLat allele (Fig. 2A). However,although the difference in tumor size between mice hetero-zygous or homozygous for the Pik3caLat allele was notstatistically significant (Fig. 2B), mice homozygous forPik3caLat/Lat died more rapidly than their heterozygouscounterparts (Fig. 2A) most likely due to an earlier onsetof the tracheal invasion.Human thyroid malignancies are characterized by altera-

tions in marker expression such as galactin-3 (Gal-3) andCK-19 (13). We, therefore, stained BRAFV600E/PIK3-CAH1047R–expressing thyroid tumors for expression of theseproteins (Fig. 2C). First, we noted that BRAFV600E/PIK3-

CAH1047R–expressing thyroid tumors displayed areas ofPTC and ATC. The regions of PTC were generally positivefor Gal-3 andCK-19 as described previously (7). In contrast,regions of ATC retainedGal-3 but lackedCK-19 expression.In addition, ATC-like lesions displayed low/no expression ofTTF-1 or E-cadherin, increased Vimentin expression and ahigher proliferative index (Ki67).As an alternative strategy to confirm collaboration of

BRAFV600E with alterations in the PI3K pathway, wegenerated Thyro::CreER; BRaf CA; Ptenlox/lox mice to initiateBRAFV600E/PTENnull thyroid tumors. Compared withBRAFV600E alone, these mice displayed increased thyroidtumor burden as soon as 1.5 months after initiation(Fig. 3B). This required complete silencing of PTENexpression because the enhanced tumorigenesis was notnoted inThyro::CreER;BRafCAmice heterozygous for Ptenlox

(Fig. 3B). As anticipated, mice with BRAFV600E/PTENnull

thyroid tumors developed end-stage disease more rapidlythan mice with BRAFV600E expression alone (Fig. 3A, P <0.01). It was notable that Thyro::CreER; BRaf CA miceheterozygous for Ptenlox tended to reach endpoint morerapidly than mice with BRAFV600E expression alone, pre-sumably because of loss-of-heterozygosity of the remainingPtenWT allele (Fig. 3B).At the histologic level, BRAFV600E/PTENnull thyroid

tumors displayed similar characteristics as BRAFV600E/

Figure 2. Thyroid cancerdevelopment in mice wild-type,heterozygous or homozygous forPik3caLat. A, Kaplan–Meiersurvival curves comparingtamoxifen-treated: 1. Thyro::CreER; BRafCA/þ, Thyro::CreER; 2.Thyro::CreER;BRafCA/þ; Pik3caLat/þ

or; 3. Thyro::CreER; BRafCA/þ;Pik3caLat/Lat mice after tumorinitiation (��, P � 0.01; log-ranktest). B, tumor burdenmeasured by ultrasonography(�, P � 0.05; ��, P � 0.01; t test). C,immunofluorescence staining forexpression of TTF-1, E-cadherin,Vimentin, Ki67, and Cytokeratin 19in a histologic section of thyroidfrom Thyro::CreER; BRafCA/þ;Pik3caLat-HR/þ focusing on areas ofPTC or ATC area as indicated.Nuclear DNA was stained bluewith DAPI.

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PIK3CAH1047R–expressing thyroid tumors. AlthoughBRAFV600E/PTENWT thyroids presented expected PTCfeatures after 9.5 months (Fig. 3C), BRAFV600E/PTENNull

thyroid tumors displayed a clear PTC phenotype as earlyas 3.5 months after initiation (Fig. 3D, left). In addition,we also detected evidence of tracheal invasion (Fig. 3D,middle) and progression to ATC (Fig. 3D, right) ofBRAFV600E/PTENnull tumors at time points 3.5 to 8monthsafter tamoxifen induction.To explore biochemical collaboration between

BRAFV600E and PI30-kinase signaling in more mechanisticdetail, we used two human BRAF-mutated ATC cell lines:8505c and Ocut2 (14). Whereas Ocut2 expresses bothBRAFV600E and PIK3CAH1047R, 8505c is BRAFV600E/TP53null. Cells were treated with inhibitors of eitherMEK1/2 (PD325901; ref. 15) or class I PI30-kinases

(GDC-0941; ref. 16) either alone or in combination witheffects on cell proliferation assessed using Crystal Violetstaining (Fig. 4A). Although both PD325901 and GDC-0941 displayed antiproliferative effects, in general, MEK1/2inhibition had a more potent inhibitory effect on cellproliferation than inhibition of class I PI30-kinases. More-over, combined blockade of both pathways had a morestriking effect on cell proliferation compared with the anti-proliferative activity of the single agents.In parallel with the cell proliferation assays, cells were

treated with PD325901 or GDC-0941, either alone or incombination, for 4 or 24 hours at which time cell extractswere analyzed by immunoblotting (Fig. 4B). At both timepoints, pathway blockade was highly selective in thatPD325901-inhibited pERK1/2 with no effect on pAKT-andGDC-0941–inhibited pAKTwith no effect on pERK1/

Figure 3. PTEN silencing cooperates with BRAFV600E for anaplastic thyroid cancer progression. A, Kaplan–Meier survival curves comparing survival oftamoxifen-treated:Thyro::CreER;BRafCA/þ; Ptenþ/þ (BRAFV600) Thyro::CreER;BRafCA/þ; Ptenlox/þ (BRAFV600E PTENhet) orThyro::CreER;BRafCA/þ; Ptenlox/lox

(BRAFV600E PTENnull) mice after tumor initiation (��, P � 0.01; log-rank test). B, thyroid tumor burden measured by ultrasonography (�, P � 0.05; t test) 1.5months after tumor induction. C, H&E staining of histologic section of thyroid tumors observed in Thyro::CreER; BRafCA/þ; Ptenþ/þ (BRAFV600E) at�40 (top)and �200 (bottom) magnifications. D, H&E staining of histologic section of thyroid tumors observed in Thyro::CreER; BRafCA/þ; Ptenlox/lox mice (BRAFV600E

PTENnull) displaying areas of PTC (left), tracheal airway invasion (middle), or ATC area (right) at �40 (top row) and �200 (bottom row) magnifications.






2. Because these pathways are reported to coordinatelyregulate themachinery that regulates the initiation of proteinsynthesis, we assessed the effects of PD325901 and GDC-0941 on the phosphorylation of ribosomal protein S6 (rpS6)and 4E-BP1, that latter of which directly regulates Cap-dependent protein translation (17).Phosphorylation of rpS6 and 4E-BP1 seemed more

sensitive to PI30-kinase inhibition at 4 or 24 hours inOcut2 cells compared with 8505c cells, which is likely areflection of the fact that Ocut2 cells expressPIK3CAH1047R. Moreover, p-RPS6 and p-4E-BP1 weresensitive to MEK1/2 inhibition in both cell types but thiswas not observed until 24 hours after inhibitor addition.However, in Ocut2 cells, we detected cooperative effectsof combined MEK1/2 plus PI30-kinase inhibition on p-RPS6 at 4 and 24 hours after inhibitor addition. This wasalso true for p-4E-BP1 24 hours after inhibitor additionbut not at 4 hours. We also detected evidence of coop-erative inhibitory effects on p-RPS6 and p-4E-BP1 in8505c cells 24 hours after inhibitor addition, but theeffects were not as striking as those observed in Ocut2cells. Overall, these data suggest that BRAFV600E and PI30-kinase signaling cooperate for sustained thyroid cancer cell

proliferation and that mutational activation of PIK3CAmay predict for more potent antiproliferative and signalingeffects following inhibition of class I PI30-kinases.

DiscussionAnaplastic thyroid carcinoma is a difficult disease to treat

because it is generally inoperable and resistant to currentregimens of chemotherapy. Although there are provocativehints from small numbers of patients from the phase Ivemurafenib clinical trial, it remains to be unequivocallydemonstrated that BRAFmutational status is prognostic fortherapeutic benefit from BRAFV600E inhibitors. However,the potential clinical benefit of vemurafenib might beinfluenced by altered signaling through ERBB receptors forEGF ligands as demonstrated by others (18). Furthermore, itwas recently demonstrated that blockade of BRAFV600E

signaling with a MEK1/2 inhibitor (AZD6244) can restorethe expression of the sodium-iodide symporter (NIS), andthereby enhance the efficacy of radioiodine therapy in bothGEMmodels and in patients (19, 20). These data, therefore,suggest that combined pathway-targeted and conventionalradiotherapy may be a successful strategy for treating late-stage thyroid cancer.

Figure 4. BRAFV600E and PI30-kinase signaling cooperate forhuman thyroid cancer cellproliferation and regulation ofphospho-rpS6 and 4E-BP1. A,8505c (BRAFV600E/PIK3CAWT) andOcut2 (BRAFV600E/PIK3CAH1047R)human anaplastic thyroid cancercells were treated with an inhibitorofMEK1/2 (PD325901, 1mmol/L) orclass I PI30-kinases (GDC-0941,2.5 mmol/L) either alone or incombination for 5 days at whichtime the cells were fixed andstainedwith Crystal violet. B andC,8505c (BRAFV600E/PIK3CAWT) andOcut2 (BRAFV600E/PIK3CAH1047R)human ATC cells were treated withan inhibitor of MEK1/2 (PD325901,1 mmol/L) or class I PI30-kinases(GDC-0941, 2.5 mmol/L) eitheralone or in combination for either4 (B) or 24 (C) hours at which timethe cells were lysed and proteinexpression/phosphorylationassessed by immunoblotting asindicated.

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There are a large number of inhibitors of BRAFV600E andPI30-kinase signaling being developed for the treatment of awide range of human cancers. However, it remains unclearwhether mutational activation of BRAF, PIK3CA, or silenc-ing of PTEN will serve as strong prognostic biomarkers forthyroid cancer patient responses to pathway-targeted ther-apy, in part because of effects of these agents on intracellularfeedback signaling (18). Indeed, although BRAFmutation isprognostic of response in melanoma, that is not true forpatients with colorectal cancer (21, 22). The availability ofGEM models of a wide range of thyroid cancer types,including the new model of ATC based on a foundationof mutational activation of BRAF plus either mutation ofPIK3CA or silencing of PTEN described here, will allowpreclinical modeling of combination pathway-targeted, con-ventional or immunomodulatory cancer therapy. It wasnotable that expression of mutationally activatedPIK3CAH1047R in thyrocytes was largely without effect inthe mouse but that it cooperated strongly with mutationallyactivated BRAFV600E. This is not without precedent becausePIK3CAH1047R expression fails to initiate tumorigenesis inmelanocytes and in the lung and pancreatic epithelium (23–25). In contrast, mutationally activated BRAFV600E is suf-ficient to initiate tumorigenesis in all of the above targettissues. Hence, we propose that mutationally activatedBRAFV600E is a potent initiator of tumorigenesis that is alsofrequently required for tumor maintenance. In contrast,mutationally activated PIK3CAH1047R is a weak initiator oftumorigenesis but greatly promotes tumorigenesis initiatedby other oncogenic events (25). This may explain why, todate, pathway-targeted inhibition of PI30-kinase signalinghas not proven successful in patients, even those whosetumors are silenced for PTEN or mutated for PIK3CA.TRP53 is also frequently mutated in anaplastic thyroid

carcinoma (50%of the cases). Indeed, separately,McFaddenand colleagues have demonstrated that combined expressionof BRAFV600E with silencing of TP53 also elicits anaplasticthyroid carcinoma (26). It has also been showed that thecombination of TRP53 and PTEN knockouts could lead toATC in another mouse model (27). This model has a similarlatency to the model described here even if it using anembryonic onset of the cre-recombinase (TPO-Cre). Thisshows that even if cannot exclude p53 alteration in theBRAFV600E PIK3CAH1047R model, subsequent p53 alter-ation resulting in ATC progression is very unlikely.The cooperation observed between BRAFV600E and PI30-

kinase signaling inGEMmodels was recapitulated, at least inpart, by analysis of bona fide human ATC-derived cell lines.It was clear that combined inhibition of both BRAFV600E

and PI30-kinase signaling had more potent antiproliferativeeffects than that of single-agent inhibition. Moreover, reg-

ulation of key nodes in protein translation, most notably thephosphorylation of 4E-BP1 was clearly under the dualcontrol of both pathways, suggesting that efficient Cap-dependent protein synthesis requires inputs from at least twopathways in ATC-derived cell lines. Although there was asuggestion that the PIK3CAH1047R-expressing Ocut2 cellswere more sensitive to PI30-kinase, an extended analysis of alarger panel of thyroid cancer cell lines will be required toconfirm if there is a genotype–drug response phenotype inthis disease.In conclusion, we describe a new mouse model of ana-

plastic thyroid carcinoma that recapitulates key features ofthe genetics and pathobiology of the cognate human disease.We propose that this GEM model will be useful for futurepreclinical studies and to understand the mechanisms bywhich these pathways cooperate to promote progression ofthyroid cancer from an indolent to a lethal disease

Disclosure of Potential Conflicts of InterestM. McMahon has received commercial research grant from Novartis, Glaxo-

Smith-Kline, and Plexxicon, and is a consultant/advisory board member of Abbvie,Sutro Biopharma, and Igenica Inc. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: R.-P. Charles, M. McMahonDevelopment of methodology: R.-P. CharlesAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): R.-P. Charles, J. Silva, W.A. PhillipsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): R.-P. Charles, J. Silva, G. Iezza, M. McMahonWriting, review, and/or revision of the manuscript: R.-P. Charles, M. McMahonStudy supervision: M. McMahon

AcknowledgmentsThe authors thank all of the members of the McMahon laboratory, especially

Christy Trejo, Teruyuki Muraguchi, Victoria Marsh, Shon Green, Anny Shai, AllisonLandman, Eric Collisson, Joseph Juan, and J. Edward van Veen for advice, guidance,and support throughout this project. The authors thank the support for mousehusbandry from Allen Villanueva. The authors also thank Bill Hyun and Jane Gordonfrom the UCSF Laboratory for Cell Analysis for assistance with microscopy, CynthiaCowdrey of the BTRCTissue Core for processing of formalin-fixed samples and ByronHann and colleagues of the UCSF Preclinical Therapeutics Core for assistance withultrasound imaging. The authors thank Dr. Lori Friedman (Genentech) for GDC-0941, Prof. J.A. Fagin (MSKCC) for providing the 8505c and Ocut-2 cells and Drs.David McFadden and Tyler Jacks (MIT) for ongoing discussion and communicatingresults prior to publication.

Grant SupportR.-P. Charles was supported by a Swiss National Science Foundation Fellowship,

J. Silva was supported by Institutional Research and Academic Career DevelopmentAward (IRACDA), W.A. Phillips was supported by project grants from the NationalHealth and Medical Research Council of Australia, and M. McMahon was supportedby the National Cancer Institute (CA131261).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMarch 27, 2014; accepted April 14, 2014; publishedOnlineFirst April 25,2014.

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Charles et al.




2014;12:979-986. Published OnlineFirst April 25, 2014.Mol Cancer Res Roch-Philippe Charles, Jillian Silva, Gioia Iezza, et al. Anaplastic Thyroid CarcinogenesisActivating BRAF and PIK3CA Mutations Cooperate to Promote

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