Thyrotrophin receptor signaling dependence ofBraf-induced thyroid tumor initiation in miceAime T. Francoa,1, Roberta Malaguarneraa,1, Samuel Refetoffb, Xiao-Hui Liaob, Emma Lundsmitha, Shioko Kimurac,Catrin Pritchardd, Richard Maraise, Terry F. Daviesf, Lee S. Weinsteing, Min Cheng, Neal Rosenh,i, Ronald Ghosseinj,Jeffrey A. Knaufa, and James A. Fagina,h,2

aHuman Oncology and Pathogenesis Program, hDepartments of Medicine, jPathology, and iMolecular Pharmacology and Chemistry, Memorial Sloan–Kettering Cancer Center, New York, NY 10065; bDepartment of Medicine and Program in Genetics, University of Chicago, Chicago, IL 60637; cLaboratory ofMetabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; dDepartment of Biochemistry, University of Leicester, LeicesterLEI7RH, United Kingdom; eInstitute for Cancer Research, London SW3 6JB, United Kingdom; fDivision of Endocrinology and Metabolism, Mount Sinai Schoolof Medicine, New York, NY 10468; and gMetabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, MD 20892

Edited by Lewis Clayton Cantley, Beth Israel Deaconess Medical Center, Boston, MA, and approved December 20, 2010 (received for review October 29, 2010)

Mutations of BRAF are found in ∼45% of papillary thyroid cancersand are enriched in tumors with more aggressive properties. Wedeveloped mice with a thyroid-specific knock-in of oncogenic Braf(LSL-BrafV600E/TPO-Cre) to explore the role of endogenous expres-sion of this oncoprotein on tumor initiation and progression. Incontrast to other Braf-induced mouse models of tumorigenesis(i.e., melanomas and lung), in which knock-in of BrafV600E inducesmostly benign lesions, Braf-expressing thyrocytes become trans-formed and progress to invasive carcinomas with a very shortlatency, a process that is dampened by treatment with an alloste-ric MEK inhibitor. These mice also become profoundly hypothyroiddue to deregulation of genes involved in thyroid hormone bio-synthesis and consequently have high TSH levels. To determinewhether TSH signaling cooperates with oncogenic Braf in this pro-cess, we first crossed LSL-BrafV600E/TPO-Cre with TshR knockoutmice. Although oncogenic Braf was appropriately activated in thy-roid follicular cells of these mice, they had a lower mitotic indexand were not transformed. Thyroid-specific deletion of the Gsαgene in LSL-BrafV600E/TPO-Cre/Gnas-E1fl/fl mice also resulted in anattenuated cancer phenotype, indicating that the cooperation ofTshR with oncogenic Braf is mediated in part by cAMP signaling.Once tumors were established in mice with wild-type TshR, sup-pression of TSH did not revert the phenotype. These data demon-strate the key role of TSH signaling in Braf-induced papillarythyroid cancer initiation and provide experimental support for re-cent observations in humans pointing to a strong association be-tween TSH levels and thyroid cancer incidence.

Ras | G protein | pax8

Thyroid follicular cells are among a select group of cell types,which includes other endocrine cell lineages, melanocytes, and

Schwann cells, in which the second messenger cAMP helps pro-mote DNA synthesis and cell proliferation. In thyroid cells thispathway is engaged via constitutive and ligand-induced activationof the TSH receptor (TSHR), which, however, requires concom-itant activation of receptor tyrosine kinase signaling for growth toensue (1–3). It is therefore fitting that distinct subtypes of thyroidneoplasms are associated with oncogenes encoding effectors of theTSH–TSH receptor–adenylyl cyclase pathway (i.e., TSHR andGNAS) (4, 5) or with proteins that signal along canonical receptortyrosine kinase pathways. Thus, rearrangements of genes encodingthe receptor tyrosine kinases RET or NTRK, as well as pointmutations of all three RAS genes and of the serine kinase BRAF,are found in a mutually exclusive manner in papillary thyroidcancers (PTC), the most prevalent form of the disease (6–8). Theactivating point mutation BRAFT1799A, which encodes forBRAFV600E, is the most common genetic abnormality in papillarythyroid cancer and constitutively activates the MEK–ERK path-way. Thyroid cancers with BRAF mutations have distinctivepathological and phenotypic features: i.e., they are more fre-

quently invasive, have higher recurrence rates, are relatively re-fractory to radioiodine therapy, and have a higher disease-specificmortality (9–11).Mutations of BRAF are also found with high frequency in be-

nign nevi and in melanomas (12, 13) and to a lesser extent in lungcancers (14, 15). Conditional endogenous expression of mutantBraf in mice with a latent BrafT1799A allele results in melanocytehyperplasia and development of nevi, which show features con-sistent with senescence (16, 17). The two mouse models in whichexpression of BrafV600E at physiological levels was conditionallytargeted to melanocytes differed in one respect: Melanoma de-velopment was encountered in only one of them without addi-tional genetic manipulations (16), whereas in the other metastaticmelanoma developed only when the tumor suppressor Pten wasalso genetically inactivated (17). Similarly, endogenous expres-sion of BrafV600E in lung alveolar epithelial cells is insufficient byitself to induce lung cancers (18).Here we examined the effects of physiological levels of on-

cogenic Braf expression on mouse thyrocyte tumorigenesis. Asopposed to that seen in other lineages, these animals developedinvasive papillary thyroid cancers with very short latency. Thepenetrance, extent, and latency of these cancers depended onthe presence of an intact TSH signaling pathway, primarily at thetime of tumor initiation. These findings may help explain theincreased risk of thyroid cancer conferred by higher TSH levelsin patients with thyroid nodules, which has recently beenreported in several epidemiological studies (19–21).

ResultsTo investigate the role of endogenous expression ofBrafV600E in thepathogenesisof thyroid cancerweestablishedmicewitha thyrocyte-specific knock-in of the oncogene, by crossing LSL-BrafV600Emice,in which a latent Braf mutant allele can be activated by Crerecombinase through excision of a floxed STOPcassette, withTPO-Cremice, which express Cre under the control of the human thyroidperoxidase (TPO)promoter (Fig. S1A). TPO is expressed in thyroidfollicular cells starting at embryonic day (E)14.5. Recombinationefficiencywas notmaximal until 7–10 d postnatally (Fig. S1B).LSL-

Author contributions: A.F., R. Malaguarnera, J.A.K., and J.A.F. designed research; A.F.,R. Malaguarnera, S.R., X.-H.L., E.L., and J.A.K. performed research; S.R., X.-H.L., S.K.,C.P., R. Marais, T.F.D., L.S.W., M.C., and N.R. contributed new reagents/analytic tools; A.F.,R. Malaguarnera, R.G., J.A.K., and J.A.F. analyzed data; and A.F., R. Malaguarnera, andJ.A.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1A.F. and R. Malaguarnera contributed equally to this work.2To whom correspondence should be addressed. E-mail: faginj@mskcc.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015557108/-/DCSupplemental.

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BrafV600E/TPO-Cre animals were born at the expected Mendelianfrequency. However, they weighed ≈50% less than wild-type lit-termates by weaning (Fig. S2A).

Endogenous Expression of BrafV600E in Thyroid Tissue InducesHypothyroidism. Because overexpression of oncogenic effectorsthat activate MAPK has previously been shown to impair thyroidhormone production in vivo (22, 23), we first sought to determinewhether the growth retardation was due to hypothyroidism. LSL-BrafV600E/TPO-Cremice were euthyroid at day 3 (Fig. S3B), yet by5 wk of age serum TSH levels were ∼500-fold greater than thoseof age-matched wild-type (WT) littermates, and serum T4 levelswere markedly decreased (Fig. S2B). Thyroglobulin (Tg), sodiumiodide symporter (Nis), and Tpo mRNA levels were markedlydown-regulated at 5 wk in LSL-BrafV600E/TPO-Cre mice. Theexpression of TshR was also significantly inhibited (Fig. S2C).Pax8 mRNA, which encodes a key transcription factor that isrequired for expression of several thyroid-specific genes, was alsodecreased. Hence, endogenous expression of BrafV600E was suf-ficient to profoundly deregulate thyroid-specific gene expressionand thyroid hormonogenesis.

Induction of Invasive PTCs by Oncogenic Braf. Braf activation led tothe development of classic infiltrative PTC with complete pen-etrance by 5 wk (Fig. 1).The tumors, which encompassed theentire thyroid gland, had features characteristic of aggressivehuman PTC with papillae lined by tall cells, with increasednumber of mitoses, nuclear clearing, and pseudonuclear inclu-sions (Fig. 1F). The malignant phenotype was further establishedbecause tumor cells frequently invaded perithyroidal tissues (Fig.1G). Thus, 8/12 (66%) PTCs invaded into surrounding skeletalmuscle by 5 wk, and vascular invasion was commonly observed(Fig. 1H). There was no difference in the severity, penetrance, orlatency of the disease between female and male mice.

Knockout of TshR or Gsα Impairs Braf-Induced PTC Development. AsTSH is a thyroid cell mitogen, we next examined whether TSHand Braf cooperated in the initiation of Braf-induced thyroidtumors. For this we crossed LSL-BrafV600E/TPO-Cre with TshR-KO mice to genetically ablate TSH signaling. As Cre is driven bythe TPO promoter in our model, and thyroid cells from TshR-KOmice have decreased Tpo expression (24), we confirmed thatrecombination efficiency of the targeted Braf gene locus wasmaintained in mice lacking TshR (Fig. S1C). Thyroid glands fromTshR-KO mice were smaller and had hypotrophic thyroid fol-licles compared with WT littermates. By 3 wk, all LSL-BrafV600E/TPO-Cre mice developed PTC (Fig. 2B). By contrast, geneticablation of TSH signaling partially blocked Braf-induced thyroidgrowth (Fig. 2I). Thyroid glands from LSL-BrafV600E/TPO-Cre/TshR-KO were smaller than those from LSL-BrafV600E/TPO-Cremice (Fig. 2I), and although the architecture of the follicles wasdistorted compared with that of TshR-KO thyroids, the histo-pathology was uniformly benign and lacked the characteristicnuclear features of PTC (Fig. 2D vs. 2C). They also exhibiteda markedly lower mitotic index than LSL-BrafV600E/TPO-Cremice (Fig. 2H vs. 2F). However, by 9 wk thyroid cells of LSL-BrafV600E/TPO-Cre/TshR-KO mice escaped the dependence uponTSH signaling and developed low-grade PTCs. The tumors weresmaller, lacked the characteristic tall cell features seen in tumorswith WT TshR, and were less invasive (Fig. 2 J and K). Hence,thyroid cells lacking TshR can be transformed by Braf aftera longer latency and are phenotypically less aggressive.TSH-induced thyroid cell growth is mediated in part by TSHR

activation of adenylyl cyclase via Gsα, the product of the Gnasgene. To further define the role of TSH signaling in thyroid tumorinitiation by oncogenic Braf, we crossed LSL-BrafV600E/TPO-Cremice with Gnas-E1fl/fl mice, in which the targeted Gsα allele isinactivated by Cre-mediated recombination. There was no dif-

ference in the thyroid histology of Gnas-E1fl/fl/TPO-Cre micecompared with WT littermates. LSL-BrafV600E/Gnas-E1fl/fl/TPO-Cre developed smaller tumors (Fig. 3A) with greatly attenuatedhistological features (Fig. 3). Whereas Braf-induced PTCs in micewith intact Gsα had characteristic tall cells (Fig. 3D), their Gsα-null counterparts were cuboidal (Fig. 3E), with scant mitoses andfew cells with nuclear clearing. These characteristics resembleindolent PTCs in humans, whereas LSL-BrafV600E/TPO-Cretumors displayed histological features of aggressive PTCs thatoften progress to poorly differentiated disease.

Fig. 1. Thyroid-specific activation of Braf leads to the development ofthyroid cancer with short latency. (A) Gross anatomical images of wild-typeand LSL-BrafV600E/TPO-Cre thyroid glands from mice at 5 wk. (B) Thyroidweight is significantly increased in both male and female LSL-BrafV600E/TPO-Cre mice compared with wild-type littermates. Bars represent mean ± SEM.(C–H) Representative H&E images from thyroid tissues of wild-type and LSL-BrafV600E/TPO-Cre mice at 5 wk. (C and D) Low (40×) and high (100×) mag-nification, respectively, of normal thyroid lobe of a WT mouse. Thyroidfollicles are filled with colloid. Arrows points to thyroid lobe (Th) and tra-cheal cartilage (Tr). (E) Magnification (40×) of markedly enlarged thyroidlobe in a LSL-BrafV600E/TPO-Cre mouse. Thyroid architecture is disrupted,and no colloid material is evident. (F) Papillary formations (P) and nuclearpseudoinclusion (N). (Magnification, 100×.) (G) Malignant follicle (T) in-vading into surrounding skeletal muscle (M). Desmoplasia (D) is evident,indicative of muscle invasion. (H) Tumor cell thrombus (T) surrounded byendothelial cells (E) hanging in the lumen of a blood vessel (V).

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Suppression of TSH Secretion Postnatally Does Not Prevent PTCDevelopment or Block Disease Progression. TshR signaling can alsobe dampened by suppressing pituitary secretion of the ligandthroughadministration of a supraphysiological doseof levothyroxine(L-T4). We next examined whether TSH suppression, beginningsoon after birth, could prevent or delay the development of PTC. At3 d, thyroid glands of WT and LSL-BrafV600E/TPO-Cre mice wereindistinguishable histologically and had a comparable proliferativerate (Fig. S3A). Administration of exogenous L-T4 for 3 wk at a dosethat completely suppressedTSH(Fig. S3E) didnot prevent theonsetor alter the characteristics of the Braf-induced PTCs and hence didnot phenocopy TshR deletion (Fig. S3F). Moreover, treatment ofLSL-BrafV600E /TPO-Cremice with L-T4 for 3 wk beginning at 5 wkof age, when all had florid PTC, did not decrease the mitotic rate oralter the severity or progression of the Braf-induced PTCs.

Endogenous Expression of Oncogenic Hras Does Not PhenocopyEffects of Braf on Thyroid Hormone Biosynthesis or Tumorigenesis.Mutations of NRAS and HRAS are common in human follicular-variant PTCs and follicular carcinomas (25, 26). Overexpression

of HRASG12V also down-regulates expression of genes requiredfor thyroid hormone biosynthesis in vitro (27), an effect that hasbeen shown to be dose dependent (28). To determine whetherendogenous expression of HrasG12V is sufficient to induce thyroiddysfunction and thyroid cell transformation, we crossed FR-HrasG12V mice, which have a latent Hras oncogenic allele ex-pressed under the control of its native promoter, with TPO-Cremice. Offspring were of normal weight compared with littermates,and serum levels of TSH and T4 were unaffected (Fig. S4A).Accordingly, there was also no difference in transcript abundanceof thyroid-specific genes (Fig. S4B). In contrast to BrafV600E,endogenous expression of HrasG12V did not induce thyroid neo-plasms through 12 mo of follow-up (29). To determine whethersupraphysiological levels of TSH might cooperate with activatedHras to induce thyroid tumors we treated FR-HrasG12V/TPO-Cremice with the goitrogen 6-propyl-2-thiouracil (PTU) for up to 20wk. Despite significant increases in TSH and the development ofbenign goiters, none of the FR-HRasG12V/TPO-Cre mice de-

Fig. 2. Braf-induced PTC development requires TshR. (A–D) H&E images from representative thyroid sections of animals of the indicated genotype at 3 wk.Thyroids from LSL-BrafV600E/TPO-Cre (B) mice are significantly increased in size compared with WT (A). LSL-BrafV600E/TPO-Cre mouse thyroid (B) shows a floridPTC, which is highly cellular. (C) Thyroid lobe of a TshR-KO animal is markedly smaller than WT (A), but retains normal follicular structures. (D) Thyroid lobe ofLSL-BrafV600E/TPO-Cre/TshR-KO is comparatively larger than that of TshR-KO and has disrupted follicular architecture. The cells lack the characteristic nuclearfeatures pathognomic of PTC. (E–H) Ki67 immunohistochemical (IHC) staining of representative thyroid lobes of the same animal groups. The mitotic index ismarkedly higher in the LSL-BrafV600E/TPO-Cre thyroid (F) compared with WT (E). There are virtually no detectable Ki67-positive cells in the thyroid sections ofTshR-KO (G) and LSL-BrafV600E/TPO-Cre /TshR-KO (H) mice. (I) Thyroid weight of wild-type, TshR−/+, TshR−/−, LSL-BrafV600E/TPO-Cre, LSL-BrafV600E/TPO-Cre/TshR−/+,and LSL-BrafV600E/TPO-Cre/TshR−/− mice at 3 wk. (J and K) Magnification (40× and 200×) of thyroid sections of LSL-BrafV600E/TPO-Cre /TshR-KO mice at 9 wk ofage. Note increase in size of thyroid in J compared with 3-wk-old animal (E). Magnification (200×) in K shows characteristic papillary structures lined byirregular nuclei; however, tumors were of lower grade and had no extrathyroidal invasion.

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veloped thyroid cancer or showed a significant increase in Ki67staining relative to controls (Fig. S5).

Selective MEK Inhibitor PD325901 Reduces Growth of Braf-InducedPTC. Human thyroid cancer cell lines harboring BRAF mutationsare sensitive to MEK inhibitors in vitro and in xenograft tumormodels (30–32). Arguably, PTCs developing following endoge-nous expression of BrafV600E in mice represent a more physio-logical context in which to evaluate this therapeutic approach.We treated animals with the specific allosteric MEK1/2 inhibitorPD325901 for 3 wk beginning at 3 wk of age. Levels of phos-phorylated ERK, a downstream target of MEK, were profoundlyinhibited 6 h after the last dose (Fig. 4 A and B). Thyroid tumorvolume, determined by MRI before and after treatment, wasdecreased in drug-treated mice (Fig. 4C) and was associated witha modest reduction in the proliferative index of the tumors (Fig.4D). However, there was no difference in the apoptotic index orin the histopathological appearance of the lesions.

DiscussionIn this study we show that endogenous expression of BrafV600E inthyroid cells was sufficient to induce PTCs with full penetrance by3 wk of age. The tumor cells had the typical nuclear features ofPTC and were enriched for tall cells, which are characteristic of

Fig. 3. Loss of Gsα attenuates the phenotype of PTC induced by endogenousexpression of BrafV600E. (A) Thyroid weight of wild-type, Gnas-E1fl/+/TPO-Cre,Gnas-E1fl/fl/TPO-Cre, LSL-BrafV600E/TPO-Cre, LSL-BrafV600E/Gnas-E1fl/+/TPO-Cre,and LSL-BrafV600E/Gnas-E1fl/fl/TPO-Cre mice at 3 wk. Bars represent mean ±SEM. (B–E) Representative H&E-stained sections of thyroid tissue at 40× and200× magnification of LSL-BrafV600E/TPO-Cre and BrafV600E/TPO-Cre/Gnas-KOmice. (B) LSL-BrafV600E/TPO-Cre tumors have dense cellularity, and the wholefield is occupied by the carcinoma. (C) Thyroid section of LSL-BrafV600E/Gnas-E1fl/fl/TPO-Cre mouse shows areas with relatively preserved follicular struc-tures (arrow) coexisting with a low-grade PTC. (Magnification, 40×.) (D)Areas of tall cell growth (arrow), characteristic of aggressive BRAF-positivePTCs in humans, are present only in the Gnas WT mice. (E) PTCs in LSL-BrafV600E/Gnas-E1fl/fl/TPO-Cre mice are composed of smaller papillae consist-ing of cuboidal cells (arrow).

Fig. 4. Treatment with the Mek inhibitor PD0325901 inhibits Braf-inducedPTC growth. LSL-BrafV600E/TPO-Cre mice were treated with PD0325901 (25mg·kg−1·d−1) or vehicle for 3 wk, beginning at 3 wk of age. (A) Represen-tative H&E sections of thyroids treated with vehicle or PD0325901. (B) pERKIHC of PTCs of vehicle- or PD0325901-treated mice. Animals were euthanized6 h after administration of the last treatment dose. (C) Thyroid volumemeasured by MRI in vehicle- or PD0325901-treated mice before and after3 wk of therapy (P = 0.0002). (D) Proliferative index measured by percentageof Ki67-positive thyrocytes in vehicle- and PD0325901-treated mice. *P = 0.01

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PTCs with BRAF mutations in humans (9). The evidence for ma-lignancy was supported by their propensity to invade extrathyroidalstructures and by the presence of vascular invasion. In humansthere is no evidence of a stepwise adenoma–carcinoma transitionleading to PTC. Instead, it is likely that a subset of papillarymicrocarcinomas, which are highly prevalent, represents pre-cursors of the clinically significant forms of the disease. About 25%of micro-PTCs harbor BRAF mutations, which has been taken asevidence that this oncogene may be a tumor-initiating event (9,33). Previously, PTC was described after high-level overexpressionof BRAFV600E in transgenic mice (23). The fact that the diseasecan be recapitulated by endogenous expression of the oncogenedemonstrates convincingly that oncogenic Braf is an initiatingevent in PTC development and sufficient to drive the process.The Braf-expressing mice were profoundly hypothyroid due to

the shutdown of expression of key genes required for iodinetransport and thyroid hormone biosynthesis. This result is con-sistent with observations in humans, in which PTCs harboringBRAF mutations have lower expression of Tg, TPO, and NIScompared with PTCs that do not have this mutation (34) andaccordingly are more refractory to 131I therapy (10). The effectsof oncogenic BRAF on thyroid gene expression in vitro takesplace within hours (35); however, the LSL-BrafV600E/TPO-Cremice did not become hypothyroid until 3 or 4 wk after the onsetof TPO, and hence Cre, expression, which begins at E14.5. Thiswas most likely due to the fact that thyroid cells do not expressTPO in a synchronous fashion, and hence Cre-mediated Brafactivation likely took place gradually. In this event, hypothy-roidism would ensue only when a sufficient proportion of cellsactivated the oncoprotein and impaired thyroid hormone bio-synthesis, in a way that could no longer be compensated for bythe reservoir of unrecombined thyroid cells.Experiments with immortalized thyroid cell lines have con-

sistently shown that Ras oncogenes inhibit differentiation (27),due in part to interference with Ttf1 activity (36) and decreasedexpression and transcriptional activity of Pax8 (37). However,a threshold of Ras oncogene expression must be present forthese effects to become apparent (28). Our data indicated thatendogenous levels of HrasG12V were not sufficient to inducehypothyroidism or to negatively impact thyroid hormonogenesis,which may be due to insufficient MAPK activation (29), whichour data strongly implicate in this process (38).Physiological expression of BrafV600E in lung alveolar epithelial

cells and melanocytes of genetically modified mice results in aninitial burst of proliferation, followed by growth arrest associatedwith activation of markers of senescence (17, 18). By contrast, themitotic rate in Braf-expressing thyroid cells remained high at alltimes from birth through at least 3 wk of age, which is inconsistentwith senescence. It may be that the timing of activation of Brafis a determining factor in transformation. Interestingly, inductionof BrafV600E expression during embryogenesis using a tyrosinase-driven Cre results in embryonic lethality and rapid trans-formation of melanoblasts, suggesting that oncogenic activationat an earlier stage of differentiation is associated with greatermalignant potential (39).Themarked elevationofTSHseen postnatally inLSL-BrafV600E/

TPO-Cre mice raised the possibility that this hormone may becooperating with oncogenic Braf in transformation, perhaps ac-counting for the high penetrance and short latency of the pheno-type. However, treatment with TSH-suppressive doses of thyroxinebeginning postnatally, at a time when thyroid histology was stillnormal, did not prevent or dampen the phenotype. This outcome islikely due to the fact that Braf-expressing cells may have becomelargely refractory toTSHaction (35). In addition, as theunligandedTSH receptor retains significant constitutive activity (40), simpleabsence of ligand may have been insufficient to block the pathway.Activation of the TSH/TSHR pathway occurs in mice at E15.

Although TSH signaling is required for expression of a subset of

thyroid-specific genes during development, thyroid gland size andmitotic rate are independent of TSH action prenatally, as pre-viously reported in TshR-KO mice and in mice lacking TSH (i.e.,pitdw/pitdwmice, which express a pit1 transcription factor defectivein DNA binding) (24). This result is also consistent with our data,in which we found normal mitotic activity in TshR-KO mice atbirth. Taken together, this result indicates that deletion of TshR isunlikely to have depleted a putative thyroid progenitor cell pop-ulation, whose existence has not been definitively established, andwhose characteristics have not been fully defined (41). However,TshR-KO cells cannot be viewed as normal, and it is possible thatthey may have a weaker response to the transforming effects ofthe oncogene because of an as yet undefined differentiationhandicap, such as a block in cell cycle progression that cannot beovercome by constitutive MAPK activation.The mechanisms accounting for the requirement for TshR for

Braf-induced transformation are unknown. There is good evi-dence that TSH primes thyroid cells to undergo cell cycle pro-gression in response to growth factors, primarily insulin-likegrowth factor I (IGF-I) or insulin (2). This effect is associated withincreased IRS-2 and PI3K phosphorylation and enhancedMAPKactivity (42). TSH also increases insulin, but not IGF-I receptorabundance in human thyroid cells (43). In many of these modelsexposure to TSH must precede growth factor stimulation for thesynergy to fully manifest (2). Our data suggest that this TSH sig-naling requirement is also important for Braf-induced tumori-genesis. The potential significance of TSH action in the initialevents associated with Braf-induced thyroid cell transformation isfurther supported by the attenuated phenotype seen in mice withthyroid-specific deletion of Gsα. However, the fact that Gsα de-letion did not prevent development of PTCs is consistent with theevidence that other TSH-activated downstream pathways, such asGq/G11, also play a critical role in thyroid cell growth (44).Although treatment of Braf-induced PTCs with PD0325901

inhibited mitotic activity, the MEK inhibitor did not revert thetumors. It should be noted that although MEK and mTORC1inhibitors prevented development of melanomas caused by in-ducible activation of Braf in the context of Pten deletion, they didnot induce tumor regression in established tumors (17), which isconsistent with our data on Braf-induced murine PTCs. It maybe that the effects of PD0325901 on MAPK signaling were notsufficiently profound or sustained to achieve a complete ther-apeutic benefit.Several recent large epidemiological studies have shown a strong

association between serum TSH levels and risk of malignancy inthyroid nodules (19–21).Remarkably, a fourfold increase in thyroidcancer was found in patients whose TSH levels were in the upperquartile compared with those in the lower quartile of the normalrange. Our data suggest that the activity of the TSH signaling path-waymaypredispose thyroid cells toBRAF-induced transformation,which if confirmed in humans will provide impetus for further de-lineation of the signaling effectors thatmediate this interaction andprovide strategies for cancer prevention and therapy.

Materials and MethodsExperimental Animals. LSL-BrafV600E mice harbor a latent oncogenic Brafknock-in allele, which following Cre-mediated recombination results in en-dogenous expression of the oncoprotein (45). TPO-Cre mice express Crerecombinase under the control of the thyroid peroxidase gene promoter,which is active only in thyroid follicular cells beginning at E14.5 (46) (Fig.S1A). FR-HrasG12V mice conditionally express a latent HrasG12V allele underthe regulatory control of its endogenous gene promoter (29). TshR-KO miceharbor a germ-line deletion of the TSH receptor (24, 47). The gene encodingGsα was conditionally deleted by using the Gsα-floxed mouse line Gnas-E1fl/fl,in which loxP recombination sites surround Gsα exon 1 at positions −1601and +419 relative to the translational start site (48). Mice were in mixedgenetic backgrounds. Genotypes were determined by PCR using previouslydescribed primers. All procedures were approved by the Institutional AnimalCare Committee of Memorial Sloan–Kettering Cancer Center.

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Histology and Immunohistochemistry. Thyroid tissues were fixed in 4%paraformaldehyde and embedded in paraffin. Five-micrometer-thick sectionswere prepared and stained with hematoxylin and eosin or appropriateantibodies described in SI Materials and Methods. Histological diagnosis wasperformed by a thyroid pathologist (R.G.) blinded to the genotype and thetreatment status of the animal. Where indicated, immunohistochemicalstaining was quantitated using Metamorph imaging software.

Real-Time Reverse Transcription–PCR. Sequences for β-actin, Tg, NIS, TPO,pax8, ttf1, and TshR are listed in SI Materials and Methods.

Drug Administration. PD352901 was administered by gavage as detailed in SIMaterials and Methods. TSH was suppressed in mice by administration ofsupraphysiological doses of L-T4 (Sigma).

Thyroid Imaging. Thyroid imaging was done by high-resolution MR, as de-tailed in SI Materials and Methods.

Radioimmunoassays (RIA). Serum TSH and T4 levels were performed as pre-viously described (SI Materials and Methods).

Statistical Analysis. Mann–Whitney U and Student’s t tests were used forstatistical analyses of intergroup comparisons. Significance was defined asa P value <0.05.

ACKNOWLEDGMENTS. We thank Miriam Benezra for technical assistance.We thank the following Memorial Sloan–Kettering Cancer Center Cores forassistance: Molecular Cytology, Genetically Engineered Mouse GenotypingService, Laboratory of Comparative Pathology, and Small Animal Imaging.This project was supported by National Institutes of Health (NIH) GrantsCA50706, CA72597, and DK17050; the Margot Rosenberg Pulitzer Founda-tion; and partially supported by the Intramural Research Program of theNational Institute of Diabetes and Digestive and Kidney Diseases. A.F. wassupported by NIH Grant F32CA136178. The Small Imaging Core is supportedby Small Animal Imaging Research Program Grant R24 CA83084, NIH CenterGrant P30 CA08748, and NIH Prostate Specialized Program of ResearchExcellence Grant P50-CA92629.

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