The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7- and ∼2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tall-cell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.

Papillary thyroid carcinoma (PTC) is the most common type of differentiated thyroid carcinoma (1). Many of the genetic events involved in the initiation of PTC have been identified (2). Notable among them are the RET/PTC oncogenes, which until recently were the most characteristic oncogenic event in this tumor type, particularly in pediatric cancers and in patients with prior exposure to ionizing radiation (3). Recently, an activating mutation of BRAF was found in 36% of PTC, making it the most common oncogene thus far identified in sporadic forms of the disease (4). The mutation was exclusively a thymine-to-adenine transversion at position 1799, previously designated as 1796, leading to a valine-to-glutamate substitution at residue 600 (V600E), formerly designated as V599 (5). The BRAF mutations are unique to PTC and not found in other types of well-differentiated thyroid follicular neoplasm. The initial observations have now been confirmed by other reports finding a prevalence of this mutation in 29% to 83% of PTCs (4, 613). Overall, of the 916 PTC reported to date, 42% were positive for the BRAFT1799A mutation.

There are three isoforms of the serine-threonine kinase RAF in mammalian cells: ARAF, BRAF, and CRAF or RAF1. CRAF is expressed ubiquitously, whereas BRAF is expressed at higher levels in hemopoietic cells, neurons, and testes (14). BRAF is also the predominant isoform in thyroid follicular cells.6

6

L. Zhang and J.A. Fagin, unpublished observations.

Although all RAF isoforms activate mitogen-activated protein (MAP)/extracellular signal–regulated kinase (ERK) kinase (MEK), they are differentially activated by oncogenic RAS. In addition, BRAF has higher affinity for MEK1 and MEK2 and is more efficient in phosphorylating MEKs than other RAF isoforms (15). The initial discovery of BRAF mutation indicated a high prevalence of this event in malignant melanomas (16) and in a smaller subset of colorectal and ovarian cancers (16). Recent resolution of the crystal structure of the wild type and BRAFV600E kinase domains helps understand the mechanisms of mutational activation of the protein (17). BRAF exhibits the characteristic bilobar structure of protein kinases. In its inactive conformation, BRAF residues G597-V601 in the activation loop form hydrophobic interactions with residues G465-V472 in the ATP binding site (P loop), resulting in a structure that is not aligned for binding to ATP or substrate. Oncogenic mutations in the activation loop or the P loop disrupt their interaction and destabilize the inactive conformation. Most, but not all of known oncogenic BRAF substitutions allow the formation of new interactions that fold the kinase into a catalytically competent structure (reviewed in refs. 18, 19). Paradoxically, some of the oncogenic BRAF mutants impair in vitro kinase activity (17). Despite this, these low-activity kinase BRAF mutants induce ERK phosphorylation, which is due to activation of CRAF, presumably by heterodimerization (17).

Several groups have examined PTCs for the presence of RET/PTC, BRAF, and RAS mutations, all of which can activate the MAP kinase (MAPK) signaling pathway (4, 6, 20). Altogether, 177 PTCs were studied, and one of these alterations was present in about 70% of tumors. However, there was no single PTC with a mutation involving more than one of these genes. The lack of concordance provides compelling genetic evidence for a requirement of mutation of MAPK signaling components for transformation to PTC. This is consistent with evidence that RET/PTC-induced dedifferentiation (21) and thyroid-stimulating hormone (TSH)–independent growth (22) are dependent on activation of the MAPK pathway in thyroid cell lines.

It is now clear that PTCs with BRAF mutations have distinct phenotypic and biological properties. Although most have a classic papillary architecture, almost all tall-cell variant PTCs are positive for BRAFT1799A (8, 20). PTCs with a BRAFT1799A mutation present more commonly at an advanced stage of the disease (8, 10) and with extrathyroidal extension (8). Undifferentiated (anaplastic) and poorly differentiated carcinomas arising from preexisting PTCs have a significant prevalence of BRAF mutations, whereas those arising from preexisting follicular carcinoma do not (8, 10, 23). In addition, a subset of microscopic PTC, thought to be early precursors of these cancers, harbors BRAF mutations, indicating that this oncogene may be activated during tumor initiation (8). These data suggest that BRAF mutations may be a tumor-initiating event in PTC and associated with tumor dedifferentiation and more aggressive behavior. To test this in an animal model, BRAFV600E expression was targeted to thyroid follicular cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Here we provide the detailed characterization of two lines with thyroid-specific expression of BRAFV600E.

Creation of Tg-BRAF mice. The human BRAFT1799A cDNA with an NH2-terminal myc tag (gift from Richard Marais, Institute of Cancer Research, United Kingdom) was cloned into the BamHI site of pSG5 downstream of the β-globin intron. The ClaI/SalI fragment from pGS5/myc-BRAFT1799A containing the β-globin intron and myc-BRAFT1799A, was cloned downstream of the bovine thyroglobulin promoter into the ClaI/SalI site of pSKbTg (Fig. 1A; ref. 24). The SpeI/SalI fragment was injected into fertilized FVB/N mouse eggs, which were implanted into pseudopregnant female mice. The pups were confirmed by PCR and Southern blotting to have integrated the transgene. Lines were then generated from founder animals by crossing them with wild-type FVB/N mice.

Figure 1.

Tg-BRAF mice show expression of transgene in the thyroid and have increased pERK levels. A, map of the Tg-BRAF transgene. A SpeI/SalI fragment containing the bovine thyroglobulin promoter, rabbit β-globin intron 2, and the myc-tagged BRAFT1799A cDNA containing the endogenous human BRAF polyadenylation signal was used for injection. Upper arrow, transcription start site; checkered boxes, 5′ untranslated regions. B, quantitative RT-PCR using RNA prepared from thyroid glands of Tg-BRAF2 and Tg-BRAF3 mice was done as described in Materials and Methods. The primers are within the 5′ untranslated region of the mRNA and span the β-globin intron. Columns, relative expression of myc-tagged BRAFV600E after normalization to β-actin. There was no detectable expression in thyroid tissue from wild-type mice. C, Western blots containing protein extracts prepared from thyroid glands of Tg-BRAF mice (+) and age/sex-matched wild-type littermates (−) probed with antibodies to pERK1/2, BRAF, or total ERK1/2. Phospho-ERK levels are increased in the Tg-BRAF2 transgenic line, which is more pronounced in male animals. In Tg-BRAF3 mice, a marginal increase in pERK levels was observed.

Figure 1.

Tg-BRAF mice show expression of transgene in the thyroid and have increased pERK levels. A, map of the Tg-BRAF transgene. A SpeI/SalI fragment containing the bovine thyroglobulin promoter, rabbit β-globin intron 2, and the myc-tagged BRAFT1799A cDNA containing the endogenous human BRAF polyadenylation signal was used for injection. Upper arrow, transcription start site; checkered boxes, 5′ untranslated regions. B, quantitative RT-PCR using RNA prepared from thyroid glands of Tg-BRAF2 and Tg-BRAF3 mice was done as described in Materials and Methods. The primers are within the 5′ untranslated region of the mRNA and span the β-globin intron. Columns, relative expression of myc-tagged BRAFV600E after normalization to β-actin. There was no detectable expression in thyroid tissue from wild-type mice. C, Western blots containing protein extracts prepared from thyroid glands of Tg-BRAF mice (+) and age/sex-matched wild-type littermates (−) probed with antibodies to pERK1/2, BRAF, or total ERK1/2. Phospho-ERK levels are increased in the Tg-BRAF2 transgenic line, which is more pronounced in male animals. In Tg-BRAF3 mice, a marginal increase in pERK levels was observed.

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Real-time reverse transcription-PCR. Thyroid lobes were surgically removed, weighed, and immediately placed in liquid N2. RNA was isolated using Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH) and 0.6 μg was reverse transcribed with SuperScript III (Invitrogen, Carlsbad, CA) in the presence of random hexamers to generate cDNA. Quantitative reverse transcription-PCR (RT-PCR) was done using QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) and primer pairs for β-actin (5′-ctgaaccctaaggccaaccgtg-3′ and 5′-ggcatacagggacagcacagcc-3′); myc-tagged BRAF (5′-caagcttatcgatcctgagaact-3′ and 5′-ggctccatgtccccgttgaa-3′); thyroglobulin (5′-tgtcccaccaagtgtgaaaa-3′ and 5′-ccaaggaaagcttgttcagc-3′); sodium iodide symporter, NIS (5′-gctcagtctcgctcaaaacc-3′ and 5′-cgtgtgacaggccacataac-3′); thyroid peroxidase, TPO (5′-tgacttccaggagcacacag-3′ and 5′-gcaagttcagtgatgccaga-3′); and the TSH receptor, TSHR (5′-ctctcttacccgagccactg-3′ and 5′-ttgtcacccggatcttcttc-3′). The cycle threshold values for β-actin and the target gene were determined using Lightcycler (Cepheid, Sunnyvale, CA) and used to calculate the normalized relative expression using the QGENE program (25).

Western blots. Thyroid tissue was placed in buffer B [20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 1 mmol/L EGTA, 1.0% Triton X-100, 50 mmol/L NaF, 1 mmol/L Na orthovanadate, and protease inhibitor cocktail; Sigma, St. Louis, MO] and homogenized using a polytron. Protein lysates were centrifuged, supernatant collected, and protein concentration determined using micro-bicinchoninic acid (Pierce, Rockford, IL). Protein lysates were subjected to SDS PAGE, transferred to nitrocellulose membranes and probed with antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) to phospho-ERK1/2, total ERK, or BRAF. Membranes were hybridized with species-specific horseradish peroxidase–conjugated antibodies (Santa Cruz Biotechnology), and bands visualized with Enhanced Chemiluminescence as directed by manufacturer (Amersham, Piscataway, NJ).

RIAs. Blood from mice was collected immediately after euthanasia with CO2. Blood was incubated on ice for 1 hour, centrifuged at 4°C for 15 minutes, serum removed, and stored at −70°C until assayed. Serum TSHs were determined as previously described (26). The serum levels of total thyroxine and triiodothyronine were measured by solid-phase RIAs (Diagnostic Products, Los Angeles, CA) and the free thyroxine was estimated from the total thyroxine and the resin thyroxine uptake ratio and expressed as the free thyroxine index (FT4I; ref. 27).

Histology and immunohistochemistry. Thyroid tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Five-micrometer-thick sections were prepared and stained with H&E. Immunostaining was done on paraffin sections using avidine-streptavidin immunoperoxidase method with rabbit antimouse thyroglobulin immunoglobulin G (a gift from Paul Kim, University of Cincinnati) at a dilution of 1:100 on automated ES system (Ventana Medical Systems, Inc.,Tuscon, AZ) without additional antigen retrieval.

Statistical analysis. Difference between male and female transgenic animals in the frequency of histologic features was assessed by Fisher's exact test. Difference in survival was assessed by log-rank test. For all others, a two-tailed t test was used.

Of seven original Tg-BRAFV600E founders showing integration of the intact transgene as determined by Southern blotting, four had transgenic litters with low or undetectable expression of myc-tagged BRAF in thyroid tissue. One founder was runted and died at 6 weeks of age without progeny, despite treatment with thyroid hormone given on the presumptive diagnosis of hypothyroidism. Examination of this animal revealed a large goiter, papillary thyroid cancer and elevated TSH levels. The remaining two lines, Tg-BRAF2 and Tg-BRAF3, with thyroid-specific expression of BRAFV600E were propagated and characterized in detail. Quantitative RT-PCR with primers spanning the β-globin intron (Fig. 1A) and thus specific to the BRAFV600E transcript, showed the presence of BRAFV600E mRNA in thyroid tissues of both Tg-BRAF2 and Tg-BRAF3 mice, with significantly higher levels of expression in Tg-BRAF2 animals (Fig. 1B). Western blots of protein extracts from thyroid tissues of Tg-BRAF2 mice confirmed the increase in BRAF levels, with males having higher expression than females (Fig. 1C). The robust increase in phospho-ERK (pERK) levels (males > females) confirms expression of a functional protein. The weaker increase in pERK levels in Tg-BRAF3 mice was consistent with lower expression of BRAFV600E in this line (Fig. 1C).

Because mice with thyroid-specific expression of RET/PTC1 (28, 29), an upstream activator of BRAF,7

7

Mitsutake et al., unpublished observations.

were found to have stunted growth due to hypothyroidism, we monitored growth in animals with thyroid-specific expression of BRAFV600E. For this, weight of transgenic mice and wild-type littermates was followed for 12 weeks. Tg-BRAF3 mice had growth rates indistinguishable from controls (Fig. 2A). Tg-BRAF2 mice initially grew marginally slower than nontransgenic littermates. The difference in body weight was more pronounced in males than in females and proved to be transient, as it was not observed past 8 weeks (Fig. 2A). To determine the effects of BRAFV600E expression on thyroid function, serum levels of TSH, total triiodothyronine, total thyroxine, and free thyroxine levels were determined. At 5 weeks, TSH levels in male Tg-BRAF2 mice were on average 80-fold greater than those of age/sex-matched wild-type littermates. TSH levels markedly declined, although they still remained significantly elevated at 8 and 12 weeks (Table 1). In female Tg-BRAF2 mice, serum TSH levels were 5- to 8-fold above those of age/sex-matched wild-type littermates and did not vary significantly with time. Tg-BRAF3 mice had a 2-fold increase in serum TSH with no significant difference between males and females. Despite the elevated TSH levels, serum total thyroxine and free thyroxine in Tg-BRAF2 and Tg-BRAF3 mice were not significantly lower than sex- and age-matched wild-type littermates at any time point (Table 1). Serum total triiodothyronine levels trended lower at all time points in both male and female Tg-BRAF2 mice but were only statistically significant in 8-week-old male animals (Table 1). Triiodothyronine levels in Tg-BRAF3 mice were not statistically different from sex- and age-matched wild-type littermates.

Figure 2.

Tg-BRAF mice have increased serum TSH levels and reduced expression of thyroid-specific genes required for hormone biosynthesis. A, total body weight of animals was determined from weaning (age, 3 weeks) to 12 weeks of age. Changes in total body weight of Tg-BRAF mice and age/sex-matched wild-type littermates. Lines, average total body weight of at least nine animals. B, quantitative RT-PCR analysis of mRNA levels of TPO, NIS, and thyroglobulin (Tg) in thyroid glands of male Tg-BRAF2 and Tg-BRAF3 mice and their age/sex-matched wild-type littermates. Columns, average relative expression, after normalization to β-actin, of the indicated TSH-regulated genes. *, P < 0.05 versus wild type.

Figure 2.

Tg-BRAF mice have increased serum TSH levels and reduced expression of thyroid-specific genes required for hormone biosynthesis. A, total body weight of animals was determined from weaning (age, 3 weeks) to 12 weeks of age. Changes in total body weight of Tg-BRAF mice and age/sex-matched wild-type littermates. Lines, average total body weight of at least nine animals. B, quantitative RT-PCR analysis of mRNA levels of TPO, NIS, and thyroglobulin (Tg) in thyroid glands of male Tg-BRAF2 and Tg-BRAF3 mice and their age/sex-matched wild-type littermates. Columns, average relative expression, after normalization to β-actin, of the indicated TSH-regulated genes. *, P < 0.05 versus wild type.

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Table 1.

Serum TSH, thyroxine, and triiodothyronine levels, and free thyroxine index in Tg-BRAF mice and sex/age-matched wild-type littermates

Tg-BRAF2
Tg-BRAF2
Tg-BRAF3
Female
Male
Male
Female
Male
5 wks
8 wks
12 wks
5 wks
8 wks
12 wks
7 wks
7 wks
WtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAF
TSH (milliunits/L) 56.5 ± 7.9 339 ± 42 53.3 ± 3.9 224 ± 49 39.2 ± 11.4 300 ± 61 149 ± 16 12,091 ± 3,931 111 ± 21 1,149 ± 203 128 ± 11 962 ± 182 42.8 ± 9.3 93.9 ± 16.2 105 ± 20 240 ± 39 
P0.000  0.002  0.001  0.007  0.000  0.003  0.018  0.009  
n 10 10 10 10 
Thyroxine (μg/dL) 2.9 ± 0.1 2.7 ± 0.1 2.4 ± 0.2 2.8 ± 0.2 2.9 ± 0.1 3.3 ± 0.2 2.7 ± 0.1 3.0 ± 0.1 3.1 ± 0.2 3.1 ± 0.1 2.9 ± 0.3 3.4 ± 0.2 3.0 ± 0.2 3.9 ± 0.2 3.3 ± 0.2 3.6 ± 0.3 
P0.019  0.208  0.166  0.066  0.845  0.100  0.021  0.347  
n 10 10 10 10 
Triiodothyronine (ng/dL) 194 ± 20 170 ± 5 248 ± 21 193 ± 12 217 ± 21 191 ± 9 177 ± 5 163 ± 7 224 ± 14 167 ± 4 254 ± 29 229 ± 32 171 ± 8 216 ± 23 194 ± 14 299 ± 68 
P0.256  0.119  0.285  0.117  0.037  0.586  0.090  0.134  
n 10 10 10 10 
RUR 1.48 ± 0.07 1.48 ± 0.06 1.43 ± 0.07 1.46 ± 0.07 1.69 ± 0.15 1.56 ± 0.10 1.27 ± 0.02 1.16 ± 0.05 1.22 ± 0.02 1.28 ± 0.12 1.16 ± 0.04 1.17 ± 0.02 1.50 ± 0.12 1.44 ± 0.04 1.25 ± 0.03 1.17 ± 0.03 
P0.940  0.785  0.501  0.039  0.504  0.966  0.676  0.119  
n 10 10 10 10 
FT4I 4.33 ± 0.16 3.99 ±.21 3.43 ± 0.31 3.93 ± 0.30 4.91 ± 0.59 5.07 ± 0.26 3.40 ± 0.16 3.51 ± 0.29 3.67 ± 0.20 3.84 ± 0.17 3.35 ± 0.30 3.97 ± 0.23 4.41 ± 0.44 5.58 ± 0.41 4.08 ± 0.26 4.33 ± 0.38 
P0.226  0.343  0.813  0.734  0.599  0.126  0.078  0.584  
n 10 10 10 10 
Tg-BRAF2
Tg-BRAF2
Tg-BRAF3
Female
Male
Male
Female
Male
5 wks
8 wks
12 wks
5 wks
8 wks
12 wks
7 wks
7 wks
WtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAFWtBRAF
TSH (milliunits/L) 56.5 ± 7.9 339 ± 42 53.3 ± 3.9 224 ± 49 39.2 ± 11.4 300 ± 61 149 ± 16 12,091 ± 3,931 111 ± 21 1,149 ± 203 128 ± 11 962 ± 182 42.8 ± 9.3 93.9 ± 16.2 105 ± 20 240 ± 39 
P0.000  0.002  0.001  0.007  0.000  0.003  0.018  0.009  
n 10 10 10 10 
Thyroxine (μg/dL) 2.9 ± 0.1 2.7 ± 0.1 2.4 ± 0.2 2.8 ± 0.2 2.9 ± 0.1 3.3 ± 0.2 2.7 ± 0.1 3.0 ± 0.1 3.1 ± 0.2 3.1 ± 0.1 2.9 ± 0.3 3.4 ± 0.2 3.0 ± 0.2 3.9 ± 0.2 3.3 ± 0.2 3.6 ± 0.3 
P0.019  0.208  0.166  0.066  0.845  0.100  0.021  0.347  
n 10 10 10 10 
Triiodothyronine (ng/dL) 194 ± 20 170 ± 5 248 ± 21 193 ± 12 217 ± 21 191 ± 9 177 ± 5 163 ± 7 224 ± 14 167 ± 4 254 ± 29 229 ± 32 171 ± 8 216 ± 23 194 ± 14 299 ± 68 
P0.256  0.119  0.285  0.117  0.037  0.586  0.090  0.134  
n 10 10 10 10 
RUR 1.48 ± 0.07 1.48 ± 0.06 1.43 ± 0.07 1.46 ± 0.07 1.69 ± 0.15 1.56 ± 0.10 1.27 ± 0.02 1.16 ± 0.05 1.22 ± 0.02 1.28 ± 0.12 1.16 ± 0.04 1.17 ± 0.02 1.50 ± 0.12 1.44 ± 0.04 1.25 ± 0.03 1.17 ± 0.03 
P0.940  0.785  0.501  0.039  0.504  0.966  0.676  0.119  
n 10 10 10 10 
FT4I 4.33 ± 0.16 3.99 ±.21 3.43 ± 0.31 3.93 ± 0.30 4.91 ± 0.59 5.07 ± 0.26 3.40 ± 0.16 3.51 ± 0.29 3.67 ± 0.20 3.84 ± 0.17 3.35 ± 0.30 3.97 ± 0.23 4.41 ± 0.44 5.58 ± 0.41 4.08 ± 0.26 4.33 ± 0.38 
P0.226  0.343  0.813  0.734  0.599  0.126  0.078  0.584  
n 10 10 10 10 

Abbreviation: RUR, resin thyroxine uptake ratio.

*

Compared with sex- and age-matched transgenic littermates.

Oncogenic activation of the MAPK pathway results in decreased expression of thyroglobulin, NIS, and TPO in clonal well-differentiated thyroid cell lines (21, 30, 31). In addition, the decrease in thyroid function in Tg-RET/PTC mice has been attributed, at least in part, to reduced expression of NIS (28). Consistent with these observations, in 5-week-old male Tg-BRAF2 mice TPO, thyroglobulin, and NIS mRNA levels were 45%, 75%, and 95% lower than those found in wild-type littermates, respectively (Fig. 2B). Similar findings were obtained in 8-week male and 5-week female Tg-BRAF2 mice (data not shown). The decrease in mRNA levels of these genes is not due to changes in expression of the TSH receptor, as TSH receptor mRNA levels were not lower than wild-type littermates in 5-week-old male Tg-BRAF2 mice, which have the highest serum TSH levels (data not shown). In 5-week-old Tg-BRAF3 mice, expression of these gene products did not vary significantly from wild-type littermates, although there was a trend towards a decrease in NIS and TPO (Fig. 2B). Taken together, these data indicate that Tg-BRAF mice were euthyroid, having compensated for the BRAF-induced thyroid dysfunction through increased TSH levels, and as shown below, goiter development.

Thyroid glands of Tg-BRAF2 mice, and to a lesser extent Tg-BRAF3 mice, were markedly larger than age- and sex-matched wild-type littermates (Fig. 3). Histologic examination of thyroid glands from 12- and 22-week-old Tg-BRAF2 mice revealed multifocal tumors typically involving both lobes of the gland and having mixed papillary and follicular growth pattern (Fig. 4A). The tumor cells showed nuclear features characteristic of human papillary carcinomas, including nuclear enlargement, overlapping and crowding, irregular nuclear contours, and occasional chromatin clearing and nuclear grooves (Fig. 4B and C). In addition, almost all tumors in Tg-BRAF2 mice had focal areas showing well-defined tall-cell features (Fig. 4D; Table 2). Approximately 50% of the 12- and 22-week-old animals revealed focal areas of dedifferentiation composed of solid sheets of spindle cells lacking characteristic nuclear features of papillary carcinoma and showing no evidence of follicular architecture or colloid formation (Fig. 4E and F). Mitotic figures were frequently seen in these areas. Microscopic appearance of these areas was comparable with those of human poorly differentiated carcinoma, whereas no severe nuclear atypia or tumor necrosis, characteristic of human anaplastic (undifferentiated) carcinoma, was observed. Areas of dedifferentiation were frequently surrounded by well-differentiated tumor with prominent tall-cell appearance. Thyroglobulin staining of thyroid section showed reduced staining in the poorly differentiated areas (data not shown); however, the reduction in thyroglobulin levels did not correlate with the degree of morphologic dedifferentiation. The tumors in Tg-BRAF2 mice showed aggressive malignant features as they frequently invaded blood vessels and thyroid gland capsule with extrathyroidal extension into adjacent adipose tissue and skeletal muscle (Fig. 4G-I; Table 2). The frequency of dedifferentiation and vascular invasion were higher in male than in female animals (P = 0.026 and 0.0052, respectively). In Tg-BRAF3 mice, 3 of 12 and 4 of 9 animals at 12 and 22 weeks, respectively, had small tumor foci with follicular architecture and cytologic features of PTC (Table 2). One of the PTC foci presented with focal tall-cell features. None of the tumors showed extrathyroidal extension, vascular invasion, or progression to poorly differentiated carcinoma.

Figure 3.

Goiter development in Tg-BRAF mice. The insert shows a thyroid gland from a 12 week male Tg-BRAF2 mouse (right) and a male wild-type littermate (left). The bars in the graph represent the average weight of the thyroid gland in Tg-BRAF3 and their wild-type littermates at 5 weeks, and Tg-BRAF2 and their wild-type littermates at 5 and 12 weeks. *, P < 0.05 vs. wild type.

Figure 3.

Goiter development in Tg-BRAF mice. The insert shows a thyroid gland from a 12 week male Tg-BRAF2 mouse (right) and a male wild-type littermate (left). The bars in the graph represent the average weight of the thyroid gland in Tg-BRAF3 and their wild-type littermates at 5 weeks, and Tg-BRAF2 and their wild-type littermates at 5 and 12 weeks. *, P < 0.05 vs. wild type.

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Figure 4.

Microscopic features of Tg-BRAF2 mice. A, low-magnification view of most of the thyroid lobe occupied by a tumor displaying several well-formed papillae (arrow). Magnification, ×40. B, a papillae lined by tumor cells with nuclear overlapping and crowding. Magnification, ×200. C, high-magnification view of characteristic nuclear features found in human PTC, including nuclear enlargement, crowding and overlapping, irregularity of nuclear contours, and occasional nuclear groves (arrow). Magnification, ×400. D, area of the tumor with prominent tall-cell appearance, characterized by cells that are two times as tall as they are wide. Magnification, ×400. E, focus of poorly differentiated carcinoma (PD) located adjacent to well-differentiated tumor (WD). Magnification, ×100. F, higher magnification of poorly differentiated area composed of sheets of spindle cells without follicular architecture or colloid formation and adjacent well-differentiated area containing cells with tall-cell features (arrow). Magnification, ×200. G, tumor invasion through the capsule of the thyroid gland (arrows). Magnification, ×100. H, vascular invasion manifested as a focus of tumor cells within the pericapsular vein which is attached to the vessel wall (arrow). Magnification ×100. I, tumor invasion into the adjacent skeletal muscle. Magnification, ×100. All sections were stained with H&E.

Figure 4.

Microscopic features of Tg-BRAF2 mice. A, low-magnification view of most of the thyroid lobe occupied by a tumor displaying several well-formed papillae (arrow). Magnification, ×40. B, a papillae lined by tumor cells with nuclear overlapping and crowding. Magnification, ×200. C, high-magnification view of characteristic nuclear features found in human PTC, including nuclear enlargement, crowding and overlapping, irregularity of nuclear contours, and occasional nuclear groves (arrow). Magnification, ×400. D, area of the tumor with prominent tall-cell appearance, characterized by cells that are two times as tall as they are wide. Magnification, ×400. E, focus of poorly differentiated carcinoma (PD) located adjacent to well-differentiated tumor (WD). Magnification, ×100. F, higher magnification of poorly differentiated area composed of sheets of spindle cells without follicular architecture or colloid formation and adjacent well-differentiated area containing cells with tall-cell features (arrow). Magnification, ×200. G, tumor invasion through the capsule of the thyroid gland (arrows). Magnification, ×100. H, vascular invasion manifested as a focus of tumor cells within the pericapsular vein which is attached to the vessel wall (arrow). Magnification ×100. I, tumor invasion into the adjacent skeletal muscle. Magnification, ×100. All sections were stained with H&E.

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Table 2.

Prevalence and histologic characteristics of PTCs developing in thyroid tissues from Tg-BRAF2 and Tg-BRAF3 mice at 12 and 22 wks

LineAge (wk)PTC prevalence (%)Histologic characterization (%)
Tall cellPoorly differentiatedInvasion
Inflammation
CapsularVascularMuscleMacrophagesLymphocytic infiltration
Tg-BRAF2 12 14/15 (93) 87 47 73 47 27 87 33 
Tg-BRAF2 22 13/14 (93) 79 50 93 50 36 93 50 
Tg-BRAF3 12 3/12 (25) 
Tg-BRAF3 22 4/9 (44) 11 56 67 
LineAge (wk)PTC prevalence (%)Histologic characterization (%)
Tall cellPoorly differentiatedInvasion
Inflammation
CapsularVascularMuscleMacrophagesLymphocytic infiltration
Tg-BRAF2 12 14/15 (93) 87 47 73 47 27 87 33 
Tg-BRAF2 22 13/14 (93) 79 50 93 50 36 93 50 
Tg-BRAF3 12 3/12 (25) 
Tg-BRAF3 22 4/9 (44) 11 56 67 

The aggressive cancer in Tg-BRAF2 mice was associated with a 30% decrease in survival at 22 weeks. No significant change in survival was found for Tg-BRAF3 mice (Fig. 5). To determine whether the decreased survival of the Tg-BRAF2 mice was due to distant metastasis, we examined lungs and hearts, two organs found to be a common site of thyroid cancer metastasis in another animal model for thyroid cancer (32). However, no evidence of metastasis to these tissues was found. The lungs from the deceased animals showed microscopic features of acute asphyxiation, suggesting that their death may have been caused by upper airway compression.

Figure 5.

Survival of Tg-BRAF2 and Tg-BRAF3 mice. Kaplan-Meier curve shows a significant increase the death rate of Tg-BRAF2 mice (P = 0.03).

Figure 5.

Survival of Tg-BRAF2 and Tg-BRAF3 mice. Kaplan-Meier curve shows a significant increase the death rate of Tg-BRAF2 mice (P = 0.03).

Close modal

The BRAFT1799A mutation is the most common oncogenic event thus far identified in PTC (4, 613). This mutation can occur early in tumor development, as it is present in papillary microcarcinomas, which are thought to represent an early stage of PTC (8). The lack of overlap between RET/PTC, RAS, and BRAF mutations in PTC (4, 6, 20) indicates that a single oncogenic hit along the MAPK pathway provides sufficient activation of this pathway that, when combined with other changes, results in transformation. Despite this, the nature of the oncogenic event has clear effects on the phenotypic features and biological behavior of the resulting cancer. For example, PTC with RAS mutations are almost exclusively of the follicular variant (33), whereas those with RET/PTC3 rearrangements are associated with the solid variant of the disease (34). PTC with BRAFT1799A are associated with a classic histotype and with tall-cell variant, which has been associated with more aggressive tumor behavior (8, 20). Furthermore, RET/PTC rearrangements are rarely if ever found in poorly differentiated or anaplastic thyroid carcinomas. By contrast, BRAFT1799A is commonly found in poorly differentiated and undifferentiated carcinomas arising from preexisting PTC (8), suggesting that this oncogenic event promotes tumor dedifferentiation and confers patients with a significantly worse prognosis.

Some of the histologic features associated with a particular oncogenic hit in human PTC have also been found in their corresponding mouse models. Thus, mice with thyroid-specific expression of RET/PTC3 develop solid variant PTC (35), and those with expression of RET/PTC1 develop tumors with a more classic PTC architecture (36). Consistent with the human data, PTC that develop in either Tg-RET/PTC1 or Tg-RET/PTC3 mice do not progress to poorly differentiated carcinomas (35, 36) unless crossed with p53−/− mice (37, 38). We show here that mice with thyroid-specific expression of BRAFV600E develop PTCs that closely recapitulate the properties BRAF-positive human PTC (i.e., classic PTC architecture, tall-cell features, and high potential for invasiveness). Moreover, in the majority of the mice from the Tg-BRAF2 line there is progression to poorly differentiated carcinomas. These results indicate that BRAFV600E can serve as a tumor initiator and promote progression to poorly differentiated carcinomas. By contrast to human poorly differentiated or anaplastic cancers that typically exhibit almost complete loss of thyroglobulin-immunoreactive cells (39), the poorly differentiated foci present in the Tg-BRAF2 mice still expressed thyroglobulin, albeit at lower levels than surrounding well-differentiated areas (data not shown). However, this difference may simply represent a limitation of this particular mouse model. As transgene expression is driven by the thyroglobulin gene promoter and dedifferentiation results in reduction in thyroglobulin promoter activity, this will be associated with a corresponding decrease in BRAFV600E expression. It is tempting to speculate that persistence of thyroglobulin expression indicates a continued requirement for BRAFV600E even in the poorly differentiated cancers.

Lower body weight in the younger Tg-BRAF2 mice suggests that the animals may have been hypothyroid at some point during development. However, despite markedly elevated TSH levels, serum thyroxine and free thyroxine levels were normal in both transgenic mouse lines at all times. There was a subtle decrease in triiodothyronine levels at one of the time points, but this was not sustained. The most likely explanation for the elevated TSH is that the Tg-BRAF mice were markedly hypothyroid in late gestation and in the early neonatal period. This was likely compensated by gradual thyroid enlargement, which by 5 weeks of age was at least 6-fold greater than nontransgenic littermates. As we did not measure free thyroid hormone levels at these early time points, the period of hypothyroidism was not detected. Thus, the data are consistent with gradual development of a compensated hypothyroid state, arising because of BRAF-induced impairment of TSH action in thyroid follicular cells.7 Higher TSH levels are required to reach a new steady state, which is arrived at in part through development of goiter. Perhaps, successful compensation was possible in Tg-BRAF animals because TSHR gene expression was unaffected hence allowing for adequate TSH responsiveness. This is in contrast to what occurs in mice with haploinsufficiency of TTF1 (also called TITF1, T/EBP, and NKX2.1), a transcription factor containing a homeobox domain that is required for normal thyroid development (40). These animals have decreased TSHR, and also have higher TSH levels, which is not sufficient to make these animals euthyroid. It is tempting to speculate that this is because these animals do not have enlarged thyroid glands, which would be required to return them to euthyroidism.

The transgenic line with higher penetrance of PTC also had higher TSH levels, and it is likely that TSH was an important contributing factor to the development of these cancers. This is because of the following: (a) Expression of the transgene is driven by the thyroglobulin promoter. Thus, the higher the TSH, the higher the expression of the mutant BRAF. (b) The growth promoting effects of TSH. TSH cooperates with growth factors or oncoproteins that activate the MAPK pathway to promote thyroid cell growth (41). This is of clinical importance, because even physiologic levels of TSH are believed to promote PTC progression and recurrence (42, 43). Indeed, TSH increments within the physiologic range are an independent variable that increases relative risk of PTC in humans (44). Hence, the Tg-BRAF model of PTC results from an accentuation of growth promoting signals that are normally involved in development of these cancers in humans. The higher intensity of these signals likely accounts for the high penetrance and the short latency.

In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC with tall-cell features, which later transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAFV600E-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis. Indeed, the similarity between the histopathologic features of the Tg-BRAFV600E PTC and their human counterparts is striking. It is likely that this animal model will serve as an important tool for further understanding of molecular events associated with dedifferentiation of PTC. This is of clinical significance because poorly differentiated and anaplastic thyroid carcinomas are uniformly associated with high mortality and there are no reliable clinical or pathologic indicators that predict predisposition to undergo progression to poorly differentiated forms of the disease. In addition, this model should be useful for testing potential therapeutic strategies for treatment of PTC as well as those aimed to prevent tumor dedifferentiation.

Grant support: NIH grants CA50706 (J.A. Fagin) and DK15070 (S. Refetoff), American Cancer Society grant RSG-03-027-01-CCE (Y.E. Nikiforov), American Cancer Society Ohio Chapter grant 06001011 (J.A. Knauf), Nakayama Foundation for Human Science (N. Mitsutake), and SUMITOMO Life Social Welfare Services Foundation (N. Mitsutake).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Richard Marais and Gilbert Vassart for providing the BRAFV600E cDNA and bovine thyroglobulin promoter constructs, respectively.

1
Hundahl SA, Cady B, Cunningham MP, et al. Initial results from a prospective cohort study of 5583 cases of thyroid carcinoma treated in the united states during 1996. U.S. and German Thyroid Cancer Study Group. An American College of Surgeons Commission on Cancer Patient Care Evaluation study.
Cancer
2000
;
89
:
202
–17.
2
Fagin JA. How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy.
J Endocrinol
2004
;
183
:
249
–56.
3
Santoro M, Melillo RM, Carlomagno F, Fusco A, Vecchio G. Molecular mechanisms of RET activation in human cancer.
Ann N Y Acad Sci
2002
;
963
:
116
–21.
4
Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma.
Cancer Res
2003
;
63
:
1454
–7.
5
Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage.
Nat Rev Mol Cell Biol
2004
;
5
:
875
–85.
6
Soares P, Trovisco V, Rocha AS, et al. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC.
Oncogene
2003
;
22
:
4578
–80.
7
Trovisco V, Vieira dC I, Soares P, et al. BRAF mutations are associated with some histological types of papillary thyroid carcinoma.
J Pathol
2004
;
202
:
247
–51.
8
Nikiforova MN, Kimura ET, Gandhi M, et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas.
J Clin Endocrinol Metab
2003
;
88
:
5399
–404.
9
Fukushima T, Suzuki S, Mashiko M, et al. BRAF mutations in papillary carcinomas of the thyroid.
Oncogene
2003
;
22
:
6455
–7.
10
Namba H, Nakashima M, Hayashi T, et al. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers.
J Clin Endocrinol Metab
2003
;
88
:
4393
–7.
11
Xu X, Quiros RM, Gattuso P, Ain KB, Prinz RA. High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines.
Cancer Res
2003
;
63
:
4561
–7.
12
Cohen Y, Xing M, Mambo E, et al. BRAF mutation in papillary thyroid carcinoma.
J Natl Cancer Inst
2003
;
95
:
625
–7.
13
Kim KH, Kang DW, Kim SH, Seong IO, Kang DY. Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population.
Yonsei Med J
2004
;
45
:
818
–21.
14
Daum G, Eisenmann-Tappe I, Fries HW, Troppmair J, Rapp UR. The ins and outs of Raf kinases.
Trends Biochem Sci
1994
;
19
:
474
–80.
15
Peyssonnaux C, Eychene A. The Raf/MEK/ERK pathway: new concepts of activation.
Biol Cell
2001
;
93
:
53
–62.
16
Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer.
Nature
2002
;
417
:
949
–54.
17
Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.
Cell
2004
;
116
:
855
–67.
18
Garnett MJ, Marais R. Guilty as charged; B-RAF is a human oncogene.
Cancer Cell
2004
;
6
:
313
–9.
19
Mercer KE, Pritchard CA. Raf proteins and cancer: B-Raf is identified as a mutational target.
Biochim Biophys Acta
2003
;
1653
:
25
–40.
20
Frattini M, Ferrario C, Bressan P, et al. Alternative mutations of BRAF, RET and NTRK1 are associated with similar but distinct gene expression patterns in papillary thyroid cancer. Oncogene 2004.
21
Knauf JA, Kuroda H, Basu S, Fagin JA. RET/PTC-induced dedifferentiation of thyroid cells is mediated through Y1062 signaling through SHC-RAS-MAP kinase.
Oncogene
2003
;
22
:
4406
–12.
22
Castellone MD, Cirafici AM, De Vita G, et al. Ras-mediated apoptosis of PC CL 3 rat thyroid cells induced by RET/PTC oncogenes.
Oncogene
2003
;
22
:
246
–55.
23
Begum S, Rosenbaum E, Henrique R, Cohen Y, Sidransky D, Westra WH. BRAF mutations in anaplastic thyroid carcinoma: implications for tumor origin, diagnosis and treatment.
Mod Pathol
2004
;
17
:
1359
–63.
24
Nguyen LQ, Kopp P, Martinson F, Stanfield K, Roth SI, Jameson JL. A dominant negative CREB (cAMP response element-binding protein) isoform inhibits thyrocyte growth, thyroid-specific gene expression, differentiation, and function.
Mol Endocrinol
2000
;
14
:
1448
–61.
25
Muller PY, Janovjak H, Miserez AR, Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR.
Biotechniques
2002
;
32
:
1372
–9.
26
Pohlenz J, Maqueem A, Cua K, Weiss RE, Van SJ, Refetoff S. Improved radioimmunoassay for measurement of mouse thyrotropin in serum: strain differences in thyrotropin concentration and thyrotroph sensitivity to thyroid hormone.
Thyroid
1999
;
9
:
1265
–71.
27
Robin NI, Hagen SR, Collaco F, Refetoff S, Selenkow HA. Serum tests for measurement of thyroid function.
Hormones
1971
;
2
:
266
–79.
28
Cho JY, Sagartz JE, Capen CC, Mazzaferri EL, Jhiang SM. Early cellular abnormalities induced by RET/PTC1 oncogene in thyroid-targeted transgenic mice.
Oncogene
1999
;
18
:
3659
–65.
29
Jhiang SM, Cho JY, Furminger TL, et al. Thyroid carcinomas in RET/PTC transgenic mice.
Recent Results Cancer Res
1998
;
154
:
265
–70.
30
Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M, Vecchio G. One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.
Mol Cell Biol
1987
;
7
:
3365
–70.
31
Missero C, Pirro MT, Di Lauro R. Multiple ras downstream pathways mediate functional repression of the homeobox gene product TTF-1.
Mol Cell Biol
2000
;
20
:
2783
–93.
32
Suzuki H, Willingham MC, Cheng SY. Mice with a mutation in the thyroid hormone receptor β gene spontaneously develop thyroid carcinoma: a mouse model of thyroid carcinogenesis.
Thyroid
2002
;
12
:
963
–9.
33
Zhu Z, Gandhi M, Nikiforova MN, Fischer AH, Nikiforov YE. Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations.
Am J Clin Pathol
2003
;
120
:
71
–7.
34
Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA. Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children.
Cancer Res
1997
;
57
:
1690
–4.
35
Powell DJJ, Russell J, Nibu K, et al. The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids.
Cancer Res
1998
;
58
:
5523
–8.
36
Jhiang SM, Sagartz JE, Tong Q, et al. Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas.
Endocrinology
1996
;
137
:
375
–8.
37
La Perle KM, Jhiang SM, Capen CC. Loss of p53 promotes anaplasia and local invasion in ret/PTC1-induced thyroid carcinomas.
Am J Pathol
2000
;
157
:
671
–7.
38
Powell DJJ, Russell JP, Li G, et al. Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53−/− mice.
Oncogene 2001 May
31
;
20
:
3235
–46.
39
Carcangiu ML, Steeper T, Zampi G, Rosai J. Anaplastic thyroid carcinoma. A study of 70 cases.
Am J Clin Pathol
1985
;
83
:
135
–58.
40
Moeller LC, Kimura S, Kusakabe T, Liao XH, Van SJ, Refetoff S. Hypothyroidism in thyroid transcription factor 1 haploinsufficiency is caused by reduced expression of the thyroid-stimulating hormone receptor.
Mol Endocrinol
2003
;
17
:
2295
–302.
41
Kimura T, Van KA, Golstein J, Fusco A, Dumont JE, Roger PP. Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models.
Endocr Rev
2001
;
22
:
631
–56.
42
Mazzaferri EL. Long-term outcome of patients with differentiated thyroid carcinoma: effect of therapy.
Endocr Pract
2000
;
6
:
469
–76.
43
Mandel SJ, Brent GA, Larsen PR. Levothyroxine therapy in patients with thyroid disease.
Ann Intern Med
1993
;
119
:
492
–502.
44
Boelaert K, Horacek Y, Daykin J, Sheppard M, Franklyn J. Gender, age, goitre type and serum TSH level predict thyroid neoplasia in 1500 patients with thyroid enlargement investigated by FNAC, 23rd Joint Meeting of the British Endocrine Societies with the European Federation of Endocrine Societies, March 22-24 2004; p. 757(Abstract).