Activating BRAF and PIK3CA Mutations Cooperate to Promote Anaplastic Thyroid Carcinogenesis

Abstract

Thyroid malignancies are the most common type of endocrine tumors. Of the various histological sub-types, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF proto-oncogene is detected in ~40% of papillary thyroid cancers (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3-kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAF^V600E, we previously developed a model of PTC. However, as in humans, BRAF^V600E-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CA^H1047R is unable to drive transformation on its own, when combined with BRAF^V600E in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAF^V600E cooperates with either PIK3CA^H1074R or with silencing of the tumor suppressor PTEN, to promote development of anaplastic thyroid cancer.

INTRODUCTION

Thyroid cancers derived from follicular epithelial cells are histologically classified into papillary (PTC), follicular or anaplastic thyroid cancers (ATC) [1]. While PTC is indolent and readily managed by surgical resection combined with radio-iodine therapy, ATC is highly aggressive with more than 50% of ATC patients dying within one year after diagnosis [2]. Although metastasis is observed in 10-20% of ATC patients, most ATC patients succumb to locally invasive, inoperable disease that is largely refractory to conventional chemotherapy [3].

Mutationally activated BRAF (commonly T1799→A in exon 15) encoding BRAF^V600E is detected in ~40% of PTC and 25% of ATC [4]. BRAF^V600E is a constitutively active protein kinase that activates the ERK1/2 MAP kinase pathway [5]. The importance of mutated BRAF in thyroid cancer maintenance is suggested by responses of thyroid cancer patients to vemurafenib, a pharmacological inhibitor of BRAF^V600E [6]. Moreover, conditional, thyrocyte-specific expression of BRAF^V600E in genetically engineered mouse (GEM) models results in PTC [7]. However, as in humans, PTC in this model is indolent and 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- or KRAS (20-30%), PIK3CA (15-25%) and CTNNB1 (60-65%) [8]. Hence, by analogy to other cancer types, it is likely that progression to more aggressive disease is due to cooperative interactions between these various genetic abnormalities. To test this, we generated mice with thyrocyte-specific expression of BRAF^V600E in conjunction with expression of mutationally activated PIK3CA^H1047R, a constitutively activated form of the p110 catalytic subunit of PI3’-kinase-α [9]. Expression of PIK3CA^H1047R, which is detected in many cancer types, is predicted to promote elevated PI3’-lipid production leading to activation of AKT protein kinases and other PI3’-lipid effectors in the cell [10]. In brief, whereas adult-onset, thyrocyte-specific expression of PIK3CA^H1047R had no detectable effect on the thyroid, it cooperated dramatically with BRAF^V600E such that mice developed rapidly lethal ATC. Similar observations were also made with thyrocyte-specific expression of BRAF^V600E combined with PTEN silencing. Using cultured human thyroid cancer cell lines, we demonstrated that these pathways cooperate to regulate the activity of mTOR and the phosphorylation of 4E-BP1. Hence, we propose that this GEM model of ATC, which recapitulates key features of the human disease, will be useful in understanding thyroid cancer progression and modeling the effects of pathway-targeted therapy in the pre-clinical setting.

MATERIALS AND METHODS

Mouse breeding and manipulation

BRaf^CA, Pik3ca^lat-1047R, Pten^lox and Thyroglobulin::CreER^T2 mice were described previously [7,11] [9,12]. BRaf^CA mice have been backcrossed in FVB/N in the lab for more than 10 generations; all the others have been obtained in C57BL/6 F129 mixed background and crossed in FVB/N since obtained. All the mice considered here are predominantly FVB/N. Thyrocyte specific activation of CreER^T2 activity was achieved by intraperitoneal injection of 1mg of Tamoxifen dissolved in peanut oil into 4 week old mice.

Cell lines

8505c line was culture as directed in RPMI complemented with 10% FCS (and validated by STR profiling, performed by Microsynth, Switzerland). Ocut-2 in DMEM complemented with 10% FCS and non-essential amino acids, STR profiles showed that this cell lines was not presenting mouse or human contamination and was of female origin as expected from the literature. STR profile of Ocut-2 did not present any relevant similarities to any registered cell lines of the American Type Culture Collection (ATCC).

Histology and Immunofluorescence of mouse thyroid tissue sections

Animal experiments were carried out in accordance with protocols approved by the University of California, San Francisco (San Francisco, CA) Institutional Animal Care and Use Committee (IACUC). Mice were anesthetized by intraperitoneal injection prior to tissue dissection, and then euthanized by section of the abdominal aorta. Thyroids were removed, rinsed in ice cold PBS and fixed for 4 hours in Z-Fix (Anatech, MI, USA). 4-5μm sections of formalin fixed, paraffin embedded tissues were stained with Hematoxilin & Eosin or processed for immunofluorescence. Epitope unmasking was performed by boiling slides for 10 minutes in a buffer comprising 10mM Tris, 0.5mM EGTA pH 9.0. Primary antibodies were obtained from the listed commercial sources and diluted as follow: anti-TTF-1 (1:200, Santa Cruz), anti-Ki67 (1:300, Abcam), anti-CK19 (1:300, TROMA-III, Hybridoma bank, University of Iowa) Vimentin (cell signaling, 1-200) and anti-Galectin-3 (Abcam, 1:200). Antigen-antibody complexes were detected using either Goat anti-rabbit Alexa-488 (1:500) or Goat anti-rat Alexa-488 (1:500) (Molecular Probes) with slides counterstained with DAPI to visualize nuclear DNA.

Ultrasound imaging

Mice were anesthetized using 2%(v/v) isofluorane at which time fur around the neck was removed using Veet® depilatory cream. Ultrasound images were collected weekly using the Vevo770 system (VisualSonics). Thyroid size was assessed by counting pixels at the largest diameter of the thyroid and converting pixel count into area (mm²) using scaling software internal to the device in combination with ImageJ (NIH).

RESULTS

Pik3ca^lat-1047R (Pik3ca^Lat hereafter) mice carry a conditional allele that expresses normal PIK3CA prior to Cre-mediated recombination after which mutationally activated PIK3CA^H1047R is expressed from the normal chromosomal locus at physiologically relevant levels of expression [9]. To test the effects of thyrocyte-specific expression of PIK3CA^H1047R we treated compound adult Thyro::CreER; Pik3ca^Lat mice with tamoxifen and monitored prospectively for palpable goiter. Even one year after tamoxifen administration, mice with thyrocyte-specific expression of PIK3CA^H1047R displayed no obvious thyroid abnormalities. Hence, unlike BRAF^V600E, mutationally activated PIK3CA^H1047R is insufficient to initiate thyroid hypertrophy or tumorigenesis [7].

To test whether PIK3CA^H1047R would influence BRAF^V600E-induced PTC, we initiated tumorigenesis in adult Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ and monitored them prospectively for disease (Fig. 1A). At 6.5 months, 2 mice out of 14 became emaciated, displayed labored breathing and had readily palpable thyroid tumors that were ≥1cm³ requiring euthanasia. Over the next two months 71% of mice (10/14) presented developed similar end-stage disease requiring euthanasia. The experiment was stopped 12.5 months after tumor initiation, a time at which 85% (12/14) of the Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ mice had been retired. By contrast, only 37% (4 out of 11) of an independent cohort of initiated Thyro::CreER; BRaf^CA/+ mice reached end-stage and required euthanasia ( tumors ≥1cm³). Kaplan-Meier survival analysis clearly demonstrated the shortened survival of mice bearing BRAF^V600E/PIK3CA^H1047R vs. BRAF^V600E expressing thyroid tumors (Fig. 1A, p<0.01). To further evaluate BRAF^V600E/PIK3CA^H1047R cooperation in thyroid tumorigenesis, tumor growth was measured in two additional cohorts of Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ or Thyro::CreER; BRaf^CA/+ mice using ultrasound imaging two months after initiation (Fig. 1B). Mice bearing BRAF^V600E/PIK3CA^H1047R thyroid tumors displayed a ~60% greater tumor burden compared to BRAF^V600E alone tumors.

Figure 1. Combined thyrocyte-specific expression of BRAF^V600E and PIK3CA^H1047R result in lethal thyroid carcinogenesis.

A: Kaplan-Meier survival curves comparing mice survival of tamoxifen-treated Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ and Thyro::CreER; BRaf^CA/+ mice (**: P≤0.01; Log-Rank Test)

B: Thyroid tumor burden in tamoxifen-treated Thyro::CreER; BRaf^CA/+; Pik3ca^Lat-HR/+ andThyro::CreER; BRaf^CA/+ mice measured by ultrasonography (**: P≤0.01; t-Test) 2.5 months after tumor induction.

C: H&E staining of histological section of thyroid from control BRaf^CA/+ (WT): Thyro::CreER; BRaf^CA/+(BRAF^V600E) and Thyro::CreER; Pik3ca^Lat/+ (PI3KCA^H1047R) mice, at 40× (Upper row) and 200× (Lower row) magnification.

D: H&E staining of histological section of thyroid Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ (BRAF^V600E PI3KCA^H1047R) showing an area of PTC (Left), tracheal airway invasion (Middle) and ATC area (Right), at 40× (Upper row) and 200× (Lower row) magnification.

To assess the effects of oncogene expression on tumor histology, thyroid specimens were prepared from normal mice or mice with thyrocyte specific expression of PIK3CA^H1047R, BRAF^V600E or combined BRAF^V600E/PIK3CA^H1047R (Figs. 1C-D). Both normal and PIK3CA^H1047R expressing thyroid (Figs. 1C) tissue displayed a characteristic follicular architecture composed of a monolayered cuboidal epithelium of thyrocytes. Indeed, even 12 months after PIK3CA^H1047R expression, no alteration in thyroid architecture was observed (data not shown). BRAF^V600E expression elicited PTC displaying the characteristic papillary thyroid architecture at 6-9 months after initiation as described previously [7] (Fig. 1C middle). By contrast, when BRAF^V600E and PIK3CA^H1047R were co-expressed, we observed clear evidence of PTC within 3-6 months that extended throughout the entire thyroid (Fig. 1D left). Moreover these PTC lesions displayed aggressive characteristics with evidence of invasion into surrounding tissues, most notably the trachea (Fig. 1D middle). Such invasion resulted in reduced airway diameter that was observed in 70% of the animals represented in the Kaplan-Meier survival curve (Fig. 1A). In addition, the thyroid tumors in 80% of these animals displayed polygonal giant cells and/or spindle-like cells (Fig. 1D right), which is a characteristic of human ATC. Such features (local invasion and aberrant tumor cytology) were not observed in PTCs induced by expression of BRAF^V600E alone [7].

To further characterize BRAF^V600E/PIK3CA^H1047R collaboration in this model, we generated Thyro::CreER; BRaf^CA mice that were either wild-type, heterozygous or homozygous for the conditional Pik3ca^Lat allele. 2.5 months after initiation, Thyro::CreER; BRafCA; Pik3ca^Lat/+ mice displayed the expected increase in tumor size compared to mice lacking the conditional Pik3ca^Lat allele. As expected, Thyro::CreER; BRafCA; Pik3ca^Lat/Lat mice also displayed an increase in tumor size (~37%) compared to mice lacking the Pik3ca^Lat allele (Fig. 2A). However, although the difference in tumor size between mice heterozygous or homozygous for the Pik3ca^Lat allele was not statistically significant (Fig. 2B), mice homozygous for Pik3ca^Lat/Lat died more rapidly than their heterozygous counterparts (Fig. 2A) most likely due to an earlier onset of the tracheal invasion.

Figure 2. Thyroid cancer development in mice wild-type, heterozygous or homozygous for Pik3ca^Lat.

A: Kaplan-Meier survival curves comparing tamoxifen treated: 1. Thyro::CreER; BRaf^CA/+, Thyro::CreER; 2. Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/+ or; 3. Thyro::CreER; BRaf^CA/+; Pik3ca^Lat/Lat mice after tumor initiation (**: P≤0.01; Log-Rank Test).

B: Tumor burden measured by ultrasonography (*P≤0.05; **: P≤0.01; t-Test).

C: Immunofluorescence staining for expression of TTF-1, E-Cadherin, Vimentin, Ki67 and Cytokeratin 19 in a histological section of thyroid from Thyro::CreER; BRaf^CA/+; Pik3ca^Lat-HR/+ focusing on areas of papillary (PTC) or anaplastic thyroid carcinoma (ATC area) as indicated. Nuclear DNA was stained blue with DAPI.

Human thyroid malignancies are characterized by alterations in marker expression such as Galactin-3 (Gal-3) and CK-19 [13]. We therefore stained BRAF^V600E/PIK3CA^H1047R expressing thyroid tumors for expression of these proteins (Fig. 2C). First, we noted that BRAF^V600E/PIK3CA^H1047R expressing thyroid tumors displayed areas of PTC and ATC. The regions of PTC were generally positive for Gal-3 and CK-19 as described previously [7]. By contrast, regions of ATC retained Gal-3 but lacked CK-19 expression. In addition, ATC like lesions displayed low/no expression of TTF-1 or E-Cadherin, increased Vimentin expression and a higher proliferative index (Ki67).

As an alternative strategy to confirm collaboration of BRAF^V600E with alterations in the PI3-kinase pathway we generated Thyro::CreER; BRaf^CA; Pten^lox/lox mice to initiate BRAF^V600E/PTEN^null thyroid tumors. Compared to BRAF^V600E alone, these mice displayed increased thyroid tumor burden as soon as 1.5 months after initiation (Fig. 3B). This required complete silencing of PTEN expression since the enhanced tumorigenesis was not noted in Thyro::CreER; BRaf^CA mice heterozygous for Pten^lox (Fig. 3B). As anticipated, mice with BRAF^V600E/PTEN^null thyroid tumors developed end-stage disease more rapidly than mice with BRAF^V600E expression alone (Fig. 3A p<0.01). It was notable that Thyro::CreER; BRaf^CA mice heterozygous for Pten^lox tended to reach end-point more rapidly than mice with BRAF^V600E expression alone, presumably because of loss-of-heterozygosity of the remaining Pten^WT allele (Fig. 3B).

Figure 3. PTEN silencing cooperates with BRAF^V600E for anaplastic thyroid cancer progression.

A: Kaplan-Meier survival curves comparing survival of tamoxifen treated: Thyro::CreER; BRaf^CA/+; Pten^+/+ (BRAF^V600) Thyro::CreER; BRaf^CA/+; Pten^lox/+ (BRAF^V600E PTEN^het) or Thyro::CreER; BRaf^CA/+; Pten^lox/lox (BRAF^V600E PTEN^null) mice after tumor initiation (**: P≤0.01; Log-Rank Test).

B: Thyroid tumor burden measured by ultrasonography (*:P≤0.05; t-Test) 1.5 months after tumor induction.

C: H&E staining of histological section of thyroid tumors observed in Thyro::CreER; BRaf^CA/+; Pten^+/+ (BRAF^V600E) at 40× (Up) and 200× (Low) magnification.

D: H&E staining of histological section of thyroid tumors observed in Thyro::CreER; BRaf^CA/+; Pten^lox/lox mice (BRAF^V600E PTEN^null) displaying areas of PTC (Left), tracheal airway invasion (Middle) or ATC area (Right) at 40× (Upper row) and 200× (Lower row) magnification.

At the histological level, BRAF^V600E/PTEN^null thyroid tumors displayed similar characteristics as BRAF^V600E/PIK3CA^H1047R expressing thyroid tumors. While BRAF^V600E/PTEN^WT thyroids presented expected PTC features after 9.5 months (Fig. 3C), BRAF^V600E/PTEN^Null thyroid tumors displayed a clear PTC phenotype as early as 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) of BRAF^V600E/PTEN^null tumors at time points 3.5 - 8 months after tamoxifen induction.

To explore biochemical collaboration between BRAF^V600E and PI3’-kinase signaling in more mechanistic detail we employed two human BRAF mutated ATC cell lines: 8505c and Ocut2 [14]. Whereas Ocut2 expresses both BRAF^V600E and PIK3CA^H1047R, 8505c is BRAF^V600E/TP53^null. Cells were treated with inhibitors of either MEK1/2 (PD325901) [15] or class 1 PI3’-kinases (GDC-0941) [16] either alone or in combination with effects on cell proliferation assessed using Crystal Violet staining (Fig. 4A). Although both PD325901 and GDC-0941 displayed anti proliferative effects, in general, MEK1/2 inhibition had a more potent inhibitory effect on cell proliferation than inhibition of class 1 PI3’-kinases. Moreover, combined blockade of both pathways had a more striking effect on cell proliferation compared to the anti-proliferative activity of the single-agents.

Figure 4. BRAF^V600E and PI3’-kinase signaling cooperate for human thyroid cancer cell proliferation and regulation of phospho-rpS6 and 4E-BP1.

A: 8505c (BRAF^V600E/PIK3CA^WT) and Ocut2 (BRAF^V600E/PIK3CA^H1047R) human anaplastic thyroid cancer cells were treated with an inhibitor of MEK1/2 (PD325901, 1μM, PD) or class 1 PI3’-kinases (GDC-0941, 2.5μM, GDC) either alone or in combination for 5 days at which time the cells were fixed and stained with Crystal Violet.

B&C: 8505c (BRAF^V600E/PIK3CA^WT) and Ocut2 (BRAF^V600E/PIK3CA^H1047R) human ATC cells were treated with an inhibitor of MEK1/2 (PD325901, 1μM, PD) or class 1 PI3’-kinases (GDC-0941, 2.5μM GDC) either alone or in combination for either 4 (B) or 24 (C) hours at which time the cells were lysed and protein expression/phosphorylation assessed by immunoblotting as indicated.

In parallel with the cell proliferation assays, cells were treated with PD325901 or GDC-0941, either alone or in combination, for 4 or 24 hours at which time cell extracts were analyzed by immunoblotting (Fig. 4B). At both time points, pathway blockade was highly selective in that PD325901 inhibited pERK1/2 with no effect on pAKT and GDC-0941 inhibited pAKT with no effect on pERK1/2. Since these pathways are reported to coordinately regulate the machinery that regulates the initiation of protein synthesis, 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 appeared more sensitive to PI3’-kinase inhibition at 4 or 24 hours in Ocut2 cells compared to 8505c cells, which is likely a reflection of the fact that Ocut2 cells express PIK3CA^H1047R. Moreover, p-RPS6 and p-4E-BP1 were sensitive to MEK1/2 inhibition in both cell types but this was not observed until 24 hours after inhibitor addition. However, in Ocut2 cells we detected cooperative effects of combined MEK1/2 plus PI3’-kinase inhibition on p-RPS6 at 4 and 24 hours after inhibitor addition. This was also true for p-4E-BP1 24 hours after inhibitor addition but not at four hours. We also detected evidence of cooperative inhibitory effects on p-RPS6 and p-4E-BP1 in 8505c cells 24 hours after inhibitor addition, but the effects were not as striking as those observed in Ocut2 cells. Overall, these data suggest that BRAF^V600E and PI3’-kinase signaling cooperate for sustained thyroid cancer cell proliferation and that mutational activation of PIK3CA may predict for more potent anti-proliferative and signaling effects following inhibition of class 1 PI3’-kinases.

Discussion

Anaplastic thyroid carcinoma is a difficult disease to treat because it is generally inoperable and resistant to current regimens of chemotherapy. Although there are provocative hints from small numbers of patients from the phase 1 vemurafenib clinical trial, it remains to be unequivocally demonstrated that BRAF mutational status is prognostic for therapeutic benefit from BRAF^V600E inhibitors. However, the potential clinical benefit of vemurafenib might be influenced by altered signaling through ERBB receptors for EGF ligands as demonstrated by others [18]. Furthermore, it was recently demonstrated that blockade of BRAF^V600E signaling with a MEK1/2 inhibitor (AZD6244) can restore the expression of the sodium-iodide symporter (NIS) and thereby enhance the efficacy of radioiodide therapy in both GEM models and in patients [19] [20]. These data therefore suggest that combined pathway-targeted and conventional radiotherapy may be a successful strategy for treating late-stage thyroid cancer.

There are a large number of inhibitors of BRAF^V600E and PI3’-kinase signaling being developed for the treatment of a wide range of human cancers. However, it remains unclear whether mutational activation of BRAF, PIK3CA or silencing of PTEN will serve as strong prognostic biomarkers for thyroid cancer patient responses to pathway-targeted therapy, in part because of effects of these agents on intracellular feedback signaling [18]. Indeed, although BRAF mutation is prognostic of response in melanoma, that is not true for patients with colorectal cancer [21] [22]. The availability of GEM models of a wide range of thyroid cancer types, including the new model of ATC based on a foundation of mutational activation of BRAF plus either mutation of PIK3CA or silencing of PTEN described here, will allow pre-clinical modeling of combination pathway-targeted, conventional or immunomodulatory cancer therapy. It was notable that expression of mutationally activated PIK3CA^H1047R in thyrocytes was largely without effect in the mouse but that it cooperated strongly with mutationally activated BRAF^V600E. This is not without precedent since PIK3CA^H1047R expression fails to initiate tumorigenesis in melanocytes and in the lung and pancreatic epithelium [23-25]. By contrast, mutationally activated BRAF^V600E is sufficient to initiate tumorigenesis in all of the above target tissues. Hence, we propose that mutationally activated BRAF^V600E is a potent initiator of tumorigenesis that is also frequently required for tumor maintenance. By contrast, mutationally activated PIK3CA^H1047R is a weak initiator of tumorigenesis but greatly promotes tumorigenesis initiated by other oncogenic events [25]. This may explain why, to date, pathway-targeted inhibition of PI3’-kinase signaling has not proven successful in patients, even those whose tumors are silenced for PTEN or mutated for PIK3CA.

TRP53 is also frequently mutated in anaplastic thyroid carcinoma (50% of the cases). Indeed, separately, McFadden and colleagues have demonstrated that combined expression of BRAF^V600E with silencing of TP53 also elicits anaplastic thyroid cancer [26]. It has also been showed that the combination of TRP53 and PTEN knockouts could lead to ATC in another mouse model [27]. This model has a similar latency to the model described here even if it using an embryonic onset of the cre-recombinase (TPO-Cre). This shows that even if cannot exclude p53 alteration in the BRAF^V600E PIK3CA^H1047R model, subsequent p53 alteration resulting in ATC progression is very unlikely.

The cooperation observed between BRAF^V600E and PI3’-kinase signaling in GEM models was recapitulated, at least in part, by analysis of bona fide human ATC derived cell lines. It was clear that combined inhibition of both BRAF^V600E and PI3’-kinase signaling had more potent anti-proliferative effects that single agent inhibition. Moreover, regulation of key nodes in protein translation, most notably the phosphorylation of 4E-BP1 was clearly under the dual control of both pathways suggesting that efficient Cap-dependent protein synthesis requires inputs from at least two pathways in ATC-derived cell lines. Although there was a suggestion that the PIK3CA^H1047R expressing Ocut2 cells were more sensitive to PI3’-kinase, an extended analysis of a larger panel of thyroid cancer cell lines will be required to confirm if there is a genotype-drug response phenotype in this disease.

In conclusion, we describe a new mouse model of anaplastic thyroid cancer that recapitulates key features of the genetics and pathobiology of the cognate human disease. We propose that this GEM model will be useful for future pre-clinical studies and to understand the mechanisms by which these pathways cooperate to promote progression of thyroid cancer from an indolent to a lethal disease

Implications.

This genetically relevant mouse model of ATC will be an invaluable platform for pre-clinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma.