Thyrotrophin receptor signaling dependence of Braf-induced thyroid tumor initiation in mice

Abstract

Mutations of BRAF are found in ∼45% of papillary thyroid cancers and are enriched in tumors with more aggressive properties. We developed mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E)/TPO-Cre) to explore the role of endogenous expression of this oncoprotein on tumor initiation and progression. In contrast to other Braf-induced mouse models of tumorigenesis (i.e., melanomas and lung), in which knock-in of Braf(V600E) induces mostly benign lesions, Braf-expressing thyrocytes become transformed and progress to invasive carcinomas with a very short latency, a process that is dampened by treatment with an allosteric MEK inhibitor. These mice also become profoundly hypothyroid due to deregulation of genes involved in thyroid hormone biosynthesis and consequently have high TSH levels. To determine whether TSH signaling cooperates with oncogenic Braf in this process, we first crossed LSL-Braf(V600E)/TPO-Cre with TshR knockout mice. Although oncogenic Braf was appropriately activated in thyroid follicular cells of these mice, they had a lower mitotic index and were not transformed. Thyroid-specific deletion of the Gsα gene in LSL-Braf(V600E)/TPO-Cre/Gnas-E1(fl/fl) mice also resulted in an attenuated cancer phenotype, indicating that the cooperation of TshR with oncogenic Braf is mediated in part by cAMP signaling. Once tumors were established in mice with wild-type TshR, suppression of TSH did not revert the phenotype. These data demonstrate the key role of TSH signaling in Braf-induced papillary thyroid cancer initiation and provide experimental support for recent observations in humans pointing to a strong association between TSH levels and thyroid cancer incidence.

Fig. 1.

Thyroid-specific activation of Braf leads to the development of thyroid cancer with short latency. (A) Gross anatomical images of wild-type and LSL-Braf^V600E/TPO-Cre thyroid glands from mice at 5 wk. (B) Thyroid weight is significantly increased in both male and female LSL-Braf^V600E/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-Braf^V600E/TPO-Cre mice at 5 wk. (C and D) Low (40×) and high (100×) magnification, respectively, of normal thyroid lobe of a WT mouse. Thyroid follicles are filled with colloid. Arrows points to thyroid lobe (Th) and tracheal cartilage (Tr). (E) Magnification (40×) of markedly enlarged thyroid lobe in a LSL-Braf^V600E/TPO-Cre mouse. Thyroid architecture is disrupted, and no colloid material is evident. (F) Papillary formations (P) and nuclear pseudoinclusion (N). (Magnification, 100×.) (G) Malignant follicle (T) invading into surrounding skeletal muscle (M). Desmoplasia (D) is evident, indicative of muscle invasion. (H) Tumor cell thrombus (T) surrounded by endothelial cells (E) hanging in the lumen of a blood vessel (V).

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-Braf^V600E/TPO-Cre (B) mice are significantly increased in size compared with WT (A). LSL-Braf^V600E/TPO-Cre mouse thyroid (B) shows a florid PTC, 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 of LSL-Braf^V600E/TPO-Cre/TshR-KO is comparatively larger than that of TshR-KO and has disrupted follicular architecture. The cells lack the characteristic nuclear features pathognomic of PTC. (E–H) Ki67 immunohistochemical (IHC) staining of representative thyroid lobes of the same animal groups. The mitotic index is markedly higher in the LSL-Braf^V600E/TPO-Cre thyroid (F) compared with WT (E). There are virtually no detectable Ki67-positive cells in the thyroid sections of TshR-KO (G) and LSL-Braf^V600E/TPO-Cre /TshR-KO (H) mice. (I) Thyroid weight of wild-type, TshR^−/+, TshR^−/−, LSL-Braf^V600E/TPO-Cre, LSL-Braf^V600E/TPO-Cre/TshR^−/+, and LSL-Braf^V600E/TPO-Cre/TshR^−/− mice at 3 wk. (J and K) Magnification (40× and 200×) of thyroid sections of LSL-Braf^V600E/TPO-Cre /TshR-KO mice at 9 wk of age. Note increase in size of thyroid in J compared with 3-wk-old animal (E). Magnification (200×) in K shows characteristic papillary structures lined by irregular nuclei; however, tumors were of lower grade and had no extrathyroidal invasion.

Fig. 3.

Loss of Gsα attenuates the phenotype of PTC induced by endogenous expression of Braf^V600E. (A) Thyroid weight of wild-type, Gnas-E1^fl/+/TPO-Cre, Gnas-E1^fl/fl/TPO-Cre, LSL-Braf^V600E/TPO-Cre, LSL-Braf^V600E/Gnas-E1^fl/+/TPO-Cre, and LSL-Braf^V600E/Gnas-E1^fl/fl/TPO-Cre mice at 3 wk. Bars represent mean ± SEM. (B–E) Representative H&E-stained sections of thyroid tissue at 40× and 200× magnification of LSL-Braf^V600E/TPO-Cre and Braf^V600E/TPO-Cre/Gnas-KO mice. (B) LSL-Braf^V600E/TPO-Cre tumors have dense cellularity, and the whole field is occupied by the carcinoma. (C) Thyroid section of LSL-Braf^V600E/Gnas-E1^fl/fl/TPO-Cre mouse shows areas with relatively preserved follicular structures (arrow) coexisting with a low-grade PTC. (Magnification, 40×.) (D) Areas of tall cell growth (arrow), characteristic of aggressive BRAF-positive PTCs in humans, are present only in the Gnas WT mice. (E) PTCs in LSL-Braf^V600E/Gnas-E1^fl/fl/TPO-Cre mice are composed of smaller papillae consisting of cuboidal cells (arrow).

Fig. 4.

Treatment with the Mek inhibitor PD0325901 inhibits Braf-induced PTC growth. LSL-Braf^V600E/TPO-Cre mice were treated with PD0325901 (25 mg·kg⁻¹·d⁻¹) or vehicle for 3 wk, beginning at 3 wk of age. (A) Representative H&E sections of thyroids treated with vehicle or PD0325901. (B) pERK IHC of PTCs of vehicle- or PD0325901-treated mice. Animals were euthanized 6 h after administration of the last treatment dose. (C) Thyroid volume measured by MRI in vehicle- or PD0325901-treated mice before and after 3 wk of therapy (P = 0.0002). (D) Proliferative index measured by percentage of Ki67-positive thyrocytes in vehicle- and PD0325901-treated mice. *P = 0.01