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 histologic subtypes, 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 protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in 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 carcinoma.

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

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.

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.

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.

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.