Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice

Abstract

Context: Thyroid cancer is the most common form of endocrine cancer, and it is a disease whose incidence is rapidly rising. Well-differentiated epithelial thyroid cancer can be divided into papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although FTC is less common, patients with this condition have more frequent metastasis and a poorer prognosis than those with PTC.

Objective: The objective of this study was to characterize the molecular mechanisms contributing to the development and metastasis of FTC.

Design: We developed and characterized mice carrying thyroid-specific double knockout of the Prkar1a and Pten tumor suppressor genes and compared signaling alterations observed in the mouse FTC to the corresponding human tumors.

Setting: The study was conducted at an academic research laboratory. Human samples were obtained from academic hospitals.

Patients: Deidentified, formalin-fixed, paraffin-embedded (FFPE) samples were analyzed from 10 control thyroids, 30 PTC cases, five follicular variant PTC cases, and 10 FTC cases.

Interventions: There were no interventions.

Main outcome measures: Mouse and patient samples were analyzed for expression of activated cAMP response element binding protein, AKT, ERK, and mammalian target of rapamycin (mTOR). Murine FTCs were analyzed for differential gene expression to identify genes associated with metastatic progression.

Results: Double Prkar1a-Pten thyroid knockout mice develop FTC and recapitulate the histology and metastatic phenotype of the human disease. Analysis of signaling pathways in FTC showed that both human and mouse tumors exhibited strong activation of protein kinase A and mTOR. The development of metastatic disease was associated with the overexpression of genes required for cell movement.

Conclusions: These data imply that the protein kinase A and mTOR signaling cascades are important for the development of follicular thyroid carcinogenesis and may suggest new targets for therapeutic intervention. Mouse models paralleling the development of the stages of human FTC should provide important new tools for understanding the mechanisms of FTC development and progression and for evaluating new therapeutics.

Figure 1.

DRP-TpoKO tumors show consistent features of aggressive FTC and develop FTC-derived lung metastases. Macroscopic images of representative WT (A) and DRP-TpoKO (C) thyroids. Representative hematoxylin and eosin staining of high-magnification images of WT (B) and DRP-TpoKO (D) thyroids. Evidence of capsular (E, arrow) and vascular (F, arrow) invasion in DRP-TpoKO tumors. Representative photomicrographs of DRP-TpoKO follicular thyroid carcinoma lung metastases (arrows) stained with hematoxylin and eosin (F) and thyroglobulin (H). Scale bar (B, applies to D), 125 μm, (E, applies to F, G, and H), 500 μm.

Figure 2.

Activation of FTC-related pathways in DRP-TpoKO, R1a-TpoKO, and Pten-TpoKO tumors. Immunohistochemical staining of WT thyroids (A–E), Pten-TpoKO tumors (F–J), R1a-TpoKO tumors (K–O), and DRP-TpoKO tumors (P–T) for pCREB (A, F, K, and P), pAkt (B, G, L, and Q), pErk (C, H, M, and R), phospho-p70S6k (D, I, N, and S), and pmTOR (E, J, O, and T). Scale bar (A, applies to all), 500 μm.

Figure 3.

Activation of cancer pathways in human FTC. Immunohistochemical staining of human normal thyroid (A–D), PTC (E–H), FVPTC (I–L), and FTC (M–P) for pCREB (A, E, I, and M), pERK (B, F, J, and N), pAKT (C, G, K, and O), and phospho-p70S6K (D, H, L, and P). Scale bar (A, applies to all), 500 μm.

Figure 4.

Activation of PKA and mTOR occurs in the same cells in DRP-TpoKO tumors. Immunohistochemical staining of pCREB (A), pmTOR (B), and phospho-p70S6K (C) in DRP-TpoKO tumors is shown. IF staining of 4′,6′-diamino-2-phenylindole (DAPI; D), pCREB (E, red), pmTOR (F, green) in DRP-TpoKO tumors (G is the merged image of D–F). T, FTC; C, capsule. Arrow indicates capsular invasion. Scale bar (A, applies to B and C), 125 μm; (D, applies to E–G), 250 μm.

Figure 5.

Microarray data comparing Pten-, R1a-, and DRP-TpoKO tumors. Principal Component Analysis plot of all tumor microarray data (A) is shown. Circles group each of the tumor genotypes. Heat map of genes identified as having significant linear trend effect between the three data sets (B) is shown. Red bar denotes linear trend genes down-regulated in DRP-TpoKO tumors, yellow bar denotes those down-regulated in malignant tumors, green bar denotes those up-regulated in DRP-TpoKO tumors, and the blue bar denotes those up-regulated in malignant tumors. DRP WT, R1a WT, and Pten WT denote the WT littermates used for comparison for each model.