Deletion of Rap1b, but not Rap1a or Epac1, Reduces Protein Kinase A-Mediated Thyroid Cancer

Abstract

Background: Thyroid cancer is an emerging health problem in the United States and worldwide. With incidence rates of thyroid cancer rapidly rising, the need to develop new treatment options is becoming a priority, and understanding the molecular mechanisms of this disease is crucial to furthering these efforts. Thyroid growth is driven by the TSH/cAMP/PKA signaling pathway, and it has previously been shown that activation of PKA through genetic ablation of the regulatory subunit Prkar1a (Prkar1a KO) is sufficient to cause follicular thyroid cancer in mouse models. cAMP also activates the Epac proteins and their downstream effectors, Rap1a and Rap1b.

Methods: Previously, the authors' laboratory generated a mouse model of follicular thyroid cancer by conferring thyroid-specific deletion of Prkar1a (R1a-TpoKO). To probe the roles of other components of the PKA signaling system in the development of thyroid cancer, this study deleted Rap1 and Epac1 in the setting of the Prkar1a knockout.

Results: Deletion of Rap1 significantly decreases thyroid size and cancer incidence in Prkar1a KO thyroids. Further, isoform-specific ablation of Rap1a and Rap1b implicates Rap1b as the downstream effector of PKA during thyroid carcinogenesis. In vivo modeling provides definitive evidence that Epac1 plays little role in thyroid proliferation and is dispensable for thyroid carcinogenesis arising from the deletion of Prkar1a.

Conclusions: This study demonstrate that PKA signaling to Rap1b is a key signaling node for follicular thyroid carcinogenesis, while Epac1 activity is not required for tumor development. This work sheds new light on the pathways involved in FTC development and identifies a possible target for the development of new therapies in the treatment of FTC.

Keywords: Epac; PKA; PRKAR1A; Rap1; follicular thyroid cancer.

FIG. 1.

Thyroid-specific deletion of Rap1 in Tpo-R1aKO mice reduces thyroid size. (A) Western blot showing total Rap1 expression in wild-type (WT), Tpo-R1aKO, and Tpo-R1aRap1KO thyroids. Three-dimensional (3D) rendering of ultrasonographic images of (B) Tpo-R1aKO and (C) Tpo-R1aRap1KO thyroids at nine months of age. (D) Thyroid volumes determined by 3D ultrasonography at three-month intervals of Tpo-R1aKO (blue line; n = 18), Tpo-R1aRap1KO (red line; n = 30), and Cre-negative littermate control (black line; n = 25) mice. (E) Scatter plot showing the range of Tpo-R1aRap1KO thyroid volumes over time (p = 0.077, ns). (F–H) 10 × images of hematoxylin and eosin–stained thyroids from Tpo-R1aKO (G), Tpo-R1aRap1KO (H), and cre-negative littermate controls (F) at nine months of age. **p ≤ 0.001 at both six- and nine-month time points. Graphs show mean and standard error of the mean (SEM).

FIG. 2.

Decreases in thyroid size in Tpo-R1aRap1KO mice is mediated by Rap1b. (A) Thyroid volumes determined by ultrasonography of Tpo-R1aRap1AKO (red line; n = 25, 22, and 3), Cre-negative littermate control (black line; n = 15), and age-matched Tpo-R1aKO (blue line; n = 18) mice at indicated time points. (B) Thyroid volumes of Tpo-R1aRap1BKO (red line; n = 18, 17, and 9), Cre-negative littermate control (black line; n = 20), and age-matched Tpo-R1aKO (blue line; n = 18) mice at the indicated time points. (C and D) Scatter plots showing thyroid volumes of Tpo-R1aRap1AKO (C) (p = 0.0024) and Tpo-R1aRap1BKO (D) (p = 0.0021) mice. (E and F) 3D renderings of ultrasonographic images from Tpo-R1aRap1AKO (E) and Tpo-R1aRap1BKO (F) mouse thyroids. **p ≤ 0.001 at six and nine months of age. *p ≤ 0.05 at six and nine months of age. Graphs show mean and SEM.

FIG. 3.

Tpo-R1aRap1BKO mice have a lower incidence rate of thyroid carcinoma due to increases in apoptosis relative to Tpo-R1aRap1A mice. (A and B) 20 × images of hematoxylin and eosin–stained thyroid sections from Tpo-R1aRap1A (A) and Tpo-R1aRap1B (B) mice. (C) Percent incidence of thyroid carcinoma in Tpo-R1aKO (n = 11 at nine months of age), Tpo-R1aRap1KO (n = 18 at nine months of age), Rap isoform-specific Tpo-R1aRap1AKO (n = 15 at six to nine months of age), and Tpo-R1aRap1BKO (n = 18 at nine months of age) mice. *p ≤ 0.05 compared to Tpo-R1aKO; #p ≤ 0.05 compared to Tpo-R1aRap1AKO. (D) Quantification of cleaved caspase-3 staining represented as a percentage of the total area of the image (n = 8 mice per genotype). **p ≤ 0.001 relative to Tpo-R1aRap1AKO. (E and F) Diaminobenzidine immunohistochemistry for cleaved caspase-3 (black arrows) on thyroid tissue sections from Tpo-R1aRap1AKO (E) and Tpo-R1aRap1BKO (F) at 40 × magnification.

FIG. 4.

Epac1 is expressed in Tpo-R1aKO tumors. (A and B) Diaminobenzidine immunohistochemistry for Epac1 on thyroid tissue sections from WT (A) and Tpo-R1aKO (B) mice at 40 × magnification. (C) Western blot showing Epac1 and Epac2 expression in WT and Tpo-R1aKO mouse thyroids; Hela cells are shown as a positive control for Epac2 expression, FRTL5 cells are shown as a positive control for both Epac1 and Epac2 expression. (D) Quantitative polymerase chain reaction showing fold change in Epac2 gene expression in WT, Tpo-R1aKO, and Epac1^–/– thyroids; WT mouse brain tissue was used as a positive control.

FIG. 5.

Epac1^–/– mice have normal thyroids. (A) Hematoxylin and eosin staining of Epac1^–/– thyroid at 20 × magnification. (B) Comparison of thyroid volumes from age-matched WT (black line), Epac1^–/– (red line), and Tpo-R1aKO (blue line) mice measured by 3D ultrasound. (C) Body weights of Epac1^–/– (red line) and WT (blue line) mice at 6 and 12 months of age. (D) T4 hormone levels of WT and Epac1^–/– mice (p = 0.571). **p ≤ 0.001 at six and nine months of age Tpo-R1aKO versus Epac1KO or WT. *p ≤ 0.05 at both 6 and 12 months of age.

FIG. 6.

Epac1 is not required for tumor formation in TpoR1aKO mice. (A and B) 3D rendering of thyroids based on volumes determined by ultrasonography of Tpo-R1aKO (A) and TpoR1a-EpacKO (B) mice at nine months of age. (C) Thyroid volumes determined by ultrasound of TpoR1a-EpacKO (red line; n = 27), and littermate Tpo-R1aKO (blue line; n = 6) and cre-negative Prkar1a^loxP/loxP;Epac1^–/– control mice (black line; n = 23). (D) Scatter plot depicting thyroid volumes of TpoR1a-EpacKO mice at the indicated time points (p < 0.0001). *p ≤ 0.001. Graphs show mean and SEM.

FIG. 7.

Epac1 is not involved in thyrotropin (TSH)-mediated thyroid hyperplasia. (A) Serum TSH levels in untreated and methimazole-treated mice (p = 0.0002 WT treated vs. untreated; p = 0.0009 Epac^–/– treated vs. untreated; p = 0.415 WT treated vs. Epac^–/– treated). (B and C) Hematoxylin and eosin–stained thyroid sections from WT (B) and Epac1^–/– (C) mice after six months of continuous methimazole treatment. **p ≤ 0.001 compared to no treatment.