Mouse Prkar1a haploinsufficiency leads to an increase in tumors in the Trp53+/- or Rb1+/- backgrounds and chemically induced skin papillomas by dysregulation of the cell cycle and Wnt signaling

Abstract

PRKAR1A inactivation leads to dysregulated cAMP signaling and Carney complex (CNC) in humans, a syndrome associated with skin, endocrine and other tumors. The CNC phenotype is not easily explained by the ubiquitous cAMP signaling defect; furthermore, Prkar1a(+/-) mice did not develop skin and other CNC tumors. To identify whether a Prkar1a defect is truly a generic but weak tumorigenic signal that depends on tissue-specific or other factors, we investigated Prkar1a(+/-) mice when bred within the Rb1(+/-) or Trp53(+/-) backgrounds, or treated with a two-step skin carcinogenesis protocol. Prkar1a(+/-) Trp53(+/-) mice developed more sarcomas than Trp53(+/-) mice (P < 0.05) and Prkar1a(+/-) Rb1(+/-) mice grew more (and larger) pituitary and thyroid tumors than Rb1(+/-) mice. All mice with double heterozygosity had significantly reduced life-spans compared with their single-heterozygous counterparts. Prkar1a(+/-) mice also developed more papillomas than wild-type animals. A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt signaling as the main pathway activated by abnormal cAMP signaling, along with cell cycle abnormalities; all changes were confirmed by qRT-PCR array and immunohistochemistry. siRNA down-regulation of Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation and Prkar1a(+/-) mouse embryonic fibroblasts and arrested both cell lines at the G0/G1 phase of the cell cycle. In conclusion, Prkar1a haploinsufficiency is a relatively weak tumorigenic signal that can act synergistically with other tumor suppressor gene defects or chemicals to induce tumors, mostly through Wnt-signaling activation and cell cycle dysregulation, consistent with studies in human neoplasms carrying PRKAR1A defects.

Figure 1.

Prkar1a haploinsufficiency reduced survival in Trp53^+/− and RB1^+/− mice and increased papilloma development during a two-step skin carcinogenesis protocol. (A) Prkar1a^+/− Trp53^+/− mice exhibited significantly decreased survival relative to Trp53^+/− and Prkar1a^+/− mice (P < 0.0001). (B) A reduced survival was also observed in Prkar1a^+/− Rb1^+/− mice when compared with Rb1^+/− and Prkar1a^+/− mice (P= 0.01). (C) Prkar1a^+/− mice were more susceptible to papilloma formation than WT mice during a 20-week treatment protocol with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (P =0.004). *P-values were calculated by the log rank test.

Figure 2.

Prkar1a haploinsufficiency increased tumor development in different tissue-specific tumor models. (A) Gross pathology (hind leg and cranium) and histology (H&E staining 20x) of sarcomas found in Prkar1a^+/− Trp53^+/− mice. At 1 year, Prkar1a^+/− mice bred into the Trp53^+/− background developed more sarcomas than Trp53^+/− mice. (B) Pituitary tumors were more frequent and larger in Prkar1a^+/− Rb1^+/− mice than in Rb1^+/− mice. A single moderately sized adenoma of the adrenal cortex (arrow) was observed in a Prkar1a^+/− Rb1^+/− mouse (H&E staining 20x). (C) Thyroid tumors had also a larger size and were more frequently bilateral in Prkar1a^+/− Rb1^+/− mice than in Rb1^+/− mice. Thyroid tumors were positive for calcitonin staining in both Rb1^+/− and Prkar1a^+/− Rb1^+/− groups. (D) Prkar1a^+/− mice developed more papillomas than WT mice after the application of a skin carcinogenesis protocol. Papilloma histology (H&E staining 20x).

Figure 3.

Cell cycle pathway expression in sarcomas, papillomas, pituitary and thyroid tumors. (A) A qRT–PCR array that included 84 genes involved in the cell cycle demonstrated that key genes were over-expressed in pituitary tumors and sarcomas from double heterozygous mice, as well as in papillomas from Prkar1a^+/− mice. (B) Strong staining for E2f1 in Prkar1a^+/− papillomas and pituitary and thyroid tumors from Prkar1a^+/− Rb1^+/− mice. p16 staining was similar in pituitary tumors from Rb1^+/− and Prkar1a^+/− Rb1^+/− mice. (C) Cyclin D1 over-expression at protein level in Prkar1a^+/− papillomas, Prkar1a^+/− Trp53^+/− sarcomas and pituitary and thyroid tumors from Prkar1a^+/− Rb1^+/− mice⁻. (D) Downstream targets of PKA (Akt and c-fos) were over-expressed in double heterozygous tumors and in Prkar1a^+/− papillomas. *P < 0.05.

Figure 4.

Wnt pathway expression in sarcomas, papillomas, pituitary and thyroid tumors. (A) Wnt signaling pathway genes were over-expressed in Prkar1a^+/− sarcomas, pituitary and thyroid tumors, papillomas from all three experiments, as demonstrated by a qRT–PCR array study that included 84 genes from this pathway. (B) Strong staining for Ctnnb1 in Prkar1a^+/− papillomas, Prkar1a^+/− Trp53^+/− sarcomas and Prkar1a^+/− Rb1^+/− pituitary tumors. (C) Wnt3 immunoreactivity was higher in Prkar1a^+/− papillomas than in WT papillomas. (D) Lrp5 protein was over-expressed in Prkar1a^+/− papillomas and double heterozygous tumors. *P < 0.05.

Figure 5.

siRNA disruption of Wnt3, Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation (Carney cells) and Prkar1a^+/− MEFs. (A) The siRNA effects on the target genes were demonstrated by western blot in Carney cells and Prkar1a^+/− MEFs. (B) The treatment with siRNA for Wnt3, Ctnnb1, E2f1 and Cdk4 led to significant decrease in cell proliferation of both cell lines; intra-cellular localization of siRNA in Carney cells and MEFs after transfection with fluorescein-labeled siRNA. (C) siRNA disruption of Wnt3, Ctnnb1, E2f1 and Cdk4 arrested Carney cells and MEFs at the G0/G1 phase of the cell cycle. *P < 0.05.

Figure 6.

Cross-talk between PKA and the Wnt and cell cycle genes in Prkar1a^+/− tumors. Prkar1a haploinsufficiency is associated with an increase in the expression of Wnt3, Lrp5 and β-catenin leading to Wnt signaling activation. Additionally, R1α haploinsufficiency in human lymphocytes and mouse models causes an increase in total cAMP-stimulated kinase activity and enhances MAPK activity, which promotes activation of the transcription factors c-myc and c-fos. R1α deficiency also promotes activation of cyclin D1, Cdk4 and E2f1, which facilitates cell cycle progression. PKA, protein kinase A; PRKAR1A, 1-α regulatory (RIα) subunit of PKA.