Insulin-like growth factor--phosphatidylinositol 3 kinase signaling in canine cortisol-secreting adrenocortical tumors.

BACKGROUND: Hypercortisolism is a common endocrine disorder in dogs, caused by a cortisol-secreting adrenocortical tumor (AT) in approximately 15% of cases. In adrenocortical carcinomas of humans, activation of the phosphatidylinositol 3 kinase (PI3K) signaling pathway by insulin-like growth factor (IGF) signaling represents a promising therapeutic target. OBJECTIVES: To investigate the involvement of PI3K signaling in the pathogenesis of ATs in dogs and to identify pathway components that may hold promise as future therapeutic targets or as prognostic markers. ANIMALS: Analyses were performed on 36 canine cortisol-secreting ATs (11 adenomas and 25 carcinomas) and 15 normal adrenal glands of dogs. METHODS: mRNA expression analysis was performed for PI3K target genes, PI3K inhibitor phosphatase and tensin homolog (PTEN), IGFs, IGF receptors, IGF binding proteins and epidermal growth factor receptors. Mutation analysis was performed on genes encoding PTEN and PI3K catalytic subunit (PIK3CA). RESULTS: Target gene expression indicated PI3K activation in carcinomas, but not in adenomas. No amino acid-changing mutations were detected in PTEN or PIK3CA and no significant alterations in IGF-II or IGFR1 expression were detected. In carcinomas, ERBB2 expression tended to be higher than in normal adrenal glands, and higher expression of inhibitor of differentiation 1 and 2 (ID1 and ID2) was detected in carcinomas with recurrence within 2.5 years after adrenalectomy. CONCLUSIONS AND CLINICAL IMPORTANCE: Based on these results, ERBB2 might be a promising therapeutic target in ATs in dogs, whereas ID1 and 2 might be valuable as prognostic markers and therapeutic targets.

Upregulation of the PI3K/Akt pathway in the tumorigenesis of canine thyroid carcinoma.

BACKGROUND: Information on the genetic events leading to thyroid cancer in dogs is lacking. HYPOTHESIS/OBJECTIVES: Upregulation of the PI3K/Akt pathway has an important role in the tumorigenesis of thyroid carcinoma in dogs. ANIMALS: Fifty-nine dogs with thyroid carcinoma and 10 healthy controls. METHODS: Quantitative RT-PCR was performed for VEGFR-1, VEGFR-2, EGFR, PIK3CA, PIK3CB, PDPK1, PTEN, AKT1, AKT2, COX-2, and CALCA. Mutation analysis was performed for known hotspots of RAS (N, K, H), PIK3CA, BRAF, RET, and for the entire coding region of PTEN. RESULTS: Forty-three dogs (73%) had follicular cell thyroid carcinoma (FTC) and 16 dogs (27%) had medullary thyroid carcinoma (MTC). The relative mRNA expressions of VEGFR-1 (P < .001), VEGFR-2 (P = .002), PDPK1 (P < .001), AKT1 (P = .009), and AKT2 (P < .001) were increased in FTC, and those of EGFR (P < .001), VEGFR-1 (P = .036), and PIK3CA (P = .019) were increased in MTC when compared to normal thyroid glands. Mutation analysis of K-RAS identified 2 activating missense mutations, which also have been described in thyroid cancer of humans. A G12R substitution was present in 1 FTC and an E63K substitution was present in 1 MTC. No functional mutations were found in the sequenced regions of H-RAS, N-RAS, PIK3CA, BRAF, RET, and PTEN. CONCLUSIONS AND CLINICAL IMPORTANCE: The increased expression of several genes associated with PI3K/Akt signaling suggests the involvement of this pathway in the pathogenesis of thyroid carcinoma in dogs, warranting further research on pathway activation and gene amplification. The mutations most frequently associated with thyroid cancer in humans are rare in dogs.

Expression of steroidogenic factor 1 in canine cortisol-secreting adrenocortical tumors and normal adrenals.

We report on a screening for the relative messenger RNA (mRNA) and protein expression of steroidogenic factor 1 (SF-1) in normal canine adrenals (n = 10) and cortisol-secreting adrenocortical tumors (11 adenomas and 26 carcinomas). The relative mRNA expression of SF-1 was determined by quantitative real-time polymerase chain reaction analysis and revealed no differences between normal adrenals, adenomas, and carcinomas. Immunohistochemistry demonstrated SF-1 protein expression in a nuclear pattern throughout the normal adrenal cortex and a predominantly nuclear staining pattern in adrenocortical tumors. Of the 15 dogs available for follow up, 7 dogs developed hypercortisolism within 2.5 yr after adrenalectomy, with metastatic disease in 6 dogs and adrenocortical tumor regrowth in 1 dog. The relative SF-1 mRNA expression in dogs with early recurrence was greater (2.46-fold, P = 0.020) than in dogs in remission for at least 2.5 yr after adrenalectomy. In conclusion, we demonstrated the presence of SF-1 expression in normal canine adrenals and adrenocortical tumors. The high SF-1 mRNA expression in carcinomas with early recurrence might indicate its value as a prognostic marker, as well as its potential for therapeutic development.

Expression of angiogenesis-related genes in canine cortisol-secreting adrenocortical tumors.

The aim of this study was to evaluate the expression of angiogenesis-related genes in canine cortisol-secreting adrenocortical tumors (ATs). Quantitative RT-PCR analysis revealed mRNA encoding for vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, angiopoietin 1 and 2 (ANGPT1 and ANGPT2), the splice variant ANGPT2443, the ANGPT-receptor Tie2, and basic fibroblast growth factor in 38 canine cortisol-secreting ATs (26 carcinomas and 12 adenomas) and 15 normal adrenals. The relative expression of both ANGPT2 and ANGPT2443 was higher in adenomas (P = 0.020 for ANGPT2 and P = 0.002 for ANGPT2443) and carcinomas (P = 0.003 for ANGPT2 and P < 0.001 for ANGPT2443) compared with normal adrenals, and this enhanced expression was also detected with Western blot analysis. Immunohistochemistry indicated expression of ANGPT2 protein in AT cells and in vascular endothelial cells of carcinomas, whereas Tie2 was mainly present in the tumor vascular endothelial cells. The ANGPT2-to-ANGTPT1 ratio, a marker for a proangiogenic state, was higher in both adenomas (P = 0.020) and carcinomas (P = 0.043). With the use of the human H295R cortisol-producing adrenocortical carcinoma cell line, we were able to demonstrate that the ANGPT2 expression was stimulated by cyclic adenosine monophosphate and progesterone but not by cortisol. In conclusion, canine cortisol-secreting ATs have enhanced ANGPT2 expression with a concomitant shift toward a proangiogenic state. On the basis of this information, treatment modalities may be developed that interfere with ANGPT2 expression, including inhibition of the cyclic adenosine monophosphate/protein kinase A pathway, or of the effect of ANGPT2, by using specific ANGPT2 inhibitors.

Activating mutations of GNAS in canine cortisol-secreting adrenocortical tumors.

BACKGROUND: Cushing's syndrome or hypercortisolism is a common endocrinopathy in dogs. In approximately 15% of cases, the disorder is caused by adrenocorticotropin (ACTH)-independent hypersecretion of cortisol by an adrenocortical tumor (AT). Without other explanation, the cortisol hypersecretion has been referred to as autonomous. OBJECTIVES: To investigate whether ACTH-independent hypersecretion of cortisol may be associated with aberrant activation of the melanocortin 2 receptor (MC2R)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway. ANIMALS: All analyses were performed on 44 cortisol-secreting ATs (14 adenomas and 30 carcinomas) derived from dogs diagnosed with ACTH-independent hypercortisolism. METHODS: Mutation analysis was performed of genes encoding the stimulatory G protein alpha subunit (GNAS), MC2R, and PKA regulatory subunit 1A (PRKAR1A) in all ATs. RESULTS: Approximately one-third of all ATs harbored an activating mutation of GNAS. Missense mutations, known to result in constitutive activation, were present in codon 201 in 11 ATs, in codon 203 (1 AT), and in codon 227 (3 ATs). No functional mutations were found in MC2R and PRKAR1A. CONCLUSIONS AND CLINICAL IMPORTANCE: Activation of cAMP signaling is a frequent event in canine cortisol-secreting ATs and may play a crucial role in both ACTH-independent cortisol production and tumor formation. To the best of our knowledge, this is the first report of potentially causative mutations in canine cortisol-secreting ATs.

Expression of the ACTH receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors.

Studies of human adrenocortical tumors (ATs) causing Cushing's syndrome suggest that hypersecretion of cortisol is caused by altered expression of steroidogenic enzymes and that steroidogenesis can only be maintained when there is expression of the ACTH receptor (ACTH-R). Here we report the screening for the mRNA expression of the ACTH-R, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, 21-hydroxylase (all in 38 cortisol-secreting ATs), 17α-hydroxylase, and 11ß-hydroxylase (both in 28 cortisol-secreting ATs). Real-time PCR (RT-PCR) was applied in all samples and was compared with that in normal canine adrenal glands. Messenger-RNA encoding StAR, steroidogenic enzymes, and ACTH-R were present in both normal adrenal glands and cortisol-secreting ATs. The amounts of mRNA encoding StAR and enzymes of the steroidogenic cluster needed for cortisol production did not differ significantly between either adenomas or carcinomas and normal adrenal glands. The amount of mRNA encoding ACTH-R was significantly lower in carcinomas than in normal adrenal glands (P = 0.008). In conclusion, RT-PCR analysis revealed no overexpression of StAR and steroidogenic enzymes in canine cortisol-secreting ATs. Significant downregulation of ACTH-R in carcinomas might be associated with the malignant character of the AT.