1A1A).). The vast majority of ACCs display overexpression of IGF2 gene transcripts, whereas the H19 selleck chemicals Tofacitinib [a micro-RNA negatively regulating IGF2 expression (16,17)] and CDKN1C (encoding the cell cycle dependent kinase inhibitor, p57kip2) genes are down-regulated, suggesting an imprinting defect or loss of heterozygosity of this chromosomal region, similar to that commonly observed in BWS. To validate these microarray results, quantitative RT-PCR was performed on RNA isolated from three randomly selected ACAs and three ACCs (Fig. 1B1B).). We found a greater than 60-fold increase of IGF2 transcripts in all three ACC samples when compared with IGF2 levels in ACA samples. Further analysis of active IGF signaling with these six human tumor samples was performed by immunoblotting for levels of total IGF-1R protein and phosphorylated AktSer473, a downstream mediator of active IGF signaling (Fig.

1C1C).). Expression of IGF-1R was observed in all six tissues, whereas two ACC samples possessed far greater levels of the receptor. Immunoblotting for phospho-AktSer473 suggested active IGF signaling in all three ACC samples and in only one ACA sample. To further validate the observation that IGF-mediated signaling was specifically increased in ACC compared with normal and adenomatous samples, tissue microarray slides containing 24 ACC, 22 ACA, and four normal adrenals were stained for phospho-IGF-1R and phospho-AktSer473 (Fig. 1D1D).). A marked increase in signal intensity of phospho-IGF-1R and phospho-AktSer473 was observed in ACC samples compared with ACA and normal adrenal tissue, represented here as a shift in the percentage of ACC samples with high intensity staining for these activated proteins.

In summary, molecular profiling of human adrenal tumors demonstrated overexpression of two critical components (IGF2 and IGF-1R) of the IGF signaling cascade and concomitant activation of the downstream effector, Akt. These results are consistent with the IGF pathway playing a critical role in ACC pathogenesis. Figure 1 Up-regulation of IGF2, overexpression of IGF-1R, and active IGF signaling in human ACCs in comparison with normal and adenoma tissues. A, Snapshot heat map of the 11p15.5 chromosomal region generated from Affymetrix U133A 2.0 Plus oligonucleotide array. …

Expression of IGF pathway members and downstream signaling in ACC cell lines To begin to determine the biologic relevance of IGF-1R in ACC, we first examined the endogenous expression profiles of IGF ligands, IGF-1R, and downstream effectors of IGF-mediated signaling in five ACC cell lines (Fig. 22).). We chose two mouse lines (Y1 and ST5) and three human Anacetrapib lines derived from invasive primary adrenocortical carcinomas (NCI-H295, SW13, and RL251). Using RNA purified from cells maintained in serum-free or serum-containing media, RT-PCR was performed to detect IGF1 and IGF2 gene expression.