Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation

Abstract

The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor A (ERA). Treatment with exogenous 17B-estradiol (E 2 ) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E 2 /ERA-dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP-responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I-dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1-dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors. [Cancer Res 2007;67(17):8368-77]

Inhibition of Cyclooxygenase-2 Down-regulates Aromatase Activity and Decreases Proliferation of Leydig Tumor Cells

“…Aromatase transcription in Leydig cells is mainly controlled by transcription factors pCREB and SF-1. In a previous study (4) we showed that SF-1 transcriptional regulation is controlled by a transduction pathway that does not involve PKA directly. This indicates that, even in the presence of very low levels of pCREB, as is the case after treating cells with H89, SF-1 can still drive aromatase transcription maintaining the gene expression.…”

“…The relative gene expression levels were normalized to a calibrator that was chosen to be the basal, untreated sample. Final results were expressed as n-fold differences in gene expression relative to 18 S rRNA and calibrator, calculated following the ⌬⌬C t method, as published previously (4).…”

“…COX-2 Silencing and COX-2 Inhibitor, NS398, Decrease pCREB and Aromatase Expression-We have previously shown that expression of aromatase, SF-1, and the phosphorylated form of CREB are elevated in R2C and in rat Leydig tumor cells (4). The altered activation of CREB, together with high SF-1 expression, may explain the increase in aromatase levels observed in tumor Leydig cells.…”

“…Transgenic mice overexpressing aromatase, the enzyme responsible for the conversion of androgens to estrogens (2), present enhancement of circulating 17␤-estradiol (E2) 3 levels, which have been associated with Leydig cell hyperplasia and tumors (3). These observations and our recent data (4,5) suggest that estrogens can elicit proliferative effects in human and rat tumor Leydig cells through an autocrine mechanism. Aromatase activity is regulated primarily at the level of gene expression and is present in testicular somatic cells and during the maturative phases of male germ cells (6).…”

“…Specific response elements appear to be involved in rat aromatase expression: a nuclear receptor half site binding SF-1/LRH-1 (8) and CRE-like sequences binding CREB/ATF protein family members (9,10). In a recent study, we have shown that Leydig cell tumors exhibit increased SF-1 expression and CREB phosphorylation, which is an indicator of elevated transcriptional activity (4). In the same study, we explained that Leydig tumor cells produce high levels of insulin-like growth factor I, which, through the activation of phosphatidylinositol 3-kinase and Akt pathways, leads to increased steroidogenic factor-1 (SF-1) transcription.…”