Elevated levels of the steroidogenic factor 1 are associated with over-expression of CYP19 in an oestrogen-producing testicular Leydig cell tumour
ABSTRACT Testicular Leydig cell tumours (LCTs) are rare, steroid-secreting tumours. Elevated levels of aromatase (CYP19 or CYP19A1) mRNA have been previously described in LCTs; however, little is known about the mechanism(s) causing CYP19 over-expression. We report an LCT in a 29-year-old male with elevated plasma oestradiol caused by enhanced CYP19 transcription.
First, we measured the intra-tumour expression of CYP19 and determined the use of CYP19 promoters by qPCR. Secondly, we explored CYP19 and promoter II (PII) for gene amplifications and activating mutations in PII by sequencing. Thirdly, we analysed intra-tumour expression of steroidogenic factor 1 (SF-1 (NR5A1)), liver receptor homologue-1 (LRH-1 (NR5A2)) and cyclooxygenase-2 (COX2 (PTGS2)). Finally, we analysed SF-1 for promoter mutations and gene amplifications.
Similar to what has been recorded in normal Leydig cells, we first found the bulk of tumour CYP19 transcripts to be PII derived, excluding promoter shift as a cause of enhanced transcription. Secondly, we excluded CYP19 and PII gene amplifications, and activating mutations in PII, as causes of elevated CYP19 mRNA. We found SF-1 mRNA to be up-regulated in the tumour, while LRH-1 and COX2 were down-regulated. The finding of elevated SF-1 levels in the tumour was confirmed by immunohistochemistry. The elevated level of SF-1 was not due to promoter mutations or amplifications of the SF-1 gene.
Our results strongly suggest that the elevated levels of SF-1 have induced PII-regulated CYP19 transcription in this tumour. These findings are of relevance to the understanding of CYP19 up-regulation in general, which may occur in several tissues, including breast cancer.
SourceAvailable from: Per Eystein Lønning[Show abstract] [Hide abstract]
ABSTRACT: Breast cancers reveal elevated E2 levels compared to plasma and normal breast tissue. Previously, we reported intra-tumour E2 to be negatively correlated to transcription levels of 17β-HSD2 but positively correlated to 17β-HSD7. Here, we explored these mechanisms further by analysing the same breast tumours for 17β-HSD2 and -7 SNPs, as well as 17β-HSD7 gene copy number. Among the SNPs detected, we found 17β-HSD2 rs4445895_T to be associated with lower intra-tumour mRNA (p = 0.039) and an elevated intra-tumour E2 level (p = 0.006). In contrast, we found the 17β-HSD7 rs1704754_C allele to be associated with elevated mRNA (p = 0.050) but not to E2 levels in breast tumour tissue. Surprisingly, 17β-HSD7–gene copy number was elevated in 19 out of 46 breast tumours examined. Elevated copy number was associated with an increased mRNA expression level (p = 0.013) and elevated tumour E2 (p = 0.025). Interestingly, elevated 17β-HSD7–gene copy number was associated with increased expression not only of 17β-HSD7, but the 17β-HSD7_II pseudogene as well (p = 0.019). Expression level of 17β-HSD7 and its pseudogene was significantly correlated both in tumour tissue (rs = 0.457, p = 0.001) and in normal tissue (rs = 0.453, p = 0.002). While in vitro transfection experiments revealed no direct impact of 17β-HSD7 expression on pseudogene level, the fact that 17β-HSD7 and 17β-HSD7_II share a 95.6% sequence identity suggests the two transcripts may be subject to common regulatory mechanisms. In conclusion, genetic variants of 17β-HSD2 and 17β-HSD7 may affect intra-tumour gene expression as well as breast cancer E2 levels in postmenopausal women.The Journal of steroid biochemistry and molecular biology 09/2014; 143. DOI:10.1016/j.jsbmb.2014.02.003 · 4.05 Impact Factor
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ABSTRACT: Leydig cell tumors are the most common tumors of the gonadal stroma and represent about 3% of all testicular neoplasms. In most cases, Leydig cell tumors are benign, however, if the tumor is malignant, no effective treatments are currently available.We have recently reported that Farnesoid X Receptor (FXR) is expressed in R2C Leydig tumor cells, and it reduces the estrogen-dependent cell proliferation by negatively regulating aromatase expression. Here, we demonstrated that treatment with GW4064, a specific FXR agonist, markedly reduced Leydig tumor growth in vivo by inhibiting proliferation and inducing apoptosis. Indeed, the tumors from GW4064-treated mice exhibited a decrease in the expression of the proliferation marker Ki-67 and aromatase along with an increase in the apoptotic nuclei. FXR activation induced an enhanced PARP cleavage, a marked DNA fragmentation and a strong increase in TUNEL-positive R2C cells also in vitro. Moreover, in both in vivoand in vitromodels, FXR ligands up-regulated mRNA and protein levels of p53 and of its downstream effector p21(WAF1/Cip1.) . Functional experiments showed that FXR ligands up-regulated p53 promoter activity and this occured through an increased binding of FXR/NF-kB complex to the NF-kB site located within p53 promoter region as revealed by EMSA and ChIP analysis. Taken together, results from the current study show, for the first time, that treatment with FXR ligands induces Leydig tumor regression in vivo, suggesting that activation of FXR may represent a promising therapeutic strategy for Leydig cell tumors. © 2012 Wiley Periodicals, Inc.International Journal of Cancer 05/2013; 132(10). DOI:10.1002/ijc.27915 · 5.01 Impact Factor
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ABSTRACT: BACKGROUND: Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells. METHODOLOGYPRINCIPAL FINDINGS: Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-β1, and E2 treatment in turn restored the inhibition of TGF-β1 on GJIC. CONCLUSIONS: Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.PLoS ONE 03/2013; 8(3):e60197. DOI:10.1371/journal.pone.0060197 · 3.53 Impact Factor