Granulosa Cell Tumor

BackgroundA somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers.Methodology/principal findingsHerein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative.Conclusions/significanceIn addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.

Inhibin is a polypeptide hormone produced by the granulosa cells of the ovary, and is present in body fluids as dimers of various sizes each comprising an alpha- and beta-subunit. Free forms of the alpha-subunit also circulate, and the presently available radioimmunoassay (Monash assay) cannot distinguish these from biologically active dimeric inhibin. Recently we described a new two-site enzyme immunoassay able for the first time to measure the levels of dimeric inhibin throughout the human menstrual cycle. The sensitivity limit of this assay is 2 pg ml-1 in human serum with cross-reactivity against activin of 0.05%. The normal range of inhibin in post-menopausal women is < 5 pg ml-1, in pre-menopausal women 2-80 pg ml-1 (2-10 pg ml-1 in the follicular phase, 40-80 pg ml-1 in the luteal phase). This assay was used to determine inhibin levels in sera from 15 (five pre-menopausal and ten post-menopausal) patients with granulosa cell tumours of the ovary. It was raised in a pre-menopausal patient preoperatively (261 pg ml-1), in six post-menopausal patients (32, 43, 54, 66, 24 and 58 pg ml-1) and one pre-menopausal patient with recurrent tumour, (237 pg ml-1), all confirmed clinically. Inhibin was normal in six patients in remission. Oestradiol levels were normal in all patients. Serial levels of inhibin predicted recurrence before overt clinical relapse in two patients. In 29 patients with malignant epithelial ovarian tumours inhibin levels were modestly elevated in nine and normal in the rest. Three patients with endometrioid histology, two with undifferentiated tumours, three with mucinous adenocarcinoma and one with clear cell carcinoma had elevated inhibin levels. Functional inhibin is secreted by all granulosa cell tumours of the ovary studied and can be used as a tumour marker to determine response to therapy and predict recurrence and is superior to oestradiol. A more detailed analysis of the levels of inhibin, and its subunits in epithelial ovarian cancer is needed to identify the molecular forms of the immunoreactive material before optimised assays can be applied to this more common tumour.

BackgroundDespite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets.MethodsThe expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Total RNA was hybridised to Affymetrix U133 Plus 2 microarrays. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2.ResultsThe overexpression of wildtype and mutant FOXL2 in COV434, and the silencing of mutant FOXL2 expression in KGN, has shown that mutant FOXL2 is able to differentially regulate the expression of many genes, including two well known FOXL2 targets, StAR and CYP19A. We have shown that many of the genes regulated by mutant FOXL2 are clustered into functional annotations of cell death, proliferation, and tumourigenesis. Furthermore, TGF-β signalling was found to be enriched when using the gene annotation tools GATHER and GeneSetDB. This enrichment was still significant after performing a robust permutation analysis.ConclusionGiven that many of the transcriptional targets of mutant FOXL2 are known TGF-β signalling genes, we suggest that deregulation of this key antiproliferative pathway is one way mutant FOXL2 contributes to the pathogenesis of adult-type GCTs. We believe this pathway should be a target for future therapeutic interventions, if outcomes for women with GCTs are to improve.

BACKGROUND: The transient receptor potential vanilloid 2 (TRPV2) channel is known to have strong species-dependent activation modes and divers functions. Based on previous results, which showed expression by human ovarian granulosa cells, we studied human TRPV2 in human granulosa cell tumors (GCTs) and derived cells (KGN cells). GCTs are rare ovarian tumors, for which neither specific therapies, nor adequate markers are available. METHODS: We analyzed primary GCTs, including a panel of 63 GCT samples, and KGN cells. We performed immunohisto-/cytochemistry, RT-PCR, Western blotting and measurements of intracellular Ca2+ levels. We studied consequences of CRISPR/Cas9-based deletion of TRPV2 on cell proliferation, migration and macropinocytotic behavior, examined changes in steroid hormone production, and determined corresponding alterations of the proteome of these cells. RESULTS: We found that GCTs express TRPV2 to a large percentage (95%). To examine roles of TRPV2, we turned to the human GCT-derived KGN cell line. CRISPR/Cas9-based deletion of TRPV2 resulted in larger cell size, increases in proliferation, migration and macropinocytotic behavior, changes in steroid production, and corresponding alterations of the proteome of these cells. Deletion of TRPV2 also significantly reduced susceptibility to cell death, which was induced within hours by cannabidiol (CBD), a preferred ligand of TRPV2, in a concentration- and time-dependent manner. However, cell death was not completely abolished and the analysis of the TRPV2-interactome suggested the voltage-dependent anion channel 1 (VDAC1) as a further target for CBD. VDAC1, as part of a cascade involving formation and persistent opening of the mitochondrial permeability transition pore (mPTP), is linked to cell death. A blocker of mPTP formation, cyclosporin A, significantly decreased the vulnerability of KGN cells to CBD-induced cell death. TRPV2-depleted KGN cells treated with cyclosporin A became almost completely insensitive to the effects of CBD. CONCLUSIONS: The results reveal a role of TRPV2 in GCT cells. Thus, CBD causes cell death in KGN cells via direct TRPV2 activation and via interaction with VDAC1. Expression of TRPV2 may thus be a novel marker to distinguish subtypes of GCTs. Furthermore, TRPV2 represents a novel drug target.