Tyrosine Kinase Inhibitors as Potential Therapeutic Agents in the Treatment of Granulosa Cell Tumors of the Ovary.

OBJECTIVE: Granulosa cell tumors of the ovary (GCTs) represent a specific subset of malignant ovarian tumors, of which there are 2 distinct subtypes, the juvenile and the adult form. Aside from surgery, no reliable therapeutic options currently exist for patients with GCT. This study sought to investigate the potential role of small molecule tyrosine kinase inhibitors (TKIs) as novel therapeutics in the clinical management of GCT. MATERIALS AND METHODS: Using TKI with distinct but overlapping multitargeted specificities, cellular proliferation, viability, and apoptosis were evaluated in 2 human GCT-derived cell lines, COV434 and KGN. RESULTS: Sunitinib, which targets the imatinib-inhibited tyrosine kinases of VEGFR, KIT, PDGFR, and FLT-3, was without effect in COV434 and KGN cell lines. Sorafenib, which has a high affinity for RAF1 and BRAF, dose dependently inhibited cellular proliferation and viability in both cell lines at concentrations equivalent to that seen in other systems. A RAF1 kinase inhibitor was without effect, suggesting that sorafenib is acting via inhibition of BRAF, or that aberrant signaling originates upstream of BRAF in the MAPK pathway. In the presence of a selective Src family inhibitor (SU6656), cell proliferation and cell viability responses dissociated; that is, although SU6656 dose dependently inhibited cell viability, it had limited effect on proliferation and apoptosis. CONCLUSIONS: These findings implicate BRAF in the activated signaling responsible for the growth and viability of GCT and suggest that TKI already in clinical use may be a therapeutic option in the treatment of GCT.

TET2 is a component of the estrogen receptor complex and controls 5mC to 5hmC conversion at estrogen receptor cis-regulatory regions.

Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95-97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Adrenal gland tumorigenesis after gonadectomy in mice is a complex genetic trait driven by epistatic loci.

Postgonadectomy adrenocortical tumorigenesis is a strain-specific phenomenon in inbred mice, assumed to be caused by elevated LH secretion and subsequent ectopic LH receptor (LHR) overexpression in adrenal gland. However, the molecular mechanisms of this cascade of events remain unknown. In this study, we took advantage of the mouse strain dependency of the phenotype to unravel its genetic basis. Our results present the first genome-wide screening related to this pathology in two independent F2 and backcross populations generated between the neoplastic DBA/2J and the nonsusceptible C57BL/6J strains. Surprisingly, the postgonadectomy elevation of serum LH was followed by similar up-regulation of adrenal LHR expression in both parental strains and their crosses, irrespective of their tumor status, indicating that it is not the immediate cause of the tumorigenesis. Linkage analysis revealed one major significant locus for the tumorigenesis on chromosome 8, modulated by epistasis with another quantitative trait locus on chromosome 18. Weight gain, a secondary phenotype after gonadectomy, showed a significant but separate quantitative trait locus on chromosome 7. Altogether, postgonadectomy adrenocortical tumorigenesis in DBA/2J mice is a dominant trait that is not a direct consequence of adrenal LHR expression but is driven by a complex genetic architecture. Analysis of candidate genes in the tumorigenesis linkage region showed that Sfrp1 (secreted frizzled-related protein 1), a tumor suppressor gene, is differentially expressed in the neoplastic areas. These findings may have relevance to the human pathogenesis of macronodular adrenal hyperplasia and adrenocortical tumors in postmenopausal women and why some of them develop obesity.

Management of granulosa cell tumour of the ovary.

PURPOSE OF REVIEW: Granulosa cell tumours of the ovary are relatively uncommon malignant tumours. Although the majority of patients present with early-stage disease that is curable, approximately 80% of patients with advanced tumours die from recurrent disease. The purpose of this review is to provide the clinician with an update on recent research relevant to granulosa cell tumours. RECENT FINDINGS: The measurement of serum inhibin levels is useful in the diagnosis of granulosa cell tumours in postmenopausal women and for monitoring the disease following oophorectomy at any age. Surgery remains the most effective primary treatment, whereas hormonal therapy for patients with advanced stages or recurrent disease has resulted in varying outcomes. The presence of the gsp oncogene and high GATA-4 protein expression in juvenile granulosa cell tumours may provide new prognostic markers. In-vitro testing of a recently discovered tyrosine kinase inhibitor indicates that further developments in this area may provide a novel therapeutic target for the treatment of granulosa cell tumours. SUMMARY: Further investigation to determine the molecular changes that contribute to tumorigenesis in granulosa cells is required. Understanding the molecular pathogenesis of this disease will potentially provide novel targeted therapeutic options which will improve the survival rate of these patients.

Characterization of the inhibitor of kappaB kinase (IKK) complex in granulosa cell tumors of the ovary and granulosa cell tumor-derived cell lines.

Granulosa cell tumors of the ovary (GCT) are a distinct, hormonally active subset of ovarian cancers. Although it has recently been shown that ∼97 % of all adult GCT harbor a novel somatic missense mutation in the FOXL2 gene, given its almost universal presence, it does not explain differences in tumor stage and/or recurrence. The nuclear factor kappaB (NFκB) transcription factor is constitutively active in two human GCT-derived cell lines, COV434 and KGN, which are useful in vitro models to investigate juvenile and adult GCT, respectively. This study aimed to determine the molecular basis and pathogenetic significance of this aberrant NFκB activity. Selective chemical inhibitors were used to target candidate components of the pathway. The constitutive activity was blocked by two independent inhibitors of IκBα phosphorylation, suggesting that aberrant activation occurs upstream of this point. NFκB inhibition resulted in a dose-dependent decrease in cell proliferation and viability and a dose-dependent increase in apoptosis. Inhibitors of earlier components of the pathway were without effect. Two independent inhibitors of inhibitor of kappaB kinase (IKK)ß, a catalytic subunit of the NFκB activation complex, were unable to inhibit the constitutive activity, but surprisingly also ligand-induced activity. These findings suggest a central role for IKKß; however, no mutations or altered expression of the IKKß, IKKα, or IKKγ genes was observed in the cell lines or in a panel of human GCT samples. This study highlights unresolved issues in understanding the pathogenesis of GCT and in the use of the COV434 and KGN cells lines as model systems.

Molecular pathogenesis of granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary (GCT) comprise a distinct subset of ovarian cancers that account for approximately 5% of all ovarian malignancies. They are thought to arise from normal proliferating granulosa cells of the late preovulatory follicle and exhibit many morphological and biochemical features of these cells. GCT are distinct from other ovarian carcinomas in their hormonal activity; their ability to secrete estrogen, inhibin, and Müllerian inhibiting substance accounts for some of the clinical manifestations of the disease and also provides useful tumor markers for disease surveillance. Although considered to be of low malignant potential, GCT are commonly associated with slow, indolent disease progression, and frequent yet long delays to tumor recurrence are characteristic of this disease. Unlike the more intensely investigated epithelial ovarian tumors, relatively little is known about the molecular and genetic changes that give rise to GCT. To date, many investigations have centered around pathways known to be involved in normal granulosa cell proliferation, including those activated by FSH receptor stimulation. Most recently, the finding that approximately 97% of adult GCT harbor a somatic missense mutation in the FOXL2 gene (c.402C→G; p.C134W) represents an exciting advancement in the field of GCT research. The high frequency with which the mutation occurs in adult GCT, along with its absence from juvenile GCT and other human malignancies is suggestive of an oncogenic or gain-of-function mutation and, indeed, that the mutation is pathognomonic for adult GCT. In this review, we explore the implications of this finding and the most recent work characterizing molecular pathways of potential pathogenetic significance in GCT.

Nuclear receptor profiling of ovarian granulosa cell tumors.

Granulosa cell tumors of the ovary (GCT) represent ~5% of malignant ovarian tumors. The adult form is defined by a mutation in the FOXL2 gene. GCT exhibit many of the features of normal proliferating granulosa cells. We have profiled the expression of the 48 human nuclear receptors (NR) by quantitative RT-PCR in a panel of GCT and in two GCT-derived cell lines, COV434 and KGN. The highest level of expression is seen for COUP-TF2 with abundant expression of PPARγ, SF-1, and TR-α. Estrogen receptor (ER)-ß is the most abundant of the steroid receptors with relatively high expression also of AR, ER-α, and PR. The concordance of expression for each NR across the tumors is remarkably high with same discordance between the cell lines and the tumors, particularly the COV434 line. No significant differences were observed with respect to tumor stage for NR expression. These findings provide a full profile of NR expression in GCT which will enable full characterization of their roles and potential as therapeutic targets.

Expression status and mutational analysis of the ras and B-raf genes in ovarian granulosa cell and epithelial tumors.

OBJECTIVES: The molecular pathogenesis of granulosa cell tumors of the ovary (GCT) is not understood. Activating mutations in the K-, N-, and H-ras protooncogenes have been identified in a wide range of human cancers, including ovarian epithelial tumors. Furthermore, an apparent association has recently been reported between the presence of ras and B-raf mutations in the same cancer types. Activation of the ras/raf pathway would be predicted to be tumorigenic in granulosa cells. METHODS: Gene expression patterns of the three ras and B-raf genes were determined in a panel of GCT and ovarian epithelial tumors, and in normal premenopausal ovaries. Expression was determined by RT-PCR using gene-specific primers combined with Southern blot analysis of the PCR products using gene-specific (32)P-labeled probes. Direct sequence analysis was used to screen for known activating mutations. RESULTS: Widespread expression of the four genes was observed in all tumor types examined. Compared to the normal ovaries, none of the genes was expressed at significantly higher levels in any of the tumor types examined. A heterozygous point mutation in codon 12 of the K-ras gene was found in five of the 10 mucinous tumors. No B-raf mutations were detected in the mucinous tumors. No mutations were detected in any of the genes in the cohort of GCT. CONCLUSION: These results suggest that neither overexpression nor activating mutations of the ras or B-raf genes are associated with the development of GCT.