GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

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Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

International Journal of Molecular Sciences ◽

10.3390/ijms22042047 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2047

Author(s):

Nina Schmid ◽

Kim-Gwendolyn Dietrich ◽

Ignasi Forne ◽

Alexander Burges ◽

Magdalena Szymanska ◽

...

Keyword(s):

Cell Proliferation ◽

Granulosa Cell ◽

Granulosa Cells ◽

Drug Targets ◽

Growth Regulation ◽

Sirtuin 1 ◽

Ki 67 ◽

Granulosa Cell Tumors ◽

Novel Drug

Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1–7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.

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Ovarian granulosa cell tumors: The role of GATA4 in predicting tumor behavior

Gynecologic Oncology ◽

10.1016/j.ygyno.2016.04.458 ◽

2016 ◽

Vol 141 ◽

pp. 176-177

Author(s):

E. Saks ◽

J. Nakayama ◽

Y. Mohiuddin ◽

A. Mills ◽

L. Duska

Keyword(s):

Granulosa Cell ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell

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Role of adjuvant chemotherapy in the management of stage IC ovarian granulosa cell tumors

Gynecologic Oncology Reports ◽

10.1016/j.gore.2019.04.004 ◽

2019 ◽

Vol 28 ◽

pp. 145-148

Author(s):

Dimitrios Nasioudis ◽

Emily M. Ko ◽

Ashley F. Haggerty ◽

Robert L. Giuntoli ◽

Robert A. Burger ◽

...

Keyword(s):

Adjuvant Chemotherapy ◽

Granulosa Cell ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell

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GATA4 protects granulosa cell tumors from TRAIL-induced apoptosis

Endocrine Related Cancer ◽

10.1677/erc-10-0041 ◽

2010 ◽

Vol 17 (3) ◽

pp. 709-717 ◽

Cited By ~ 24

Author(s):

Antti Kyrönlahti ◽

Marjut Kauppinen ◽

Essi Lind ◽

Leila Unkila-Kallio ◽

Ralf Butzow ◽

...

Keyword(s):

Transcription Factor ◽

Granulosa Cell ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Granulosa Cell Tumors ◽

Trail Receptors ◽

Apoptotic Effect ◽

Induced Apoptosis

Disturbances in granulosa cell apoptosis have been implicated in the pathogenesis of human granulosa cell tumors (GCTs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cytokine that induces apoptosis in a variety of malignancies without toxic effects on benign cells. The aim of this study was to investigate the expression and functionality of the TRAIL receptors DR4 and DR5 in human GCTs. Additionally, we examined the role of GATA4, a transcription factor expressed in normal and malignant granulosa cells, in TRAIL-induced GCT apoptosis. For this purpose, a tissue microarray of 80 primary and 12 recurrent GCTs was subjected to immunohistochemistry for DR4 and DR5, and freshly isolated primary GCT cultures were utilized to evaluate the functional effects of TRAIL on GCT cells. To clarify the role of GATA4 in the regulation of TRAIL-induced apoptosis, a human GCT-derived cell line (KGN) was transduced with lentiviral vectors expressing small hairpin RNAs targeting GATA4 or transfected with adenovirus expressing either wild-type or dominant negative mutant GATA4. We found that receptors DR4 and DR5 are expressed in a vast majority of GCTs as well as in primary GCT cultures, and that TRAIL induces apoptosis in the primary GCT cultures. Moreover, we showed that overexpressing GATA4 protects GCTs from TRAIL-induced apoptosis in vitro, whereas disrupting GATA4 function induces apoptosis and potentiates the apoptotic effect of TRAIL administration. Our results demonstrate that the TRAIL pathway is functional in GCT cells, and suggest that transcription factor GATA4 may function as a survival factor in this ovarian malignancy.

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Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 are highly expressed in ovarian granulosa cell tumors

Acta Endocrinologica ◽

10.1530/eje-10-0849 ◽

2011 ◽

Vol 164 (1) ◽

pp. 115-122 ◽

Cited By ~ 21

Author(s):

Anniina Färkkilä ◽

Mikko Anttonen ◽

Jurate Pociuviene ◽

Arto Leminen ◽

Ralf Butzow ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Granulosa Cell ◽

Biologically Active ◽

Treatment Modalities ◽

Vascular Endothelial ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell ◽

Endothelial Growth Factor

ObjectiveOvarian granulosa cell tumors (GCTs) are hormonally active sex cord stromal tumors accounting for 3–5% of all ovarian cancers. These tumors are generally diagnosed at an early stage but there is a high risk of recurrence, associated with high mortality. Treatment of recurrent GCTs is difficult, and biologically targeted treatment modalities are lacking. GCTs are highly vascularized, and angiogenic factors most probably play a role in their pathology. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis, but in GCTs, the role of VEGF and its receptors VEGFR-1 (FLT1) and VEGFR-2 (KDR) remains largely unknown. Our objective is to study the expression of VEGF and its receptors in human GCTs.MethodsWe analyzed GCTs from 106 patients for the expressions of VEGF and its receptors utilizing tumor tissue microarray, tumor mRNA, and patient serum samples.ResultsWe found that VEGF and its main biologically active receptor VEGFR-2 were highly expressed in primary and recurrent GCTs, when compared with normal granulosa-lutein cells. The expression of VEGF correlated positively to tumor microvessel density and to VEGFR-2 expression at the protein (P<0.05) and mRNA (P<0.05) levels. In contrast to VEGFR-2, the expression of VEGFR-1 was weak. Tumor VEGF protein expression was not prognostic for recurrence, however, we found high levels of circulating VEGF in the serum of patients with primary GCT.ConclusionsThe results suggest an important role of VEGF and VEGFR-2 in GCT pathology and support the possibility of applying novel VEGF- or VEGFR-2-targeted treatments to patients with GCT.

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Expression Status and Mutational Analysis of the PTEN and P13K Subunit Genes in Ovarian Granulosa Cell Tumors

International Journal of Gynecological Cancer ◽

10.1111/igc.0b013e3181a1cdfd ◽

2009 ◽

Vol 19 (3) ◽

pp. 339-342 ◽

Cited By ~ 15

Author(s):

Sophie Bittinger ◽

Maria Alexiadis ◽

Peter J. Fuller

Keyword(s):

Sequence Analysis ◽

Granulosa Cell ◽

Granulosa Cells ◽

Mutational Analysis ◽

Regulatory Subunit ◽

Missense Mutations ◽

Altered Expression ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell ◽

Pathway Activation

Granulosa cell tumors (GCT) are a unique subset of ovarian tumors which have a molecular phenotype resembling that of follicle stimulating hormone (FSH)-stimulated pre-ovulatory granulosa cells. FSH acts via its receptor to stimulate signaling pathways including the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Activation of this pathway occurs in solid tumors, including ovarian epithelial tumors, through mutation of the PI3K subunit genes or inactivation of the tumor suppressor, PTEN. Activation of this pathway would be predicted to be tumorigenic in granulosa cells.Expression of the 2 PI3K subunit genes, PIK3CA, which encodes the catalytic subunit, and PIK3R1, which encodes the regulatory subunit, together with the PTEN gene was determined in a panel of GCT, 2 human GCT-derived cell lines, COV434 and KGN, and normal ovary. Direct sequence analysis was used to screen for mutations. Expression of all 3genes was observed in the GCT without evidence of overexpression for the PI3K subunit genes or loss of expression for PTEN. Sequence analysis of amplicons spanning exons 9and 20, in which greater than 75% of mutations occur in the PIK3CA gene did not identify any missense mutations. Similarly, the previously reported deletions in exons 12 and 13 of the PIK3R1 were not found in the GCT. Three amplicons spanning the entire coding sequence of the PTEN gene were sequenced; neither deletions nor mutations were identified.These findings suggest that activation of PI3K signaling through PI3K/PTEN mutation or altered expression, in contrast to many other types of solid tumor, is not associated with GCT.

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8 Role of Expression of Estrogen Receptor β, Proliferating Cell Nuclear Antigen, and p53 in Ovarian Granulosa Cell Tumors

Molecular Genetics, Gastrointestinal Carcinoma, and Ovarian Carcinoma - Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas ◽

10.1016/s1874-5784(05)80094-7 ◽

2005 ◽

pp. 371-379

Author(s):

Stefania Staibano ◽

Gaetano De Rosa

Keyword(s):

Estrogen Receptor ◽

Granulosa Cell ◽

Proliferating Cell Nuclear Antigen ◽

Estrogen Receptor Β ◽

Nuclear Antigen ◽

Granulosa Cell Tumors ◽

Ovarian Granulosa Cell ◽

Proliferating Cell

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Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

Endocrinology ◽

10.1210/en.2008-1032 ◽

2009 ◽

Vol 150 (2) ◽

pp. 929-935 ◽

Cited By ~ 55

Author(s):

Pradeep P. Kayampilly ◽

K. M. J. Menon

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Protein Kinase ◽

Mrna Expression ◽

Granulosa Cell ◽

Granulosa Cells ◽

Ampk Activation ◽

Cyclin D2 ◽

Cell Cycle Inhibitor ◽

Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.

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The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽

10.1530/rep-10-0142 ◽

2010 ◽

Vol 140 (2) ◽

pp. 295-303 ◽

Cited By ~ 8

Author(s):

Jennifer L Juengel ◽

Lisa J Haydon ◽

Brigitta Mester ◽

Brian P Thomson ◽

Michael Beaumont ◽

...

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Cell Function ◽

Antral Follicle ◽

Brushtail Possum ◽

Follicular Growth ◽

Theca Cells ◽

Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

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