Dataset Information

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors.

ABSTRACT: 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.

SUBMITTER: Schmid N

PROVIDER: S-EPMC7923107 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors.

Schmid Nina N Dietrich Kim-Gwendolyn KG Forne Ignasi I Burges Alexander A Szymanska Magdalena M Meidan Rina R Mayr Doris D Mayerhofer Artur A

International journal of molecular sciences 20210219 4

Sirtuins (SIRTs) are NAD<sup>+</sup>-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 ...[more]

PMID: 33669567

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Project description:BackgroundHuman granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions.MethodsPrimary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann-Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test.ResultsIn primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation.ConclusionsThese findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

Project description:Adult-type granulosa cell tumor (aGCT) is a rare malignant ovarian sex cord-stromal tumor, harboring recurrent FOXL2 c.C402G/p.C134W hotspot mutations in 97% of cases. These tumors are considered to have a favorable prognosis, however aGCTs have a tendency for local spread and late recurrences, which are associated with poor survival rates. We sought to determine the genetic alterations associated with aGCT disease progression. We subjected primary non-recurrent aGCTs (n?=?7), primary aGCTs that subsequently recurred (n?=?9) and their matched recurrences (n?=?9), and aGCT recurrences without matched primary tumors (n?=?10) to targeted massively parallel sequencing of ?410 cancer-related genes. In addition, three primary non-recurrent aGCTs and nine aGCT recurrences were subjected to FOXL2 and TERT promoter Sanger sequencing analysis. All aGCTs harbored the FOXL2 C134W hotspot mutation. TERT promoter mutations were found to be significantly more frequent in recurrent (18/28, 64%) than primary aGCTs (5/19, 26%, p?=?0.017). In addition, mutations affecting TP53, MED12, and TET2 were restricted to aGCT recurrences. Pathway annotation of altered genes demonstrated that aGCT recurrences displayed an enrichment for genetic alterations affecting cell cycle pathway-related genes. Analysis of paired primary and recurrent aGCTs revealed that TERT promoter mutations were either present in both primary tumors and matched recurrences or were restricted to the recurrence and absent in the respective primary aGCT. Clonal composition analysis of these paired samples further revealed that aGCTs display intra-tumor genetic heterogeneity and harbor multiple clones at diagnosis and relapse. We observed that in a subset of cases, recurrences acquired additional genetic alterations not present in primary aGCTs, including TERT, MED12, and TP53 mutations and CDKN2A/B homozygous deletions. Albeit harboring relatively simple genomes, our data provide evidence to suggest that aGCTs are genetically heterogeneous tumors and that TERT promoter mutations and/or genetic alterations affecting other cell cycle-related genes may be associated with disease progression and recurrences.

Dataset Information

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors.

Publications

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors.

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