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Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells.

ABSTRACT: BACKGROUND:Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. METHODS:qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. RESULTS:Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. CONCLUSIONS:Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

SUBMITTER: Sasagawa T

PROVIDER: S-EPMC7011436 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells.

Sasagawa Tadashi T Jinno-Oue Atsushi A Nagamatsu Takeshi T Morita Kazuki K Tsuruga Tetsushi T Mori-Uchino Mayuyo M Fujii Tomoyuki T Shibuya Masabumi M

BMC cancer 20200210 1

<h4>Background</h4>Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. ...[more]

PMID: 32041578

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Project description:BackgroundHuman placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases.MethodsThree choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2'-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry.ResultsIn the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2'-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots.ConclusionsOur findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

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Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells.

Publications

Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells.

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