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ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21

View Article: PubMed Central - PubMed

ABSTRACT

Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17)t(X;17)(p11;q25), resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1) as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow–derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated β-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP). These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

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ASPL-TFE3 induces cellular senescence in mesenchymal stem cells. (A) UE7T/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. (B) UE7T/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and cell numbers were then determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± SD. *P < .05. (C) UE7T/TR-Vec and UE7T/TR-AT cells were incubated with tetracycline for 0, 24, and 48 h and were then subjected to immunoblotting with the indicated antibodies. (D) SA-β-gal staining was performed in UE7T/TR-Vec and UE7T/TR-AT cells after tetracycline treatment for 0, 24, and 48 h. (E) Proportions of SA-β-gal-positive cells in cultures of UE7T/TR-Vec and UE7T/TR-AT cells were calculated after tetracycline treatment for 0, 24, and 48 h. Data are presented as mean ± SD of three independent experiments. **P < .01; ***P < .005.
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f0020: ASPL-TFE3 induces cellular senescence in mesenchymal stem cells. (A) UE7T/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. (B) UE7T/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and cell numbers were then determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± SD. *P < .05. (C) UE7T/TR-Vec and UE7T/TR-AT cells were incubated with tetracycline for 0, 24, and 48 h and were then subjected to immunoblotting with the indicated antibodies. (D) SA-β-gal staining was performed in UE7T/TR-Vec and UE7T/TR-AT cells after tetracycline treatment for 0, 24, and 48 h. (E) Proportions of SA-β-gal-positive cells in cultures of UE7T/TR-Vec and UE7T/TR-AT cells were calculated after tetracycline treatment for 0, 24, and 48 h. Data are presented as mean ± SD of three independent experiments. **P < .01; ***P < .005.

Mentions: Oncogene expression induces premature senescence termed oncogene-induced senescence (OIS) in certain cell types, including primary fibroblasts and progenitor/stem cells [27], [30]. Furthermore, recent studies have reported that the expression of sarcoma-associated fusion oncogenes in MSCs can lead to the development of tumors resembling sarcomas [31], [32], [33], [34], suggesting that MSCs are a potential model for analyzing the pathogenesis of sarcomas with specific fusion proteins. Therefore, we established a tetracycline-inducible system for ASPL-TFE3 in UE7T-13 human bone marrow-derived MSCs (UE7T/TR-AT cells) and confirmed the induction of ASPL-TFE3 expression by tetracycline addition using immunoblotting (Figure 4A). As shown in Figure 4B, the addition of tetracycline resulted in suppression of the proliferation of UE7T/TR-AT cells. We next examined the expression of cell cycle regulators in UE7T/TR-AT cells and found that the induction of ASPL-TFE3 expression markedly increased p21 protein expression, whereas the expression of p53, p16, and p27 showed no remarkable changes (Figure 4C). To determine whether ASPL-TFE3 expression induces cellular senescence, we performed SA-β-gal staining in UE7T/TR-AT cells following tetracycline treatment (Figure 4D). ASPL-TFE3 expression in UE7T/TR-AT cells resulted in an increase in the percentage of SA-β-gal-positive cells from 2% at 0 h to 20% at 24 h and 32% at 48 h after tetracycline treatment (Figure 4E). The proportions of SA-β-gal-positive cells were increased by 46% at 4 days after tetracycline treatment, followed by a slight decrease to 35% at 8 days after tetracycline treatment (Supplementary Figure 2).


ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21
ASPL-TFE3 induces cellular senescence in mesenchymal stem cells. (A) UE7T/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. (B) UE7T/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and cell numbers were then determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± SD. *P < .05. (C) UE7T/TR-Vec and UE7T/TR-AT cells were incubated with tetracycline for 0, 24, and 48 h and were then subjected to immunoblotting with the indicated antibodies. (D) SA-β-gal staining was performed in UE7T/TR-Vec and UE7T/TR-AT cells after tetracycline treatment for 0, 24, and 48 h. (E) Proportions of SA-β-gal-positive cells in cultures of UE7T/TR-Vec and UE7T/TR-AT cells were calculated after tetracycline treatment for 0, 24, and 48 h. Data are presented as mean ± SD of three independent experiments. **P < .01; ***P < .005.
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f0020: ASPL-TFE3 induces cellular senescence in mesenchymal stem cells. (A) UE7T/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. (B) UE7T/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and cell numbers were then determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± SD. *P < .05. (C) UE7T/TR-Vec and UE7T/TR-AT cells were incubated with tetracycline for 0, 24, and 48 h and were then subjected to immunoblotting with the indicated antibodies. (D) SA-β-gal staining was performed in UE7T/TR-Vec and UE7T/TR-AT cells after tetracycline treatment for 0, 24, and 48 h. (E) Proportions of SA-β-gal-positive cells in cultures of UE7T/TR-Vec and UE7T/TR-AT cells were calculated after tetracycline treatment for 0, 24, and 48 h. Data are presented as mean ± SD of three independent experiments. **P < .01; ***P < .005.
Mentions: Oncogene expression induces premature senescence termed oncogene-induced senescence (OIS) in certain cell types, including primary fibroblasts and progenitor/stem cells [27], [30]. Furthermore, recent studies have reported that the expression of sarcoma-associated fusion oncogenes in MSCs can lead to the development of tumors resembling sarcomas [31], [32], [33], [34], suggesting that MSCs are a potential model for analyzing the pathogenesis of sarcomas with specific fusion proteins. Therefore, we established a tetracycline-inducible system for ASPL-TFE3 in UE7T-13 human bone marrow-derived MSCs (UE7T/TR-AT cells) and confirmed the induction of ASPL-TFE3 expression by tetracycline addition using immunoblotting (Figure 4A). As shown in Figure 4B, the addition of tetracycline resulted in suppression of the proliferation of UE7T/TR-AT cells. We next examined the expression of cell cycle regulators in UE7T/TR-AT cells and found that the induction of ASPL-TFE3 expression markedly increased p21 protein expression, whereas the expression of p53, p16, and p27 showed no remarkable changes (Figure 4C). To determine whether ASPL-TFE3 expression induces cellular senescence, we performed SA-β-gal staining in UE7T/TR-AT cells following tetracycline treatment (Figure 4D). ASPL-TFE3 expression in UE7T/TR-AT cells resulted in an increase in the percentage of SA-β-gal-positive cells from 2% at 0 h to 20% at 24 h and 32% at 48 h after tetracycline treatment (Figure 4E). The proportions of SA-β-gal-positive cells were increased by 46% at 4 days after tetracycline treatment, followed by a slight decrease to 35% at 8 days after tetracycline treatment (Supplementary Figure 2).

View Article: PubMed Central - PubMed

ABSTRACT

Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17)t(X;17)(p11;q25), resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1) as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow&ndash;derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated &beta;-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP). These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

No MeSH data available.


Related in: MedlinePlus