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

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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.

No MeSH data available.


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Suppression of p21 reduces ASPL-TFE3-induced senescence. (A) UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2), and were then cultured in the presence of tetracycline (Tet +) for 48 h. Lysates of untreated UE7T/TR-AT cells (−) or siRNA-transfected cells were subjected to immunoblotting using the indicated antibodies. (B) SA-β-gal staining was performed in untreated UE7T/TR-AT cells (−) or UE7T/TR-AT cells transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2) after tetracycline treatment for 48 h. (C) Proportions of SA-β-gal-positive cells were calculated for all treatment groups. Data are presented as mean ± SD of three independent experiments. *P < .05; ***P < .005.
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f0025: Suppression of p21 reduces ASPL-TFE3-induced senescence. (A) UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2), and were then cultured in the presence of tetracycline (Tet +) for 48 h. Lysates of untreated UE7T/TR-AT cells (−) or siRNA-transfected cells were subjected to immunoblotting using the indicated antibodies. (B) SA-β-gal staining was performed in untreated UE7T/TR-AT cells (−) or UE7T/TR-AT cells transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2) after tetracycline treatment for 48 h. (C) Proportions of SA-β-gal-positive cells were calculated for all treatment groups. Data are presented as mean ± SD of three independent experiments. *P < .05; ***P < .005.

Mentions: To determine whether p21 contributes to the induction of cellular senescence by ASPL-TFE3, we suppressed p21 expression using p21 siRNAs (p21–1 and p21–2) in tetracycline-treated UE7T/TR-AT cells (Figure 5A). UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs, and SA-β-gal staining was performed (Figure 5B). After 48 h of incubation with tetracycline, 31% of control siRNA-transfected cells were positive for SA-β-gal staining, whereas the transfection of p21 siRNAs p21–1 and p21–2 decreased the proportions of SA-β-gal-positive cells to 16% and 17%, respectively (Figure 5C). These findings indicate that ASPL-TFE3-induced senescence is mediated at least in part by p21 expression.


ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21
Suppression of p21 reduces ASPL-TFE3-induced senescence. (A) UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2), and were then cultured in the presence of tetracycline (Tet +) for 48 h. Lysates of untreated UE7T/TR-AT cells (−) or siRNA-transfected cells were subjected to immunoblotting using the indicated antibodies. (B) SA-β-gal staining was performed in untreated UE7T/TR-AT cells (−) or UE7T/TR-AT cells transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2) after tetracycline treatment for 48 h. (C) Proportions of SA-β-gal-positive cells were calculated for all treatment groups. Data are presented as mean ± SD of three independent experiments. *P < .05; ***P < .005.
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f0025: Suppression of p21 reduces ASPL-TFE3-induced senescence. (A) UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2), and were then cultured in the presence of tetracycline (Tet +) for 48 h. Lysates of untreated UE7T/TR-AT cells (−) or siRNA-transfected cells were subjected to immunoblotting using the indicated antibodies. (B) SA-β-gal staining was performed in untreated UE7T/TR-AT cells (−) or UE7T/TR-AT cells transfected with either control siRNA or p21 siRNAs (p21–1 and p21–2) after tetracycline treatment for 48 h. (C) Proportions of SA-β-gal-positive cells were calculated for all treatment groups. Data are presented as mean ± SD of three independent experiments. *P < .05; ***P < .005.
Mentions: To determine whether p21 contributes to the induction of cellular senescence by ASPL-TFE3, we suppressed p21 expression using p21 siRNAs (p21–1 and p21–2) in tetracycline-treated UE7T/TR-AT cells (Figure 5A). UE7T/TR-AT cells were transfected with either control siRNA or p21 siRNAs, and SA-β-gal staining was performed (Figure 5B). After 48 h of incubation with tetracycline, 31% of control siRNA-transfected cells were positive for SA-β-gal staining, whereas the transfection of p21 siRNAs p21–1 and p21–2 decreased the proportions of SA-β-gal-positive cells to 16% and 17%, respectively (Figure 5C). These findings indicate that ASPL-TFE3-induced senescence is mediated at least in part by p21 expression.

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