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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.


ASPL-TFE3 up-regulates p21. (A) 293/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) HeLa cells were transiently transfected with either the vector control or the ASPL-TFE3 expression vector and were incubated for 24 h. Cell lysates were then subjected to immunoblotting using antibodies against FLAG, p21, and actin. (C) p21 mRNA levels were determined using real-time quantitative PCR in 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h. Relative p21 expression was analyzed using quantitative PCR and gene expression data were normalized to those for beta-actin. Data are presented as fold changes in mRNA levels relative to no treatment (0 h) from three independent experiments. Error bars show SD; ***P < .005.
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f0010: ASPL-TFE3 up-regulates p21. (A) 293/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) HeLa cells were transiently transfected with either the vector control or the ASPL-TFE3 expression vector and were incubated for 24 h. Cell lysates were then subjected to immunoblotting using antibodies against FLAG, p21, and actin. (C) p21 mRNA levels were determined using real-time quantitative PCR in 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h. Relative p21 expression was analyzed using quantitative PCR and gene expression data were normalized to those for beta-actin. Data are presented as fold changes in mRNA levels relative to no treatment (0 h) from three independent experiments. Error bars show SD; ***P < .005.

Mentions: Because cell cycle progression is regulated by complexes of cell cycle regulatory proteins that include cyclins, Cdk, and Cdk inhibitors, we analyzed the expression of cell cycle regulatory proteins in 293/TR-AT cells following tetracycline treatment. The induction of ASPL-TFE3 expression resulted in an increase in protein level of the Cdk inhibitor p21 [23], [24], whereas the expression levels of other cell cycle regulatory proteins, including p27, p16, p53, Cdk2, and Cdk4, showed no remarkable changes (Figure 2A). Up-regulated p21 protein expression was detectable as early as 2 h after tetracycline treatment, in parallel with ASPL-TFE3 protein expression (Supplementary Figure 1). We further investigated the phosphorylation level of Rb, which plays a key role during the transition from G0/G1 to S phases [25], [26], and observed a decrease in its phosphorylation level after tetracycline treatment (Figure 2A). These findings indicate that ASPL-TFE3 expression increases p21 protein level and decreases the phosphorylation level of Rb, resulting in growth arrest of 293 cells.


ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21
ASPL-TFE3 up-regulates p21. (A) 293/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) HeLa cells were transiently transfected with either the vector control or the ASPL-TFE3 expression vector and were incubated for 24 h. Cell lysates were then subjected to immunoblotting using antibodies against FLAG, p21, and actin. (C) p21 mRNA levels were determined using real-time quantitative PCR in 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h. Relative p21 expression was analyzed using quantitative PCR and gene expression data were normalized to those for beta-actin. Data are presented as fold changes in mRNA levels relative to no treatment (0 h) from three independent experiments. Error bars show SD; ***P < .005.
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f0010: ASPL-TFE3 up-regulates p21. (A) 293/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) HeLa cells were transiently transfected with either the vector control or the ASPL-TFE3 expression vector and were incubated for 24 h. Cell lysates were then subjected to immunoblotting using antibodies against FLAG, p21, and actin. (C) p21 mRNA levels were determined using real-time quantitative PCR in 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h. Relative p21 expression was analyzed using quantitative PCR and gene expression data were normalized to those for beta-actin. Data are presented as fold changes in mRNA levels relative to no treatment (0 h) from three independent experiments. Error bars show SD; ***P < .005.
Mentions: Because cell cycle progression is regulated by complexes of cell cycle regulatory proteins that include cyclins, Cdk, and Cdk inhibitors, we analyzed the expression of cell cycle regulatory proteins in 293/TR-AT cells following tetracycline treatment. The induction of ASPL-TFE3 expression resulted in an increase in protein level of the Cdk inhibitor p21 [23], [24], whereas the expression levels of other cell cycle regulatory proteins, including p27, p16, p53, Cdk2, and Cdk4, showed no remarkable changes (Figure 2A). Up-regulated p21 protein expression was detectable as early as 2 h after tetracycline treatment, in parallel with ASPL-TFE3 protein expression (Supplementary Figure 1). We further investigated the phosphorylation level of Rb, which plays a key role during the transition from G0/G1 to S phases [25], [26], and observed a decrease in its phosphorylation level after tetracycline treatment (Figure 2A). These findings indicate that ASPL-TFE3 expression increases p21 protein level and decreases the phosphorylation level of Rb, resulting in growth arrest of 293 cells.

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.