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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.

No MeSH data available.


p21 is a direct target of ASPL-TFE3. (A) Luciferase reporter plasmids containing the p21 promoter sequence were co-transfected into HeLa cells or p53-deficient KATO III cells with the indicated amount of empty vector and/or expression vector for ASPL-TFE3. Cells were harvested 24 h after transfection and luciferase activity was assayed. Data are presented as the fold changes in luciferase activity relative to that in cells transfected with the control vector. Data are presented as mean ± SD of at least three independent experiments. ***P < .005. (B) ChIP analysis of 293/TR-AT cells using PCR primers specific for the p21 promoter. Chromatin was isolated from 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h, immunoprecipitated with anti-FLAG antibody or control mouse IgG, followed by analysis by PCR amplification. Input represents the enzymatically sheared chromatin prior to immunoprecipitation.
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f0015: p21 is a direct target of ASPL-TFE3. (A) Luciferase reporter plasmids containing the p21 promoter sequence were co-transfected into HeLa cells or p53-deficient KATO III cells with the indicated amount of empty vector and/or expression vector for ASPL-TFE3. Cells were harvested 24 h after transfection and luciferase activity was assayed. Data are presented as the fold changes in luciferase activity relative to that in cells transfected with the control vector. Data are presented as mean ± SD of at least three independent experiments. ***P < .005. (B) ChIP analysis of 293/TR-AT cells using PCR primers specific for the p21 promoter. Chromatin was isolated from 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h, immunoprecipitated with anti-FLAG antibody or control mouse IgG, followed by analysis by PCR amplification. Input represents the enzymatically sheared chromatin prior to immunoprecipitation.

Mentions: Because the ASPL-TFE3 fusion oncoprotein functions as an aberrant transcription factor, we investigated whether ASPL-TFE3 activates the p21 gene promoter using luciferase reporter assays of the full-length human p21 promoter. Cotransfection of the ASPL-TFE3 expression vector and the reporter vector in HeLa cells caused a marked increase in luciferase activity compared with that following transfection with the control empty vector (Figure 3A). Because p53 is a well-known inducer of p21 [23], we determined whether p53 is required for activation of the p21 promoter by ASPL-TFE3, and found that transiently expressed ASPL-TFE3 activated the p21 gene promoter in p53-deficient (p53−/−) KATO III cells (Figure 3A). We next investigated the binding of ASPL-TFE3 to the p21 promoter using ChIP assays with an anti-FLAG antibody. After 24 h of incubation with tetracycline, DNA–protein complexes were immunoprecipitated from 293/TR-AT cells and direct binding of the FLAG-tagged ASPL-TFE3 with the endogenous p21 promoter was observed (Figure 3B). Collectively, these results indicate that ASPL-TFE3 directly binds to the p21 promoter and modulates its transcriptional activity independent of p53.


ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21
p21 is a direct target of ASPL-TFE3. (A) Luciferase reporter plasmids containing the p21 promoter sequence were co-transfected into HeLa cells or p53-deficient KATO III cells with the indicated amount of empty vector and/or expression vector for ASPL-TFE3. Cells were harvested 24 h after transfection and luciferase activity was assayed. Data are presented as the fold changes in luciferase activity relative to that in cells transfected with the control vector. Data are presented as mean ± SD of at least three independent experiments. ***P < .005. (B) ChIP analysis of 293/TR-AT cells using PCR primers specific for the p21 promoter. Chromatin was isolated from 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h, immunoprecipitated with anti-FLAG antibody or control mouse IgG, followed by analysis by PCR amplification. Input represents the enzymatically sheared chromatin prior to immunoprecipitation.
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f0015: p21 is a direct target of ASPL-TFE3. (A) Luciferase reporter plasmids containing the p21 promoter sequence were co-transfected into HeLa cells or p53-deficient KATO III cells with the indicated amount of empty vector and/or expression vector for ASPL-TFE3. Cells were harvested 24 h after transfection and luciferase activity was assayed. Data are presented as the fold changes in luciferase activity relative to that in cells transfected with the control vector. Data are presented as mean ± SD of at least three independent experiments. ***P < .005. (B) ChIP analysis of 293/TR-AT cells using PCR primers specific for the p21 promoter. Chromatin was isolated from 293/TR-AT cells cultured in the presence of tetracycline for 0 or 24 h, immunoprecipitated with anti-FLAG antibody or control mouse IgG, followed by analysis by PCR amplification. Input represents the enzymatically sheared chromatin prior to immunoprecipitation.
Mentions: Because the ASPL-TFE3 fusion oncoprotein functions as an aberrant transcription factor, we investigated whether ASPL-TFE3 activates the p21 gene promoter using luciferase reporter assays of the full-length human p21 promoter. Cotransfection of the ASPL-TFE3 expression vector and the reporter vector in HeLa cells caused a marked increase in luciferase activity compared with that following transfection with the control empty vector (Figure 3A). Because p53 is a well-known inducer of p21 [23], we determined whether p53 is required for activation of the p21 promoter by ASPL-TFE3, and found that transiently expressed ASPL-TFE3 activated the p21 gene promoter in p53-deficient (p53−/−) KATO III cells (Figure 3A). We next investigated the binding of ASPL-TFE3 to the p21 promoter using ChIP assays with an anti-FLAG antibody. After 24 h of incubation with tetracycline, DNA–protein complexes were immunoprecipitated from 293/TR-AT cells and direct binding of the FLAG-tagged ASPL-TFE3 with the endogenous p21 promoter was observed (Figure 3B). Collectively, these results indicate that ASPL-TFE3 directly binds to the p21 promoter and modulates its transcriptional activity independent of p53.

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.