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CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

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Related in: MedlinePlus

Signaling by CSF1 positively feeds back on the p53 pathway.A. H1299 tetracyline-inducible p53 P72 cells were infected with a lentivirus expressing the CSF1 receptor CSF1R or vector alone (Vector). Left panel: QRT-PCR analysis of stably-infected clones for CSF1R level. Values shown are the average of 2 independent replicates, normalized to control. Error bars mark standard deviation. Right panel: H1299 tetracycline-inducible P72 cells infected with vector (vector) or CSF1R (CSF1R) were treated with 50 ng/mL CSF1 and analyzed for phosphorylated CSF1R protein (phospho-Y723) and AKT (phospho-Ser473) after 0, 1 or 5 minutes. The data depicted are representative of 3 independent experiments. B. H1299 tetracycline-inducible p53 cells stably-infected with vector (Vector) or CSF1R (CSF1R) were serum-starved overnight and then incubated with doxycycline (0.75 ug/ml) alone or in combination with CSF1 (50 ng/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2 and GAPDH. The data depicted are representative of 3 independent experiments. C. Left Panel: QRT-PCR of RNA isolated from H1299 tet-inducible P72 cells stably-infected with CSF1R or Vector. Cells were untreated (0 hr) or treated with Doxycycline for the indicated time points. RNA was analyzed for the levels of p53; values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. Right Panel: QRT-PCR of RNA isolated from H1299 tet-inducible p53 cells (P72 variant), infected with CSF1R or vector. Cells were untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for 24 hours. RNA was analyzed for the levels of p21 (CDKN1A); values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. D. H1299 tetracycline-inducible p53 cells stably-infected with CSF1R (CSF1R) were treated with or without CSF1R inhibitor II (10 uM, Calbiochem) in combination with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), GAPDH and p21 (CDKN1A). The data depicted are representative of 3 independent experiments. E. Western analysis of lysates isolated from H1299 tetracycline-inducible p53 cells stably-infected with vector (vector) or CSF1R (CSF1R). Cells were untreated (0 hr) or were treated with doxycycline (0.75 ug/ml) and Etoposide (Etop, 50 uM) for the time points indicated. Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2, GAPDH and Cleaved Lamin A (CLA). Relative values of Ser15 p53 phosphorylation, assessed by densitometry and normalized to total GAPDH, are indicated.
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pone-0074297-g004: Signaling by CSF1 positively feeds back on the p53 pathway.A. H1299 tetracyline-inducible p53 P72 cells were infected with a lentivirus expressing the CSF1 receptor CSF1R or vector alone (Vector). Left panel: QRT-PCR analysis of stably-infected clones for CSF1R level. Values shown are the average of 2 independent replicates, normalized to control. Error bars mark standard deviation. Right panel: H1299 tetracycline-inducible P72 cells infected with vector (vector) or CSF1R (CSF1R) were treated with 50 ng/mL CSF1 and analyzed for phosphorylated CSF1R protein (phospho-Y723) and AKT (phospho-Ser473) after 0, 1 or 5 minutes. The data depicted are representative of 3 independent experiments. B. H1299 tetracycline-inducible p53 cells stably-infected with vector (Vector) or CSF1R (CSF1R) were serum-starved overnight and then incubated with doxycycline (0.75 ug/ml) alone or in combination with CSF1 (50 ng/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2 and GAPDH. The data depicted are representative of 3 independent experiments. C. Left Panel: QRT-PCR of RNA isolated from H1299 tet-inducible P72 cells stably-infected with CSF1R or Vector. Cells were untreated (0 hr) or treated with Doxycycline for the indicated time points. RNA was analyzed for the levels of p53; values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. Right Panel: QRT-PCR of RNA isolated from H1299 tet-inducible p53 cells (P72 variant), infected with CSF1R or vector. Cells were untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for 24 hours. RNA was analyzed for the levels of p21 (CDKN1A); values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. D. H1299 tetracycline-inducible p53 cells stably-infected with CSF1R (CSF1R) were treated with or without CSF1R inhibitor II (10 uM, Calbiochem) in combination with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), GAPDH and p21 (CDKN1A). The data depicted are representative of 3 independent experiments. E. Western analysis of lysates isolated from H1299 tetracycline-inducible p53 cells stably-infected with vector (vector) or CSF1R (CSF1R). Cells were untreated (0 hr) or were treated with doxycycline (0.75 ug/ml) and Etoposide (Etop, 50 uM) for the time points indicated. Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2, GAPDH and Cleaved Lamin A (CLA). Relative values of Ser15 p53 phosphorylation, assessed by densitometry and normalized to total GAPDH, are indicated.

Mentions: We next sought to assess the impact, if any, of CSF1 signaling on p53 function. In particular, as CSF1 was previously described as part of a senescence-associated cytokine signature for p53 [29], we sought to test the hypothesis that increased CSF1 transactivation might underlie part of the enhanced ability for the P72 variant to induce growth arrest and senescence, as opposed to apoptosis. Analysis of our inducible H1299 cells revealed an absence of expression of the CSF1 receptor CSF1R in this lung adenocarcinoma background. Therefore, we created a lentiviral expression vector for CSF1R, and infected H1299 tet-inducible P72 cells with this CSF1R construct or vector alone (pLKO-1); we generated pooled stably-infected cells as well as individual sub-clones, and obtained identical results with both (data not shown). To assess the integrity of this pathway in these cells, we first treated vector-infected or CSF1R-infected clones with recombinant CSF1 (50 ng/mL) and measured cFMS activation (Y723 phosphorylation) as well as downstream AKT activation (S473 phosphorylation) using phospho-specific antibodies for these proteins. These analyses suggested that the CSF1/CSF1R pathway was intact in these cells, as assessed by increased CSF1R and AKT phosphorylation after treatment with CSF1 (Fig. 4A). We next used doxycycline to induce p53 in these cells in order to analyze the impact of activation of the CSF1R receptor by exogenous CSF1 on the stabilization, phosphorylation, and transcriptional activity of p53. Interestingly, this analysis revealed that CSF1R signaling appears to feed back on the p53 pathway, leading to increased phosphorylation of p53 on serine 15, increased p53 accumulation, and increased expression of the p53 target protein p21 (CDKN1A, Fig. 4B). This occurred even in the absence of exogenous CSF1, indicating that the CSF1 induced by p53 was able to signal through this pathway (Fig. 4B, compare p53 serine 15 phosphorylation in lanes 4 and 8). The increased expression of p21 was, at least in part, at the transcriptional level, as there was a 2-fold increase in RNA for this gene in the presence of doxycycline in CSF1R cells compared to vector (Fig. 4C).


CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

Signaling by CSF1 positively feeds back on the p53 pathway.A. H1299 tetracyline-inducible p53 P72 cells were infected with a lentivirus expressing the CSF1 receptor CSF1R or vector alone (Vector). Left panel: QRT-PCR analysis of stably-infected clones for CSF1R level. Values shown are the average of 2 independent replicates, normalized to control. Error bars mark standard deviation. Right panel: H1299 tetracycline-inducible P72 cells infected with vector (vector) or CSF1R (CSF1R) were treated with 50 ng/mL CSF1 and analyzed for phosphorylated CSF1R protein (phospho-Y723) and AKT (phospho-Ser473) after 0, 1 or 5 minutes. The data depicted are representative of 3 independent experiments. B. H1299 tetracycline-inducible p53 cells stably-infected with vector (Vector) or CSF1R (CSF1R) were serum-starved overnight and then incubated with doxycycline (0.75 ug/ml) alone or in combination with CSF1 (50 ng/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2 and GAPDH. The data depicted are representative of 3 independent experiments. C. Left Panel: QRT-PCR of RNA isolated from H1299 tet-inducible P72 cells stably-infected with CSF1R or Vector. Cells were untreated (0 hr) or treated with Doxycycline for the indicated time points. RNA was analyzed for the levels of p53; values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. Right Panel: QRT-PCR of RNA isolated from H1299 tet-inducible p53 cells (P72 variant), infected with CSF1R or vector. Cells were untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for 24 hours. RNA was analyzed for the levels of p21 (CDKN1A); values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. D. H1299 tetracycline-inducible p53 cells stably-infected with CSF1R (CSF1R) were treated with or without CSF1R inhibitor II (10 uM, Calbiochem) in combination with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), GAPDH and p21 (CDKN1A). The data depicted are representative of 3 independent experiments. E. Western analysis of lysates isolated from H1299 tetracycline-inducible p53 cells stably-infected with vector (vector) or CSF1R (CSF1R). Cells were untreated (0 hr) or were treated with doxycycline (0.75 ug/ml) and Etoposide (Etop, 50 uM) for the time points indicated. Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2, GAPDH and Cleaved Lamin A (CLA). Relative values of Ser15 p53 phosphorylation, assessed by densitometry and normalized to total GAPDH, are indicated.
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pone-0074297-g004: Signaling by CSF1 positively feeds back on the p53 pathway.A. H1299 tetracyline-inducible p53 P72 cells were infected with a lentivirus expressing the CSF1 receptor CSF1R or vector alone (Vector). Left panel: QRT-PCR analysis of stably-infected clones for CSF1R level. Values shown are the average of 2 independent replicates, normalized to control. Error bars mark standard deviation. Right panel: H1299 tetracycline-inducible P72 cells infected with vector (vector) or CSF1R (CSF1R) were treated with 50 ng/mL CSF1 and analyzed for phosphorylated CSF1R protein (phospho-Y723) and AKT (phospho-Ser473) after 0, 1 or 5 minutes. The data depicted are representative of 3 independent experiments. B. H1299 tetracycline-inducible p53 cells stably-infected with vector (Vector) or CSF1R (CSF1R) were serum-starved overnight and then incubated with doxycycline (0.75 ug/ml) alone or in combination with CSF1 (50 ng/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2 and GAPDH. The data depicted are representative of 3 independent experiments. C. Left Panel: QRT-PCR of RNA isolated from H1299 tet-inducible P72 cells stably-infected with CSF1R or Vector. Cells were untreated (0 hr) or treated with Doxycycline for the indicated time points. RNA was analyzed for the levels of p53; values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. Right Panel: QRT-PCR of RNA isolated from H1299 tet-inducible p53 cells (P72 variant), infected with CSF1R or vector. Cells were untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for 24 hours. RNA was analyzed for the levels of p21 (CDKN1A); values shown are the average of 3 independent replicates, normalized to control. Error bars mark standard deviation. D. H1299 tetracycline-inducible p53 cells stably-infected with CSF1R (CSF1R) were treated with or without CSF1R inhibitor II (10 uM, Calbiochem) in combination with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), GAPDH and p21 (CDKN1A). The data depicted are representative of 3 independent experiments. E. Western analysis of lysates isolated from H1299 tetracycline-inducible p53 cells stably-infected with vector (vector) or CSF1R (CSF1R). Cells were untreated (0 hr) or were treated with doxycycline (0.75 ug/ml) and Etoposide (Etop, 50 uM) for the time points indicated. Cells were analyzed for activated CSF1R (phospho-Y723), p53 (phospho-Ser15), total p53, MDM2, GAPDH and Cleaved Lamin A (CLA). Relative values of Ser15 p53 phosphorylation, assessed by densitometry and normalized to total GAPDH, are indicated.
Mentions: We next sought to assess the impact, if any, of CSF1 signaling on p53 function. In particular, as CSF1 was previously described as part of a senescence-associated cytokine signature for p53 [29], we sought to test the hypothesis that increased CSF1 transactivation might underlie part of the enhanced ability for the P72 variant to induce growth arrest and senescence, as opposed to apoptosis. Analysis of our inducible H1299 cells revealed an absence of expression of the CSF1 receptor CSF1R in this lung adenocarcinoma background. Therefore, we created a lentiviral expression vector for CSF1R, and infected H1299 tet-inducible P72 cells with this CSF1R construct or vector alone (pLKO-1); we generated pooled stably-infected cells as well as individual sub-clones, and obtained identical results with both (data not shown). To assess the integrity of this pathway in these cells, we first treated vector-infected or CSF1R-infected clones with recombinant CSF1 (50 ng/mL) and measured cFMS activation (Y723 phosphorylation) as well as downstream AKT activation (S473 phosphorylation) using phospho-specific antibodies for these proteins. These analyses suggested that the CSF1/CSF1R pathway was intact in these cells, as assessed by increased CSF1R and AKT phosphorylation after treatment with CSF1 (Fig. 4A). We next used doxycycline to induce p53 in these cells in order to analyze the impact of activation of the CSF1R receptor by exogenous CSF1 on the stabilization, phosphorylation, and transcriptional activity of p53. Interestingly, this analysis revealed that CSF1R signaling appears to feed back on the p53 pathway, leading to increased phosphorylation of p53 on serine 15, increased p53 accumulation, and increased expression of the p53 target protein p21 (CDKN1A, Fig. 4B). This occurred even in the absence of exogenous CSF1, indicating that the CSF1 induced by p53 was able to signal through this pathway (Fig. 4B, compare p53 serine 15 phosphorylation in lanes 4 and 8). The increased expression of p21 was, at least in part, at the transcriptional level, as there was a 2-fold increase in RNA for this gene in the presence of doxycycline in CSF1R cells compared to vector (Fig. 4C).

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

Show MeSH
Related in: MedlinePlus