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Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells.

Sauer L, Gitenay D, Vo C, Baron VT - Oncogene (2010)

Bottom Line: Egr-1 increased the transcription of HB-EGF (epidermal growth factor), amphiregulin and epiregulin, resulting in autocrine activation of the EGF receptor (EGFR) and downstream MEK/ERK cascade.Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway.Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Institute of San Diego, San Diego, CA 92121, USA.

ABSTRACT
Early growth response-1 (Egr-1) is overexpressed in human prostate tumors and contributes to cancer progression. On the other hand, mutation of p53 is associated with advanced prostate cancer, as well as with metastasis and hormone independence. This study shows that in prostate cell lines in culture, Egr-1 overexpression correlated with an alteration of p53 activity because of the expression of SV40 large T-antigen or because of a mutation in the TP53 gene. In cells containing altered p53 activity, Egr-1 expression was abolished by pharmacological inhibition or RNAi silencing of p53. Although forced expression of wild-type p53 was not sufficient to trigger Egr-1 transcription, four different mutants of p53 were shown to induce Egr-1. Direct binding of p53 to the EGR1 promoter could not be detected. Instead, Egr-1 transcription was driven by the ERK1/2 pathway, as it was abrogated by specific inhibitors of MEK. Egr-1 increased the transcription of HB-EGF (epidermal growth factor), amphiregulin and epiregulin, resulting in autocrine activation of the EGF receptor (EGFR) and downstream MEK/ERK cascade. Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway. Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

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p53 regulates the expression of EGFR(Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A. Controls showing p53 and actin mRNA are the same as in figure 6A.
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Figure 7: p53 regulates the expression of EGFR(Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A. Controls showing p53 and actin mRNA are the same as in figure 6A.

Mentions: Both mutant-p53 and wt-p53 bind to the promoter of EGFR and directly regulate its transcription (Deb et al., 1994; Ludes-Meyers et al., 1996). Therefore, we looked at the effect of p53 inhibitor Pifithrin-α on EGFR expression. Pifithrin-α caused a decrease of both the protein and mRNA levels of EGFR in DU145, as shown in figure 7A. However, it has been shown previously that Egr-1 activates the transcription of EGFR in response to hypoxia in osteosarcoma cells (Nishi et al., 2002). Since inhibition of p53 also decreases Egr-1 expression, we tested the possibility that Egr-1 mediates the effect of p53 on the transcription of EGFR.


Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells.

Sauer L, Gitenay D, Vo C, Baron VT - Oncogene (2010)

p53 regulates the expression of EGFR(Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A. Controls showing p53 and actin mRNA are the same as in figure 6A.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2865566&req=5

Figure 7: p53 regulates the expression of EGFR(Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A. Controls showing p53 and actin mRNA are the same as in figure 6A.
Mentions: Both mutant-p53 and wt-p53 bind to the promoter of EGFR and directly regulate its transcription (Deb et al., 1994; Ludes-Meyers et al., 1996). Therefore, we looked at the effect of p53 inhibitor Pifithrin-α on EGFR expression. Pifithrin-α caused a decrease of both the protein and mRNA levels of EGFR in DU145, as shown in figure 7A. However, it has been shown previously that Egr-1 activates the transcription of EGFR in response to hypoxia in osteosarcoma cells (Nishi et al., 2002). Since inhibition of p53 also decreases Egr-1 expression, we tested the possibility that Egr-1 mediates the effect of p53 on the transcription of EGFR.

Bottom Line: Egr-1 increased the transcription of HB-EGF (epidermal growth factor), amphiregulin and epiregulin, resulting in autocrine activation of the EGF receptor (EGFR) and downstream MEK/ERK cascade.Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway.Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Institute of San Diego, San Diego, CA 92121, USA.

ABSTRACT
Early growth response-1 (Egr-1) is overexpressed in human prostate tumors and contributes to cancer progression. On the other hand, mutation of p53 is associated with advanced prostate cancer, as well as with metastasis and hormone independence. This study shows that in prostate cell lines in culture, Egr-1 overexpression correlated with an alteration of p53 activity because of the expression of SV40 large T-antigen or because of a mutation in the TP53 gene. In cells containing altered p53 activity, Egr-1 expression was abolished by pharmacological inhibition or RNAi silencing of p53. Although forced expression of wild-type p53 was not sufficient to trigger Egr-1 transcription, four different mutants of p53 were shown to induce Egr-1. Direct binding of p53 to the EGR1 promoter could not be detected. Instead, Egr-1 transcription was driven by the ERK1/2 pathway, as it was abrogated by specific inhibitors of MEK. Egr-1 increased the transcription of HB-EGF (epidermal growth factor), amphiregulin and epiregulin, resulting in autocrine activation of the EGF receptor (EGFR) and downstream MEK/ERK cascade. Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway. Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

Show MeSH
Related in: MedlinePlus