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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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Secretion of cytokines in prostate cancer cells(Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.
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Figure 5: Secretion of cytokines in prostate cancer cells(Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.

Mentions: The presence of autocrine growth factors in the medium of DU145 was assessed by adding the conditioned medium from DU145 onto 22Rv1 cells. Figure 5A shows that DU145-conditioned medium stimulated the phosphorylation of ERK-1/2 and AKT in 22Rv1, and increased the expression of Egr-1. Egr-1 induction was inhibited by prior incubation of 22Rv1 with EGFR inhibitor PD168393 (figure 5B), indicating that growth factors secreted by DU145 stimulate the EGFR and most likely are EGFR ligands.


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)

Secretion of cytokines in prostate cancer cells(Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.
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Related In: Results  -  Collection

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Figure 5: Secretion of cytokines in prostate cancer cells(Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.
Mentions: The presence of autocrine growth factors in the medium of DU145 was assessed by adding the conditioned medium from DU145 onto 22Rv1 cells. Figure 5A shows that DU145-conditioned medium stimulated the phosphorylation of ERK-1/2 and AKT in 22Rv1, and increased the expression of Egr-1. Egr-1 induction was inhibited by prior incubation of 22Rv1 with EGFR inhibitor PD168393 (figure 5B), indicating that growth factors secreted by DU145 stimulate the EGFR and most likely are EGFR ligands.

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