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Jab1 is a target of EGFR signaling in ERalpha-negative breast cancer.

Wang J, Barnes RO, West NR, Olson M, Chu JE, Watson PH - Breast Cancer Res. (2008)

Bottom Line: EGF treatment of cell lines resulted in increased Jab1 nuclear expression.This effect was inhibited by the ERK pathway inhibitor, PD98059.EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deeley Research Center, BC Cancer Agency, Vancouver Island Center, Victoria, BC, Canada.

ABSTRACT

Introduction: c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERalpha-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERalpha- phenotype.

Methods: MDA-MB-231 and MDA-MB-468 ERalpha-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.

Results: EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERalpha- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).

Conclusion: Jab1 is a target of EGFR signaling in ERalpha- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERalpha- breast cancer.

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Effect of Jab1 short interfering RNA (siRNA) knockdown on p27 levels with and without epidermal growth factor (EGF) treatment in MDA-MB-231 cells. (a) Western blot analysis of cell lysates collected following the indicated treatments. Relative p27 band intensities were calculated by densitometry and normalized to the loading control (GAPDH). (b) The percentage change in p27 expression for EGF-treated cells relative to untreated controls. Data shown are representative of several independent experiments, demonstrating that knockdown of Jab1 abrogates EGF-induced downregulation of p27. Bars represent mean ± standard deviation. Statistical analysis was by t test (*P < 0.0001). All EGF treatments were at 50 ng/mL for 4 hours. EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1; pEGFR, phosphorylated epidermal growth factor receptor.
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Figure 4: Effect of Jab1 short interfering RNA (siRNA) knockdown on p27 levels with and without epidermal growth factor (EGF) treatment in MDA-MB-231 cells. (a) Western blot analysis of cell lysates collected following the indicated treatments. Relative p27 band intensities were calculated by densitometry and normalized to the loading control (GAPDH). (b) The percentage change in p27 expression for EGF-treated cells relative to untreated controls. Data shown are representative of several independent experiments, demonstrating that knockdown of Jab1 abrogates EGF-induced downregulation of p27. Bars represent mean ± standard deviation. Statistical analysis was by t test (*P < 0.0001). All EGF treatments were at 50 ng/mL for 4 hours. EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1; pEGFR, phosphorylated epidermal growth factor receptor.

Mentions: To investigate whether EGFR signaling has a functional effect on Jab1 activity, we performed immunoblotting and double-immunostaining for the Jab1 downstream target, p27. In both MDA-MB-231 and MDA-MB-468 cell lines, Western blot assay showed that EGF treatment and phosphorylation of EGFR resulted in a significant decrease in p27 expression (Figure 3a). Additional observed changes following EGF treatment included increased pAKT (Figure 3a). The inverse correlation between nuclear Jab1 and p27 expression was also observed in double-immunostaining for these proteins (Figure 3b). To confirm that Jab1 was necessary for the effect of EGF on p27, we performed Jab1 knockdown using an siRNA approach in MDA-MB-231 cells in conjunction with EGF treatment. In addition to re-confirming that cells treated with EGF have reduced p27 (P < 0.05), we found that Jab1 knockdown restored p27 to EGF-untreated levels compared with cells treated with EGF and control siRNA (P < 0.0001) (Figure 4a,b). In cells treated with Jab1 siRNA, EGF had no effect on p27 levels (P = 0.68). Taken together, these results indicate not only that EGFR signaling affects Jab1 translocation but that it may regulate targets downstream of Jab1 and that the effect of EGF on p27 levels is mediated by Jab1.


Jab1 is a target of EGFR signaling in ERalpha-negative breast cancer.

Wang J, Barnes RO, West NR, Olson M, Chu JE, Watson PH - Breast Cancer Res. (2008)

Effect of Jab1 short interfering RNA (siRNA) knockdown on p27 levels with and without epidermal growth factor (EGF) treatment in MDA-MB-231 cells. (a) Western blot analysis of cell lysates collected following the indicated treatments. Relative p27 band intensities were calculated by densitometry and normalized to the loading control (GAPDH). (b) The percentage change in p27 expression for EGF-treated cells relative to untreated controls. Data shown are representative of several independent experiments, demonstrating that knockdown of Jab1 abrogates EGF-induced downregulation of p27. Bars represent mean ± standard deviation. Statistical analysis was by t test (*P < 0.0001). All EGF treatments were at 50 ng/mL for 4 hours. EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1; pEGFR, phosphorylated epidermal growth factor receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481501&req=5

Figure 4: Effect of Jab1 short interfering RNA (siRNA) knockdown on p27 levels with and without epidermal growth factor (EGF) treatment in MDA-MB-231 cells. (a) Western blot analysis of cell lysates collected following the indicated treatments. Relative p27 band intensities were calculated by densitometry and normalized to the loading control (GAPDH). (b) The percentage change in p27 expression for EGF-treated cells relative to untreated controls. Data shown are representative of several independent experiments, demonstrating that knockdown of Jab1 abrogates EGF-induced downregulation of p27. Bars represent mean ± standard deviation. Statistical analysis was by t test (*P < 0.0001). All EGF treatments were at 50 ng/mL for 4 hours. EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1; pEGFR, phosphorylated epidermal growth factor receptor.
Mentions: To investigate whether EGFR signaling has a functional effect on Jab1 activity, we performed immunoblotting and double-immunostaining for the Jab1 downstream target, p27. In both MDA-MB-231 and MDA-MB-468 cell lines, Western blot assay showed that EGF treatment and phosphorylation of EGFR resulted in a significant decrease in p27 expression (Figure 3a). Additional observed changes following EGF treatment included increased pAKT (Figure 3a). The inverse correlation between nuclear Jab1 and p27 expression was also observed in double-immunostaining for these proteins (Figure 3b). To confirm that Jab1 was necessary for the effect of EGF on p27, we performed Jab1 knockdown using an siRNA approach in MDA-MB-231 cells in conjunction with EGF treatment. In addition to re-confirming that cells treated with EGF have reduced p27 (P < 0.05), we found that Jab1 knockdown restored p27 to EGF-untreated levels compared with cells treated with EGF and control siRNA (P < 0.0001) (Figure 4a,b). In cells treated with Jab1 siRNA, EGF had no effect on p27 levels (P = 0.68). Taken together, these results indicate not only that EGFR signaling affects Jab1 translocation but that it may regulate targets downstream of Jab1 and that the effect of EGF on p27 levels is mediated by Jab1.

Bottom Line: EGF treatment of cell lines resulted in increased Jab1 nuclear expression.This effect was inhibited by the ERK pathway inhibitor, PD98059.EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deeley Research Center, BC Cancer Agency, Vancouver Island Center, Victoria, BC, Canada.

ABSTRACT

Introduction: c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERalpha-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERalpha- phenotype.

Methods: MDA-MB-231 and MDA-MB-468 ERalpha-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.

Results: EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERalpha- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).

Conclusion: Jab1 is a target of EGFR signaling in ERalpha- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERalpha- breast cancer.

Show MeSH
Related in: MedlinePlus