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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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Related in: MedlinePlus

Epidermal growth factor (EGF) stimulates increased nuclear localization of Jab1 in MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (a, b) Immunofluorescence assays. (c) Quantitative image analysis of nuclear Jab1 (bars represent mean ± standard deviation). Statistical analysis was by t test (*P < 0.01). (d) Increased expression of Jab1 in the nucleus relative to cytoplasm, detected by Western blot in MDA-MB-231 nuclear extracts. Lamin A/C was included as a positive control for the nuclear extract, and GAPDH was included as a loading control. (e) Increased Jab1 nuclear expression positively correlated to EGF dose. DAPI, 4'-6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1.
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Figure 1: Epidermal growth factor (EGF) stimulates increased nuclear localization of Jab1 in MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (a, b) Immunofluorescence assays. (c) Quantitative image analysis of nuclear Jab1 (bars represent mean ± standard deviation). Statistical analysis was by t test (*P < 0.01). (d) Increased expression of Jab1 in the nucleus relative to cytoplasm, detected by Western blot in MDA-MB-231 nuclear extracts. Lamin A/C was included as a positive control for the nuclear extract, and GAPDH was included as a loading control. (e) Increased Jab1 nuclear expression positively correlated to EGF dose. DAPI, 4'-6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1.

Mentions: Jab1 has been shown previously to exist in both the nucleus and cytoplasm of different cell types. However, it has been shown that interactions between Jab1 and many of its downstream targets are associated with translocation of Jab1 to the nucleus. These include interaction with AP-1 [31], NF-κB [32], and p27 [32]. To determine whether Jab1 translocation is affected by EGFR signaling, we first used immunofluorescence microscopy to look for changes in cellular localization of Jab1 following treatment with EGF. We observed that EGF treatment was followed by increased translocation of Jab1 to the nucleus in both MDA-MB-231 and MDA-MB-468 breast cancer cell lines (Figure 1b). This effect is particularly evident in the merged images. Quantitative analysis of Jab1 nuclear expression confirmed that Jab1 levels were approximately two-fold higher following EGF treatment compared with untreated cells (Figure 1c). This difference was statistically significant in both cell lines tested (P < 0.01). These results were confirmed by immunoblots of extracts prepared from EGF-treated MDA-MB-231 cells, which showed increased nuclear Jab1 and a corresponding decrease in cytoplasmic Jab1 following EGF treatment (Figure 1d), as well as EGF dose-response experiments that showed increased nuclear Jab1 with increased EGF treatment concentration (Figure 1e). Similar observations were made in MDA-MB-468 cells (data not shown).


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)

Epidermal growth factor (EGF) stimulates increased nuclear localization of Jab1 in MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (a, b) Immunofluorescence assays. (c) Quantitative image analysis of nuclear Jab1 (bars represent mean ± standard deviation). Statistical analysis was by t test (*P < 0.01). (d) Increased expression of Jab1 in the nucleus relative to cytoplasm, detected by Western blot in MDA-MB-231 nuclear extracts. Lamin A/C was included as a positive control for the nuclear extract, and GAPDH was included as a loading control. (e) Increased Jab1 nuclear expression positively correlated to EGF dose. DAPI, 4'-6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Epidermal growth factor (EGF) stimulates increased nuclear localization of Jab1 in MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (a, b) Immunofluorescence assays. (c) Quantitative image analysis of nuclear Jab1 (bars represent mean ± standard deviation). Statistical analysis was by t test (*P < 0.01). (d) Increased expression of Jab1 in the nucleus relative to cytoplasm, detected by Western blot in MDA-MB-231 nuclear extracts. Lamin A/C was included as a positive control for the nuclear extract, and GAPDH was included as a loading control. (e) Increased Jab1 nuclear expression positively correlated to EGF dose. DAPI, 4'-6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jab1, c-Jun activation domain-binding protein-1.
Mentions: Jab1 has been shown previously to exist in both the nucleus and cytoplasm of different cell types. However, it has been shown that interactions between Jab1 and many of its downstream targets are associated with translocation of Jab1 to the nucleus. These include interaction with AP-1 [31], NF-κB [32], and p27 [32]. To determine whether Jab1 translocation is affected by EGFR signaling, we first used immunofluorescence microscopy to look for changes in cellular localization of Jab1 following treatment with EGF. We observed that EGF treatment was followed by increased translocation of Jab1 to the nucleus in both MDA-MB-231 and MDA-MB-468 breast cancer cell lines (Figure 1b). This effect is particularly evident in the merged images. Quantitative analysis of Jab1 nuclear expression confirmed that Jab1 levels were approximately two-fold higher following EGF treatment compared with untreated cells (Figure 1c). This difference was statistically significant in both cell lines tested (P < 0.01). These results were confirmed by immunoblots of extracts prepared from EGF-treated MDA-MB-231 cells, which showed increased nuclear Jab1 and a corresponding decrease in cytoplasmic Jab1 following EGF treatment (Figure 1d), as well as EGF dose-response experiments that showed increased nuclear Jab1 with increased EGF treatment concentration (Figure 1e). Similar observations were made in MDA-MB-468 cells (data not shown).

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