<p>Supplementary Figure 4 - Supplementary Figure 4. YB-1 down-regulates HER2 expression but HER2 down-regulation does not affect the MEK/ERK pathway - PDF file 196K, (A) LNCaP and PC-3 cells were transfected with 40 nmol/L of the indicated siRNA, and incubated for 72 h. Whole-cell extracts were subjected to SDS-PAGE and western blotting analysis. (B) LNCaP and PC-3 cells were transfected with 40 nmol/L of the indicated siRNA and incubated for 72 h, and then quantitative RT-PCR was performed for HER2 and GAPDH. The target transcript expression was adjusted to GAPDH expression. Each target transcript expression level in cells transfected with control siRNA was defined as 1. Boxes, mean; bars, plus-minus s.d.; *P < 0.05. (C) LNCaP and PC-3 cells were transfected with 40 nmol/L of the indicated siRNA, incubated for 72 h, incubated with serum-free media for 24 h, and treated with 50 ng/mL of EGF for 30 min. Whole-cell extracts were subjected to SDS-PAGE and western blotting analysis</p>
ARTICLE ABSTRACT
Purpose: Y-box–binding protein-1 (YB-1) is known to conduct various functions related to cell proliferation, anti-apoptosis, epithelial–mesenchymal transition, and castration resistance in prostate cancer. However, it is still unknown how YB-1 affects cancer biology, especially its correlations with the mitogen-activated protein kinase (MAPK) signaling pathway. Therefore, we aimed to examine the interaction between YB-1 and the MAPK pathway in prostate cancer.Experimental Design: Quantitative real-time PCR, Western blotting, and co-immunoprecipitation assay were conducted in prostate cancer cells. YB-1, phosphorylated YB-1 (p-YB-1), and ERK2 protein expressions in 165 clinical specimens of prostate cancer were investigated by immunohistochemistry. YB-1, p-YB-1, and ERK2 nuclear expressions were compared with clinicopathologic characteristics and patient prognoses.Results: EGF upregulated p-YB-1, whereas MEK inhibitor (U0126, PD98059) decreased p-YB-1. Inversely, silencing of YB-1 using siRNA decreased the expression of ERK2 and phosphorylated MEK, ERK1/2, and RSK. Furthermore, YB-1 interacted with ERK2 and Raf-1 and regulated their expressions, through the proteasomal pathway. Immunohistochemical staining showed a significant correlation among the nuclear expressions of YB-1, p-YB-1, and ERK2. The Cox proportional hazards model revealed that high ERK2 expression was an independent prognostic factor [HR, 7.947; 95% confidence interval (CI), 3.527–20.508; P < 0.0001].Conclusion: We revealed the functional relationship between YB-1 and MAPK signaling and its biochemical relevance to the progression of prostate cancer. In addition, ERK2 expression was an independent prognostic factor. These findings suggest that both the ERK pathway and YB-1 may be promising molecular targets for prostate cancer diagnosis and therapeutics. Clin Cancer Res; 19(17); 4638–50. ©2013 AACR.
