Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition

Abstract

Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

FIGURE 1.

EGF induces EMT in PCa and SCCHN cells. A, a functional EGFR signaling system in ARCaP cells. Left panel, expression and phosphorylation of EGFR and neu(ErbB2) in ARCaP cells. A pan-p-EGFR antibody that detects phosphorylation at tyrosine residues 992, 1045, 1068, 1148, and 1173 was used. Right panel, EGF treatment (50 ng/ml) rapidly activates the phosphorylation of both EGFR and ErbB2 in ARCaP_E cells. B, EGF (50 ng/ml, 72 h) induces EMT in ARCaP_E cells. C, Western blot analyses of E-cadherin expression and nuclear translocation of β-catenin in ARCaP_E cells in response to EGF treatment (50 ng/ml). D, confocal microscopy showed that EGF (50 ng/ml) induces down-regulation and disassociation of EPLIN and E-cadherin, and nuclear translocation of β-catenin in ARCaP_E cells. E, EGF treatment (50 ng/ml) induces re-organization of the actin cytoskeleton in ARCaP_E cells. F, EGF (50 ng/ml, 18 h) treatment increases invasion of ARCaP_E cells through Matrigel in modified Boyden chamber. Error bars denote S.E. (n = 3). G, EGF treatment (100 ng/ml, 72 h) induces EMT-like morphological changes in 686LN cells.

FIGURE 2.

EGF induces EPLIN protein degradation in PCa and SCCHN cells. A, left panel: EGF treatment (50 ng/ml) induces progressive decrease in protein expression of both EPLIN isoforms in ARCaP_E cells. The total lysates were the same as used in Fig. 1C. Middle panel: EGF treatment (50 ng/ml) reduces protein expression of EPLIN and E-cadherin in C4-2 and PC3 cells. Right panel: EPLIN-α is the predominant EPLIN isoform in several human SCCHN cell lines, which is reduced in the more aggressive cell lines (37B, M4e) when compared with that in the low invasive cell lines (37A, 686LN) (top). EPLIN-α expression is reduced upon EGF treatment in 37A (50 ng/ml; middle) and 686LN (100 ng/ml; bottom) cells. B, the effects of EGF on the mRNA expression of EPLIN isoforms in ARCaP_E cells were assessed by regular (left panel) and real-time quantitative (middle panel) PCR and EPLIN-β reporter assay (right panel). Expression of GAPDH was used as control. Bars denote S.E. (n = 3). C, EGF (50 ng/ml) promotes protein turnover of both EPLIN isoforms, whereas treatment with a proteasome inhibitor MG132 partially rescues EPLIN in ARCaP_E cells in ARCaP_E cells. D, EGF induces polyubiquitination of EPLIN in a time-dependent manner. ARCaP_E cells were treated with 50 ng/ml EGF. Immunoprecipitation was performed at the indicated times with an anti-EPLIN antibody, and Western blot analyses were performed with an anti-polyubiquitin antibody. Input, total cell lysates; DMSO, dimethyl sulfoxide; CHX, cycloheximide.

FIGURE 3.

EGF induces serine phosphorylation and degradation of EPLIN through the ERK1/2 pathway. A, EGF (50 ng/ml) induces EPLIN phosphorylation at serine residues in ARCaP_E cells. B, EGF (50 ng/ml) activates ERK1/2 signaling in ARCaP_E cells. C, blockade of ERK1/2 signaling with PD98059 or U0126 inhibits EGF-induced EPLIN phosphorylation in ARCaP_E cells. Input: total cell lysates. D, blockade of ERK1/2 signaling inhibits EGF-induced EPLIN degradation in ARCaP_E cells. E, blockade of ERK1/2 signaling with U0126 inhibits EGF-induced EPLIN degradation in C4-2 cells. CHX, cycloheximide; DMSO, dimethyl sulfoxide.

FIGURE 4.

Ser-362 and Ser-604 are critical to EGF-induced phosphorylation and degradation of EPLIN. A, left panel: Ser-362 is an ERK substrate and adjacent to a putative PEST sequence in human EPLIN protein. Right panel: schematic diagram of the EPLIN-GFP construct and its point mutants at Ser-362, Ser-604, or Ser-362/Ser-604. B, mutation at Ser-362, Ser-604, or Ser-362/Ser-604 abrogates EGF-induced EPLIN phosphorylation in PC3 cells. Immunoprecipitation with anti-GFP antibody and subsequent blotting with p-Ser antibody were performed. Input: total cell lysates. C, mutation at Ser-362, Ser-604, or Ser-362/Ser-604 inhibits EGF-induced ubiquitination of EPLIN in PC3 cells. Immunoprecipitation with anti-GFP antibody and subsequent blotting with polyubiquitin antibody were performed. Input: total cell lysates. D, mutation at Ser-362, Ser-604, or Ser-362/Ser-604 inhibits EGF-induced EPLIN degradation in PC3 cells. Protein T½ assays were performed in PC3 cells transiently expressing wild type (WT) EPLIN or the point mutants. An anti-GFP antibody was used to detect the presence of EPLIN-GFP proteins. E, mutation at Ser-362, Ser-604, or Ser-362/Ser-604 inhibits EGF-induced down-regulation of EPLIN and E-cadherin in C4-2 and PC3 cells.

FIGURE 5.

EGF induces in vivo down-regulation of EPLIN and E-cadherin in ARCaP_E xenograft tumors. A, intraperitoneal administration of recombinant EGF does not significantly affect the subcutaneous growth of ARCaP_E tumor in athymic nude mice. (B) EGF treatment markedly reduces tissue expression of EPLIN and E-cadherin, whereas increases the expression of p-EGFR and β-catenin in subcutaneous ARCaP_E tumors.