Autocrine epiregulin activates EGFR pathway for lung metastasis via EMT in salivary adenoid cystic carcinoma

Abstract

Salivary adenoid cystic carcinoma (SACC) is characterized by invasive local growth and a high incidence of lung metastasis. Patients with lung metastasis have a poor prognosis. Treatment of metastatic SACC has been unsuccessful, largely due to a lack of specific targets for the metastatic cells. In this study, we showed that epidermal growth factor receptors (EGFR) were constitutively activated in metastatic lung subtypes of SACC cells, and that this activation was induced by autocrine expression of epiregulin (EREG), a ligand of EGFR. Autocrine EREG expression was increased in metastatic SACC-LM cells compared to that in non-metastatic parental SACC cells. Importantly, EREG-neutralizing antibody, but not normal IgG, blocked the autocrine EREG-induced EGFR phosphorylation and the migration of SACC cells, suggesting that EREG-induced EGFR activation is essential for induction of cell migration and invasion by SACC cells. Moreover, EREG-activated EGFR stabilized Snail and Slug, which promoted EMT and metastatic features in SACC cells. Of note, targeting EGFR with inhibitors significantly suppressed both the motility of SACC cells in vitro and lung metastasis in vivo. Finally, elevated EREG expression showed a strong correlation with poor prognosis in head and neck cancer. Thus, targeting the EREG-EGFR-Snail/Slug axis represents a novel strategy for the treatment of metastatic SACC even no genetic EGFR mutation.

Keywords: EGFR; EMT; EREG; SACC; lung metastasis.

Figure 1. Lung metastatic SACC-LM cells exhibit EMT characteristics

(A) The transwell migration and invasion assays established the migration and invasion capability of SACC-83 and SACC-LM cells with representative images shown. Scale bar = 200 μm. (B) Graphic representation of the percent of migrated cells from 3 separate experiments (mean ± SD). * indicates a p < 0.05. (C) Representative images of wound healing for SACC-83 and SACC-LM cells. Scale bar = 200 μm. (D) The number of migrated cells within the areas of healing surpassing the red lines was determined, and each experiment was repeated 3 times. * indicates a p < 0.05. (E) Representative images of the morphology and staining for E-cadherin, N-cadherin and vimentin in SACC-83 and SACC-LM cells. Scale bar = 200 μm. (F) Western blot analysis of E-cadherin, N-cadherin, ZO-1, vimentin, Snail and Slug protein levels in SACC-83 and SACC-LM cell lines.

Figure 2. Autocrine EREG secretion contributes to the auto-activation of EGFR in highly metastatic SACC

(A) Western blot analysis of p-EGFR, EGFR, p-AKT, AKT, p-STAT3, STAT3, p-ERK and ERK protein levels in SACC-83 and SACC-LM cell lines. (B) Immunofluorescence staining for EGFR is presented with DAPI (blue) nuclear staining. Scale bar = 200 μm. (C) Analysis of EREG mRNA levels with fold change in SACC-LM cells compared to SAC-83 cells using published chip assay data. (D) The mRNA and protein levels of EREG in SACC-83 and SACC-LM cell lines by RT-PCR and Western blot analysis, respectively. (E) The mRNA level of HB-EGF, TGF-α, AREG, EGF in SACC-83 and SACC-LM cell lines. (F) Western blot analyses of p-EGFR and EGFR from SACC-LM cells that were serum-starved as indicated. (G) Graphic representation of the ratio of p-EGFR and EGFR to GAPDH for indicated time points in SACC-LM cells. (H) Western blot analyses of p-EGFR in SACC-LM cells that were starved for 0.5h and then treated with EREG neutralizing antibody or with normal Ig G for 6 hours.

Figure 3. EREG activated EGFR/Akt/ERK/STAT3 pathways and promoted migration of SACC cells

(A) Western blot analyses for P-EGFR, EGFR, P-AKT, AKT, P-ERK, ERK, p-STAT3, STAT3 in SACC-83 cells treated with 0 to 1000 ng/mL rhEREG for 4 hours. (B) Western blot analyses for P-EGFR, EGFR, P-AKT, AKT, P-ERK, ERK, p-STAT3, STAT3 in SACC-83 cells treated with 50 ng/mL rhEREG as indicated. (C) Representative images of migration by SACC-83 cells treated with or without rhEREG (50 ng/ml) for 36 hours with representative images shown. Scale bar = 200 μm. (D) Graphic representation of the percent of migrated cells treated with rhEREG from 3 separate experiments as outlined in (C) with the mean ± SD indicated, *p < 0.05. (E) Representative images of migration by SACC-LM cells treated with EREG neutralizing antibody or with normal Ig G for 36 hours with representative images shown. Scale bar = 200 μm. (F) Graphic representation of the percent of migrated cells treated with EREG antibody from 3 separate experiments as outlined in (E) with the mean ± SD indicated, *p < 0.05.

Figure 4. EREG induces protein stabilization of snail and slug

(A) Western blot analyses for Snail and Slug in SACC-83 cells treated with or without 50 ng/mL rhEREG for 4 hours. (B) RT-PCR analyses for mRNA levels of Snail and Slug. (C) Immunofluorescence staining for Snail and Slug in SACC-83 cells treated with or without 50 ng/mL rhEREG for 4 hours. Scale bar = 200 μm. (D) Western blot analyses for Snail and Slug from SACC-83 cells treated with 50 ng/mL rhEREG as indicated. (E) Western blot analyses for Snail and Slug from SACC-83 cells treated with 0 to 1000 ng/mL rhEREG for 4 hours.

Figure 5. EREG enhanced Snail and Slug stability require EGFR activation

(A–B) SACC-83 cells were treated with or without rhEREG for 2 hr, followed by incubation with cycloheximide (CHX; 10 μM) for an extended period of time. The levels of Snail and Slug were determined by Western blot analysis. (C–D) Graphic representation of densitometry results for Snail (C) and Slug (D) after cycloheximide treatment (circle, with rhEREG; square, without rhEREG). (E) Western blot analysis of Snail, Slug, P-EGFR, EGFR, P-AKT, AKT, P-ERK, ERK, p-STAT3, STAT3 from SACC-83 cells pretreated with various inhibitors for 1 hr followed by stimulation with rhEREG for 2 hr.

Figure 6. Targeting EGFR inhibits the migration and invasion capabilities of SACC-LM cells

(A) Western blot analysis of p-EGFR and EGFR from SACC-LM cells treated with 0 to 20 uM erlotinib for 72 hours. (B) Western blot analysis of E-cadherin and vimentin levels from SACC-LM cells treated with 2 uM erlotinib for 72 hours. (C) Representative images of migration by SACC-LM cells treated with or without erlotinib (2 uM) for 24 hours. Scale bar = 200 μm. (D) Graphic representation of percent migrated cells from 3 separate experiments with mean ± SD percent indicated. * indicates a p < 0.05. (E) Representative images of wound healing by SACC-LM cells treated with or without erlotinib (2 uM) for 24 hours. Scale bar = 200 μm. (F) Graphic representation of the number of migrated cells within the areas of healing surpassing the red lines from 3 separate experiments. * indicates a p < 0.05.

Figure 7. Targeting EGFR inhibits the lung metastasis of SACC in vivo

(A) SACC-LM cells treated with or without erlotinib (2 uM) for 72 hours were injected into the tail vein of nude mice. Histopathologic analysis shows small metastatic nodules in lung tissues. (B) Graphic representation of the area of lung metastases with mean ± SD; n = 5. (C–D) Patient survival obtained from publicly available microarray data was analyzed based on EREG mRNA expression level.