EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition

Abstract

There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.

Keywords: CK13, cytokeratin 13; CSCs, cancer stem-like cells; DMSO, dimethyl sulfoxide; EMT, epithelial–mesenchymal transition; EGFR, epidermal growth factor receptor; ESCC, esophageal squamous cell carcinoma; FACS, fluorescence-activated cell sorting; IHC, immun; EGFR inhibitor; ZEB1; cancer stem cell; epithelial-mesenchymal transition; esophageal squamous cell carcinoma; organotypic 3D culture.

Figure 1.

EGFR inhibitors suppressed enrichment of CSCs induced by TGF-β1. (A) EPC2T cells and OKF6T cells were treated with or without erlotinib (2.5 μM) and TGF-β1 (5 ng/ml) for 72 hours. CD44 high expressing CSCs were enriched by TGF-β1 and the enrichment of CSCs by TGF-β1 was significantly suppressed by erlotinib. (*P < 0.05 vs. DMSO control, #P < 0.05 vs TGF-β1) (B) EPC2T cells and OKF6T cells were treated with or without cetuximab (10 μg/ml) and TGF-β1 (5 ng/ml) for 72 hours. Fold change of CD44 high expressing CSCs was shown. (*P < 0.05 vs. DMSO control, # P < 0.05 vs TGF-β1)

Figure 2.

EGFR inhibitors suppressed expression of ZEB1 and ZEB2. (A) EPC2T cells were treated with erlotinib for 72 hours and expression levels of ZEB1 and ZEB2 were determined by real-time RT-PCR. (*P < 0.05 vs. DMSO control) (B) EPC2T cells were treated with cetuximab (10 μg/ml) for 72 hours and expression levels of ZEB1 and ZEB2 were determined by real-time RT-PCR. (* P < 0.05 vs. vehicle control)

Figure 3.

Erlotinib upregulated Notch transcriptional factors and induced differentiation. EPC2T cells and OKF6T cells were treated with erlotinib (2.5 μM) for 72 hours and expression levels of indicated genes were determined by real-time RT-PCR. Notch1 and Notch3 are critical transcriptional factors in keratinocyte differentiation. CK13 and involucrin (IVL) are differentiation markers of keratinocytes. (* P < 0.05 vs. DMSO control)

Figure 4.

Erlotinib suppressed tumor growth and invasion as well as enrichment of CSCs in organotypic 3D culture. EPC2T cells and OKF6T cells were cultured in organotypic 3D culture system with or without erlotinib (5 μM). (A) Tissues were stained by hematoxylin and eosin (H&E) (upper panels), anti-phospho EGFR antibody (middle panels) and anti-E-cadherin antibody (lower panels). Scale bar indicates 100 μm. (B) Cell growth was evaluated by Ki-67 staining. Histograms show percentage of Ki-67 positive cells at the basal layer. Scale bar indicates 100 μm. (*P < 0.05 vs. DMSO control) (C) Tissues were stained by anti-CD44 antibody. Scale bar indicates 100 μm.

Figure 5.

CD44 high expressing CSCs were reduced by erlotinib in organotypic 3D culture. EPC2T cells and OKF6T cells were cultured in organotypic 3D culture system with or without erlotinib (5 μM). Then, tumor cells were isolated from the whole organotypic 3D culture tissue and expression levels of CD24 and CD44 were analyzed by FACS. (*P < 0.05 vs. DMSO control)