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. 2019 Oct;20(4):3317-3325.
doi: 10.3892/mmr.2019.10562. Epub 2019 Aug 6.

Afatinib, an EGFR inhibitor, decreases EMT and tumorigenesis of Huh‑7 cells by regulating the ERK‑VEGF/MMP9 signaling pathway

Yafei Chen  1 , Xin Chen  1 , Xiaojun Ding  1 , Yingwei Wang  1
Affiliations

Afatinib, an EGFR inhibitor, decreases EMT and tumorigenesis of Huh‑7 cells by regulating the ERK‑VEGF/MMP9 signaling pathway

Yafei Chen et al. Mol Med Rep. 2019 Oct.

Abstract

Transcatheter arterial embolization (TAE) therapy has been used in the treatment of inoperable hepatocellular carcinoma (HCC). However, tumor recurrence and metastasis are common in patients after TAE, and these processes may be caused by circulating tumor cells (CTCs). Epithelial‑mesenchymal transition (EMT) serves important roles in CTCs, and abnormal expression and activation of epidermal growth factor receptor (EGFR) is common in cancer cells. Afatinib is an EGFR‑tyrosine kinase inhibitor (TKI). The present study aimed to investigate the effects of afatinib on EMT and tumorigenesis in HCC cells. Western blot analysis suggested that afatinib was able to effectively suppress overactivation of EGFR. Moreover, the expression levels of EMT‑ and metastasis‑associated genes were found to be modulated by afatinib through EGFR inhibition. In addition, Cell Counting Kit‑8 and Transwell assays suggested that the viability, migration and invasion of HCC cells were inhibited by afatinib through EGFR inhibition. Furthermore, the activity of the ERK signaling pathway and the expression levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) were decreased following treatment with afatinib in vitro. Collectively, the present results suggested that the inhibitory effects of afatinib on EMT and tumorigenesis may be associated with the ERK‑VEGF/MMP9 signaling pathway. The present study provides new insights into understanding the mechanism underlying HCC and may facilitate the development of novel therapeutic strategies to treat HCC recurrence.

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Figures

Figure 1.
Figure 1.
Detection of the expression levels of EGFR and E-cadherin in HCC tumor samples. (A) Reverse transcription-quantitative PCR and (B) western blot analysis of EGFR and E-cadherin. (C) Densitometric analysis of the protein expression levels of EGFR and E-cadherin. n=42. *P<0.05, **P<0.01. EGFR, epidermal growth factor receptor; TAE, transcatheter arterial embolization; HCC, hepatocellular carcinoma.
Figure 2.
Figure 2.
Detection of the expression levels of EGFR, E-cadherin, Vimentin, MTA1 and TIAM1 in HCC tumor samples. (A) mRNA and (B) protein expression level of EGFR. (C) Western blot analysis of EGFR and p-EGFR. (D) mRNA and (E) protein expression levels of E-cadherin, Vimentin, MTA1 and TIAM1. (F) Western blot analysis of E-cadherin, Vimentin, MTA1 and TIAM1. n=3. *P<0.05, **P<0.01 vs. corresponding control. EGFR, epidermal growth factor receptor; MTA1, metastasis associated 1; TIAM1, T cell lymphoma invasion and metastasis 1; si-CTR, scrambled siRNA; si-EGFR, siRNA targeting EGFR; siRNA, small interference RNA; p-, phosphorylated; EGFR-TKI, afatinib treatment; TKI, tyrosine-kinase inhibitor.
Figure 3.
Figure 3.
Effect of afatinib on the proliferation, migration and invasion of HCC cells. (A) Cell Cycle Kit-8 assay was performed to investigate cell viability. Relative quantification of (B) migration and (C) invasion of HCC cells. Transwell (D) migration and (E) invasion assay for the migration and invasion ability of HCC cells. n=3. *P<0.05. EGFR, epidermal growth factor receptor; si-CTR, scrambled siRNA; si-EGFR, siRNA targeting EGFR; siRNA, small interference RNA; EGFR-TKI, afatinib treatment; TKI, tyrosine-kinase inhibitor; HCC, hepatocellular carcinoma.
Figure 4.
Figure 4.
Effect of afatinib on the ERK-VEGF/MMP9 signaling pathway. (A) Western blot and (B) densitometry analysis of VEGF and MMP9 protein expression levels. (C) Western blot and (D) densitometry analysis of ERK and p-ERK levels. The ratio of p-ERK/total ERK was evaluated. n=3. *P<0.05 vs. corresponding control. EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; siCTR, scrambled siRNA; si-EGFR, siRNA targeting EGFR; siRNA, small interference RNA; EGFR-TKI, afatinib treatment; TKI, tyrosine-kinase inhibitor; p-, phosphorylated.
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