Tissue-based quantitative proteomics to screen and identify the potential biomarkers for early recurrence/metastasis of esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is the eighth cause of cancer-related deaths worldwide. To screen potential biomarkers associated with early recurrence/metastasis (R/M) of ESCC patients after radical resection, ESCC patients were analyzed by a comparative proteomics analysis using iTRAQ with RPLC-MS to screen differential proteins among R/M groups and adjacent normal tissues. The proteins were identified by qRT-PCR, Western blotting, and tissue microarray. The protein and mRNA expression difference of PHB2 between tumor tissues of ESCC patients and adjacent normal tissues, ESCC patients with and without metastasis, four ESCC cell lines and normal esophageal epithelial cells were inspected using immunohistochemical staining, qRT-PCR, and Western blotting. The EC109 and TE1 cells were used to establish PHB2 knockdown cell models, and their cell proliferation and invasion ability were determined by cell counting method, Transwell^® assay. Thirteen proteins were selected by cutoff value of 0.67 fold for underexpression and 1.5-fold for overexpression. Seven proteins were confirmed to be associated with R/M among the 13 proteins. The potential biomarker PHB2 for early recurrence/metastasis of ESCC was identified. PHB2 expression was related to the OS of ESCC patients (P = 0.032) and had high levels in the tumor tissues and human cell lines of ESCC (P = 0.0002). Also, the high PHB2 expression promoted the metastasis of ESCC (P = 0.0075), suggesting high PHB2 expression was a potential prognostic biomarker. Experiments showed that PHB2 could significantly promote the proliferation and cell invasion ability of human ESCC cell lines and the knockdown of PHB2 suppressed the phosphorylation level of AKT, as well as the expression of MMP9 and RAC1. PHB2 could predict the early metastasis of ESCC patients.

Keywords: Esophageal squamous cell carcinoma; metastasis; proteomic; recurrence; tissue microarray.

Figure 1

Study protocol.

Figure 2

Dysregulated proteins were screened by Western blotting and qRT‐PCR. (A) Differential proteins expression in 22 clinical ESCC samples was detected by Western blotting. (B) Differential mRNA expression in 22 clinical ESCC samples was detected by qRT‐PCR. Group A: tumor tissues from patients without R/M in 2 years after radical resection; Group B: tumor tissues from patients with recurrence after radical resection; Group C: tumor tissues from patients with metastasis after radical resection; Group D: adjacent normal tissues from patients without R/M in 2 years after radical resection. Statistical analysis was performed with two‐tailed unpaired t‐tests. *P < 0.05, **P < 0.01.

Figure 3

PHB2 expression was correlated with the OS of ESCC patients. (A and B) Kaplan–Meier curves of OS (A) and DMFS (B) in relation to PHB2 expression in ESCC patients with three‐field lymphadenectomy. Log‐rank P values are indicated within the graphs.

Figure 4

PHB2 had high expression levels in the tumor tissues and human cell lines of ESCC. (A and B) The PHB2 protein expression in the adjacent normal tissues (A) and tumor tissues (B) of ESCC was detected by immunohistochemical staining. (C) The PHB2 mRNA expression was compared between adjacent normal tissues and tumor tissues of 90 ESCC patients. (D and E) The PHB2 mRNA and protein expression in four human ESCC cell lines (EC109, TE1, EC9706, EC18) and a normal human esophageal epithelial cell line were determined by qRT‐PCR (D) and Western blotting (E). Statistical analysis was performed with two‐tailed unpaired t‐tests. *P < 0.05, **P < 0.01.

Figure 5

High expression of PHB2 promoted the metastasis of ESCC. The PHB2 mRNA expression was determined between metastasis and nonmetastasis in the ESCC patients. (B) The relative metastasis rate was counted among the ESCC patients with high or low PHB2 expression after surgery. Statistical analysis was performed with two‐tailed unpaired t‐tests.

Figure 6

PHB2 promoted the proliferation ability of human ESCC cells. (A and B) The knockdown PHB2 expression in TE1 and EC109 cells was determined by Western blotting (A) and qRT‐PCR (B). (C and D) The proliferation ability of TE1 (C) and EC109 (D) cells transfected with siRNA‐PHB2 or siRNA‐Control were detected by cell counting method. Statistical analysis was performed with two‐tailed unpaired t‐tests. *P < 0.05.

Figure 7

PHB2 promoted the invasion ability of human ESCC cells through activating the AKT signaling pathway. (A and B) The invasive ability was detected by the Transwell^® invasion assay. The two cell lines were transfected with siRNA‐PHB2 or siRNA‐Control. (C) The protein expression of p‐AKT, AKT, MMP9, RAC1 and PHB2 in the EC109 and TE1 cells was detected by Western blotting. The two cell lines transfected with siRNA‐PHB2 or siRNA‐Control. Statistical analysis was performed with two‐tailed unpaired t‐tests. *P < 0.05.