Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

Abstract

Angiopoietin-like 4 (ANGPTL4) promotes cancer cell migration through vessels and has been implicated in cancer metastasis. Our previous study identified a robust increase in ANGPTL4 mRNA expression in lung-metastasized tongue cancer (TC) cells. Therefore, the present study investigated the association of ANGPTL4 with lung metastasis and outcomes of patient with TC. ANGPTL4 expression in TC cells was investigated by immunohistochemical staining. Patients were classified into 'low (0-30%)' and 'high (>30%)' ANGPTL4-expression groups based on the proportion of ANGPTL4-positive TC cells. The high ANGPTL4-expression group included 15 of 48 patients with TC. Notably, a significantly greater proportion of patients with lung metastasis exhibited a high rate of ANGPTL4-expressing cancer cells compared with patients without lung metastasis (P=0.029). The overall 5-year survival rate was lower in the high (27%) ANGPTL4-expression group compared with the low (68%) ANGPTL4-expression group. Univariate and multivariate analyses revealed that patients with high ANGPTL4 expression in TC cells exhibited significantly lower overall survival (OS) rates [hazard ratio (HR), 2.99; 95% confidence interval (95% CI), 1.34-6.69; P=0.008 and HR, 2.72; 95% CI, 1.14-6.51; P=0.024, respectively]. High plasma ANGPTL4 concentrations as measured by ELISA were associated with lung metastasis (P<0.001). The optimal cut-point for prediction of TC lung metastasis was 9.1 ng/ml (P<0.001; 95% CI, 7.2-10.9). The OS of patients with plasma ANPTL4 above the cut-point was significantly lower than that of patients with plasma ANGPTL4 ≤9.1 ng/ml (P<0.001). These results suggest that a high level of ANGPTL4 in cancer cells and plasma may predict lung metastasis and/or a poor prognosis of patients with TC.

Keywords: ANGPTL4; lung metastasis; plasma; prognosis; survival; tongue cancer.

Figure 1.

Immunohistochemical staining of ANGPTL4. Immunohisto-chemical staining for cancer tissues of patients with TC was performed using an anti-ANGPTL4 antibody. Representative images showing TC cells that were found to exhibit (A) a high (positive cell rate >30%) or a low [positive cell rate (B) 1-30% or (C) ≈0%) rate of ANGPTL4 expression. (D) Noncancerous tongue epithelial cells. Scale bars, 100 µm. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Figure 2.

Association of overall patient survival rate with ANGPTL4 expression in TC. The overall survival rate was evaluated for groups of patients with TC classified by high (n=15; solid line) or low (n=33; dashed line) rates of ANGPTL4 expression in their collected TC tissue samples. Statistical analysis was performed using the Kaplan-Meier method and verified using the log-rank test. The table of numbers at risk at the bottom of the figure indicates the number of patients living just before the time point. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Figure 3.

ANGPTL4 plasma concentrations in patients with TC with or without lung metastasis. ANGPTL4 plasma concentrations were compared between patients with TC with and without lung metastasis. Open circles indicate individual ANGPTL4 plasma concentrations of patients with TC (n=20 each). Closed circles and bars denote averages and standard deviation values, respectively. Statistical analysis was performed using an unpaired Student's t-test. *P<0.001. The dashed line indicates the optimal cut-point of the plasma ANGPTL4 concentration for prediction of TC lung metastasis. The optimal cut-point was determined by bootstrapped receiver operating characteristic analysis under Liu's method using 1,000 bootstrap samples. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Figure 4.

Correlation of overall patient survival rate with plasma ANGPTL4 concentration in TC. The overall survival rate was evaluated for groups of patients with TC classified by ANGPTL4 concentration >9.1 ng/ml (n=20; solid line) or ≤9.1 ng/ml (n=20; dashed line). Statistical analysis was performed using the Kaplan-Meier method and verified using the log-rank test. The table of numbers at risk at the bottom of the figure indicates the number of patients living just before the time point. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Figure 5.

Promotive effects of ANGPTL4 on various stages of tongue cancer cell metastasis from the tongue to lungs. Black arrows indicate the stages of metastasis promoted by ANGPTL4. ANGPTL4, angiopoietin-like 4.