Laminin-332 promotes the invasion of oesophageal squamous cell carcinoma via PI3K activation

Abstract

Laminin-332 is major component of epithelial basement membrane, and has an important role in cell migration and tumour invasion. Recently, the phosphatidylinositol 3-kinase (PI3K) activation induced by laminin-332 during carcinogenesis or tumour invasion has been highlighted in skin squamous cell carcinoma. The expression of laminin-332 in 126 resected oesophageal squamous cell carcinoma (ESCC) specimens was immunohistochemically examined to determine its associations with the clinicopathological characteristics, and the effect of laminin-332 on the invasiveness and the PI3K activation was assessed by in vitro experiments using ESCC cell lines (ESCCs). Sections with immunostaining signals in >30% cancer cells, which were observed in 55 of 126 cases, were judged to be positive for laminin-332. The positivity was significantly correlated with pTNM stage and poor prognosis. Inactivation of the PI3K pathway by laminin-332 blocking antibody suppressed the invasiveness of TE8 cell line, which secreted laminin-332 at high level and had high PI3K activity. The addition of the purified laminin-332 activated the PI3K pathway and increased the invasiveness of TE11 cell line, which secreted laminin-332 at lower level and had low PI3K activity. The deactivation of PI3K pathway using the PI3K inhibitor decreased the invasiveness of ESCCs and the secretion of laminin-332 in vitro. The expression of laminin-332 was one of the prognostic factors of ESCC. Laminin-332 could provide the autocrine positive-feedback loop through PI3K activation, contributing the invasive ability. Therefore, the inhibitor of PI3K pathway might be useful as the anticancer therapies for ESCC.

Figure 1

Immunohistochemical staining of laminin-332. Original magnification: × 200 (A,C,D) × 400 (B). (A, B) Expression of laminin-332 in normal oesophageal epithelium. Laminin-332 is expressed faintly in the cytoplasm of epithelial cells in the basal layer. (C) Positive staining of laminin-332 of ESCC is indicated. Laminin-332 is clearly shown in the cytoplasm of ESCC. (D) Negative staining of laminin-332 of ESCC is indicated.

Figure 2

(A) Disease-free survival in relation to laminin-332 expression status of ESCC patients. (B) Overall cancer-specific survival in relation to laminin-332 expression status.

Figure 3

(A) Expression of laminin-332 in the conditioned medium of each ESCCs. All five ESCCs (TE1, 8, 9, 10, 11) secreted laminin-332, but the secreted level of TE11 is low. In the reduced medium (upper panel), the 155-kD band shows the un-cleaved laminin-332 γ2 chain, and the 105-kD band shows the cleaved laminin-332 γ2 chain. In the non-reduced medium (lower panel), the upper band shows the laminin-332 heterotrimer including the un-cleaved laminin-332 γ2 chain, and the lower band shows the laminin-332 heterotrimer including the cleaved laminin-332 γ2 chain. (B) Expression of EGFR and Integrin β4 in each ESCCs. All five ESCCs express EGFR. The band intensity shows the expression at the highest level in TE8 and at the lowest level in TE1. All five ESCCs express integrin β4 approximately at the same levels. (C) The activity of PI3K pathway was evaluated by Western blot analysis using the p-Akt antibody. The band intensity of p-Akt shows expression at higher level in TE8, TE9 and TE10 than in TE1, TE11.

Figure 4

(A) The effect of the laminin-332 blocking antibody at different concentration on PI3K pathway in TE10 cell line. The laminin-332 blocking antibody suppresses the activation of PI3K pathway at the concentration of >40 μg/ml. (B) The effect of the laminin-332 blocking antibody on the invasiveness of TE10. The number of invaded cells decreases to an approximately half with the laminin-332 blocking antibody (40 μg/ml) by comparison with non-treatment or control IgG antibody (40 μg/ml). The PI3K inhibitor (Wortmannin;100 nM) decreased to an approximately quarter by comparison with non-treatment.

Figure 5

(A) The effect of the purified laminin-332 at different concentration on PI3K pathway in TE11 cell line. The addition of the purified laminin-332 activated the PI3K pathway at the concentration of >0.5 μg/ml. (B) The effect of the purified laminin-332 on the invasiveness of TE11 cell line. The number of invaded cells increases with additional laminin-332 (0.5 μg/ml) by comparison with non-treatment or control Albumin (0.5 μg/ml). However, the number of invaded cells decreased with both additional laminin-332 (0.5 μg/ml) and Wortomannin (100 nM).

Figure 6

The relationship between the secretion of laminin-332 and PI3K pathway in TE10 cell line. The PI3K inhibitor (Wortmannin) decreases the activity of PI3K pathway in a dose-dependent manner, and the amount of the secreted laminin-332 into the medium decrease in proportion to the activity of PI3K pathway.