E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade

Abstract

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Figure 1.

Cellular and molecular effects of DEcad KD in vivo. A and D–H‴ are standard confocal sections with posterior side to the right and ventral side up. E‴, F‴, G‴ and H‴ are optical cross sections (apical side up). E″, F″, G″ and H″ are zooms from E–E′, F–F′, G–G′ and H–H′, respectively (white dashed boxes). (A) Control disc marked with anti-LanA and Topro nuclear staining. B and C are schemes of Drosophila wing disc regions and respective cross sections, in which hh-Gal4 (B) and ptc-Gal4 (C) drive transgene expression. A stands for anterior and P for posterior. (D–F‴) hh-Gal4 is driving UAS-DEcad-dsRNA and UAS-GFP (D–E‴)—green in D, E, E″ and E‴—or UAS-p35 (F–F‴). (G–H‴) ptc-Gal4 is driving UAS-DEcad-dsRNA and UAS-GFP (G–G‴)—green in G, G″ and G‴—or UAS-mCherry (H–H‴)—red in H, H″ and H‴. Discs are stained with (D–D″) anti-DEcad, anti-activated Caspase-3 and Topro, which marks nuclei or (A and E–G‴) anti-LanA (red in A, E, E″, E‴, F, F″, F‴, G, G″, G″ and white in E′, F′, G′) and anti-DEcad (green in F, F″, F‴ and white in D′, F′) or (H–H‴) anti-βPS integrin (green in H, H″, H‴ and white in H′). Yellow arrowheads highlight LanA or βPS accumulation at the DEcad+/DEcad– interface.

Figure 2.

Ecad+ versus Ecad– human gastric cancer cells in vitro. (AA‴) By analysing cell lysates, we observe that AGS cells negative for Ecad (A) express higher levels of LM-γ2 (A′), as well as integrin β1 (A″) and β4 (A‴). (B–B‴) In an Ecad inhibition model, Ecad-defective MKN28 cells (B) express higher levels of LM-γ2 (B′), integrin β1 (B″) and integrin β4 (B″) than those transfected with a non-targeting siRNA control. Graphs correspond to the average of protein expression levels obtained by band quantification. SE is represented as the error bar. Values were normalized to tubulin and to the respective control (AGS parental cells or MKN28 siCtr). (*) stands for P ≤ 0.05, (**) for P ≤ 0.01 and (***) for P ≤ 0.001.

Figure 3.

LM-γ2 functional significance. Inhibition of LM-γ2 (A) reduced cell invasion (B) and Src activity (C). (D) LM-γ2 induces resistance to apoptosis, as evidenced by a decrease in Caspase-3 levels in control cells when compared with those transfected with siRNA for LM-γ2. (E) pJNK underexpression and (F) pAkt upregulation in gastric cancer cells expressing LM-γ2 suggest that they are involved in the control of survival. Graphs correspond to protein expression levels obtained by band quantification or percentage of invasive cells. Values were normalized to tubulin and AGS parental cells transfected with non-targeting control siRNA (AGS siCtr). For the experiment in which apoptosis was stimulated, values were normalized by AGS siCtr treated with DMSO. Average ± SE is presented. (*) represents a significant difference of P ≤ 0.05, (**) of P ≤ 0.01 and (***) of P ≤ 0.001.

Figure 4.

Ecad and LM-γ2 expression in human gastric cancer samples. (A) Histological samples of normal versus tumour tissue from the Human Protein Atlas database are representative of Ecad downregulation and LM-γ2 overexpression in gastric carcinomas. (B) Data from the Human Protein Atlas database demonstrate an overall and concurrent tendency for Ecad intensity and quantity to decrease and for LM-γ2 to increase in gastric cancer tissues, when compared with normal mucosa. Manual annotation of each sample was performed by a board of certified pathologists or specially educated personnel, using a simplified classification scheme for the immunohistochemical outcome.

Figure 5.

Working model for the events that follow Ecad loss in wild-type epithelia. Cells with reduced Ecad are committed to die. However, ectopic expression of Laminin and its integrin receptors enables Ecad-defective cells to evade cell death/elimination from the epithelia (through Akt activation and JNK inhibition) and invade adjacent tissues (via pSrc phosphorylation).