Snail levels control the migration mechanism of mesenchymal tumor cells

Abstract

Cancer cells use two major types of movement: Mesenchymal, which is typical of cells of mesenchymal origin and depends on matrix metalloproteinase (MMP) activity, and amoeboid, which is characteristic of cells with a rounded shape and relies on the activity of Rho-associated kinase (ROCK). The present authors previously demonstrated that, during neoplastic transformation, telomerase-immortalized human fibroblasts (cen3tel cells) acquired a ROCK-dependent/MMP independent mechanism of invasion, mediated by the downregulation of the ROCK cellular inhibitor Round (Rnd)3/RhoE. In the present study, cen3tel transformation was also demonstrated to be paralleled by downregulation of Snail, a major determinant of the mesenchymal movement. To test whether Snail levels could determine the type of movement adopted by mesenchymal tumor cells, Snail was ectopically expressed in tumorigenic cells. It was observed that ectopic Snail did not increase the levels of typical mesenchymal markers, but induced cells to adopt an MMP-dependent mechanism of invasion. In cells expressing ectopic Snail, invasion became sensitive to the MMP inhibitor Ro 28-2653 and insensitive to the ROCK inhibitor Y27632, suggesting that, once induced by Snail, the mesenchymal movement prevails over the amoeboid one. Snail-expressing cells had a more aggressive behavior in vivo, and exhibited increased tumor growth rate and metastatic ability. These results confirm the high plasticity of cancer cells, which can adopt different types of movement in response to changes in the expression of specific genes. Furthermore, the present findings indicate that Rnd3 and Snail are possible regulators of the type of invasion mechanism adopted by mesenchymal tumor cells.

Keywords: MMP; ROCK; Rnd3/RhoE; Snail; amoeboid movement; mesenchymal movement.

Figure 1.

Sna1 expression in cen3tel cells at different stages of transformation. (A) Results of microarray analysis. Sna1 expression in cen3tel cells is indicated relative to that in cen3 primary fibroblasts (log₂ of the ratio). (B) Western blot analysis of Snail levels. γ-tubulin was used as loading control. Ph, phase.

Figure 2.

Western blot analysis of the expression of (A) recombinant green fluorescent protein-Snail protein and (B) the mesenchymal markers vimentin and fibronectin in Snail-transfected clones and in mock-transfected cells (C1, lanes 7A and 8B). γ-tubulin was used as loading control. WT, wild-type; GFP, green fluorescent protein.

Figure 3.

Effect of the Rho-associated kinase inhibitor Y27632 and the matrix metalloproteinase inhibitor Ro 28–2653 on the invasion capacity of phase III tumorigenic cells, mock-transfected cells (C1 and C2 clones) and Snail-expressing clones. Invasion was measured using Boyden chambers. Grey columns, Ro 28–2653 (0.1 mM); black columns, Y27632 (10 mM). Invasion of cells exposed to inhibitors is shown as percentage of invasion in untreated cells (white columns). Data are presented as the mean ± standard deviation of 2–4 independent experiments. In cen3tel, C1 and C2 cells, the P-value refers to Y27632-treated cells vs. control cells, while in Snail clones, the P-value refers to Ro 28–2653-treated cells vs. control cells (*P<0.05; **P<0.005; ***P<0.0005). WT, wild-type.

Figure 4.

In vivo tumorigenic and metastatic capacity of Snail-expressing clones. (A) Growth curves of tumors obtained in SCID mice (n=3) after subcutaneous inoculation of mock-transfected phase III tumorigenic cen3tel cells (C1) or Snail-expressing clones (Snail-wt 6 and 9). Vertical bars indicate the standard error. (B) Number of lung metastases obtained upon intravenous injection of mock-transfected phase III tumorigenic cen3tel cells (C1) or Snail-expressing clones (Snail-wt 6 and 9) into SCID mice. In two mice inoculated with Snail-wt 9 cells, the precise number of metastases could not be determined due to their large abundance, and was arbitrarily assigned a value of 200. Horizontal bars represent the median number of metastases. WT, wild-type; SCID, severe combined immunodeficiency.