Targeting ROCK2 rather than ROCK1 inhibits Ewing sarcoma malignancy

Abstract

Understanding the molecular processes characterizing Ewing sarcoma (EWS) cell migration is crucial to highlight novel therapies for patients with disseminated disease. In this study we analyzed the role of ROCK kinases in the regulation of cell migration, growth and differentiation of EWS cells. Overexpression of ROCK promotes invasion and metastasis in many solid tumors. However, the effect of ROCK in EWS has not been extensively investigated. Expression of ROCK1 and ROCK2 was analyzed by western blotting in a representative panel of human EWS cell lines, in comparison with the parameters of in vitro malignancy. We investigated the effects of a ROCK2 specific inhibitor toward those of a pan-ROCK inhibitor on the growth, migration and differentiation of two EWS cell lines. ROCK2 but not ROCK1 expression was found to be associated with in vitro cell migration and anchorage‑independent growth capabilities. Exposure of EWS cells to ROCK inhibitors significantly reduced migration and growth, while favoring morphology changes and neural differentiation. These effects were more striking when cells were specifically deprived of ROCK2 activity. Our findings lead to consider ROCK2, rather than ROCK1, as a possible molecular target for the treatment of EWS.

Figure 1.

ROCK2, rather than ROCK1, affects Ewing sarcoma (EWS) malignancy. (Α) Densitometric analysis of ROCK1 and ROCK2 expression evaluated by western blotting in a representative panel of EWS cell lines. ROCK1/actin and ROCK2/actin ratios are expressed as adjusted volume optical density (OD/mm²) and each column represents mean ± SE of at least three separate experiments. (Β) Migration ability of EWS cell lines. Each column represents mean ± SE of at least two separate experiments. (C) Anchorage-independent growth of EWS cell lines. Each column represents mean ± SE of at least two separate experiments. (D) Western blotting of ROCK2 and ROCK1 expression in STA-ET 2.1 and STA-ET 2.2 EWS cell lines. Equal loading was monitored by anti-actin blotting. (E) Migration ability (left panel) and anchorage-independent growth (right panel) of the STA-ET 2.1 and STA-ET 2.2 EWS cell lines. Each column represents mean ± SE of at least two separate experiments. *P≤0.05; **P≤0.001, paired Students t-test.

Figure 2.

ROCK2 inhibition impairs the migratory properties and anchorage-independent growth capabilities of human Ewing sarcoma (EWS) cell lines. Effect of the Y27632 inhibitor (10 µM) and the Stemolecule™ ROCKII Inhibitor (10 µM) on (A) migration and (B) growth in soft agar of 6647 (left panels) and SKES-1 (right panels) cell lines. Each column represents the mean ± SE of at least two separate experiments performed in triplicate. *P≤0.05; **P≤0.001, paired Students t-test.

Figure 3.

ROCK2 inhibition affects the differentiation properties of Ewing sarcoma (EWS) cells. (A) Effect of the Y27632 inhibitor (2 µM and 10 µM) and the Stemolecule™ ROCKII Inhibitor (2 µM and 10 µM) on neurite outgrowth. Each column represents the mean ± SE of the percentage of cells bearing neurites from at least five randomly selected fields. The numbers above each bar represent the ratio between the total number of cells bearing neurites and the total number of cells counted. ***P≤0.0001, paired Students t-test. (B) Immunostaining of βIII-tubulin in the 6647 (upper panel) and SKES-1 cells (lower panel) treated with Y27632 inhibitor (2 µM and 10 µM) or the ROCK2 inhibitor (2 µM and 10 µM). Digital images were taken in identical conditions using the image analysis software NIS-Elements (Nikon Instruments S.p.A.). Magnification, ×600; scale bar, 20 µm. (C) Effect of the Y27632 inhibitor (2 µM and 10 µM) and the ROCK2 inhibitor (2 µM and 10 µM) on 6647 (upper panel) and SKES-1 (lower panel) cell morphology. Magnification, ×100.

Figure 4.

ROCK2 inhibition and proliferation of Ewing sarcoma (EWS) cells. Effect of the Y27632 inhibitor (10 µM) and the ROCK2 inhibitor (10 µM) on the growth in monolayer cell culture of 6647 and SKES-1 cell lines.