Prognostic value and functional role of ROCK2 in pediatric Ewing sarcoma

Abstract

Ewing's sarcoma (EWS) is a highly aggressive bone cancer that affects children and adolescents. Despite advances in multimodal management, 5-year event-free survival rates for patients presenting with metastases at diagnosis remain at 25%. As key regulators of actin organization, the Rho-associated coiled-coil containing protein kinases, ROCK1 and ROCK2, have been associated with cancer dissemination and poorer prognosis. Recently, in vitro data indicating ROCK2 as a molecular target for the treatment of EWS has been presented. Nonetheless, a deeper exploration of the contribution of this kinase dysregulation in EWS is still necessary. In this regard, the present study aimed to evaluate the expression of ROCK1 and ROCK2 in 23 pediatric tumor samples and to verify the prospect of using their pharmacological inhibition through functional assays. Our results showed positive immunostaining for ROCK1 and ROCK2 in the majority samples (75 and 65%, respectively). A significantly increased risk of incomplete remission in patients with positive immunostaining for ROCK2 was found (P=0.026), though no correlations with other prognostic features (huvos classification, FLI1/EWS status, relapse, metastasis or death) were observed. Associations with survival were merely suggestive. Apparent protein expression of both kinases was also found in EWS cell lines (SK-ES-1 and RD-ES). Treatments with selective ROCK inhibitors did not alter cell viability or migration in vitro. However, a significant increase in invasion was observed after treatment with SR3677 (ROCK2 inhibitor) and hydroxyfasudil (pan-inhibitor). Consequently, even though the majority of EWS samples included in our study showed positivity for ROCK1 and ROCK2, the lack of significant associations with prognosis and absence of appropriate responses to their inhibition in vitro does not support their prospective use as therapeutic targets for the treatment of this metastatic tumor. Larger cohort studies might provide more evidence on whether there is a specific role of ROCK kinases in EWS physiopathology.

Keywords: Ewing sarcoma; ROCK; expression; inhibition; target.

Figure 1.

Representative microphotographs of immunodetection staining for ROCK1 and ROCK2 in pediatric EWS and patient's survival curves according to ROCK1 and ROCK2 protein profiles. (A) ROCK1 and ROCK2 positive and negative patterns, respectively. (B) Overall survival curves. (C) Event-free survival curves. Kaplan Meier curves. Log-rank test. Original magnification, ×200. ROCK, Rho-associated coiled-coil containing protein kinases; EWS, Ewing's sarcoma.

Figure 2.

Relative ROCK1 and ROCK2 expression in EWS cell lines. (A) Relative mRNA expression levels of ROCK1 and ROCK2 in primary osteoblast cell lines and the EWS cell lines RD-ES and SK-ES-1. ROCK2, but not ROCK1 was found with significantly higher expression (P=0.03) when compared to primary osteoblast cell lines with a 1.97 fold-change for RD-ES and 1.68 for SK-ES-1. Each column represents the mean ± standard deviation. (B) Protein expression levels of ROCK1, ROCK2 and the endogenous GAPDH in SK-ES-1 and RD-ES cell lines. The expression levels of both kinases were found comparable. ROCK, Rho-associated coiled-coil containing protein kinases; EWS, Ewing's sarcoma; FC, fold-change.

Figure 3.

Cell growth of RD-ES and SK-ES-1 cell lines was not affected by ROCK1 and ROCK2 inhibition. (A) Cell viability relative to control (DMSO) after treatment with the drugs GSK429286, SR3677 and hydroxyfasudil for 48, 72 and 96 h in the RD-ES and SK-ES-1 cell lines. (B) Cell cycle analysis of the RD-ES and SK-ES-1 cell lines after 24 h of treatment with the drugs GSK429286, SR3677 and hydroxyfasudil. (C) Analysis of the clonogenic capacity of RD-ES and SK-ES-1 cell lines after 48 h of treatment with drugs GSK429286, SR3677 and hydroxyfasudil. Each column represents the mean ± standard deviation from at least three independent experiments. ROCK, Rho-associated coiled-coil containing protein kinases; DMSO, dimethyl sulfoxide.

Figure 4.

Migration and invasive capacity of the SK-ES-1 cell line was not affected by ROCK1 and ROCK2 inhibition. (A) Migration capacity through wound healing assay at time 0 and after 24 h of treatment with the control (DMSO) and the drugs GSK429286, SR3677 and hydroxyfasudil. Gap closure was measured through the Motic Images Plus v2.0 software (Motic China Group Co., Ltd.). (B) Invasion capacity through Matrigel assay after 24 h of treatment. Increased invasion was observed after treatment with SR3677 (50 nM) and hydroxyfasudil (8 µM) suggesting a stimulating effect after ROCK2 inhibition. The data represents mean ± standard deviation from at least three independent experiments. Statistical analysis was performed through Student's t-test. (^#) Complete clinicopathological data was not available; (*) significant P-value (Fisher's exact test, 2-tailed). ROCK, Rho-associated coiled-coil containing protein kinases; DMSO, dimethyl sulfoxide.