ROCK inhibition with Fasudil induces beta-catenin nuclear translocation and inhibits cell migration of MDA-MB 231 human breast cancer cells

Tumor aggressiveness is usually associated with metastasis. MDA-MB 231, a triple-negative breast cancer (TNBC), is an aggressive type of breast cancer and associated with early metastasis. The Rho/ROCK pathway is a key regulator of cell motility involving cytoskeleton regulation through stabilization of actin filaments and stress fiber formation. In this study we show that Fasudil, a ROCK inhibitor, inhibited the migration of MDA-MB 231 and A549 cells, without altering the viability of these cells at the concentration of 10 μM, modified tumor cell morphology, with disorganization of stress fibers and promotes activation of the canonical-Wnt/beta-catenin pathway. Therefore, Fasudil present a promising approach to the prevention of breast cancer metastasis through a different mechanism of action from the well-known one.

including myosin light chain (MLC), myosin light chain phosphatase (MLCP), LIM kinase (LIMK), all of which are involved in cytoskeleton regulation through stabilization of actin filaments and stress fiber formation 7 .
The Wnt signaling pathway is an evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. Perturbation of Wnt signaling with aberrant expression of Wnt factors, their receptors, or downstream signaling molecules may lead to the development of several human cancers 8 . Recently our group demonstrated that the disorganization of cholesterol enriched-lipid rafts leads to Wnt signaling resulting in reduced tumor cells migration 9 .
For the design of rational therapies, it is crucial to understand mechanisms that underlie the metastatic behaviour of TNBC cells and to characterise high risk metastasis. Recent studies identify ROCK as a promising candidate for a therapeutic target that could treat patients with highly metastatic cancer 10 . However, the function of ROCK particularly during the migration of TNBC cells is unclear, which hampers the precise interpretation of this target. Here, we show that Fasudil, a ROCK-inhibitor, induces a non-migratory phenotype in MDA MB 231 cells, with disorganization of stress fibers and activation of the canonical-Wnt/beta-catenin pathway. The collection of our data identifies a TNBC-specific mechanism of ROCK and beta-catenin and demonstrates the relevance of a cell-type specific background for the cancer-type-specific role of a protein kinase.

Results
Cell viability. To evaluate the effects of Fasudil on cell viability we performed a MTT-based and a lactate desidrogenase (LDH)-based assay. We analysed the viability of the cells after 24 and 48 h of treatment with increasing concentrations of Fasudil (0.1, 1, 10, 50 and 100 µM). The results of the MTT assay showed that from 0.1 to 50 µM of Fasudil cell viability was not altered after 24 or 48 h of treatment, whereas 100 µM of Fasudil reduced cell viability in both 24 h (25% reduction) and 48 h (10% reduction) of incubation in the MTT assay (Fig. 1A). When analysing LDH liberation by cells incubated with same concentrations of Fasudil we observed that even higher concentration (100 µM) of Fasudil did not induce liberation of the enzyme (Fig. 1B). To rule out a possible cell-specific effect we performed the same assays using a lung tumor cell line (A549). In this context, no alteration was observed in the release of LDH nor MTT conversion (data not shown).
Cell migration quantified by the cell-based scratch assay. To analyse whether Fasudil could alter cell migration, we performed a cell-based scratch assay followed by the quantification of the wounded area. Cells were cultured up to 90-100% confluence and then scratched wound lines were created with a micropipette tip. Some cultures were treated for 24 h with Fasudil (0.1, 1, 10, 50 or 100 µM). Interestingly, untreated cells nearly covered the scratched areas of the dish in 24 h, whereas in Fasudil-treated cultures relatively large empty areas were visible in the culture dishes after 24 h of treatment in all tested concentrations ( Fig. 2A and B). Empty areas varied from 25% to 50%, as compared to control untreated cultures ( Fig. 2A and B).
To discard a possible cell-specific effect of fasudil against MDA-MB231 cell we decided to further analyse the effects of the drug against a lung tumor cell line (A549). In this regard, cells were incubated with 10 µM of Fasudil. Although we did not observe a complete coverage of the scratched areas of the dish in control cells after 24 h incubation, Fasudil-treated groups caused a reduction in coverage area with almost 50% empty areas ( Fig. 3A and B).
Fasudil-treatment induces changes in cell morphology. Since we found that treatment of MDA-MB 231 cells with 10 µM of Fasudil for 24 h was able to reduce the cell migration without interfering in cell viability (Figs 1 and 2), we decided to use this concentration of Fasudil and duration of treatment for all the subsequent experiments to analyse its effects in cell morphology. Interestingly, we found that Fasudil induces changes in cell morphology. Control cells were well spread over the dishes and displayed many ruffled membranes and lamellipodia, whereas in Fasudil-treated cells membrane protrusions were less prominent and most of the cells displayed spindle shaped morphology with long and fine membrane extensions ( Fig. 4A and B). These differences are clearly observed when cells were labelled with phalloidin, which stains filamentous actin, showing stress fiber in control untreated cells. Fasudil treatment induced a partial stress fiber disorganization ( Fig. 5A and B).
Same protocol was performed using A549 cell line. Similarly to the results obtained with MDA-MB 231 cells, Fasudil (10 μM) also caused morphological alterations in this tumor cell. Actin disruption with stress fibre disorganization was observed after 24 h incubation (Fig. 6).

Fasudil-treatment activates the canonical-Wnt pathway in MDA-MB 231 but not in A549 cells.
To test a possible involvement of the Wnt/beta-catenin pathway in observed effects of Fasudil, we analysed beta-catenin localization using immunofluorescence microscopy. In control cells (Fig. 7A,B,C) beta-catenin was localized in the cytosol (Fig. 7A,C). However, after 24 h of treatment with Fasudil (10 µM) (Fig. 7D,E,F), beta-catenin was found within the nuclei of MDA-MB 231 cells (Fig. 7D,F).
When we analysed the activation of the Wnt canonical pathway in the A549 line, we did not observe the translocation of the beta-catenin to the nucleus, suggesting that the reduction of the cell migration in the A549 line is following the non-canonical pathway of Wnt, with alterations in the actin cytoskeleton and consequently in cell morphology (Fig. 8).

Discussion
It is well known that cancer progression and metastasis require cell motility 11 , and cell migration is a key step in angiogenesis. Fasudil markedly reduced MDA-MB 231 cell migration and lead to partial actin filaments disorganization, resulting in a reduced in vitro invasive ability of TNBC MDA-MB 231 cells. The current study showed that Fasudil did not alter human TNBC MDA-MB 231 cell viability at 10 µM. The IC50 concentration for killing MDA-MB 231 cells was higher than 100 μM at 24 and 48 h. Fasudil was found to be effective at reducing migration of the MDA-MB 231 cell line in vitro, as evidenced through a cell-based scratch assay. This effect was not cell specific since when we used another tumor cell line originated from lung (A549) similar effects against cell migration was observed. Fasudil also caused the disorganization of stress fibers. In addition, beta-catenin was found within the nuclei which suggests that the Wnt/beta-catenin signalling pathway was activated by Fasudil. These results indicate that Fasudil is an agent in the prevention of metastasis of breast tumor cells of highly aggressive strains such as TNBC MDA-MB 231. Differently, lung tumor cells (A549) did not translocate β-catenin to nucleus suggesting that the reduction in cell migration can occurs due to non-canonical Wnt pathway, resulting alterations in cytoskeleton and consequently in cell morphology.
To the best of our knowledge, this is the first evidence of a correlation between both Wnt pathways (canonical and non-canonical) in MDA-MB 231 cells. This relationship has not yet been well elucidated and our results with both tumor cell lines (from breast and lung) may help to clarify the interaction between these Wnt pathways.
The small GTPases of the Rho family (i.e. RhoA, Rac1 and Cdc42) are known regulators of the actin cytoskeleton 12 . The GTPase RhoA activates ROCK, which is a major regulator of actin cytoskeleton dynamics 13 . Fasudil signalling might change stress fiber organization through the inhibition of ROCK in MDA-MB 231 cells. These  Previous experiments of our group have shown the involvement of the Wnt/beta-catenin pathway in the modulation of cell migration in MDA-MB 231 cells 9 . Thus, we decided to study the possible involvement of the Wnt/ beta-catenin pathway in the reduction in cell migration observed after Fasudil treatment. The canonical Wnt/ beta-catenin pathway can regulate transcription factors that control cell movement/invasion. Wnt binds to specific cell-surface receptors Frizzled and this disrupts the destruction complex of beta-catenin which translocate to the nucleus where it activates TCF/Lef1 transcription complex. beta-catenin also promotes cell-to-cell adhesion by accumulating in cell-cell contact sites, namely the adherens junctions. Increased cytoplasmic and nuclear beta-catenin is frequently found in different cancer types, but its impact on the individual tumour pathology can differ strikingly. β-catenin signalling decreases the migration of melanocytes and melanoma cell lines in vitro but promotes lung metastases in the NRAS-driven melanoma murine model 15 and also in a murine model, it has  already been observed that ROCK activates beta-catenin and causes epidermal hyperplasia in murine skin by actomyosin contractility and increased epidermal cell proliferation 16 . Also, in liver cancer nuclear beta-catenin is correlated with invasion, enhanced metastasis, poor prognosis and reduced disease-free survival 17,18 . In an earlier study, it was demonstrated that ROCK inhibition induces MCF-7 dormant breast cancer cells to disseminate through the disintegration of cell junctions concomitant with increased cell proliferation, migration and invasion through reduced expression of E-cadherin, beta-catenin, and actin filament bundles at the cell membrane. Interesting, we show here the opposite, ROCK inhibition by Fasudil induces beta-catenin signalling and the inhibition of cell migration of MDA-MB 231 cells. The cell-type specificity or developmental stage of cancer cells might explain the conflicting roles of ROCK in cancer cells. Further studies are needed to clarify the relationship between ROCK and Wnt/beta-catenin during breast tumor cell migration.

Synthesis of Fasudil.
Fasudil was synthesized as shown in Fig. 9, following the procedure previously described 19 . Isoquinoline sulfonyl chloride hydrochloride was slowly added to a saturated sodium bicarbonate solution. The mixture was kept at constant pH 5-6. The solution was stirred for 30 min and extracted with dichloromethane (DCM). The organic phase was dried with anhydrous sodium sulphate and evaporated under reduced pressure. The residue was dissolved in DCM and dropwise added to a solution of homopiperazine in an ice-cold bath. The resulting mixture was stirred at room temperature for 4 h and solvent was evaporated. The remaining oil was purified by flash chromatography (methanol/ethyl acetate 1/1, V/V). The purified free base was dissolved in DCM and hydrochloric acid (HCl) was bubbled in the ice-cold DCM solution to afford Fasudil hydrochloride. 1 H Figure 7. Analysis of the activation of the canonical-Wnt pathway after treatment with Fasudil. MDA-MB 231 cells were grown for 24 h cultures and treated with Fasudil (10 µM) for 24 h. Cells were analysed 24 h after Fasudil treatment by immunofluorescence microscopy for beta-catenin (red, A,D) and the nuclear stain DAPI (blue, B,E). Merged images show β-catenin/DAPI (C,F). Note the distribution of the beta-catenin throughout the cytoplasm of control cells and within the nuclei in cells treated with Fasudil. Panels G and H represents images in higher magnification from areas indicated in panels C and F, respectively. Scale bars represent 10 μm. Figure 8. Analysis of the activation of the canonical-Wnt pathway after treatment with Fasudil. A549 cells were grown for 24 h cultures and treated with Fasudil (10 µM) for 24 h. Cells were analysed 24 h after Fasudil treatment by immunofluorescence microscopy for beta-catenin (red) and the nuclear stain DAPI (blue). Merged images show beta-catenin/DAPI. Scale bars represent 10 μm.

Statistical analysis.
All the values are represented as the means ± standard error. Statistical analysis was performed with one-way ANOVA with Newman-Keuls post-test and statistical significance was defined as *p < 0.05.