Key components of the Rho/ROCK pathway

The Rho family of small GTPases regulate a diverse array of cellular processes, including cytoskeletal dynamics, cell polarity, membrane transport and gene expression, which are integral for the growth and metastatic potential of cancer cells (Ref. Reference Rath and Olson7). The three best characterised members of this family are Rho (A, B and C), Rac (1, 2 and 3) and Cdc42 (Ref. Reference Rath and Olson7). They cycle between a GTP-bound active state and GDP-bound inactive state which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), as illustrated in Figure 1 (Refs Reference Cherfils and Zeghouf12, Reference Hart13). In their active state, they act on one of over 60 downstream targets which include Rho-associated coiled-coil containing protein kinase (ROCK), mDia (Ref. Reference Tominaga14), serine/threonine p21-activating kinases 4-6 (Ref. Reference Jin15), Par6 (Ref. Reference Johansson16) and Wiskott-Aldrich Syndrome Protein (Ref. Reference Prehoda17). In addition, through interaction with various well characterised pathways, including the phosphoinositide 3-kinase, focal adhesion kinase, Src, LIM domain kinase (LIMK) and mitogen-activated protein kinase/Erk protein networks, Rho GTPase activation ultimately leads to actin cytoskeleton remodelling, increased cell motility, changes in proliferation and cell survival (Refs Reference Sanz-Moreno10, Reference Kusuyama18, Reference Samarakoon19, Reference Ohashi20). ROCK, a downstream effector of Rho, phosphorylates MYPT1, the targeting subunit of myosin phosphatase, resulting in decreased myosin phosphatase activity and thereby increased phosphorylation of the regulatory myosin light-chain 2 (MLC2) protein (Ref. Reference Ito21). Both ROCK/MYPT1/MLC2 and ROCK/LIMK/cofilin signalling axes are heavily involved in stress fibre assembly, cell adhesion and motility (Fig. 1). Further, the ROCK family contains two members, ROCK1 and ROCK2, which share 65% overall identity and 92% identity in the kinase domain (Ref. Reference Hahmann and Schroeter22) and are thus believed to also share more than 30 immediate downstream substrates, including MYPT1, MLC, and LIMK (Ref. Reference Rath and Olson7). Some differences in the activation of specific isoforms of ROCK have also been reported. For example, induction of pressure overload cardiac hypertrophy in mice leads to elevated ROCK1, but not ROCK2, expression (Ref. Reference Hahmann and Schroeter22) and specific activation of the Rho/ROCK1/c-Jun N-terminal kinase (JNK) signalling in hypertrophic cardiomyocytes (Ref. Reference Jin23). Similarly, ROCK2 has been implicated as the relevant isoform in a mouse model of acute ischaemic stroke (Ref. Reference Lee24). Finally, emerging evidence suggests potential distinct roles of ROCK1 and ROCK2 in regulating stress-induced actin cytoskeleton reorganisation and cell detachment in mouse embryonic fibroblasts (Ref. Reference Shi25) and migrating neurons (Ref. Reference Newell-Litwa26).

Figure 1. Key components of the Rho/ Rho-associated coiled-coil containing protein kinase (ROCK) signalling pathway. Various extracellular stimuli (growth factors and hormones) bind to cell membrane receptors, which subsequently act upon guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) to regulate activation of Rho GTPase proteins. Once in its GTP-bound ‘active’ state, Rho GTPase binds to ROCK (ROCK1/2) to stimulate key downstream effectors (Refs Reference Rath and Olson7, Reference Cherfils and Zeghouf12, Reference Ito21). ROCK-mediated phosphorylation of myosin light-chain (MLC) promotes phosphorylation of myosin and increased actomyosin contraction. Activation of LIMK by ROCK leads to phosphorylation and inactivation of the actin-depolymerising protein cofilin, altering actin filament organisation. Collectively, activation of key downstream effectors of Rho causes changes in motility, proliferation and other essential cellular processes.

Moreover, ROCK can be effectively targeted by (non-isoform) specific inhibitors including Y-27632, fasudil and new generation compounds, which prevent activation of ROCK by competing with ATP for binding to the kinase (Refs Reference Breitenlechner27, Reference Patel28, Reference Vogel29). Interestingly, fasudil has been shown to be safe for use in humans for the treatment of cerebral vasospasm with an acceptable side effect profile, making it an attractive drug for clinical study (Ref. Reference Liu30).

Exploring the effects of inhibiting Rho/ROCK in cancer: the pre-clinical evidence

Numerous studies have thus far investigated the therapeutic efficacy of Rho/ROCK inhibition in in vitro and in vivo models of cancer (Table 1, (Refs Reference Itoh5, Reference Patel28, Reference Vogel29, Reference Abe31, Reference Deng32, Reference Genda33, Reference Horiuchi34, Reference Igishi35, Reference Lane36, Reference Liu37, Reference Nakabayashi and Shimizu38, Reference Nakajima39, Reference Nakajima40, Reference Nakajima41, Reference Ogata42, Reference Ohta43, Reference Pille44, Reference Routhier45, Reference Somlyo46, Reference Somlyo47, Reference Takamura48, Reference Teiti49, Reference Ueno50, Reference Voorneveld51, Reference Wermke52, Reference Xia53, Reference Yang54, Reference Ying55, Reference Zhang56, Reference Zhu57, Reference Zohrabian58). As summarised in Table 1, blocking Rho/ROCK signalling in cancer cells can effectively reduce cellular proliferation, invasion and angiogenesis in vitro and reduce tumour growth and metastasis formation in vivo. Interestingly, the effects on proliferation are heterogeneous, with several studies reporting no effect at all (Refs Reference Ito21, Reference Patel28, Reference Nakajima39, Reference Ogata42, Reference Somlyo46, Reference Teiti49), one study demonstrating an anti-proliferative effect when fasudil was used at a supraphysiological concentration (Ref. Reference Nakabayashi and Shimizu38) and several more recent studies suggesting marked effects on cell growth (Refs Reference Vogel29, Reference Abe31, Reference Ohta43, Reference Wermke52, Reference Zhang56) that can be further enhanced when ROCK inhibition is combined with chemotherapy (Refs Reference Igishi35, Reference Ohta43). Further, when efficacy of ROCK inhibitors was examined in the context of tumour cell motility, migratory and invasive characteristics, more consistent findings were observed across a variety of cancer models examined (Refs Reference Itoh5, Reference Patel28, Reference Nakajima39, Reference Ogata42, Reference Teiti49). Several groups have also shown that inhibition of ROCK and its stimulated signalling might prove to be a promising strategy for restraining tumour progression in vivo, for example by slowing down primary tumour growth (Refs Reference Routhier45, Reference Wermke52, Reference Ying55) and formation of metastases (Refs Reference Liu37, Reference Takamura48, Reference Teiti49, Reference Voorneveld51, Reference Zhang56). The potential differences observed between the in vitro [two-dimensional (2D) observations] and in vivo findings may be partially explained by the different models examined, origin of the inhibitors used (Table 1), or the critical role RhoA plays in cellular invasion and metastasis (Ref. Reference Timpson59). Perhaps, this discrepancy could also be more reflective of the complex involvement Rho/ROCK has in cellular processes in cancer that cannot be accurately recapitulated in simple 2D assays (Ref. Reference McGhee60). A deeper understanding of Rho/ROCK signalling activation in vivo is necessary to fully characterise the importance of inhibiting this pathway in cancer medicine as has recently been achieved for its prototype partner Rac GTPase (Ref. Reference Johnsson61).

Table 1. The therapeutic efficacy of RhO/ROCK inhibitors (ROCKi) in various models of cancer.

^a Indicates pharmaceutical collaboration.

The Rho/ROCK pathway is critical in angiogenesis

Sustained angiogenesis is one of the key hallmarks of tumour progression (Ref. Reference Hanahan and Weinberg62) that incorporates abnormal signalling cues from key cell types within the complex tumour microenvironment (Ref. Reference Hanahan63). It is well documented that in response to tissue hypoxia, angiogenesis is constantly stimulated resulting in a highly abnormal vasculature (Ref. Reference Fukumura64). These vessels are immature, tortuous, have increased permeability and lead to intratumoural hypoxia, which can mediate resistance to anti-cancer therapies (Ref. Reference De Bock65). Moreover, the tumour-associated angiogenic vasculature, growth-promoting trophic factors that are expressed and secreted by the endothelial cells and prolonged hypoxia can collectively drive hyper-proliferation and development of a more aggressive tumour phenotype with increased propensity to metastasise (Ref. Reference Carmeliet and Jain66). Angiogenesis is a complex process, which is largely controlled by Vascular endothelial growth factor (VEGF) and its membrane receptors. To initiate the angiogenic process, endothelial cells (ECs) lose junctional integrity and increase permeability (Ref. Reference Gavard and Gutkind67). Subsequent degradation of the basement membrane and remodelling of the ECM enables ECs to migrate, proliferate and ultimately undergo morphogenesis in order for new vessels to develop (Ref. Reference Liu and Senger68).

The Rho/ROCK pathway has been shown to be an integral part of VEGF-mediated angiogenesis and is not only implicated in VEGF signalling, but also in numerous processes necessary for angiogenesis to occur, including EC migration, survival and cell permeability (Ref. Reference Yin69) (Fig. 2). It has been shown that adherin junctions between ECs need to be loosened in order for EC migration and proliferation to occur (Ref. Reference Carmeliet and Jain66). Rho/ROCK signals via p-MLC break down intracellular junctions and thereby increase vascular permeability (Ref. Reference Bryan and D'Amore70). In order for ECs to invade surrounding tissue and form new vessels, the basement membrane (BM) and ECM must be disrupted via matrix metalloproteinase (MMP) secretion (Ref. Reference Zeng71). Rho/ROCK activation has been shown to directly stimulate MMP-9 secretion (Ref. Reference Turner72) and is also associated with increased MMP expression in tumours (Refs Reference Xue73, Reference Zhang74). Once the BM and ECM are disrupted, EC migration and tube formation can occur. van Nieuw Amerongen et al. (Ref. Reference van Nieuw Amerongen75) used human umbilical vein endothelial cells (HUVECs) to show that not only do VEGF-induced changes in the EC cytoskeleton depend on RhoA, but also that growth of human microvascular endothelial cells (hMVECs) into a fibrin matrix in response to VEGF is inhibited by Y-27632, suggesting that the Rho/ROCK pathway is necessary for ingrowth of ECs. Bryan et al. (Ref. Reference Bryan76) showed that disruption of the Rho/ROCK pathway inhibits VEGF-mediated changes to the cytoskeleton in ECs and also that ECs treated with Y-27632 failed to assemble into recognisable vessel structures, highlighting the importance of the Rho/ROCK pathway in vasculogenesis. Hoang and Uchida (Refs Reference Hoang77, Reference Uchida78) both demonstrated that inhibiting Rho/ROCK prevented ECs from forming organised vascular structures by suppressing cellular motility. As the Rho/ROCK pathway has been established as being critical to multiple steps in angiogenesis, many studies have attempted to elucidate the importance of its involvement in the cancer setting. Croft et al. (Ref. Reference Croft79) used a conditionally active form of ROCK2 in colon carcinoma cells to show that increased ROCK signalling promoted tumour angiogenesis and tumour cell invasion in vivo. Using HUVEC and glioma cell co-culture techniques, Nakabayashi et al. (Ref. Reference Nakabayashi and Shimizu38) further showed that the ROCK inhibitor fasudil suppressed tumour-induced angiogenesis and the migration of HUVEC cells through transwell plates. Moreover, the same group showed that the growth of T98G glioma xenografts was significantly inhibited when tumour-bearing mice were treated daily with fasudil (Ref. Reference Nakabayashi and Shimizu38). ROCK inhibitors also showed significant promise as anti-angiogenic agents in additional in vivo models, for example Nakajima et al. (Ref. Reference Nakajima40) showed that administration of the ROCK inhibitor Wf-536 reduced the number of spontaneous metastases and impaired angiogenesis in a Lewis lung carcinoma model. Further, Somlyo et al. (Ref. Reference Somlyo47) showed that mice bearing xenotransplants of PC3 cells had a reduction in tumour volume and increased survival when treated with a combination of Wf-536 and Marimastat (an MMP inhibitor). ROCK inhibitors have not been evaluated in human trials to date. However, considerable clinical data exists regarding the effects of VEGF inhibitors on various cancer subtypes. Although anti-angiogenic therapies have shown variable efficacy in cancer treatment, a deeper understanding of the mechanisms of action has highlighted the potential importance of timing of administration on the anti-cancer effects. This hypothesis is an interesting new strategy to explore and test.

Figure 2. Rho/ Rho-associated coiled-coil containing protein kinase (ROCK) signalling and the tumour microenvironment: unexplored treatment opportunities. (a) Schematic illustrating key events that lead to tumour progression and metastasis. (b) In the presence of ROCK inhibitors, invasion and metastasis are impaired: the Rho/ROCK pathway as a mediator and therapeutic target of cancer metastasis. Within cancer cells, ROCK inhibitors prevent the phosphorylation of LIMK and p- myosin light-chain (MLC) which results in impaired actin-myosin filament bundling. This in turn affects cellular proliferation, morphology, adhesion, motility and gene transcription. ROCK is essential in cancer-associated fibroblasts (CAF) associated invasion and also in cell- extracellular matrix (ECM) signalling.

Rho/ROCK inhibitors as vascular normalising agents

Clinical use of anti-angiogenic agents has generated disappointing results when used as monotherapy (Ref. Reference Giantonio80), but more success has been had when these agents are combined with cytotoxic chemotherapy (Ref. Reference Tabernero81). A potential explanation for this may include acquired resistance mechanisms because of continual VEGF inhibition (Refs Reference Bergers and Hanahan82, Reference Casanovas83), intrinsic vascular heterogeneity within tumours (Ref. Reference Varkaris84) and/or impaired drug delivery because of excessive reduction in tumour vasculature, which ultimately shifts the net balance towards hypoxia-driven rebound angiogenesis (Ref. Reference Jain85). VEGF inhibition leads to increased tumour oxygenation when administered in a transient manner, a process called vascular normalisation (Refs Reference Hoang86, Reference Rao87, Reference Tong88) (Fig. 2). Exploiting this process to improve the efficacy of standard cytotoxic therapies is attractive and several pre-clinical and clinical studies have explored this concept thus far. Lee et al. (Ref. Reference Lee89) demonstrated that blocking VEGF in glioblastoma or colon adenocarcinoma compensates for hypoxia-induced radiation resistance. The authors further showed that using an anti-VEGF antibody resulted in greater tumour growth delay when combined with radiation, than radiation alone. Blocking VEGF signalling was subsequently found to lead to pruning of immature vessels and generation of a morphologically ‘normalized’ vascular network within tumours, allowing deeper penetration of molecules, such as chemotherapeutics into the cancer (Ref. Reference Tong88). Most recently, Coutelle et al. (Ref. Reference Coutelle90) showed that dual targeting of VEGF and Angiopoietin-2 in addition to reducing tumour growth and sprouting angiogenesis significantly improved vascular normalisation parameters, including leakiness, hypoxia and perfusion as prerequisites for improved access for chemotherapy. Importantly, in the same study, the authors also showed for the first time, that the formation of vascular basement membrane sleeves that facilitate the rapid vascular regrowth associated with resistance to VEGF-targeting drugs can be eliminated by such dual targeting strategies. Falcon et al. (Ref. Reference Falcon91) similarly demonstrated that platelet-derived growth factor (PDGF) beta blockade in Lewis lung carcinoma tumours increased tumour vessel efficiency in vivo. Further, they found that the combination of imatinib with cyclophosphamide improved the delivery of cyclophosphamide to the tumour and the tumour burden was reduced in vivo.

The Rho/ROCK pathway has been specifically examined in this context: Ader et al. (Ref. Reference Ader92) performed in vivo induction of dominant negative Rho (RhoBN19) to show that inhibiting Rho decreased tumour cell survival after irradiation and moreover, tumours had improved oxygenation and decreased vessel density. The critical aspect of optimal timing of administration of combinations involving anti-VEGF therapies and cytotoxic agents was further explored by Winkler et al. (Ref. Reference Winkler8). Treatment of glioblastoma xenografts with an anti-VEGF receptor (VEGFR) 2 monoclonal antibody resulted in a significant reduction in tumour hypoxia on day 2, with maximal reduction on day 5. By day 8, tumour hypoxia had started to increase. Further, radiation therapy produced a synergistic effect when given on days 4–6. This suggests that after VEGFR blockade, there is an initial increase in tumour oxygenation during which, the effects of radiotherapy are increased, but importantly with continual VEGFR blockade, the tumour becomes hypoxic again and the synergism with radiation is lost. Several randomised trials have shown that the addition of bevacizumab to chemotherapy and radiotherapy improves progression free survival in patients with central nervous system malignancies (Refs Reference Lai93, Reference Narayana94) and a phase I trial specifically testing the vascular normalisation strategy has shown this holds considerable promise in patient care (Ref. Reference Willett95). Here, patients with rectal cancer receiving neoadjuvant chemotherapy plus radiation were exposed to the VEGF inhibitor, bevacizumab. Interestingly, bevacizumab treatment led to normalisation of the tumour vasculature, increased tumour cell apoptosis and resulted in a complete pathological response in two patients (Ref. Reference Willett95). Therefore, it would be interesting to examine whether Rho/ROCK pathway inhibitors may prove effective vascular normalising agents, increasing efficacy of cytotoxic therapies by modulating key components of the VEGF signalling pathway. However, the transient nature of vascular normalisation means that the window of opportunity for drug delivery is temporary, may be difficult to predict and therefore apply in the clinical setting. These issues are yet to be systematically examined.

Rho/ROCK inhibitors as provascular agents

In addition to normalising the tumour vasculature, a provascular strategy may also be a promising treatment approach, where transient vasodilation by targeted therapy improves blood supply and exposure of tumour cells to circulating chemotherapeutics and/or sensitivity to radiation. As most vasodilators dilate both the tumour and systemic vasculature, there can be unpredictable effects on the tumour vasculature. If the tumour vessels are in series with the systemic circulation, systemic vasodilation can increase tumour blood flow, however if the tumour vasculature is in parallel, then systemic vasodilation will cause a reduction in tumour blood flow (vascular steal phenomenon) (Ref. Reference Sonveaux96). An ideal provascular agent would therefore, be one that preferentially targets the tumour vascular bed. A number of studies have shown some success with this strategy, suggesting the idea has merit. Gallez and Sonveaux (Refs Reference Gallez97, Reference Sonveaux98) both demonstrated the possibility of increasing tumour blood flow using vasodilators. Jordan and Stewart (Refs Reference Jordan99, Reference Stewart100) further showed that in vivo administration of nitric oxide not only increased tumour blood flow, but sensitised tumours to the effects of radiation. Given the critical interplay between tumour hypoxia and angiogenesis, modulation of tumour-oxygen sensing has also proven an effective strategy to improve blood flow to the tumour. A systematic review of clinical trials assessing the effects of improving tumour oxygenation to radiosensitise tumours, suggests there may be clinical benefit, finding a 23% improvement in locoregional control and a 13% improvement in overall survival (Ref. Reference Overgaard101). In terms of improving the delivery of chemotherapy, studies by Masunaga et al. (Ref. Reference Masunaga102) and Martinive et al. (Ref. Reference Martinive103) observed significant improvements in the uptake of selected chemotherapies when tumour-bearing mice were injected with nicotinamide or an endothelin-1 receptor antagonist, respectively. Most recently, Wong et al. (Ref. Reference Wong104) demonstrated that treatment combining low-dose Cilengitide, an αvβ3/ αvβ5 integrin receptor inhibitor, with a calcium channel blocker, Verapamil, significantly improved efficacy of chemotherapeutic, gemcitabine, in in vivo models of lung and pancreatic cancer. In the same study, detailed analysis of pre- and post-treatment material revealed that the cyclical administration of the dual vascular modulator-chemotherapy combination led to increased tumour vascular function and intratumoural drug delivery while reducing hypoxia and desmoplasia in these models. Finally, by comparing the ability of capillary ECs isolated from normal versus tumour microvasculature to sense and respond to physical cues in their ECM, Ghosh et al. (Ref. Reference Ghosh105) demonstrated that tumour-derived ECs exhibit different sensitivities to various mechanical cues in vitro and that these abnormal responses, which may be implicated in the loss of normal structure in the tumour microvasculature, are because of aberrant and increased Rho signalling.

With this in mind, exploration of the vasodilatory effects of ROCK inhibitors in cancer may be an interesting treatment approach. ROCK inhibitors reduce vasospasm via reduction in smooth muscle contraction and down-regulation of endothelial nitric oxide synthase, leading to their use in the treatment of ischaemic stroke (Ref. Reference Liu30), with significant efficacy in reducing post stroke cerebral vasospasm and an acceptable side effect profile. Importantly, no statistically significant differences in the side effects reported by patients were observed when fasudil was compared with placebo. ROCK inhibitors have been shown to normalise smooth muscle contraction and suppress vascular lesion formation, making them a therapy of interest in hypertension, pulmonary hypertension, hypertensive vascular disease and ischaemic heart disease (Refs Reference Oka3, Reference Shimokawa and Rashid4). It is therefore plausible to hypothesise that Rho/ROCK inhibitors may act as provascular agents, improving tumour blood flow and increasing exposure of cells to chemotherapy and/or sensitising cells to the effects of radiation (Fig. 2). However, as outlined for vascular normalisation, the timing and dosing of provascular agents are likely to be critical in determining success and this concept is yet to be systematically examined.

Rho/ROCK signalling within the complex tumour microenvironment

The dynamic and complex interplay between tumour cells, stromal cells and the ECM affect cancer initiation, progression, metastasis and also, chemoresistance (Refs Reference Samuel106, Reference Tredan107). Recent data indicate that carcinogenesis and tumour angiogenesis result not only from the interaction of cancer cells with ECs of various origin (as discussed above), but that surrounding ‘normal’ stromal and inflammatory cells also have a crucial role in directing the formation of the blood vessels that nourish a developing tumour (Ref. Reference Chen108). In addition, loss of normal tissue homeostasis during tumourigenesis initiates a stromal remodelling cascade which leads to fibroblast activation (i.e. myofibroblasts/cancer-associated fibroblasts or CAFs) and production of biomechanically and biochemically altered ECM (Ref. Reference Malik109). Increased deposition and modification of the ECM mediated through CAF-expressed biochemical signalling molecules, including Rho/ROCK, Caveolin-1, Syndecans and Hippo pathway members YAP/TAZ (Refs Reference Malik109, Reference Provenzano and Keely110) can then lead to activation of signal transduction pathways that promote tumour cell growth, proliferation and survival.

Rho GTPases have been shown to be implicit in a number of stromal processes that contribute to the invasiveness and metastatic potential of cancer cells (Refs Reference Wyckoff11, Reference Timpson59). It has been long understood that the presence of high density stroma in breast tissue confer an increased risk of developing breast cancer (Ref. Reference Boyd111). Women with high mammographic densities have increased proliferation of stromal or epithelial tissue on histological examination and this has been correlated with an increased risk of breast cancer (Ref. Reference Boyd111). It was further hypothesised that interactions between the stroma and epithelium ultimately lead to cancer formation (Ref. Reference Boyd111). In an effort to better understand this phenomenon, Lisanti et al. (Ref. Reference Lisanti112) conducted genome-wide transcriptional profiling of low density (LD) breast fibroblasts, compared with high density (HD) breast fibroblasts, revealing differences in several key processes including stress response, inflammation, stemness and signal transduction. The authors postulated that the presence of HD fibroblasts could be considered a pre-cancerous phenotype and Rho GTPase activation (along with increased JNK1, inducible nitric oxide synthase, fibroblast growth factor receptor, epidermal growth factor receptor and PDGF receptor signalling) was identified as a key biological process in this setting (Ref. Reference Lisanti112). Moreover, in an in vitro system of tumour explants embedded in collagen gels, activation of Rho/ROCK was shown to be essential for contractility-dependent collagen realignment, whereas inhibition of Rho/ROCK led to a substantial reduction of contact guidance tracks, an early step in the invasion process (Ref. Reference Provenzano113). Goetz et al. (Ref. Reference Goetz114) further demonstrated that high levels of stromal Caveolin-1, an activator of Rho/ROCK signalling (Ref. Reference Joshi115), can initiate ECM re-organisation in the tumour and in the cancer-associated stroma, promoting metastatic behaviour in a Rho–ROCK-dependent manner. Conversely, in the same study, down-regulation of Caveolin-1 blocked Rho/ROCK activity, leading to altered ECM topography and reduced cell contractility (Ref. Reference Goetz114).

Further work in breast cancer has shown that breast cancer cells grown in a 3D floating matrix differentiate into tubular structures, however if the same matrix is attached to the dish, the cells do not differentiate, but proliferate and spread (Ref. Reference Wozniak116). In the same study, differentiation could be disrupted by increasing the density of the matrix. Interestingly, it was also shown that tubulogenesis required contraction of the 3D matrix which was dependent on the Rho/ROCK pathway and that RhoA activity was down-regulated in differentiated cells (Ref. Reference Wozniak116). Subsequently, p190RhoGAP-B was shown to mediate down-regulation of RhoA activity and inhibition of ductal morphogenesis. RhoA activity was reduced at cell-cell adhesions versus activity at cell-ECM adhesions (Ref. Reference Ponik117). These studies highlight the important role the Rho/ROCK pathway has in how cancer cells interact with their environment, and how this environment in turn, affects tumour cell behaviour.

The stromal compartment of tumours has long been thought to contribute to the aggressive phenotype of cancers, and CAFs have been found to provide tumour cells with proliferative and anti-apoptotic signals affecting angiogenesis and ECM remodelling. Specifically, Cadamuro et al. (Ref. Reference Cadamuro118) showed that PDGF-D plays a major role in CAF recruitment and activates Rho/ROCK to promote fibroblast migration. Further, increased palladin expression in CAFs is associated with increased growth and metastasis of pancreatic cancer cells by increasing their ability to remodel the ECM, thereby promoting tumour invasion (Ref. Reference Goicoechea119). Gaggioli et al. (Ref. Reference Gaggioli120) demonstrated that squamous cell carcinoma (SCC) cells required fibroblasts to invade into a 3D organotypic matrix. Moreover, they showed that inhibition of Rho/ROCK signalling specifically in the fibroblasts (not in the SCC cells) reduced invasion of the SCC cells. In addition, Scott et al. (Ref. Reference Scott121) showed that LIMK signalling, downstream of ROCK, is required for path generation during cancer cell invasion by both leading tumour cells and stromal cells. These findings suggest that the presence of fibroblasts is necessary for cancer cell invasion and that the Rho/ROCK activation is critical in this context. Similarly, Sanz-Moreno et al. (Ref. Reference Sanz-Moreno10) demonstrated a role for cytokine signalling through GP130-IL6ST/JAK1 in the regulation of ROCK-dependent actomyosin contraction, which drives matrix remodelling by CAFs and migration of melanoma cells. Interestingly, the ROCK-induced actomyosin contractility was found to further stimulate JAK1/STAT3 signalling, indicating that there is a self-reinforcing positive feedback loop (Ref. Reference Sanz-Moreno10). Therefore, inhibition of Rho/ROCK signalling in this context may block both intrinsic and microenvironment-derived extrinsic signals that promote CAF-facilitated cancer invasion, and could potentially have a sustained effect by breaking the positive feedback loop.

Migration and invasion are important elements of the growth of the primary tumour, but also play a critical role in the development of metastasis. In vivo, cells must breach the endothelial barrier to metastasise (Refs Reference Hanahan and Coussens122, Reference Jain123). The process of intercalation is where cancer cells first adhere to ECs, open the EC junctions, stimulate EC retraction and then insert into the endothelial monolayer. It has been shown that Cdc42 depletion impairs intercalation in PC3 cells and also that Cdc42, RAC1 and RhoA impair EC junction opening. Mice injected with Cdc42 depleted PC3 cells developed fewer metastases, highlighting the importance of the Rho GTPases in intercalation (Ref. Reference Reymond124). Collectively, these studies indicate a critical role for the Rho/ROCK pathway in modulating relevant cross-talk between tumour cells and their surrounding microenvironment, particularly in the context of driving cellular migration, invasion and metastasis (Fig. 2).

Conclusions and the long road to clinical translation

The Rho/ROCK pathway has been a popular field of study for cancer researchers. However, despite ROCK inhibitors being demonstrated to be safe for human use, these agents have not yet been translated to the cancer clinic. These compounds have well documented effects on cellular proliferation, however their effects on cell invasion, tumour growth and metastasis appear to be more robust. Large scale cancer genome sequencing studies have revealed that mutations in the Rho GTPase family are rare (Refs Reference Biankin125, Reference Kandoth126), where generally aberrant activation of this pathway occurs through overexpression of Rho GTPases or by changes in the levels of regulators of Rho activity, including increased activation of GEFs and inactivation or loss of GAPs or GDIs. Importantly, it should be noted that increased expression of Rho/ROCK signalling components may not necessarily correlate with an increase in total activity of these proteins, as this process is also tightly regulated through subcellular localisation of Rho and downstream effectors and by their interaction with key regulatory molecules (Refs Reference Timpson59, Reference Johnsson61, Reference Pajic127). Thus, although this is an active area of research, there are currently no effective predictive biomarkers of treatment response to Rho/ROCK inhibition.

In addition to their effects on tumour cell proliferation and motility, ROCK inhibitors modulate angiogenesis and vascular tone and thus could potentially improve the delivery and efficacy of chemotherapy or other novel targeted agents (Refs Reference Vogel29, Reference Horiuchi34, Reference Somlyo47). The Rho/ROCK pathway is also important in regulating the dynamic cross-talk between tumour cells and their microenvironment which may also be therapeutically exploited to inhibit metastasis formation. Finally, the therapeutic potential of ROCK inhibitors as an adjunct to cytotoxic chemotherapy is yet to be systematically examined.

As differences in the activation of the two ROCK isoforms have been reported in cardiovascular or CNS disorders, with ROCK1 implicated as the predominant mechanism for the hypotensive effects of pan-ROCK inhibitors, one could hypothesise that there may be isoform-specific regulation of cancer cell behaviour, interactions within the tumour microenvironment and control of carcinogenesis and metastasis. From this, targeting ROCK2 could potentially lead to less toxicity compared with pan-ROCK inhibition. Attempts to produce more specific and clinically suitable ROCK inhibitors are ongoing, with increased focus on isoform-specific targeting (Ref. Reference Green128). On the other hand, given that tumours are highly adaptive and rapidly acquire resistance when exposed to therapy, hitting multiple oncogenic signalling nodules or hallmarks of cancer with non-isoform selective ROCK inhibitors, may overall represent a more effective treatment strategy, as recently highlighted by Hanahan D (Ref. Reference Hanahan63).

Further understanding of Rho signalling in the various tumour compartments will determine whether the inhibitors of this complex pathway may serve as effective treatments for newly diagnosed or recurrent tumours and will establish the optimum combinations with radiation, cytotoxic chemotherapy, and other targeted molecular compounds. Importantly, these agents may improve the delivery of chemotherapy to the tumour, perhaps enhancing efficacy, reducing the effective dose required or overcoming some mechanisms of chemoresistance.

Research in progress and outstanding research questions

This review highlights a number of avenues for further research when examining the clinical utility of ROCK inhibitors to treat cancer. Some interesting areas of research include closely examining how the Rho/ROCK pathway is implicated in tumour stromal signalling, particularly in cancers where tumour stroma is highly prominent such as pancreatic cancer. Studying the stroma for potential biomarkers of tumour response may provide additional important insights rather than solely focusing research on the tumour itself. State of the art molecular imaging techniques such as Forster resonance energy transfer (FRET) imaging can provide relevant information into the dynamic and spatiotemporal regulation of cell signalling behaviour under physiological and disease conditions. Transgenic mice expressing Rho GTPase FRET biosensors will provide detailed knowledge of the normal physiological roles this pathway plays at the cellular level. In addition, crossing these mouse strains with other disease models will allow us to examine, in an intact 3D system, how this pathway is involved in cancer initiation, progression, chemotherapy responsiveness and chemoresistance mechanisms. This knowledge will allow further biomarker development, examination of the effects of ROCK inhibition in primary versus metastatic lesions and in pre-cancerous lesions.

ROCK inhibitors have yet to make an appearance in the clinical setting to treat patients with cancer. They have been shown previously to have an acceptable side effect profile when used to treat post cerebrovascular accident vasospasm, but these patients had a short, continuous infusion and were monitored in intensive care. Patients with cancer will need long term exposure and ideally, take an oral preparation. Before trials examining the anti-cancer effects of these drugs can be planned, further phase I studies need to be conducted to determine the most appropriate dosing schedule and with chronic dosing in mind. Protracted infusion with a pump, such as that used for 5-fluorouracil in the oxaliplatin, 5-fluorouracil and folinic acid (FOLFOX) chemotherapy combination for colon cancer is possible, but could potentially considerably increase the cost of the treatment as well as patient morbidity. Hypotension is the most predictable side effect for patients (Ref. Reference Liu30), and it may mean that elderly patients would be less likely to tolerate this drug well, which could be an issue in the management of pancreatic cancer. In our pre-clinical trials laboratory, our early data indicate that mice are able to tolerate a daily, oral preparation of a ROCK inhibitor and this is associated with measurable anti-tumour effects. Further systematic in vivo studies are needed to exactly predict the optimal sequence of administration of these drugs in conjunction with chemotherapy or other targeted therapeutics.

An interesting challenge remains in determining which Rho GTPase family members are the most promising druggable targets and how significant the beneficial effects of targeting this signalling network, in combination with other targeted agents and/ or conventional chemotherapeutics, will be. Further studies are necessary to accurately ascertain the effects this pathway has in cancer and in cancer stroma and if possible, identify potential biomarker(s) of response. Refining exactly which patients are most likely to benefit and which combinations dosing schedules are most effective is the key goal for further research.