Globally, lung cancer is the most prevalent type of cancer, affecting 2.09 million individuals and was responsible for 1.76 million associated deaths in 2018 (1). Smoking is the leading cause of lung cancer, as 86% of individuals with the disease have a history of smoking (2). Lung cancer can be divided into 2 major types, which are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for >85% of all lung cancer cases and can be classified into adenocarcinoma (50%), lung squamous cell carcinoma (SCC) (30-40%) and large cell carcinomas (10%) (3). NSCLC is often diagnosed at a late stage, resulting in poor therapeutic responses and high mortality rates (4,5). The 1-year survival rate for patients with lung cancer is 44%, and the 5-year survival rate is only 17% (6). The treatment options for lung cancer are surgery, radiotherapy, platinum-based chemotherapy and neoadjuvant chemotherapy. However, the efficacy of these treatments for some patients remains unsatisfactory, as lung cancer is a heterogeneous disease. Moreover, these treatments can have deleterious side-effects, while patients with metastatic tumors are vulnerable to developing a post-treatment resistance to such medications, rendering the treatment of lung cancer difficult (7). However, improvements in effectiveness and survival rates have been observed when histology-guided chemotherapy, maintenance therapy, or vascular endothelial growth factor (VEGF)-targeted therapy is combined with platinum doublet therapy, which is the standard therapy for unresectable and metastatic lung cancer (8,9). Nowadays, novel therapies targeting molecular aberrations or driver mutations have emerged as a therapy of choice due to the excellent effectiveness and lower side-effects, owing to the completely sequenced human genome allowing the identification of novel mutations that play a key role in lung carcinogenesis. An example of such a therapy, which has been tested and has yielded promising results, is tyrosine kinase therapy, that specifically targets the epidermal growth factor receptor (EGFR), a type of protein commonly altered in NSCLC (10). However, this novel treatment spectrum only includes a minority of patients that harbor the mutation (11). Therefore, targeting oncogenic pathways that play a central role in cancer is a sensible strategy, as it is likely to be effective. One such pathway that has recently become of interest in cancer therapy is the Rho-associated kinase signaling pathway (ROCK).

ROCK plays an essential role in carcinogenesis, particularly in promoting cancer cell motility that causes metastasis. ROCK is an effector of the small GTPase Rho and has been studied in various malignancies, such as breast (12), skin (13), liver (14) and lung cancer (15). Studies on lung cancer usually use NSCLC cell lines or tissue biopsies from patients with NSCLC to assess changes in the proliferation, migration, and growth of cancer following the inhibition of knockdown of ROCK (16-21). These studies have found that ROCK is responsible for promoting lung cancer growth if upregulated or overexpressed, and this has led researchers to suggest that ROCK may be a novel target for the treatment of lung cancer.

In the present review, the role of ROCK in lung cancer is discussed and the published evidence from in vitro and in vivo studies that were performed to decode the function of ROCK in lung cancer is summarized.

A literature search on ROCK and studies on lung cancer was conducted from January 1, 2020, to November 15, 2020 using Scopus, PubMed and Web of Science, with the following keywords: 'ROCK OR Rho' OR 'Rho-associated kinase' AND 'Lung cancer' OR 'NSCLC' OR 'Non-small cell lung cancer' OR 'SCLC' OR 'small-cell lung cancer'. Only original research articles on the roles of ROCK and ROCK inhibition in studies on lung cancer written in the English language were selected for reading. The full text of the articles concerned was retrieved following the screening of the titles and abstracts.

ROCK is a member of the protein kinase A/protein kinase G/protein kinase C (AGC) serine/threonine kinases family that plays an essential role in promoting cell motility by facilitating cytoskeleton contractility (22,23). As shown in Fig. 1, ROCK is activated by binding of the Rho GTPase (i.e., RhoA and RhoC) to its Rho-binding domain (RBD), which leads to the activation by phosphorylation of the myosin-binding subunit of the myosin light chain phosphatase (MYPT) (24), myosin light chain (MLC) (24) and LIM kinases (LIMK) (25,26). The activation of the MLC substrate leads to the activation of myosin II motor activity, leading to the crosslinking of myosin to filamentous actin (F-actin), enhancing actomyosin cytoskeleton contractility (27,28). On the other hand, the activation of MYPT inhibits MLC dephosphorylation, and the activation of LIMK phosphorylates cofilin, which renders it inactive and unable to polymerize F-actin (29). In brief, the phosphorylation of MLC and MYPT leads to increased levels of phospho-MLC, and thus promotes actomyosin contractility, which alters the migratory behavior of cells. Enhanced contractility resulting from ROCK activation also facilitates cancer cell proliferation and regulates cell adhesion (28,30-32).

There are 2 ROCK homologs, namely ROCK1 and ROCK2. Collectively they are referred to as ROCK. ROCK1 and ROCK2 consist of 1,354 and 1,388 amino acids, respectively, and both contain an N-terminally located kinase domain, a coiled-coil region followed by a Rho-binding domain (33). The homologs share ~65% similarity in their overall amino acid sequences, and the sequence similarity increases to 92% if only their kinase domains are compared (22). Although both ROCK proteins share the same function, which is to regulate cytoskeleton contractility and exert a redundant effect on MLC and MYPT phosphorylation (34), they have also been found to differ from each other in terms of tissue distribution and subcellular localization. ROCK1 is ubiquitously expressed in non-neuronal tissues such as the lungs, liver, thymus, stomach, spleen, kidneys, testes, placenta and embryo (22,28). ROCK1 has been found at the microtubule-organizing center, cytoplasm, plasma membrane, and cell-cell adhesion sites (19,35,36). Moreover, ROCK2 is abundant in the brain, muscle, placenta, lungs and heart (22,28). It has been found to be localized at the nuclei and pre-synapses, including active zones (31). Notably, different types of cancer seem to have various needs for the ROCK proteins, specifically expressing both ROCK1 and ROCK2, or either alone (37-40). According to Kümper et al (34), both ROCK isoforms are independently important to the cells since the cells that lack any of the ROCK isoforms are still capable of proliferating. However, the cells that lack both ROCK isoforms are unable to contract and become flattened out. Eventually, cell division and growth are attenuated. This evidence has demonstrated the essential role of ROCK in maintaining cell survival, and the heterogeneity of ROCK expression in different types of cancer has particularly suggested the need for the development of a ROCK isoform-specific cancer therapy.

Since the use of the first approved ROCK inhibitor (fasudil) for the treatment of cerebral vasospasms (98), immense efforts have been made to repurpose the therapeutic benefits of this agent for the treatment of cancer (81,99-101). Apart from fasudil, a wide range of compounds has also been tested against lung cancer, and as ROCK overexpression or upregulation is associated with an enhanced growth of lung cancer as discussed above, the current treatment modality is to inhibit or reduce ROCK expression. Researchers have been testing several inhibitors that may target ROCK upstream effectors or downstream substrates in lung cancer, such as RhoA inhibitor (64), RhoC inhibitor (85,102), ROCK pan-inhibitor (Y27632) (103,104) and LIMK inhibitor (BMS-5) (105). As these targets are intertwined in one signaling pathway, the inhibition of RhoA or LIMK, for example, also yields a preferable result, as with the inhibition of ROCK. Thus, the present review also includes studies on ROCK-associated substrate inhibitors instead of only studies on ROCK inhibition. Novel natural compounds have also been tested for their inhibitory action against ROCK in lung cancer, with promising results (46,80). Examples of the natural compounds studied are zerumbone derived from ginger (80), glabridin from licorice (106), plumbagin from chitrak (46), deguelin from cork bush (107), p-escin from horse chestnut (108), and XAP from Muruwa (102). In recent years, natural compounds have attracted the attention of researchers in developing novel cancer treatment, since they are believed to exert a less toxic effect on normal cells (109). p-escin (a RhoA/ROCK inhibitor) for instance, has been evaluated to not cause significant body weight loss or histologic cytotoxicity on normal mice after 34 weeks of treatment, which indicates that this natural compound is unable to jeopardize general health (108). As reviewed by Surien et al (110), a number of natural compounds have been proven to be beneficial in the treatment of lung cancer at pre-clinical studies. From the literature search, the present review identified 6 different studies that evaluated ROCK expression following treatment with natural compounds (46,80,102,106-108). Notably, these natural compounds have been documented to be capable of reducing RhoA, RhoC and ROCK expression, all of which promote cell proliferation, migration, invasion and angiogenesis.

The specificity of ROCK inhibition should also be considered, as some ROCK inhibitors, such as Y-27632 have additional off-target inhibitory activity against mitogen- and stress-activated protein kinase 1 (MSK1) (111). Therefore, a more potent and specific ROCK inhibitor should be developed to resolve these issues. Some researchers have begun to investigate this possibility by developing OXA-06, a potent ATP-competitive ROCK inhibitor that is structurally distinct from Y-27632, a ROCK1/2 inhibitor (15) and a Rho-kinase inhibitor (RKI) (112), both of which have been tested and proven to exhibit less in vitro off-target protein kinase inhibitory activity. OXA-06 also has the ability to block anchorage-independent cancer cell growth by causing cell cycle arrest in G0/G1 (15). siRNA and short hairpin RNA (shRNA) have also been used for the inhibition of ROCK activation in lung cancer studies. These short double-strand RNA molecules are a promising therapeutic approach that can be programmed to silence a specific target. Their use, either in vitro or in vivo, has allowed researchers to compare the effects of ROCK inhibition with those of their novel compounds and has enhanced our understanding of the underlying mechanisms that regulate this pathway. For example, siRNA targeting either ROCK1 or ROCK2 alone is capable of inhibiting ~90% of NSCLC anchorage-independent cells. Moreover, non-specific siRNA that targets both ROCKs effectively inhibits tumor growth as reflected by near-complete colony formation suppression. These findings demonstrate that siRNAs can specifically discriminate against ROCK homologs and give a clear image of their inhibition effects on a specific ROCK homolog (15). Moreover, shRNA has been used to target RhoC (85) and ROCK1 (37) in lung cancer studies. These studies reported that the inhibition of RhoC and ROCK1 by shRNA significantly suppressed the EMT process and the migration of lung cancer, as with other ROCK inhibitors.

The use of microRNAs (miRNAs or miRs) for the treatment of lung cancer has also been documented. miRNAs are small, non-protein-coding RNA molecules that have been shown to be involved in carcinogenesis as either tumor suppressors or oncogenes (113). miRNAs are regarded as convenient biomarkers due to their better stability compared to mRNAs. As reviewed by Iqbal et al (114), several miRNAs have been identified to play a prominent role in lung cancer, and a few have been identified to target RhoA/ROCK. Throughout the literature, only 4 studies on miRNAs have been found that elucidate ROCK expression. These miRNAs have been reported to be downregulated in NSCLC tissue biopsies and their restoration in vitro has been reported to reduce the proliferation, migration and invasion of the A549, H1299 and SPC-A1 cell lines by decreasing ROCK expression (19,20,115,116). Furthermore, a previous meta-analysis and Kaplan-Meier data by Yang et al (117) and Wu et al (118) demonstrated that other miRNAs, such as miRNA-21, miRNA-155, miRNA-19b and miRNA-146a, can be used to predict recurrence, as a prognostic biomarker and to demonstrate association with the survival rate of patients with lung cancer.

Generally, studies on ROCK and lung cancer in literature can be divided into 2 major categories: Kinase-targeting ROCK inhibition (Table I) and non-kinase-targeting ROCK inhibition (Table II). Kinase-targeting ROCK inhibitors target upstream effectors, downstream substrates, or ROCK, whereas non-kinase-targeting ROCK inhibitors target signaling pathways other than ROCK, such as proteins, oncogenes and non-coding RNAs, but can still affect ROCK expression. The targets of the 24 ROCK inhibitors identified in the literature are presented in Fig. 3. Collectively, these inhibitors can inhibit MLC/LIMK phosphorylation, which activates cofilin and promotes F-actin depolymerization. As a result, crosslinking between F-actin and myosin is reduced, leading to a decrease in actomyosin contractility, which plays an essential role in the transformation of malignant phenotype in cancer cells (119). It was found that the majority of the kinase-targeting ROCK inhibition studies were conducted in vitro using NSCLC cell lines, while there is a lack of studies conducted in vivo or by using tissue biopsies of lung cancer patients. The most commonly used cell line is human A459 lung adenocarcinoma cells, followed by H1299 and H460 cells, which are also categorized as NSCLC.

Targeting proteins, oncogenes, or non-coding RNAs that coordinate Rho may also be a promising approach, as presented in non-kinase-targeting ROCK inhibition studies (Table II). Generally, these studies have reported that NSCLC tissue has a higher expression of DEK (73), keratin14 (KRT14) (120), chromodomain helicase DNA-binding protein 4 (CHD4) (74) and long non-coding RNA (lncRNA) NORAD (121); the depletion or disruption of these has been observed to reduce RhoA, ROCK and phospho-myosin expression in NSCLC cell lines. It has also been demonstrated that the depletion of these proto-oncogenes is capable of inhibiting NSCLC cell growth by promoting apoptosis and attenuating their proliferation, migration and invasion (73,74,120,121). In addition, a high DEK and CHD4 expression has been shown to be associated with poor survival rates among patients with NSCLC, as analyzed using Kaplan-Meier data, and to be positively associated with TNM staging, differentiation and nodal status (73,74). Therefore, it is suggested that DEK and CHD4 expression may be critical factors and potential biomarkers of NSCLC progression. Therefore, studies on DEK, CHD4, KRT14 and lncNORAD expression in NSCLC tissues provide insight into the possibility of these proteins to be translated into potential therapeutic targets for treating lung cancer.

Some studies have found inconsistent effectiveness in treating cancer either by targeting both ROCK isoforms, or only one of them. Furthermore, the characterization of the role of ROCK1/2 in lung cancer carcinogenesis is therefore essential. However, ROCK1 tends to be more significant than ROCK2 in lung cancer studies (34,37). ROCK1 is also the preferred isoform in most of the kinase-targeting ROCK inhibition studies, as shown in Fig. 3. It may be due to the predominant role of ROCK1 over ROCK2 in regulating cytoskeletal organization, which indicates isoform-specific regulation in lung cancer. ROCK1 has been regarded as a positive regulator of cell migration and invasion in several solid tumors, such as osteosarcoma (38), breast (12), pancreatic (122), gastric (123) and lung cancer (37,124,125). In addition, some ROCK inhibitors, such as Y-27632, which can target both ROCK isoforms, have been found to be more potent against ROCK1 than against ROCK2 (126). However, this should not be the reason to discount the role of ROCK2 in lung cancer growth. Further evidence is needed to demarcate the role of ROCK2 in lung cancer. Overall, the aim of these ROCK inhibition studies, either kinase-targeting or non-kinase-targeting ROCK inhibition, is to determine the effects of ROCK inhibition that elucidates the ROCK function in lung carcinogenesis. The majority of the studies included cell migration assays to investigate cellular motility changes following ROCK inhibition or knockdown, since it is a key mediator that regulates cytoskeletal rearrangement that affects cellular motility (37,104,116,125). Examples of the migration assays employed are Transwell, collagen invasion, tube formation and scratch wound healing assays. Their findings suggested that the ROCK signaling pathway should be considered as a potential therapeutic target for inhibiting lung cancer development, as ROCK action is diffuse and its activation can promote multiple hallmarks of cancer.

Identifying the genetic aberrations of lung cancer, such as KRAS and EGFR mutations is important in tailoring the appropriate therapy for the disease. NSCLC harboring KRAS mutations has been found to be more vulnerable to RhoA inhibitor-induced apoptosis compared to wild-type NSCLC (127). As KRAS mutations are common in lung cancer, occurring in 30% of adenocarcinoma and 5% of SCC cases, the inhibition if RhoA appears to be an excellent treatment option for NSCLC (45,128). Furthermore, the KRAS-mutant NSCLC cell line is also vulnerable to a combination of drug pairs that inhibit polo-like kinase 1 (PLK1), a synthetic lethal partner of Ras oncogene, and ROCK that exhibit marked apoptosis induction and colony form inhibition (45). Another common gene aberration that has been studied as a drug target is EGFR mutations, which occur in 39% of adenocarcinoma and 58% of the SCC subtype of lung cancer (129). EGFR is a tyrosine kinase that plays an essential role in SCC pathogenesis by dimerizing its receptor (130). Therefore, inhibiting EGFR dimerization can inhibit the pathogenesis, and inhibiting RhoA by using lovastatin has been shown to yield the same effect as an EGFR inhibitor, as RhoA is necessary for EGFR localization and activation (131). Inhibiting EGFR activation can also reduce programmed death-ligand 1 (PD-L1) expression associated with the p-ERK1/2/p-c-Jun pathway (132). Reducing PD-1 and its interaction with the ligand is important in restoring the proliferation of T-cells and promoting the cytotoxic activity of immune cells to cancer cells (133). Importantly, an increased PD-L1 expression has been reported to be associated with EGFR mutations (134,135) and is currently the only approved biomarker for NSCLC immune checkpoint inhibitors (136).

Targeting proteins that regulate the ROCK signaling pathway has also been suggested to yield promising results. Osteopontin (OPN), a major non-collagenous bone matrix protein, has been found to be responsible for activating ROCK1, and to subsequently increase LIMK and cofilin phosphorylation in NSCLC cell lines, thereby promoting cancer cell migration and invasion (103). It is suggested that OPN is a worthy target as its inhibition can significantly reduce the tumor weight and volume of NSCLC as studied by Cho et al (137). In addition, OPN can reduce lamellipodia formation and actin polymerization via the ROCK signaling pathway following suppression with zerumbone and plumbagin, respectively (46,80). Since OPN plays an essential role in mediating tumor-stroma interaction and contributes to tumor growth and metastasis (138), targeting OPN may hold promise for the prevention of lung cancer metastasis. In addition, caveolin-1 (CAV1), an ECM-associated oncogenic membrane protein that can activate the ROCK signaling pathway, may also be a potential druggable target. High levels of stromal CAV1 have been identified in various types of cancer, including lung cancer (139,140) and its interactions with Rho-GTPases have been demonstrated to promote metastasis through Src, Ras and Erk activation (141). This interaction promotes cell migration and invasion by regulating CAV1 tyrosine phosphorylation, which can lead to the regulation of focal adhesion dynamics (142).

The treatment of lung cancer has improved substantially over the years, involving various strategies and modalities, such as surgery, radiotherapy, chemotherapy, immunotherapy and molecular-targeted therapy (143). The search for a suitable target candidate to treat lung cancer is still ongoing. Herein, the ROCK signaling pathway is suggested as one of the potential targets that can be utilized for the treatment of lung cancer, since its inhibition has resulted in promising outcomes to reduce cancer cell proliferation, migration, and invasion in pre-clinical studies. Repurposing the use of already licensed drugs, such as fasudil for the treatment of lung cancer is a good start, as it provides a rapid translation of pre-clinical data into effective therapies for lung cancer patients. However, the use of fasudil is still associated with certain drawbacks, such as the off-target effect. On the other hand, other novel compounds may have insufficient efficacy apart from the concerns of side-effects and the selective binding of ROCK inhibitors, as the ROCK signaling pathway also plays an essential role in normal cell homeostasis. Nevertheless, the expression of ROCK in cancer is higher if compared to normal cells or tissues; thus, the use of ROCK inhibitors in cancerous and normal cells or tissues may yield different outcomes in term of expression following treatment. Specific pathway inhibition, limited to cancer cells, is preferable to prevent undesirable side-effects caused by current chemotherapeutic drugs. Obtaining a range of effective doses without causing adverse effects in patients is also another challenge that requires a comprehensive analysis of safety, efficacy and toxicity to be made. The majority of the ROCK and lung cancer studies were found to be performed in vitro only, while there is a lack of in vivo studies or the use of tissue biopsies from patients with lung cancer. Therefore, further research should also employ these more complex settings to fully elucidate the mechanisms of the ROCK signaling pathway in lung cancer.

To date, to the best of our knowledge, no single inhibitor targeting the ROCK signaling pathway has been approved for use in clinical trials against lung cancer. Nevertheless, defactinib, a drug that can inhibit FAK, a mechanosensor that can detect changes in the ECM and activate ROCK, has been tested in a clinical trial for the treatment of KRAS mutant NSCLC. However, defactinib has been shown to only yield modest and contrasting results from pre-clinical studies (144). This may be due to the insufficient efficacy and it being an unspecific target of FAK inhibitor. Therefore, it is suggested that the targeting of ROCK should be performed to yield a more profound impact in regulating cellular phenotypes, as discussed herein, thus treating lung cancer. A combination of agents in the treatment of lung cancer should also be considered in future research, as supported by better outcomes in pre-clinical studies targeting ROCK with EFGR or PLK1 (45,104). Moreover, an agent that can affect multiple oncogenic pathways and fine-tuning treatment strategies based on molecular aberrations can provide more effective treatment strategies, since cancer is highly adaptive and can acquire resistance rapidly (145,146). Targeting ROCK can also help to solve several issues, such as drug resistance seen in hypoxic tumors. According to Murakami et al (147), gefitinib was found to be ineffective in hypoxic EGFR mutation-positive NSCLC due to a vascular inadequacy that dampens the bioavailability of the drug in the target area. Therefore, targeting ROCK along with gefitinib can help in restoring the role of the blood vessel to deliver oxygen and increase the drug bioavailability. Taken together, the ROCK signaling pathway plays a critical role in the carcinogenesis of lung cancer, and is therefore suggested as a potential therapeutic target in the treatment of lung cancer. Further in-depth research is urgently required to enhance our understanding of this pathway, and further attempts should be made to elucidate the biological mechanisms between ROCK and lung cancer.