Journal of Clinical & Experimental OncologyISSN: 2324-9110

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Review Article, J Clin Exp Oncol Vol: 6 Issue: 4

Rho-Kinases in Oral Squamous Cell Carcinoma: A Review

Anjali P Ganjre*, Sangamithra S and Asmita Kharche

Department of Oral Pathology and Microbiology, Dr. D.Y Patil Dental College and Hospital, Dr. D.Y Patil Vidyapeeth, Pimpri, Pune 411018 Maharashtra, India

*Corresponding Author : Anjali P Ganjre
Department of Oral Pathology and Microbiology, Dr. D.Y Patil Dental College and Hospital, Dr. D.Y Patil Vidyapeeth, Pimpri, Pune 411018 Maharashtra, India
Tel: 9970722995
E-mail: [email protected]

Received: April 10, 2017 Accepted: April 20, 2017 Published: April 27, 2017

Citation: Ganjre AP, Sangamithra S, Kharche A (2017) Rho-Kinases in Oral Squamous Cell Carcinoma: A Review. J Clin Exp Oncol 6:4. doi: 10.4172/2324-9110.1000189

Abstract

OSCC is most common and lethal malignancy worldwide. Global estimated death is to be more than 10,000 annually. Metastasis is the deadly consequence associated with OSCC because of homing of cancer cells to lymph nodes. Motility of cells is accomplished by important signaling molecule known as Rho kinases. Rhokinases modulates cytoskeleton of ?transformed? malignant cell to govern distant metastasis. Interaction of Rho kinases and signaling molecules assists in driving the tumor cell through extracellular matrix and blood vessels to reach the destined location. By focusing and systemizing on the molecular aspect of Rhokinases will help in divulging the problem of the mobility of OSCC cells and will also aid in designing novel anticancer therapies.

Keywords: Rho-kinases; Oral cancer; Metastasis

Introduction

Rho-Kinases belong to the AGC family of serine/threonine protein kinases. It consists of protein kinases A, G, and C. Increase in ROCK signaling assists in tumor motility and metastasis which accountable for aggressiveness of cancer cells [1]. ROCK proteins support the generation of stress fibers and focal adhesions [2]. They are central regulator of the actin cytoskeletal (AC) organization and downstream regulator of the small GTPases Rho [1]. They involve numerous alterations in the AC and extracellular matrix which determines metastatic process [3].

Somatic mutation in RHO kinase genes have been identified in human cancer. Mutation in ROCK genes causes increase in kinase activity due absence of auto-inhibition. Increased in ROCK protein expression is correlated with aggressive behavior and poor survival in various types of cancers [4].

Homing of cancer cells in lymph nodes is the key problem associated with OSCC as it directly affects the survival and prognosis of the patient. Altering or disrupting the activity of the RHO kinase signaling would achieve commendable results in restraining the metastatic process. So aim of our paper is to review the various biomarkers which play important role in modulating ROCK activity in OSCC. It will aid in tailoring new treatment plans for the patients.

Discussion

Rhokinases plays vital role in metastasis of the oral cancer [5]. It acts via various signal transduction pathways and molecules. It has a coordinated effect on controlling actin cytoskeletal and dissolving cell-cell junction to achieve lymph node metastasis. It guides cancer cells in invading extracellular matrix and blood vessels to reach the destined site [4].

Till date, various molecular markers have been applied to study the molecular unrevealed aspect of invasion of OSCC. In our review, we have analyzed those molecular markers which could help in determining the movement of cancer cells to achieve metastasis (Table 1).

Sr No. Studies done Molecules Expression of Rhokinases Methods
1 Torre et al. [8] CD 44/HA Increase Rho Immunoblotting assay on cell lines
2 Bourguignon et al. [9] CD44-LARG Activate RhoA Cell culture, immunoblotting
3 Tanaka  et al. [11] Pim-1 High positive expression Ihc, Immunoblotting assay
4 Jiang et al. [15] mRNA 138 Reduced expression RT-PCR, western blot, assays
5 Zhao et al. [16] Lovastatin Expression of Rho Immunoflourescence microscopy, west blot, gel electrophoresis, Rho activity assays
6 Islam et al. [3] Atorvastatin Inhibition of Rho Western blot, various assays, confocal microscope
7 Yao-yin Li et al. [17] Snail Activate RHO PCR
8 Su et al. [18] DEPDC1B No Effect on RhoA Western blot, assays
9 Goncalves et al. [20] melatonin Inhibition of RHO in SS9, no effect on SS25 cell lines RT-PCR, IHC, assays
10 Miyamoto et al. [21] Fasudil  Inhibition of  ROCK /RHOA PCR-RT, In vivo
11 Tumur et al. [23] EGFR RHO overexpressed in HNSCC Cellculture, western blot, assays, immnoflourecence staining
12 Tsai et al. [24] SHSP 2 Depletion Immunoblotting, assays
13 Pan et al. [25] PKC elevated RT-PCR, western blot analysis, cell lines assay
14 Xu et al. [26] CCR7 Overexpressed, positively correlated with HNSCC assay Western blotting,
IHC, assay
15 Iyoda et al. [28] Epithelial cell transforming sequence overexpressed RT-PCR

Table 1: Showing studies done on Rho kinases and its effect on the various molecules in OSCC.

Hyaluronan (HA) is a protein interacts with surface receptor CD 44 for degradation of extracellular matrix. External portion of CD 44 binds with HA while internal portion is engages with the RHO activated RHO kinases. These interactions are responsible for cytoskeletal modulation to acquire pro-metastatic specific phenotype of the cancer cells [6]. Thus HA and CD 44 were found to be accountable for the invasion and mobility of the cancer cells by activating Rho kinases [6].

Phosphatidylinositol 3 kinase is proficient in catalyzing the translation of phosphatidylinositol 3,4-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) which results in activation of AKT pathway. Rho kinases found to increase the activity of PI-3 kinases which enhances the survival mechanism and the chemo-resistance of cancer cells [7].

Torre et al. found that specific inhibitors of Rho kinase and PI-3 kinases signaling were helpful in cessation of progression of HNSCC cell. It was found that RHO kinase activity was critical for HA-CD44- mediated phosphatase phosphorylation. By inhibiting Rho kinase activity would aid in diminishing the phosphatase phosphorylation which in turn reduces the ability of HA to accelerate the migration of cancerous cells. Moreover, it was revealed that RHO kinase signaling was responsible for inducing the expression of Matrix metalloproteinase (MMP) via enhancing the expression of membrane type I- MMP for ECM degradation [8].

The leukemia-associated Rho guanine nucleotide exchange factor (LARG) was found in patients with acute myeloid leukemia. It acts as a GDP/GTP exchange protein for the Rho subfamily of GTPases such as RhoA. It was demonstrated that CD44/HA induces LARG mediated RhoA activation for the invasion of Head and neck squamous cell carcinoma (HNSCC). The DH domain of LARG exihibits GDP/GTP exchange activity for RhoA signaling. Thus, HACD44 interaction serves as an upstream activator of LARG function. Moreover, study proved that HA/ CD44 interacts with EGFR and leads to stimulation of phospholipases Cs (PLC) mediated PI 3(Rho kinaseand phosphatidylinositol 3) production. HA-CD44 later on attaches to PI3 receptor results in the intracellular calcium mobilization, cytoskeleton activation and reorganization of actin filament which results in invasion of tumor cells [9].

Human Pim-1(a homologue of murine Pim-1 which encodes serine/threonine kinase) has a role in the cancerous transformation of blood cells. It is pro-oncogenic gene. Research has proven that Pim-1 was implicated in the Oncogenesis of T-cell lymphoma in cooperation with c-Myc. It was responsible for proliferation of cells by inhibiting apoptotic pathways [10].

Tanaka et al showed the correlation of Pim and cell motility in OSCC. They had proved that OSCC cells with high expressing Pim have a potential for metastasis. Pim increases the Rho associated protein in cancer cells. It results in formation of lamedoodia and invadopodia by modulating organization of cytoskeleton filament [11].

SiRNA is a functional molecule. It is produced by Dicer cleavage of 200-500 nucleotides length dsRNA duplexes. SiRNAs forms complexes with RISC (RNA-induced silencing complex) which are involved in gene silencing, viral defense and transposon control. It was found that siRNA can systemically silence carcinogenic genes by specifically targeting tumor cells [12]. Zhou J revealed the role of RhoA in tongue squamous cell carcinoma cell lines. Silencing of endogenous RhoA gene has an inhibitory action on RhoA activity and thus shown to be potential target in controlling metastasis of OSCC [13].

Micro RNA is non-coding microRNAs. At the post transcription level, it regulates the target gene expression. Reduce expression or function of microRNA was related with tumorigenic process and metastasis. It was found that specific miRNA has a specific role in invasion and metastasis. MiR-222 suppresses the tongue squamous cell carcinoma cell invasion [14].

Jiang et al proved that inhibition of miR-138 level was linked with enhanced metastatic potential of cancer cells. It was found that two putative miR-138 precursor genes; pre-miR-138-1 and premiR- 138-2 and its human homologs were found to be located on chromosome 3p21.33 and 16q13, respectively. Loss of heterozygosity (LOH) at both chromosomal loci was related with enhanced cell invasion. Experimental data proved that miRNA-138 responsible for migration, invasion, cell shape, and regulates migration and invasion stress fiber formation via RhoC activation. Study found out that Rho directly target miRNA138 gene at specific site and controls cell migration and invasion. MiRNA was found to target Guanine nucleotide exchange factors (ARHGEF) in Rho signaling pathway which helps in achieving cessation of metastasis [15].

Statins are powerful inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase which involved in mevalonate pathway (MP). MPs end products are responsible for formation of Geranylgeranyl transferase (GGPP) and farnesyl transferase (FPP). They serve as membrane anchors for post-translational modifications of small guanosine-50-triphosphate-binding proteins for regulation of proliferation of cell, intracellular trafficking and motility of cell. MP prevents the activity of Geranylgeranyl pyrophosphate (GGPP) which is responsible for isoprenylation of Rho proteins. Zoa et al. has corelated the effect of Lovastatin with EGFR and Rho kinases in oral squamous carcinoma cells. It was found that Lovastatin’s induces actin disorganization by geranylgeranylation of the Rho family of proteins which results in the inhibition of Epidermal growth factor receptor (EGFR) dimerization, commencement and downstream signaling. It helps in cessation of proliferation and movement of cancer cells. Moreover, activation of Rho has a direct effect on cell cycle by cyclin D1 up regulation. Lovastatin has a dose-dependent effect on cyclin D1 protein [16].

Islam et al. showed the correlation of Atorvastatin and RhoC in cell invasion and motility. Atorvastatin modulates Rho C function. It decreases the mobility, invasiveness, stress fiber integrity and proliferation of malignant cells which helps in cessation of anchorage dependent colony formation at distant site. It reduces phosphorylation of ERK1/2 and STAT3 which responsible for modulation of actin cytoskeleton. Study demonstrated that Atorvastatin disrupts actin cytoskeleton re-arrangement and controls the directional movement of metastatic cells by altering the functional activity of Rho C. It was also shown that atorvastatin causes inhibition in translocation of RhoC from cytosol to membrane position so there was no downstream signal transduction from membrane receptors [3].

Snail-related zinc-finger transcriptional repressors (Snail/SNAI1 and Slug/SNAI2) plays the role by controlling cell morphology, cellcell interactions via Cdc42. Deletion of the Snail 1Gene (SNAI1) aids in interfering the regulation of cell morphology and cell-cell interactions via Cdc42.

The ezrin/ radixin/moesin (ERM) family is a cross-linker between plasma membranes and actin cytoskeleton. Rho GTPases and ERM family are involved in tumor metastasis. Snail silencing causes significant decrease in the level of Cdc42 activity and in filopodia formation by inhibiting premature assembly of stress fibers. Also, RhoA and Cdc42 regulates downstream of Snail. Rho A increases Cdc42 activity and enhances the association of ERM with activated Cdc42, promoting Snail-mediated cell motility. Yo Yin Li was found that Snail regulates cell motility through RhoA/ Cdc42/p-ERM pathway in OSCC. Study demonstrated that Snailsilencing suppressed the motility of OSCC cells by disorganization of cytoskeleton via RhoA/Cdc42/p-ERM pathway [17].

The DEP domain is a globular domain consists of 90 amino acids and founds to implicate in membrane localization and controlling broad range of cellular activities. The DEPDC1B protein contains 2 conserved domains (DEP and RhoGAP) that are involved in Rho GTPases signaling. It regulates the cell motility, growth, differentiation, cytoskeletal reorganization and cell cycle progression via Rac1 expression. It affects Rac1 GTP loading and supports ERK1/2 activity. Overexpression of DEPDC is associated with invasion and cell motility. Su et al showed that DEPDC1B regulates anchorageindependent growth of oral cancer cells by regulating Rhokinases via ERK1/2 pathway [18].

Melatonin has oncostatic, antiangiogenic and antimetastatic properties in several types of neoplasms. Melatonin decreases migration and invasiveness of cancer cells by increasing the expression of cell surface adhesion proteins, E-cadherin and beta (1)-integrin. ROCK and melatonin in collaboration with each other participates in inhibition of migration of cancer cells by changing cytoskeletal organization of leader cell. It was found that melatonin increases the number of focal contacts through ROCK [19] Goncalvels analyses the action of melatonin on expression of HIF-1α, Vascular endothelial growth factor (VEGF) and ROCK-1 gene protein. Study found out that melatonin inhibits the expression ROCK-1 genes in OSCC. Thus acts as an antimetastatic agent [20].

Fausudil is a Rho kinase inhibitor and has capacity of restoring BRAK which is an antitumor cytokines in fibroblasts. Research has found that as Fausudil is a ROCK inhibitor which suppresses the growth of fibrosarcoma by increasing BRAK expression. It modulates myosin light chain phosphorylation in fibroblasts and thus controlled the directional movement of cancer cells. Similarly, several studies have proved that Fasudil stimulates the expression of BRAK proteins in cancer cells. Thus Fasudil via “RhoA/ROCK pathway inhibitor” suppresses tumor progression by increasing the expression of BRAK gene.21 M Moreira Carboni Sde et al. demonstrated the inhibitory role of HA-1077 (Fausil) on Rho kinase. In HNSCC, Fausil acts as a RHO kinase inhibitor and can be used as a therapeutic purpose for cessation of metastasis [21].

EGFR expression is directly correlated with prognosis of HNSCC patients. Increase in EGFR results in poor prognosis. It was found that EGFR family and its downstream signaling pathways such as PI3K/ Akt/GSK3β upregulated snail expression and directly decreases E cadherin level [22].

Tumur et al. demonstrated the role of Rhokinases in HNSCC and correlates it with E cadherin expression via EGFR/ PI3K/Akt/GSK3β signaling pathway. It was revealed that activation of Rho signaling, following EGFR phosphorylation, causes significant down-regulation of cell surface E-cadherin level in HNSCC cells. Also, it activates RhoC via PI3K signaling which stabilizes snail and down-regulates E-cadherin, results in EGF-induced cell invasion [23].

Src homolog domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a positive signal transducer between receptor tyrosine kinase and ERK pathway. They are responsible for cell proliferation and cell migration. They involved in invadopodia formation and directional movement of cancer cells. Tsai et al demonstrated that SHP2 modulates invadopodia formation by suppressing RHO signaling in HNSCC. Study has shown that SHP2 dephosphorylation causes activation of Rho GTPases-activating protein (GAPs) results in cessation of metastasis and invasion [24].

Protein kinase family (PKC) is a serine/threonine kinase and responsible for proliferation, differentiation, apoptosis, and migration of cancer cells. “In vitro” study demonstrated that epidermal PKC was responsible for metastasis of cancer cells in transgenic mice in response to 12-O-tetradecanoylphorbol-13-acetate stimulation. It also modulates hepatocyte growth factor/c-Met signaling which causes angiogenesis, tumorigenesis, and metastasis in HNSCC.25 Pan et al demonstrated that disruption of PKC results in inactivation of Rhokinases. PKCε-Rho GTPases signaling axis is critical for modulating an invasive and motile phenotype in HNSCC tumor cells. Study demonstrated that disruption of PKC pathway results in inhibition of Rhokinases [25].

Chemokines are responsible for the dynamics of cytoskeleton and thus responsible for directional migration of cancer cells. CCR 7 helps in metastasis of HNSCC cells. It was found that increased expression of CCR7 in HNSCC activate phosphoinositide-3 kinase (PI3K) Cdc42, Pyk2, mTOR and NF-κB pathways. This pathway encourages cancer cells to invade into lymph nodes and at the same time they help in sustenance of their survival. Xu et al. revealed that RHO kinase was activated by CCR7 via P13K and results in metastasis of HNSCC cells [26].

Rho family of RhoA, Rac and Cdc42 regulates activity of actin cytoskeleton which helps in motility of cells. Rac1 promotes membrane ruffle and lamellipodia formation while Cdc42 generate filopodia in the leading edge of migrating cells.

Patel el al. “in vitro” demonstrated the status of activation of RhoA, Rac and Cdc42 in HNSCC cell lines. It was proved that the EGFR/ Vav2/Rac1 axis was crucial pathway for the acquisition of mobility and invasive attributes of HNSCC cells. Ras and RhoA displayed a constantly active EGFR/Vav2/Rac1 signaling axis which participates in tumor invasion and metastasis. Rac1 was found to coordinate the activation of “signaling molecules” involved in remodeling of the actin cytoskeleton [27].

ECT2 is a guanine nucleotide exchange factor (GEF) for Rho GTPases family and responsible for cytokinesis in tumor cells. GEFs activate RHO GTPases in signal transduction pathway by catalyzing the exchange of GDP for GTP. Iyoda et al. unrevealed the action done by ECT2 in OSCC cell lines. It was found that knockdown of ECT2 results in impaired activation of Rho GTPases and downregulation of cyclin D1, cyclin E, and CDK4 which leads to cell cycle arrest at the G1 phase and thus accountable for cessation of cancer cell progression [28].

Conclusion

Mobilization of malignant cells toward distant site is a lethal process in OSCC. Rho kinases are allocated as a crucial player for the migration of malignant cells at distant site. Interaction of array of molecules with Rho kinases deals with the modulation of cytoskeleton which responsible for invadopodia formation in the cancer cells. These molecules directly or indirectly influence the activity of Rho kinases. Different preclinical and clinical trials have been carried out to assess the role of Rho kinase signaling as an anticancer therapy. Many positive clinical outcomes targeted for Rho kinase activity were found to be beneficial for controlling metastatic potential of cancer cells. By acknowledging the importance of interaction of these molecules with Rho kinases would provide a better opportunity in improving and designing novel treatment program for OSCC patients.

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