Rho-Associated Coiled-Coil Research Articles

391-P: Targeted Deletion of Rho-Associated Coiled-Coil–Containing Protein Kinase 2 in Kidney Tubules Suppresses Expression and Function of Mineralocorticoid Receptor in Mice

391-P: Targeted Deletion of Rho-Associated Coiled-Coil–Containing Protein Kinase 2 in Kidney Tubules Suppresses Expression and Function of Mineralocorticoid Receptor in Mice

Inhibition of Rho-Associated Coiled-Coil Containing Protein Kinases with Belumosudil Mesylate Shows Anti-Tumor and Immune Modulatory Properties in Models of Multiple Myeloma

Background: Myeloma patient outcomes have continuously improved with the introduction of proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), monoclonal antibodies (mAbs) recognizing cell surface proteins and, most recently, B-cell maturation antigen (BCMA)-targeted bispecifics and chimeric antigen receptor (CAR)-guided T-cells. However, the prognosis in triple-class refractory (TCR) disease that is refractory to PIs, IMiDs, and CD38 mAbs remains poor and, since BCMA-targeted CAR-Ts and bispecifics are not curative, quadruple-class refractory (QCR) disease that progresses during or after these therapies is an emerging challenge, indicating a persistent need for novel agents. Rho-associated coiled-coil containing protein kinases (ROCK1 and ROCK2) may represent novel potential targets since ROCK1 has been associated with a plasma cell leukemia-like transcriptional profile and as a myeloma dependency. Moreover, ROCK2 mediates expression of Interferon regulatory factor 4 (IRF4) and c-MYC, as well as Th17 responses that may contribute to an immune suppressive microenvironment. Methods: To examine the central hypothesis that targeting ROCK1/2 could be a novel and effective approach to myeloma therapy, we performed pre-clinical studies with the ROCK1/2 inhibitor belumosudil mesylate using drug-naïve and drug-resistant myeloma cell lines in vitro. Also, in vivo studies with immune correlates were performed in the Vk*MYC immune-competent syngeneic myeloma model, which has been reported to have a 67% positive predictive value for clinical efficacy in humans. Results: Belumosudil mesylate as a single agent reduced the viability of a panel of myeloma cell lines representing different molecular subtypes in a time- and concentration-dependent manner, with median inhibitory concentrations from 618 to 1,114 nM at 72 hours. These represent clinically relevant concentrations as the mean steady state C max for belumosudil mesylate given with food in studies of chronic graft-versus-host disease patients was 4.36 mM. Staining of myeloma cells with Annexin V and propidium iodide (PI) demonstrated that belumosudil induced appearance first of Annexin +/PI - (early apoptotic) cells, followed later by Annexin +/PI + (late apoptotic) and Annexin -/PI + (necrotic) cells, in a concentration-dependent manner. Studies of myeloma resistance mechanisms showed belumosudil largely overcame adhesion-mediated drug resistance, and was active against MM.1S and RPMI 8226 cells that are considered daratumumab-resistant. Moreover, belumosudil showed equal or even, in some cases, higher potency against bortezomib-, carfilzomib-, dexamethasone-, iberdomide-, lenalidomide-, melphalan-, and mezigdomide-resistant cell lines compared to their drug-naïve counterparts. Belumosudil induced in vitro killing of myeloma cells in a time- and dose-dependent manner independent of the presence of natural killer (NK) cells. Notably, when used in combination with the CD38 mAb isatuximab and in the presence of NK cells, belumosudil enhanced myeloma cell killing and prevented isatuximab-induced loss of CD38 expression. Mechanistically, belumosudil induced a reduction in c-MYC, IRF4, and phospho-STAT3 levels, consistent with its action on the IRF4/c-MYC axis. Dosing of belumosudil in vivo at 50 mg/kg twice weekly for three weeks in Vk*MYC mice with paraprotein levels consistent with active myeloma produced a consistent decline in disease burden as determined by serum protein electrophoresis monitoring ( A) compared to vehicle controls. NanoString analysis of tumor samples from these mice ( B) found down-regulation of XBP1 (X-box binding protein 1) and SLAMF7 (Signaling lymphocytic activation molecule family member 7), consistent with the anti-tumor efficacy of belumosudil mesylate. In addition, up-regulation was seen of c- JUN and c- FOS, consistent with promotion of T-cell activation and clonal expansion, as well as effector differentiation of antigen-activated CD8 + T cells. Conclusions: These pre-clinical in vitro and in vivo data support the hypothesis that targeting of ROCK1 and ROCK2 with belumosudil mesylate may be a promising strategy for relapsed/refractory myeloma and provide a rationale for its translation to the clinic. Currently, clinical trials of belumosudil are planned.

Simultaneous Effects of a Selective EP2 Agonist, Omidenepag, and a Rho-Associated Coiled-Coil Containing Protein Kinase Inhibitor, Ripasudil, on Human Orbital Fibroblasts.

Purpose: To assess the combined effects of omidenepag (OMD), a selective EP2 agonist, and ripasudil (Rip), an inhibitor of rho-associated coiled-coil containing protein kinases, on the human orbital adipose tissue, two-dimensional (2D) or three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were employed. Methods: Cellular metabolic functions (2D), physical (3D), lipid staining (3D), and quantitative polymerase chain reaction for adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) molecules, including collagen (COL)1, 4, and 6, and fibronectin (FN) (3D) were evaluated in the presence of OMD (100 nM) and/or Rip (10 μM). Results: Real-time metabolic analyses revealed that the adipogenic differentiation (DIF+) with OMD significantly shifted an energetic state toward energetic, whereas DIF+ with Rip significantly shifted that toward quiescent. In the case of both drugs upon DIF+, the metabolic effect of OMD was predominant. DIF+ induced enlargement and stiffed 3D spheroid with increased lipid staining and mRNA expression of adipogenesis-related genes, COL4 and COL6, and decreased the expression of COL1. In the presence of OMD and/or Rip to DIF+, (1) the sizes were further increased by Rip and the stiffness was significantly decreased by OMD or Rip and (2) COL4 or AP2 expression was substantially increased by OMD or Rip, respectively. Conclusion: The results presented herein indicate that the metabolic effects of OMD and Rip exerted opposing effects and the effects of OMD toward Ap2 and ECM expressions were distinct from those of Rip, but the effects of OMD toward the physical aspects and adipogenesis of the 3D cultured HOFs were similar to the effects of Rip.

Structural Insights into the Interactions of Belumosudil with Rho-Associated Coiled-Coil Containing Protein Kinases 1 and 2 Based on Molecular Docking, Molecular Dynamics Simulations, and Free Energy Calculations

Rho-associated coiled-coil containing protein kinases (ROCKs) are members of the cyclic adenosine monophosphate-dependent protein kinase/protein kinase G/protein kinase C family that participates in a variety of important physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, migration, and inflammatory responses. In this study, we focused on ROCK1 and ROCK2, which are targets of the Food and Drug Administration-approved inhibitor 2-(3-(4-((1[Formula: see text]-Indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-[Formula: see text]-isopropylacetamide (belumosudil). We constructed four representative belumosudil/ROCK complex structures by molecular docking. The interactions between belumosudil and ROCK were then investigated via molecular dynamics simulations and binding free energy calculations. It was found that belumosudil showed a stronger binding affinity toward ROCK2 than toward ROCK1. Binding free energy calculations and free energy decompositions suggested that the modification of various regions of the belumosudil structure may enhance its binding affinity with ROCK, in addition to improving the selectivity between ROCK1 and ROCK2. This work therefore provides useful information to aid future drug design.

Rock1 is a novel host dependency factor of human enterovirus A71: Implication as a drug target.

Human enterovirus A71 (EV-A71) is the major causative agent of hand-foot-and-mouth disease (HFMD) commonly associated with severe neurological diseases, particularly in children under 5 years of age. Several investigational therapeutic agents and vaccine candidates are being developed. However, no approved drug against EV-A71 infection is available, and no proven drug target has been identified. Since host kinases are key regulators of multiple signaling pathways in response to viral infections, here we screened a kinase inhibitor library and identified potent inhibitors against EV-A71 infection. Among the hits, GSK269962A, a Rho Associated Coiled-Coil Containing Protein Kinase (Rock) inhibitor with potent antiviral activity, was selected for further analysis. We found that this Rock inhibitor not only efficiently suppressed the replication of EV-A71 in RD cells, but also in human intestinal organoids, in a dose-dependent manner. Interestingly, small interfering RNA depletion of Rock1, but not Rock2, significantly restricted viral replication in RD cells, indicating that Rock1 is a novel host dependency factor for EV-A71 replication and can serve as a target for the development of anti-EV-A71 therapeutics.

Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

BackgroundChemoresistance limits the therapeutic effect of cisplatin (DDP) on non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) function as important regulators in chemoresistance. This study aimed to explore the regulation of circRNA Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Alpha (circ_PIP5K1A) in DDP resistance.MethodsThe expression analysis of circ_PIP5K1A, micoRNA-493-5p (miR-493-5p) and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) was conducted through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell sensitivity was determined using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell proliferation and cell viability were evaluated by colony formation assay and MTT assay, respectively. Cell cycle and apoptosis detection was performed via flow cytometry. Cell motility was examined by transwell migration or invasion assay. Dual-luciferase reporter assay was applied to confirm the target binding. ROCK1 protein level was assayed via Western blot. In vivo assay was carried out using xenograft model in mice.ResultsCirc_PIP5K1A level was abnormally increased in DDP-resistant NSCLC tissues and cells. Silencing circ_PIP5K1A reduced DDP resistance, proliferation, cell cycle progression and cell motility in DDP-resistant NSCLC cells. Circ_PIP5K1A directly interacted with miR-493-5p in NSCLC cells. The function of circ_PIP5K1A was dependent on the negative regulation of miR-493-5p. MiR-493-5p directly targeted ROCK1 and circ_PIP5K1A regulated the ROCK1 level via acting as a sponge of miR-493-5p. Overexpression of miR-493-5p inhibited chemoresistance and cancer progression by downregulating ROCK1 expression in DDP-resistant NSCLC cells. Circ_PIP5K1A regulated DDP sensitivity in vivo via the miR-493-5p/ROCK1 axis.ConclusionThese findings suggested that circ_PIP5K1A upregulated the ROCK1 expression to promote DDP resistance and cancer progression in NSCLC by sponging miR-493-5p.

Exosomes of Mesenchymal Stem Cells as a Proper Vehicle for Transfecting miR-145 into the Breast Cancer Cell Line and Its Effect on Metastasis.

Background Despite recent advances in scientific knowledge and clinical practice, management, and treatment of breast cancer, as one of the leading causes of female mortality, breast cancer remains a major burden. Recently, methods employing stem cells and their derivatives, i.e., exosomes, in gene-based therapies hold great promise. Since these natural nanovesicles are able to transmit crucial cellular information which can be engineered to have robust delivery and targeting capacity, they are considered one of the modes of intercellular communication. miR-145, one of the downregulated microRNAs (miRNAs) in various cancers, can regulate tumor cell invasion, metastasis, apoptosis, and proliferation and stem cell differentiation. Objectives The aim of this study was to investigate the role of exosomes secreted from adipose tissue-derived mesenchymal stem cells (MSCs) for miR-145 transfection into breast cancer cells in order to weaken their expansion and metastasis. Methods Here, we exploited the exosomes from adipose tissue-derived mesenchymal stem cells (MSC-Exo) to deliver miR-145 in the T-47D breast cancer cell line. Lentiviral vectors of miR-145-pLenti-III-enhanced green fluorescent protein (eGFP) and empty pLenti-III-eGFP as the backbone were used to transfect MSCs and T-47D cells. In order to find the efficiency of exosomes as a delivery vehicle, the expression level of some miR-145 target genes, including Rho-Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Matrix Metalloproteinase 9 (MMP9), and Tumor Protein p53 (TP53), was compared in all treatment groups (T-47D cells treated by miR-145-transfected MSCs and their derivatives or their backbone) and control group (untransfected T-47D cells) using real-time PCR. Results The obtained data represented the inhibitory effect of miR-145 on apoptosis induction and metastasis in both direct miR-treated groups. However, exosome-mediated delivery caused an improved anticancer property of miR-145. Conclusion Restoration of miR-145 using MSC-Exo can be considered a potential novel therapeutic strategy in breast cancer in the future.

Upregulation of miR-496 Rescues Propofol-induced Neurotoxicity by Targeting Rho Associated Coiled-coil Containing Protein Kinase 2 (ROCK2) in Prefrontal Cortical Neurons.

Early exposure to general anesthesia in children might be a potentially highrisk factor for learning and behavioral disorders. The mechanism of neurotoxicity induced by general anesthesia was not defined. miR-496 could regulate cerebral injury, while the roles of miR- 496 in neurotoxicity were not elucidated. Therefore, we aimed to investigate the effects of miR- 496 in neurotoxicity induced by propofol. Primary Prefrontal Cortical (PFC) neurons were isolated from neonatal rats and treated with propofol to induce neurotoxicity. Cell viability was detected by (3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The target relationship of miR-496 and Rho Associated Coiled-Coil Containing Protein Kinase 2 (ROCK2) was explored using luciferase assays. Propofol decreased cell viability, promoted cell apoptosis, and decreased the expression of miR-496 in PFC neurons in a dose-dependent manner. Overexpression of miR-496 attenuated neurotoxicity induced by propofol in PFC neurons. ROCK2 was a target of miR-496, and miR-496 oppositely modulated the expression of ROCK2. Besides, propofol increased the expression of ROCK2 through inhibiting miR-496 in PFC neurons. Overexpression of miR-496 attenuated propofol- induced neurotoxicity by targeting ROCK2 in PFC neurons. miR-496 was decreased in PFC neurons treated with propofol, and overexpression of miR-496 attenuated propofol-induced neurotoxicity by targeting ROCK2. miR-496 and ROCK2 may be promising targets for protecting propofol-induced neurotoxicity.

Combretastatin A-4 disodium phosphate and low dose gamma irradiation suppress hepatocellular carcinoma by downregulating ROCK1 and VEGF gene expression.

Hepatocellular carcinoma (HCC) is a tough opponent. HCC contributes to 14.8% of all cancer mortality in Egypt. There are many choices for management of HCC; however tumor relapse has been reported in animal and clinical studies. This study was conducted to investigate the impact of low dose γ-irradiation (LDR) and combretastatin A-4 disodium phosphate (CA-4DP) on HCC recurrence. HCC was induced in male Wistar albino rats by oral administration of N-nitrosodiethylamine (NDEA) for 17 weeks. We evaluated the expression of the endothelial cell marker (CD31) by immunostaining. Expression of Rho Associated Coiled-Coil Containing Protein Kinase 1(ROCK1) and Vascular endothelial growth factor (VEGF) expression was assessed by real-time PCR after (6, 24 and 48 h). Our results showed that expression of CD31 and gene expression of ROCK1 and VEGF was significantly repressed at all-time intervals by combination therapy ofLDR and CA-4DP as compared with untreated NDEA/HCC group and NDEA/HCC groups treated with either LDR or CA-4DP alone, (P < 0.05). Our study demonstrated the additive effect of LDR in combination with CA-4DP in suppression of HCC.

Long Noncoding RNA X-Inactive Specific Transcript Facilitates Cellular Functions in Melanoma via miR-139-5p/ROCK1 Pathway.

PurposeAlthough X-inactive specific transcript (XIST) is known to play a critical role in the pathogenesis of melanoma, the mechanisms through which this remains unclear.MethodsRNAseq, immunohistochemistry, and qRT-PCR were used to identify the levels of XIST, miR-139-5p, and Rho-Associated Coiled-Coil Containing Protein Kinase-1 (ROCK1) in melanoma tissues and cells. A subcellular fractionation assay was used to determine the location of XIST. CCK-8 and colony formation assays were used to evaluate cellular proliferation. Cell migration and wound healing assays were used to detect the effects on cell migration. RNA pull-down was used to confirm the interaction between XIST and miR-139-5p. Besides, the xenograft tumor experiment was performed to further verify the roles of XIST in melanoma.ResultsIn this study, an increased level of XIST was revealed in melanoma tissues and cells, which was associated with higher TNM stage and positive lymph node metastasis. XIST was found to function as a “molecular sponge” of miR-139-5p to facilitate cellular functions. Moreover, these consequences could be partially reversed by inhibition of miR-139-5p. MiR-139-5p was found to target ROCK1 directly, leading to suppression of ROCK1 expression; this effect could be partially reversed by inhibiting XIST expression. Furthermore, the deletion of ROCK1 induced anti-oncogenic effects similar to those seen with knockout of XIST. Upregulation of miR-139-5p and knockdown of XIST could inhibit cell functions in melanoma.ConclusionOur findings suggested that the lncRNA XIST facilitates cellular functions in melanoma via the miR-139-5p/ROCK1 pathway.

Polydatin Alleviates Lipopolysaccharide-Triggered Inflammatory Injury Through Up-Regulating miR-125b in Chondrogenic ATDC5 Cells

Osteoarthritis is a bone-joint disease prevalent in older people characterized by joint inflammation. In traditional Chinese medicine, polydatin plays an important anti-inflammatory role. This study analyzed the potential effects and possible internal mechanisms of polydatin on osteoarthritis. First, lipopolysaccharide-induced osteoarthritis injury was established in chondrogenic ATDC5 cells. Lipopolysaccharides significantly stimulated inflammatory injuries in ATDC5 cells as exemplified by a decrease in cell viability and an increase in inflammatory cytokine secretions including interleukin-6, tumor necrosis factor-a, and interleukin-1. Moreover, lipopolysacchrides also increased Cleaved caspase-3 and Cleaved Poly (ADP-ribose) polymerase to promote cell apoptosis. Second, polydatin showed significant protective effects against lipopolysaccharide-induced inflammatory injury, again exemplified by increased cell viability, decreased inflammatory cytokines, Cleaved caspase-3, and Cleaved poly (ADP-ribose) polymerase. Lastly, miR-125b and its binding target Rho-Associated Coiled-Coil Containing Protein Kinase 1 were closely associated with regulatory effects of polydatin against lipopolysaccharide-stimulated ATDC5 cell inflammatory injuries. Polydatin alleviated lipopolysaccharide-stimulated inflammatory injuries via the down-regulation of miR-125b. The present study concludes that polydatin plays an anti-inflammatory role in lipopolysaccharide-stimulated ATDC5 cell inflammatory injuries via the down-regulation of miR-125b.

FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.

The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC. The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver. Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1). This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.

A Rho-Associated Coiled-Coil Containing Kinase Inhibitor, Y-27632, Improves Viability of Dissociated Single Cells, Efficiency of Colony Formation, and Cryopreservation in Porcine Pluripotent Stem Cells.

The establishment of porcine epiblast stem cells (pEpiSCs) and induced pluripotent stem cells (piPSCs) derived from diametrical derivations is of great importance in developing biomedical models. However, pEpiSCs and piPSCs have been technically much harder to culture than mouse embryonic stem cells, showing problematic properties such as spontaneous differentiation and apoptosis after cryopreservation. Therefore, we demonstrated that Y-27632 as a Rho-associated coiled-coil containing kinase inhibitor could prevent dissociated pEpiSCs and piPSCs from undesirable differentiation and apoptosis in cryopreservation protocols. pEpiSC 2, 8 lines, Sendai virus-induced pluripotent stem cells (Sev-iPSCs), and lentivirus-induced pluripotent stem cells were cultured with 10 μM Y-27632 before collecting dissociated cells retrieved from colonies using various enzymes. Dissociated single cells were transferred into freezing mediums (open pulled straw vitrification, STEM-CELLBANKER® (SCB), 10% dimethylsulfoxide in serum) for cryopreservation. The rates of viability and colony formation obtained from dissociated porcine stem cells after freezing/thawing were examined in the presence of Y-27632. The characteristics of pluripotency and in vitro differentiation were also examined in these stem cells treated with Y-27632 after cryopreservation. As a result, the viability and efficiency of colony formation of dissociated pEpiSCs (2, 8 lines) and Sev-iPSCs treated with 10 μM Y-27632 using the SCB cryopreservation protocol were significantly increased when compared with those of nontreated Y-27632 (p < 0.05). Pluripotency genes (OCT-3/4, NANOG, and SOX2) were positively expressed in Y-27632-treated porcine pluripotent stem cells. Also, in vitro differentiation of these stem cells was successfully induced in the presence of 10 μM Y-27632. These results indicated that treatment of Y-27632 for single-cell dissociation and the SCB cryopreservation protocol could facilitate handling porcine pluripotent stem cells and provide the widespread use of these stem cells.

Overexpression of Rho-Associated Coiled-Coil Containing Protein Kinase 2 Is Correlated with Clinical Progression and Poor Prognosis in Breast Cancer.

BackgroundRho-associated coiled-coil containing protein kinases 2 (ROCK2) is one of the best characterized targets for the small GTPase Rho. It has been reported that ROCK2 is critical for cancer cell migration and invasion. The objective of this study was to investigate the association of ROCK2 expression with clinicopathological features and overall survival of breast cancer patients.Material/MethodsThe expression of ROCK2 in breast cancer and paired adjacent normal tissues was detected and compared by immunohistochemical staining of tissue array. ROCK2 mRNA expression and clinicopathological information was extracted from the TCGA breast cancer dataset. The association of ROCK2 expression with the clinicopathological characteristics of patients with breast cancer was evaluated using univariate and multivariate Cox proportional hazards models. Overall survival was analyzed using the Kaplan-Meier method.ResultsImmunohistochemistry showed that ROCK2 expression was significantly higher in tumor tissues than in paired adjacent normal tissues [immunoreactivity score (IRS): tumor, 5.25±2.10, n=40 vs. adjacent normal 3.83±1.06, n=40, P<0.01]. The IRS was correlated to breast cancer staging. Similarly, the mRNA level of ROCK2 was correlated to tumor stage. Notably, ROCK2 mRNA expression (hazard ratio [HR] 1.665 and 95% confidence interval [CI] 1.115–2.488, P=0.013) were also associated with overall survival in a multivariate analysis.ConclusionsUpregulation of ROCK2 was associated with the progression of breast cancer. High expression of ROCK2 may predict poor overall survival rates for breast cancer patients.

Long non-coding RNA NEAT1 regulates the proliferation, migration and invasion of gastric cancer cells via targeting miR-335-5p/ROCK1 axis.

This study aimed to elucidate the roles of long non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) in gastric cancer. The expression of NEAT1 in gastric cancer tissues and cells was determined. NEAT1 was then overexpressed and suppressed in BGC-823 cells in vitro to further explore the effects of NEAT1 on cell proliferation, migration, and invasion. In addition, the regulatory relationship among NEAT1, miR-335-5p and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) was investigated. LncRNA NEAT1 was upregulated in gastric cancer tissues and cells. Upregulated NEAT1 significantly promoted the proliferation, migration and invasion of BGC-823 cells, while suppression of NEAT1 exhibited contrary results. In addition, lncRNA NEAT1 inhibited the expression of miR-335-5p, and miR-335-5p targeted ROCK1 in BGC-823 cells. miR-335-5p overexpression significantly inhibited cell proliferation, migration and invasion, which was counteracted by ROCK1 overexpression concurrently. Our findings indicate that upregulation of NEAT1 may promote proliferation, migration and invasion of gastric cancer cells via targeting miR-335-5p/ROCK1 axis. NEAT1-miR-335-5p-ROCK1 axis may be a potential therapeutic strategy for gastric cancer therapy.

Role of Rho-Associated Coiled-Coil Forming Kinase Isoforms in Regulation of Stiffness-Induced Myofibroblast Differentiation in Lung Fibrosis.

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho-associated coiled-coil forming kinase (ROCK) has been shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in a fibrotic state. There are two known ROCK isoforms in humans, ROCK1 and ROCK2, but the specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a gelatin methacryloyl hydrogel-based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix, but not soft matrix, can induce myofibroblast differentiation with high smooth muscle actin isoform (αSMA) expression. Furthermore, our data confirmed that the inhibition of ROCK signaling by a pharmacological inhibitor (i.e., Y27632) attenuates stiffness-induced αSMA expression and fiber assembly in myofibroblasts. To assess the role of ROCK isoforms in this process, we used short interfering RNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix, as opposed to soft matrix, where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fiber assembly. Moreover, complete loss of αSMA fiber assembly was seen only in the absence of both ROCK isoforms, suggesting that both isoforms are implicated in this process. Overall, our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

Rock the Rock of Atherosclerosis

The Rho-Associated Coiled-Coil Containing Protein Kinase (ROCK1 and ROCK2) were one of the downstream effectors of the small GTPase Rho. The Rho/ROCK pathway plays an important role in mediating multiple cellular processes, including endothelial dysfunction, the proliferation and migration of smooth muscle cells, foam cell formation, and arterial stiffness and aging, all of which are involved in the pathogenesis of atherosclerosis. Vascular cells (including endothelial cells, smooth muscle cells, and macrophages) undergo pathophysiological changes through the ROCK signaling pathway and ROCK inhibitors are being developed as effective therapeutic agents for atherosclerotic cardiovascular diseases. However, it is not entirely clear how ROCK isoforms are regulated, and how both isoforms contribute to the pathogenesis of atherosclerosis. A recent article from Liao’s laboratory demonstrated that deletion of the ROCK2 allele in BM-derived cells attenuates plaque formation in cholesterol-fed LDLr-/- mice. Mechanistically, ROCK2 deletion decreases foam cell formation (the hallmark of atherosclerosis) by facilitating Reverse Cholesterol Transport (RCT) in macrophages, through peroxisome proliferator-activated receptor-γ/liver X receptor-α/ATP-binding cassette transporter A1 pathway. This study provides further mechanistic insight into the therapeutic benefits of ROCK2 inhibition in preventing the development of atherosclerosis.

Effects of Rosuvastatin versus Atorvastatin on Rho-Associated Coiled-Coil Containing Protein Kinase Activity and Endothelial Function in Patients with Atherosclerosis

This randomized, double-blind, parallel-design study compared the short-term effects of rosuvastatin and atorvastatin on serum lipids and markers of inflammation and endothelial function in patients with stable atherosclerosis. Patients received either 10 mg/day rosuvastatin (n = 18) or 20 mg/day atorvastatin (n = 18), orally, for 4 weeks. Serum lipids, high-sensitivity Creactive protein (hsCRP), Rho-associated coiled-coil containing protein kinase (ROCK) activity and flow-mediated dilation (FMD) of the brachial artery were assessed before and after therapy. Both statins produced significant reductions in total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride and hsCRP levels, and significant increases in FMD. Both statins significantly reduced ROCK activity and inhibition was significantly greater with rosuvastatin. There was no correlation between ROCK activity and LDL-C level in either group. There was a significant correlation between ROCK activity and FMD for both statins, but no correlations between FMD and LDL-C or hsCRP levels. Short-term treatment with either rosuvastatin or atorvastatin inhibits ROCK activity independent of cholesterol reduction, and improves endothelium dysfunction in patients with atherosclerosis.

365. Inhibition of Rho Associated Coiled-Coil Forming Kinase Increases Efficacy of Measles Virus Infection

365. Inhibition of Rho Associated Coiled-Coil Forming Kinase Increases Efficacy of Measles Virus Infection

Letter by Settergren et al Regarding Article, “Evidence for Statin Pleiotropy in Humans: Differential Effects of Statins and Ezetimibe on Rho-Associated Coiled-Coil Containing Protein Kinase Activity, Endothelial Function, and Inflammation”

To the Editor: It is well established that statins improve the prognosis of patients with coronary artery disease. However, it is still unclear whether the protective effects of statins relate to lipid lowering only or whether other so-called pleiotropic effects may contribute. Experimental data support the pleiotropic effects of statins, but knowledge on their clinical relevance is limited. We read with great interest the recent article by Liu et al.1 The authors compared the effect of simvastatin 40 mg/d, and the combination of simvastatin 10 mg and ezetimibe 10 mg …