Rho Kinase Research Articles

Rho kinase inhibitor Y-27632 downregulates IL-1β expression in mice with experimental autoimmune myocarditis

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1β expression in the myocardial tissue of EAM mice. Notably, IL-1β expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1β expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10µM. However, 10µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adultmice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/β-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Dietary copper improves intestinal structural integrity in juvenile grass carp (Ctenopharyngodon idella) probably related to its increased intestinal antioxidant capacity and apical junction complex

This research evaluated the effects of copper (Cu) on intestinal antioxidant capacity and apical junctional complex (AJC) in juvenile grass carp. A total of 1080 healthy juvenile grass carp (11.16 ± 0.01 g) were fed six diets including different dosages of Cu, namely 0, 2, 4, 6, 8 mg/kg (Cu citrate [CuCit] as Cu source) and 3 mg/kg (CuSO4·5H2O as Cu source). The trial lasted for 9 weeks. The findings revealed that dietary optimal Cu supplementation (2.2 to 4.1 mg/kg) promoted intestinal growth, including intestinal length, intestinal length index, intestinal weight, and intestinal somatic index (P < 0.05). Furthermore, optimal Cu boosted the intestinal mucosal barrier in juvenile grass carp. On the one hand, optimal Cu reduced diamine oxidase and D-lactate levels in serum (P < 0.05), reduced levels of the oxidative damage indicators malondialdehyde, reactive oxygen species (ROS), protein carbonyl, superoxide dismutase (P < 0.05), and catalase mRNA levels were elevated (P < 0.05), thus boosting intestinal antioxidant capacity, the binding protein Keap1a/1b/Nrf2 signaling pathway might be involved. Optimal Cu had no impact on glutathione peroxidase 1b (GPx1b) gene expression (P > 0.05). On the other hand, optimal Cu increased intestinal tight junction (TJ) proteins (except for claudin 15b) and adherens junction (AJ) proteins (E-cadherin, α-catenin, β-catenin, nectin and afadin) mRNA levels (P < 0.05), which could be connected to the signaling pathway formed by the Ras homolog gene family, member A (RhoA), Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK). Finally, based on serum indicator D-lactate and intestinal oxidative damage index (ROS), Cu requirement (CuCit as Cu source) for juvenile grass carp from initial weight to final weight (from 11 to 173 g) was determined to be 4.14 and 4.12 mg/kg diet, respectively. This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Sildenafil improves hemodynamic changes caused by acute pulmonary embolism by inhibiting Rho kinase activity.

This study examined the effects of sildenafil on acute pulmonary embolism (APE) using a rat model. Sprague-Dawley rats were randomly divided into the sham, pulmonary thromboembolism (PTE), and sildenafil groups. The sham and PTE groups received normal saline once daily via gavage for 14 consecutive days, whereas the sildenafil group received sildenafil (0.5 mg/kg/day) once daily via gavage for 14 consecutive days. Autologous emboli were prepared from blood samples collected from the left femoral artery of rats in each group on day 13, and autologous emboli were injected into the jugular vein cannula of rats in the PTE and sildenafil groups on day 14. Sham-treated rats received the same volume of saline. Right systolic ventricular pressure (RVSP) and mean pulmonary arterial pressure (MPAP) were used to assess pulmonary embolism, and western blotting and enzyme-linked immunosorbent assay were used to detect relevant markers. The Rho kinase signaling pathway was significantly activated in rats with APE, and sildenafil significantly inhibited this activation. Sildenafil protected against APE through inhibiting Rho kinase activity, thereby reducing pulmonary vasoconstriction and decreasing elevated pulmonary arterial pressure. These findings might provide new ideas for the clinical treatment of acute pulmonary thromboembolism.

RhoA/rho kinase pathway activation in age-associated endothelial cell dysfunction and thrombosis.

The small GTPase RhoA and the downstream Rho kinase (ROCK) regulate several cell functions and pathological processes in the vascular system that contribute to the age-dependent risk of cardiovascular disease, including endothelial dysfunction, excessive permeability, inflammation, impaired angiogenesis, abnormal vasoconstriction, decreased nitric oxide production and apoptosis. Frailty is a loss of physiological reserve and adaptive capacity with advanced age and is accompanied by a pro-inflammatory and pro-oxidative state that promotes vascular dysfunction and thrombosis. This review summarises the role of the RhoA/Rho kinase signalling pathway in endothelial dysfunction, the acquisition of the pro-thrombotic state and vascular ageing. We also discuss the possible role of RhoA/Rho kinase signalling as a promising therapeutic target for the prevention and treatment of age-related cardiovascular disease.

Fasudil, a Rho kinase inhibitor, attenuates cardiovascular changes in an experimental rat model of metabolic syndrome via modulation of PCSK9 and BNP.

Fasudil, a Rho kinase inhibitor, attenuates cardiovascular changes in an experimental rat model of metabolic syndrome via modulation of PCSK9 and BNP.

Modeling mechanical activation of macrophages during pulmonary fibrogenesis for targeted anti-fibrosis therapy.

Pulmonary fibrosis is an often fatal lung disease. Immune cells such as macrophages were shown to accumulate in the fibrotic lung, but their contribution to the fibrosis development is unclear. To recapitulate the involvement of macrophages in the development of pulmonary fibrosis, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissues became mechanically activated. The resulting co-alignment of macrophages, collagen fibers, and fibroblasts promoted widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin αMβ2 and Rho-associated kinase 2. These results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by macrophages. The cocultured microtissue model is a powerful tool to study the immune-stromal cell interactions and the anti-fibrosis drug mechanism.

Targeting ROCK2 improves macromolecular permeability in a 3D fibrotic pancreatic cancer microenvironment model

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-β (TGFβ)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFβ or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFβ/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer.

Abstract 5914: Non-muscle myosin IIA as a promising therapeutic target in breast cancer

Abstract We aimed to determine the role of non-muscle myosin IIA (NMIIA) in breast cancer using various cancer cell lines including estrogen receptor (ER)+, HER2+ and triple negative breast cancers (TNBC). Myosin heavy chain 9 (MYH9) gene encodes for the heavy chain of the hexameric protein NMIIA. We have recently identified that NMIIA serves as a binding partner for HER3 following the inhibition of the HER family member compared to the vehicle control treatment, as observed in HER3 immunoprecipitates. We checked the basal protein and mRNA levels of NMIIA in different breast cancer cell lines and found higher levels of NMIIA in both HER2+ and TNBC cell lines compared to ER+ cell lines. These data correlate with clinical data of primary breast cancer patients with high MYH9 being less likely in ER+ breast cancer patients and more likely in the PAM50 subtypes basal and HER2 enriched breast cancer patients. By employing a pool of four siRNA targeting MYH9, our data indicated a reduction in proliferation of HER2+ and TNBC cell lines when compared to the control siRNA. We have previously found that loss of MYH9 sensitizes to the HER family inhibitor neratinib treatment as assessed by cell proliferation, colony formation on matrigel, cell migration and invasion. We aimed to determine if pharmacological inhibition of the NMIIA pathway would sensitize to neratinib. RhoA-associated kinase (ROCK) serves as a master regulator of NMIIA activity as it directly phosphorylates the regulatory light chain (RLC) of NMIIA. Pharmacological inhibition of ROCK-NMIIA pathway using the ROCK inhibitor GSK269962A alone or in combination with neratinib reduced the cell proliferation in HER2+ cell lines BT474 and MDA-MB-453 as indicated by MTT and cell proliferation assay. It is well known that HER2-targeted therapies cause the upregulation of HER3. Given our identification of NMIIA as one of the interacting partners of HER3, our focus will be on investigating the NMIIA-HER3 signaling axis to elucidate its contribution to an adaptive response to HER2 inhibition and potential treatment resistance. Furthermore, we will explore strategies to target NMIIA in breast cancer. Citation Format: Wasim Feroz, Rosalin Mishra, Mary Kate Kilroy, Joan T. Garrett. Non-muscle myosin IIA as a promising therapeutic target in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5914.

Abstract 5409: Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion

Abstract Although head and neck squamous cell carcinomas (HNSCCs), including tongue, vocal cord, and buccal cancers, are one of the most common cancers worldwide with a mortality rate of more than 40%, there have been no solid standard biomarkers for effectively diagnosing HNSCCs at early stages and no effective protocols available to support in vitro expansion of HNSCC cells. To establish a cell culture method to support in vitro self-renewal of HNSCC cells, patient-derived tumor was enzymatically digested, and the tongue cancer epithelial cells were seeded onto feeder cells with the Rho kinase inhibitor (Y-27632). Additionally, to test our hypothesis that free thiol groups in the cells, generated by a mild reduction of cell surface proteins using the reducing agent tris(2-carboxyethyl) phosphine hydrochloride (TCEP), would modulate cancer cell adhesion and migration, we assessed the expression levels of integrins, focal adhesion kinase (FAK), and phosphorylated FAK (pFAK) of tongue cancer epithelial cells cultured on both soft and rigid polyacrylamide (PAAm) hydrogel-based cell culture substrates. Patient-derived tongue cancer epithelial cells exhibited self-renewal ability and maintained cancerous phenotypes, with high expressions of P53, Ki67, E-cadherin, CD44, and AHDH1A1. Treatment with the reducing agent (TCEP) increased the degree of cell adhesion through the significantly upregulated integrin α5β1-mediated phosphorylation of FAK, resulting in inhibited single-cell and collective cell migration as well as invasion. Additionally, when these cells were cultured on stiff PAAm hydrogels mimicking the tumor microenvironment, the cells exhibited accelerated cell migration behaviors, compared to cells on soft hydrogels. These results highlight the synergetic contributions of feeder cells and Rho kinase inhibitor to support long-term self-renewal of tongue cancer patient-derived epithelial cells. Citation Format: Joo Hyun Kim, Laurensia D. Anggradita, Sung Sik Hur, Jae Hong Park, Sang-Heon Kim, Myung Jin Ban, Yongsung Hwang. Phosphorylated focal adhesion kinase by mild reduction of cell surface proteins inhibits integrin α5β1-dependent patient- derived cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5409.

Abstract 1554: LIM Domain 7 (LMO7) splice variant influences prostate cancer biology

Abstract LIM Domain 7 (LMO7) has been shown to participate in cell-cell junction formation through interaction with afadin and α-actinin. It has also been reported to collaborate with Rho kinase to regulate actin filament formation and to activate the Rho-myocardin-related transcription factor serum response factor pathway. Additionally, LMO7 itself may function as a transcription factor, regulating the expression of genes involved in Epithelial Mesenchymal Transition (EMT). Alternative splicing of LMO7 has the potential to modulate its function. Previously, we reported LMO7 as a target that is alternatively spliced in prostate cancer (PCa) between self-reported Black and White patients. In addition, by analyzing expression of LMO7 splice variants in The Cancer Genome Atlas (TCGA) data, we previously reported increased expression of a LMO7 splice variant skipping exon 12 in breast, lung and liver cancer compared with paired normal tissues. In the present study, we found that LMO7 is downregulated in metastatic PCa specimens within the Stand Up To Cancer cohort. To investigate the functional impact of expression of the LMO7 splice variant skipping exon 12 in PCa, we utilized CRISPR-Cas9 methodology to delete LMO7 exon 12 in PCa cells and assessed resulting alterations in PCa cell biology. The LMO7 exon 12 deleted PC3 PCa cells exhibited enhanced migratory potential compared with parental PCa cells. An Immunofluorescence (IF) assay showed weak staining of cell-cell junctions in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. Moreover, IF staining showed retardation in f-actin in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. As E-Cadherin is often inhibited during EMT, we assessed the level of E-Cadherin by western blot and real-time polymerase chain reaction (PCR). These analyses revealed that E-Cadherin protein as well as messenger RNA is suppressed in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. These findings suggest a role for skipping of LMO7 exon 12 in disrupting PCa cell-cell junctions, down-regulating E-Cadherin in PCa and enhancing PCa cell migratory potential, consistent with EMT. Citation Format: Muthana Al Abo, Daniel J. George, Zefeng Wang, Steven R. Patierno, Jennifer A. Freedman, Alice Jiang. LIM Domain 7 (LMO7) splice variant influences prostate cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1554.

Abstract 296: BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts

Abstract The discovery of BRAF mutations in around 60% of melanoma patients and the subsequent development of targeted inhibitors to counter the hyperactive BRAF-driven pro-tumorigenic signaling pathway have marked a significant milestone in melanoma therapy. However, despite this progress, the long-term outcomes remain unsatisfactory for many patients due to the occurrence of drug resistance. We previously reported that cancer-associated fibroblasts (CAFs) are stimulated by BRAF inhibitors (BRAFi) to drive matrix remodeling and therapeutic escape pathways in melanoma cells. However, the underlying mechanism by which CAFs are reprogrammed by BRAFi to obtain enhanced phenotypes remains elusive. Here, we present compelling evidence showing that BRAFi (PLX4032), but not CRAF inhibitor (GW5074) or pan-RAF inhibitor (RAF709), induces the accumulation of nuclear β-catenin in CAFs harboring wild-type BRAF, a process mediated by BRAF and CRAF but not ARAF isoform. BRAFi binding to BRAF and CRAF leads to robust RAF kinase dimerization, including BRAF and CRAF homodimer and BRAF: CRAF heterodimer. RAF dimers are subsequently recruited to the plasma membrane, where BRAF and CRAF are phosphorylated on residues Thr599 and Ser338, respectively, and activated in a RAS-GTP-dependent manner. RAF activation stimulates the downstream Rho kinase (ROCK) and MEK/ERK signaling pathways simultaneously. Notably, ablating RAS and RAF isoforms effectively suppressed BRAFi-induced activation of ROCK1/2 signaling in CAFs, further confirming that the ROCK pathway is a downstream effector of BRAFi-driven RAF activation and is intricately linked to increased nuclear β-catenin in CAFs. The pharmacological blockade of ROCK activity, using HA-1077, diminished BRAFi-induced nuclear β-catenin accumulation in CAFs, suggesting BRAFi-driven cytoskeletal remodeling plays a pivotal role in the nuclear shuttling of β-catenin. Using a mouse model that mimics human BRAF-mutant melanoma, we found that elevated level of nuclear β-catenin drives the conversion of fibroblasts into α-SMA-positive CAFs, leading to elevated collagen deposition and contributing to melanoma progression in vivo. In summary, BRAFi reprograms the transcriptional activity in CAFs by driving increased nuclear β-catenin to increase their ability to remodel the tumor ECM and promote melanoma cell proliferation. Collectively, the data offer new insights into the molecular mechanisms that underlie the paradoxical reprogramming of CAFs by BRAFi in melanoma therapy through the non-canonical β-catenin pathway. Citation Format: Bruna da Silva Soley, Radhika Athalye, Ryan Zhang, Thomas Andl, Yuhang Zhang. BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 296.

“Keep on ROCKIn”: Repurposed ROCK inhibitors to boost corneal endothelial regeneration

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

Efficacy and Safety of Rho Kinase Inhibitors vs. Beta-Blockers in Primary Open-Angle Glaucoma: A Systematic Review with Meta-Analysis.

Background: In order to support the positioning of Rho kinase inhibitors (Rhokis) in the European market for the treatment of glaucoma, scientific evidence comparing the efficacy and safety of Rhokis and beta-blockers (β-βs) in the treatment of open-angle glaucoma after 3 months was assembled through a systematic review and meta-analysis (meta-A) of randomized controlled trials (RCTs). Methods: Relevant articles were searched for on PubMed, EMBASE, and the Cochrane Library. Of the 251 articles found, three met all eligibility criteria. These three articles were assessed for risk of bias. Data were extracted and a random effects meta-A was performed. The studies' methods were homogeneous but there was great heterogeneity within the data (I2 = 92-93%; p < 0.001). Results: All studies had low risk of bias. The meta-A showed statistically better efficacy of β-βs, resulting in an intraocular pressure (IOP) reduction mean difference of 1.73 (1.19-2.27) at 8 a.m., 0.66 (0.19-1.15) at 10 a.m. and 0.49 mmHg (0.001-0.98) at 4 p.m., compared to Rhokis. This difference is not clinically significant as intra-operator variability of IOP measurements varies from ±2 to ±3 mmHg The adverse effects of Rhokis were essentially topical, whereas β-βs mainly caused systemic side effects. Conclusions: This Meta-A showed that Rhokis are clinically non-inferior to beta-blockers in reducing IOP. Rhokis have a better safety profile.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes

Aims/hypothesisA hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier.MethodssEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors.ResultsUsing intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell–cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state.Conclusions/interpretationDiabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.Graphical

Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells

BackgroundHypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural component of plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying extracellular vesicles (EVs). However, the role of mechanics in Cav-1-induced EV formation from VSMCs has not been reported.ResultsExposure of VSMCs to 10% mechanical stretch (0.5 Hz) for 72 h resulted in Cav-1 translocation into non-caveolar regions of the plasma membrane and subsequent redistribution of Cav-1 from the VSMCs into EVs. Inhibition of Rho-A kinase (ROCK) in mechanically-stimulated VSMCs exacerbated the liberation of Cav-1 positive EVs from the cells, suggesting a potential involvement of actin stress fibers in this process. The mineralization potential of EVs was measured by incubating the EVs in a high phosphate solution and measuring light scattered by the minerals at 340 nm. EVs released from stretched VSMCs showed higher mineralization potential than the EVs released from non-stretched VSMCs. Culturing VSMCs in pro-calcific media and exposure to mechanical stretch increased tissue non-specific alkaline phosphatase (ALP), an important enzyme in vascular calcification, activity in EVs released from the cells, with cyclic stretch further elevating EV ALP activity compared to non-stretched cells.ConclusionOur data demonstrate that mechanical stretch alters Cav-1 trafficking and EV release, and the released EVs have elevated mineralization potential.