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Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

SummaryTumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formedde novo-induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet websex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis.MethodsPEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis.ResultsPEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density.ConclusionsOur data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis., Blood vessel development depends upon endothelial cell migration and adhesion, which are regulated by Rho-GTPases. Here the authors identify Rho-GTPase guanine nucleotide exchange factors that specifically control lateral filopodial contacts and are required for lumen formation during angiogenesis.

Objective— RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results— Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho–myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion— RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers.

INTRODUCTION: Development of brain metastatic lesions requires activation of signaling pathways for cancer cell invasion. Metastatic disease is intrinsically linked to endothelial cell activation for brain colonization and angiogenic growth for disease progression. DOCK180 family of Rho guanine nucleotide exchange factors (GEFs) are implicated in invasion, and recent studies show they control blood vessel development. The study aims to elucidate the role of the Rac GEF DOCK4 in breast cancer brain metastases. METHOD: i) Patient samples were analysed for DOCK4 expression levels which were related to histological parameters in primary tumours, and expression levels in brain metastases, ii) the role of DOCK4 in the brain vasculature was studied in heterozygous Dock4 null mice and mouse model of breast cancer brain metastasis, iii) the DOCK4 signalling pathway was delineated using biochemical assays and proteomics approaches. RESULTS: i) Significant correlation was observed between DOCK4 expression and histological grade in primary breast tumours, independent of ER and LN status. There was a trend towards reduced survival in cases with moderate/ strong DOCK4 expression. Moderate/ strong DOCK4 expression was observed in brain metastases specimens. ii) There was significant decrease in brain blood vessel lumen size in Dock4 + /- mice and number of blood vessels with dilated lumens in EO771 intracranial tumours in Dock4 + /- mice. iii) DOCK4 interacts with the Cdc42 GEF DOCK9 and RhoG effector ELMO to control filopodia formation and blood vessel lumen morphogenesis. CONCLUSION: DOCK4 and interaction partners are potential targets and biomarkers in brain metastases through regulation of cancer cell invasion and angiogenesis.