Guanine Nucleotide Exchange Factor For Cdc42 Research Articles

Actin and CDC-42 contribute to nuclear migration through constricted spaces in C. elegans.

Successful nuclear migration through constricted spaces between cells or in the extracellular matrix relies on the ability of the nucleus to deform. Little is known about how this takes place in vivo. We have studied confined nuclear migration in Caenorhabditis elegans larval P cells, which is mediated by the LINC complex to pull nuclei towards the minus ends of microtubules. Null mutations of the LINC component unc-84 lead to a temperature-dependent phenotype, suggesting a parallel pathway for P-cell nuclear migration. A forward genetic screen for enhancers of unc-84 identified cgef-1 (CDC-42 guanine nucleotide exchange factor). Knockdown of CDC-42 in the absence of the LINC complex led to a P-cell nuclear migration defect. Expression of constitutively active CDC-42 partially rescued nuclear migration in cgef-1; unc-84 double mutants, suggesting that CDC-42 functions downstream of CGEF-1. The Arp2/3 complex and non-muscle myosin II (NMY-2) were also found to function parallel to the LINC pathway. In our model, CGEF-1 activates CDC-42, which induces actin polymerization through the Arp2/3 complex to deform the nucleus during nuclear migration, and NMY-2 helps to push the nucleus through confined spaces.

Substrate induced dynamical remodeling of the binding pocket generates GTPase specificity in DOCK family of guanine nucleotide exchange factors

Dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) activate two members of Rho family GTPases, Rac1/Cdc42, to exert diverse cellular processes, including cell migration. As DOCK GEFs have been critically implicated in tumour cell migration, understanding their function and specificity is imperative for designing anti-metastatic drugs. Based on their GTPase specificity they have been classified as Rac, Cdc42 and dual specific GEFs. Despite extensive structural studies, the factors that determine GTPase specificity of DOCK GEFs have remained elusive. Here, we show that subtle dynamical coupling between GEF and GTPase structures modulate the binding interface to generate mutual specificity. To cluster the dynamically coupled residues in GEF-GTPase complexes a novel intra-residue backbone-torsion-angles based mutual information (TMI) technique was employed. TMI was calculated from 4500 trajectories obtained from a total of 4.5μs molecular dynamics simulations performed on members of all the three clades of DOCK GEFs. The obtained clusters suggest a specificity generation mechanism that involves optimization of the binding pocket for the crucial divergent residue at the 56th position of Rac/Cdc42 (FCdc42/WRac1). These clusters encompass five residues from the structural segment lobe C - α10 helix of the DOCK proteins and functional SWI region of GTPase, which induce orchestrated structural modulations to generate the specificity. Even the conserved residues from SWI region are seen to augment the specificity defining dynamical rearrangements. Furthermore, the proposed dynamical GTPase- DOCK GEF specificity model was verified using mutagenesis studies on Rac1 and dual GTPase specific Dock2 and Dock6, respectively. Thus the current study provides the generic substrate specificity determinants of DOCK GEFs, which are not apparent from the conventional structural analysis.

LncRNA-p21 suppresses cell proliferation and induces apoptosis in gastric cancer by sponging miR-514b-3p and up-regulating ARHGEF9 expression.

The long non-coding RNA p21 (lncRNA-p21) was a tumor suppressor gene in most cancer types including gastric cancer (GC). We aimed to identify a specific lncRNA-p21-involved pathway in regulating the proliferation and apoptosis of GC cells. A lower lncRNA-p21 expression in tumors was associated with advanced disease stage and predicted worse survival of GC patients. LncRNA-p21 overexpression in GC cell line somatic gastric cancer (SGC)-7901 and human gastric cancer (HGC)-27 suppressed cell proliferation and enhanced apoptosis, while lncRNA-p21 knockdown caused the opposite effects. Through bioinformatics analysis and luciferase-based reporter assays, we identified miR-514b-3p as a sponge target of lncRNA-p21. Cdc42 guanine nucleotide exchange factor 9 (ARHGEF9), functioned as a tumor suppress factor in GC, was found as the downstream target of miR-514-3p, and their expressions were negatively correlated in GC tumor tissues. In addition, like lncRNA-p21 overexpression alone, miR-514-3p inactivation alone also led to decreased proliferation and increased apoptosis in SGC-7901 and HGC-27 cells, which were markedly attenuated by additional ARHGEF9 knockdown. Xenograft SGC-7901 cells with more lncRNA-p21 or ARHGEF9 expressions or with less miR-514-3p expression exhibited obviously slower in vivo growth than the control SGC-7901 cells in nude mice. Our study reveals a novel lncRNA-p21/miR-514b-3p/ARHGEF9 pathway that can be targeted for GC therapy.

Abstract 139: Dock7 regulates the AKT/mTOR pathway to promote survival and sustain the transformative properties of cancer cells

Abstract Akt is a well-known target of mitogenic signaling, which is activated by phosphoinositide 3-kinase, PDK1 and mTORC2, and is commonly regarded as a master regulator of cell survival. However, there is still a good deal to learn regarding how Akt can promote survival when cancer cells are faced by the multitude of challenges they need to overcome during the process of malignant transformation. While investigating the role of Dock7, a Cdc42 and Rac guanine nucleotide exchange factor of the Dock180 family, in tumorigenesis, we discovered an unexpected connection between Dock7, Akt and cell survival. Specifically, we found that Dock7 is necessary for cancer cells to survive under the stressful conditions that arise due to the loss of a substratum or serum deprivation, through its ability to stabilize a low, basal level of Akt kinase activity. Dock7 binds to Akt, as well as associates with the known Akt substrate, TSC2, promoting its phosphorylation. Since the phosphorylation of TSC2 eliminates its ability to work with its partner TSC1 to deactivate the Rheb GTPase, a direct activator of mTORC1, we probed whether mTORC1 function is also necessary for Dock7-dependent cancer cell survival. Indeed, we determined that Dock7, as well as its signaling partner Cdc42, is required to maintain a functional level of S6 kinase activity, a downstream target of mTORC1, which was prevented by the mTORC1 inhibitor rapamycin. We further show an association between mTOR, Rheb and Dock7, but not Raptor, the defining component of mTORC1. Additionally, Raptor is dispensable for the Dock7-dependent S6 kinase activity, despite its rapamycin sensitivity. Together, these findings elucidate a novel Dock7-Akt-mTOR signaling node which promotes cancer cell survival in the absence of classical mTORC1 function to allow cells to overcome stresses faced during the development of cancer. Citation Format: Oriana Y. Teran, Miao-chong J. Lin, Matthew R. Zanotelli, Kristin F. Wilson, Richard A. Cerione. Dock7 regulates the AKT/mTOR pathway to promote survival and sustain the transformative properties of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 139.

Repeated Winning and Losing Experiences in Chronic Social Conflicts Are Linked to RNA Editing Pattern Difference.

Winner-loser effects influence subsequent agonistic interactions between conspecifics. Previous winning experiences could strengthen future aggression and increase the chance of winning the next agonistic interaction, while previous losing experiences could have the opposite effect. Although the role of A-to-I RNA editing has been recently implicated in chronic social defeat stress and aggressive behavior, it remains to be further elucidated in chronic social conflicts in agonistic interactions, especially in the repeated aggression (winners) and repeated defeat (losers) resulted from these conflicts. In the current study, transcriptome-wide A-to-I RNA editing in the dorsal striatum was investigated in a mouse model of chronic social conflicts, and compared between mice repeatedly winning and losing daily agonistic interactions. Our analysis identified 622 A-to-I RNA editing sites in the mouse dorsal striatum, with 23 to be differentially edited in 22 genes, most of which had been previously associated with neurological, psychiatric, or immune disorders. Among these differential RNA editing (DRE) sites four missense variants were observed in neuroligin 2 (Nlgn2), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9) BLCAP apoptosis inducing factor (Blcap), and cytoplasmic FMR1 interacting protein 2 (Cyfip2), as well as two noncoding RNA sites in small nucleolar RNA host gene 11 (Snhg11) and the maternally expressed 3 (Meg3) gene. Moreover, significant changes were observed in gene functions and pathways enriched by genes with A-to-I RNA editing in losers and especially winners compared to controls. Our results demonstrate that repeated winning and losing experiences in chronic social conflicts are linked to A-to-I RNA editing pattern difference, underlining its role in the molecular mechanism of agonistic interactions between conspecifics.

Human ARHGEF9 intellectual disability syndrome is phenocopied by a mutation that disrupts collybistin binding to the GABAA receptor α2 subunit

Intellectual disability (ID) is a common neurodevelopmental disorder that can arise from genetic mutations ranging from trisomy to single nucleotide polymorphism. Mutations in a growing number of single genes have been identified as causative in ID, including ARHGEF9. Evaluation of 41 ARHGEF9 patient reports shows ubiquitous inclusion of ID, along with other frequently reported symptoms of epilepsy, abnormal baseline EEG activity, behavioral symptoms, and sleep disturbances. ARHGEF9 codes for the Cdc42 Guanine Nucleotide Exchange Factor 9 collybistin (Cb), a known regulator of inhibitory synapse function via direct interaction with the adhesion molecule neuroligin-2 and the α2 subunit of GABAA receptors. We mutate the Cb binding motif within the large intracellular loop of α2 replacing it with the binding motif for gephyrin from the α1 subunit (Gabra2-1). The Gabra2-1 mutation causes a strong downregulation of Cb expression, particularly at cholecystokinin basket cell inhibitory synapses. Gabra2-1 mice have deficits in working and recognition memory, as well as hyperactivity, anxiety, and reduced social preference, recapitulating the frequently reported features of ARHGEF9 patients. Gabra2-1 mice also have spontaneous seizures during postnatal development which can lead to mortality, and baseline abnormalities in low-frequency wavelengths of the EEG. EEG abnormalities are vigilance state-specific and manifest as sleep disturbance including increased time in wake and a loss of free-running rhythmicity in the absence of light as zeitgeber. Gabra2-1 mice phenocopy multiple features of human ARHGEF9 mutation, and reveal α2 subunit-containing GABAA receptors as a druggable target for treatment of this complex ID syndrome.

Targeting Rac and Cdc42 GEFs in Metastatic Cancer.

The Rho family GTPases Rho, Rac, and Cdc42 have emerged as key players in cancer metastasis, due to their essential roles in regulating cell division and actin cytoskeletal rearrangements; and thus, cell growth, migration/invasion, polarity, and adhesion. This review will focus on the close homologs Rac and Cdc42, which have been established as drivers of metastasis and therapy resistance in multiple cancer types. Rac and Cdc42 are often dysregulated in cancer due to hyperactivation by guanine nucleotide exchange factors (GEFs), belonging to both the diffuse B-cell lymphoma (Dbl) and dedicator of cytokinesis (DOCK) families. Rac/Cdc42 GEFs are activated by a myriad of oncogenic cell surface receptors, such as growth factor receptors, G-protein coupled receptors, cytokine receptors, and integrins; consequently, a number of Rac/Cdc42 GEFs have been implicated in metastatic cancer. Hence, inhibiting GEF-mediated Rac/Cdc42 activation represents a promising strategy for targeted metastatic cancer therapy. Herein, we focus on the role of oncogenic Rac/Cdc42 GEFs and discuss the recent advancements in the development of Rac and Cdc42 GEF-interacting inhibitors as targeted therapy for metastatic cancer, as well as their potential for overcoming cancer therapy resistance.

The UIG-1/CDC-42 guanine nucleotide exchange factor acts in parallel to CED-10/Rac1 during axon outgrowth in Caenorhabditis elegans

ABSTRACT During development of the brain, neuronal circuits are formed through the projection of axons and dendrites in response to guidance signals. Rho GTPases (Rac1/RhoA/Cdc42) are major regulators of axo-dendritic outgrowth and guidance due to their role in controlling actin cytoskeletal dynamics, cell adhesion and motility. Functional redundancy of Rho GTPase-regulated pathways in neuronal development can mask the roles of specific GTPases. To examine potential Rho GTPase redundancy, we utilized a recently isolated hypomorphic mutation in a Caenorhabditis elegans Rac1 protein – CED-10(G30E) – which reduces the GTP binding and inhibits axon outgrowth of the PVQ interneurons. Here, we show that the CDC-42-specific guanine nucleotide exchange factor UIG-1 acts in parallel to CED-10/Rac1 to control PVQ axon outgrowth. UIG-1 performs this function in a cell-autonomous manner. Further, we found that transgenic expression of CDC-42 can compensate for aberrant CED-10(G30E)-regulated signalling during PVQ axon outgrowth. Together, our study reveals a previously unappreciated function for CDC-42 in PVQ axon outgrowth in C. elegans.

Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex

SummaryNDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

Dynamic Actin Reorganization and Vav/Cdc42-Dependent Actin Polymerization Promote Macrophage Aggregated LDL (Low-Density Lipoprotein) Uptake and Catabolism.

Objective- During atherosclerosis, LDLs (low-density lipoproteins) accumulate in the arteries, where they become modified, aggregated, and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which leads to the progression of atherosclerosis. Therapeutic blockade of this process may delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment-the lysosomal synapse (LS)-aided by local actin polymerization to digest agLDL. Here, we investigated the specific regulators involved in actin polymerization during the formation of the LS. Approach and Results- We demonstrate in vivo that atherosclerotic plaque macrophages contacting agLDL deposits polymerize actin and form a compartment strikingly similar to those made in vitro. Live cell imaging revealed that macrophage cortical F-actin depolymerization is required for actin polymerization to support the formation of the LS. This depolymerization is cofilin-1 dependent. Using siRNA-mediated silencing, pharmacological inhibition, genetic knockout, and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and GEF (guanine nucleotide exchange factor) Vav in promoting actin polymerization during the formation of the LS and exclude a role for Rac1. Conclusions- These results highlight critical roles for dynamic macrophage F-actin rearrangement and polymerization via cofilin-1, Vav, and Cdc42 in LS formation, catabolism of agLDL, and foam cell formation. These proteins might represent therapeutic targets to treat atherosclerotic disease.

Local and global Cdc42 guanine nucleotide exchange factors for fission yeast cell polarity are coordinated by microtubules and the Tea1-Tea4-Pom1 axis.

ABSTRACTThe conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast Schizosaccharomyces pombe has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when scd1 function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth. Instead, cells adopt an isotropic-like pattern of growth, which we term PORTLI growth. Surprisingly, PORTLI growth is caused by spatially inappropriate activity of Gef1. Although most Cdc42 GEFs are membrane associated, we find that Gef1 is a broadly distributed cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1–Tea4–Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results suggest a new model of fission yeast cell polarity regulation, involving coordination of ‘local’ (Scd1) and ‘global’ (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks.

DOCK9 induces membrane ruffles and Rac1 activity in cancer HeLa epithelial cells

Dedicator-of-cytokinesis (DOCK) proteins are a family of guanine-nucleotide exchange factors (GEF) for Rho GTPases. The DOCK-D homology subfamily comprises DOCK9, DOCK10, and DOCK11. DOCK9 and DOCK11 are GEFs for Cdc42 and induce filopodia, while DOCK10 is a dual GEF for Cdc42 and Rac1 and induces filopodia and ruffles. We provide data showing that DOCK9, the only one of the DOCK-D members that is not considered hematopoietic, is nevertheless expressed at high levels in T lymphocytes, as do DOCK10 and DOCK11, although unlike these, it is not expressed in B lymphocytes. To investigate DOCK9 function, we have created a stable HeLa clone with inducible expression of HA-DOCK9. Induction of expression of HA-DOCK9 produced loss of elongation and polygonal shape of HeLa cells. Regarding membrane protrusions, HA-DOCK9 prominently induced filopodia, but also an increase of membrane ruffles. The latter was consistent with an increase in the levels of activation of Rac1, suggesting that DOCK9 carries a secondary ability to induce ruffles through activation of Rac1.

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis.We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release.Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

Dynamic Actin Reorganization and Vav/Cdc42‐dependent Actin Polymerization Promote Macrophage Aggregated LDL Uptake and Catabolism

During atherosclerosis, low density lipoproteins (LDL) accumulate in the arteries, where they become modified, aggregated and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which is an important component of atherosclerosis. Characterization and therapeutic blockade of this process will likely delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment, the lysosomal synapse, aided by local actin polymerization to digest agLDL. Here, we demonstrate in vivo that atherosclerotic plaque macrophages contacting cholesterol rich LDL deposits polymerize actin and form a strikingly similar compartment. Live cell imaging revealed macrophage cortical F‐actin depolymerization driven by PIP2 hydrolysis occurs in parallel to actin polymerization at the lysosomal synapse and is required for its formation. Using siRNA‐mediated silencing, pharmacological inhibition, genetic knockout and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and guanine nucleotide exchange factor Vav in promoting actin polymerization at the lysosomal synapse, and we rule out roles for Rac1 and Rac2. These data indicate that dynamic actin rearrangement and polymerization via Vav and Cdc42 aids compartment formation, catabolism of agLDL and promotes foam cell formation.Support or Funding InformationThe project described was supported by National Institutes of Health grants R37‐DK27083 and R01‐HL093324. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.K.S. is an American Heart Association Stanley Stahl Postdoctoral Fellow (ID: 15POST22990022).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microgravity simulation activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis during vasculogenesis

Gravity plays an important role in normal tissue maintenance. The ability of stem cells to repair tissue loss in space through regeneration and differentiation remains largely unknown. To investigate the impact of microgravity on blood vessel formation from pluripotent stem cells, we employed the embryoid body (EB) model for vasculogenesis and simulated microgravity by clinorotation. We first differentiated mouse embryonic stem cells into cystic EBs containing two germ layers and then analyzed vessel formation under clinorotation. We observed that endothelial cell differentiation was slightly reduced under clinorotation, whereas vascular branch morphogenesis was markedly enhanced. EB-derived endothelial cells migrated faster, displayed multiple cellular processes, and had higher Cdc42 and Rac1 activity when subjected to clinorotation. Genetic analysis and rescue experiments demonstrated that Cdc42 but not Rac1 is required for microgravity-induced vascular branch morphogenesis. Furthermore, affinity pull-down assay and mass spectrometry identified Rap1GDS1 to be a Cdc42 guanine nucleotide exchange factor, which was upregulated by clinorotation. shRNA-mediated knockdown of Rap1GDS1 selectively suppressed Cdc42 activation and inhibited both baseline and microgravity-induced vasculogenesis. This was rescued by ectopic expression of constitutively active Cdc42. Taken together, these results support the notion that simulated microgravity activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis.

PP17. RHO GTPASE SIGNALLING IN CANCER CELL INVASION AND ANGIOGENESIS IN BRAIN TUMOURS

AbstractINTRODUCTION: Rho GTPases are molecular switches that cycle between GDP-bound inactive and GTP-bound active states. They transduce signals from the extracellular environment and downstream of oncogenes to control the actin cytoskeleton, and cellular functions including cell motility, cell cycle progression, polarity, malignant transformation, invasion and metastasis. Activation of Rho proteins is controlled by guanine nucleotide exchange factors (GEFs), and GTPase activating proteins (GAPs). Rho GEFs and GAPs are frequently overexpressed in tumours and they are tractable targets for cancer therapy. Rho proteins Rac1 and Cdc42 are deregulated in GBM. Our study is focused on understanding the role of Rac GEF DOCK4 and Cdc42 GEF DOCK9 [Abraham S, Scarcia M, ..Mavria G (2015) Nat Commun. 6: 7286] in the tumour cell and stroma cell compartments of glioblastomas. METHOD: i) The role of DOCK4 and DOCK9 in GBM invasion was analysed using organotypic spheroid assays, the U251 model and patient-derived models of GBM ii) DOCK4 expression was analysed in GBM patient samples and related to expression of stem cell marker nestin. iii) Blood vessel types were characterised in GBM patient samples and related to histological parameters and patient outcome following treatments. iii) The role of DOCK4 was investigated in the vasculature of heterozygous Dock4 null mice and syngeneic GBM tumour models implanted orthotopically. RESULTS: i) There was reduced outgrowth of GBM cells in spheroid assays with DOCK4 or DOCK9 knockdown. A complex of DOCK4 with DOCK9 was identified by immunoprecipitation in U251 cells. ii) Paired DOCK4 and nestin scores of immunostaining from 19 GBM patient samples showed DOCK4 scores correlated with nestin scores using two-tailed Spearmans Rho. Oncomine microarray data shows significant increase of DOCK4 levels in cultured GBM cells isolated from tumours (n=25) compared to neural stem cells (p=8.16 x 104). iii) Hyperproliferative blood vessels in GBM show larger diameter compared to normal blood vessels and increased number of glomeruloid structures was observed in recurrences iv) Heterozygous Dock4 genetic deletion results in reduction of blood vessel diameter. CONCLUSIONS: Tumour cell invasion into adjacent normal brain and vascular proliferation are pathologic hallmarks of GBM. Our results show that DOCK4 may be implicated in both vascular growth and glioblastoma invasion. Delineating further the signalling network in which DOCK4 participates may shed considerable light in these processes and identify new targets for treatments.

Drosophila DOCK Family Protein Zizimin Involves in Pigment Cell Differentiation in Pupal Retinae.

The dedicator of cytokinesis (DOCK) family proteins are known as one of guanine nucleotide exchange factors (GEFs), that contribute to cellular signaling processes by activating small G proteins. Although mammalian Zizimin is known to be a GEF for Cdc42 of Rho family small GTPase, its role in vivo is not well understood. Here we studied in vivo function of Drosophila Zizimin (Ziz). Knockdown of Ziz in eye imaginal discs induced the rough eye phenotype accompanied with fusion of ommatidia, loss of bristles and loss of pigments. Immunostaining analyses revealed that Ziz mainly localizes in the secondary pigment cells (SPCs) and tertiary pigment cells (TPCs) in pupal retinae. Ziz-knockdown induced SPC- and TPC-like cells with aberrant morphology in the pupal retina. Delta (Dl), a downstream target of EGFR signaling is known to regulate pigment cell differentiation. Loss-of-function mutation of Dl suppressed the rough eye phenotype and the defect in differentiation of SPCs and TPCs in Ziz-knockdown flies. Moreover, Ziz-knockdown increased Dl expression level especially in SPCs and TPCs. In addition, mutations of rhomboid-1 and roughoid that are activators of EGFR signaling pathway also suppressed both the rough eye phenotype and the defect in differentiation of SPCs and TPCs in Ziz-knockdown flies. Activation of EGFR signaling in Ziz-knockdown flies were further confirmed by immunostaining with anti-diphospho ERK IgG. These results indicate that Ziz negatively regulates the Dl expression in SPCs and TPCs to control differentiation of pigment cells and this regulation is mediated by EGFR signaling pathway.Key words: Zizimin, DOCK, EGFR signaling pathway, pigment cell, Drosophila.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl’s big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

OP52THE ROLE OF DOCK4 IN CANCER CELL INVASION AND ANGIOGENESIS IN BREAST CANCER BRAIN METASTASES

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.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

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.