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Cell division cycle 42 (CDC42) is a small Rho GTPase, which serves as a fundamental intracellular signal node regulating actin cytoskeletal dynamics and several other integral cellular processes. CDC42-associated disorders encompass a broad clinical spectrum including Takenouchi-Kosaki syndrome, autoinflammatory syndromes and neurodevelopmental phenotypes mimicking RASopathies. Dysregulation of CDC42 signaling by genetic defects in either DOCK6 or ARHGAP31 is also considered to play a role in the pathogenesis of Adams-Oliver syndrome (AOS). Here, we report a mother and her child carrying the previously reported pathogenic CDC42 variant c.511G>A (p.Glu171Lys). Both affected individuals presented with short stature, distinctive craniofacial features, pectus deformity as well as heart and eye anomalies, similar to the recently described Noonan syndrome-like phenotype associated with this variant. Remarkably, one of the patients additionally exhibited aplasia cutis congenita of the scalp. Multi-gene panel sequencing of the known AOS-causative genes and whole exome sequencing revealed no second pathogenic variant in any disease-associated gene explaining the aplasia cutis phenotype in our patient. This observation further expands the phenotypic spectrum of CDC42-associated disorders and underscores the role of CDC42 dysregulation in the pathogenesis of aplasia cutis congenita.

Fatty acid binding protein 5 (FABP5) is a promising target for development of inhibitors to help control pain and inflammation. In this work, computer-based docking (DOCK6 program) was employed to screen ∼2 M commercially available compounds to FABP5 based on an X-ray structure complexed with the small molecule inhibitor SBFI-26 previously identified by our group (also through virtual screening). The goal was discovery of additional chemotypes. The screen resulted in the purchase of 78 candidates, which led to the identification of a new inhibitor scaffold (STK-0) with micromolar affinity and apparent selectivity for FABP5 over FABP3. A second similarity-based screen resulted in three additional hits (STK-15, STK-21, STK-22) from which preliminary SAR could be derived. Notably, STK-15 showed comparable activity to the SBFI-26 reference under the same assay conditions (1.40 vs 0.86 μM). Additional molecular dynamics simulations, free energy calculations, and structural analysis (starting from DOCK-generated poses) revealed that R enantiomers (dihydropyrrole scaffold) of STK-15 and STK-22 have a more optimal composition of functional groups to facilitate additional H-bonds with Arg109 of FABP5. This observation suggests enantiomerically pure compounds could show enhanced activity. Overall, our study highlights the utility of using similarity-based screening methods to discover new inhibitor chemotypes, and the identified FABP5 hits provide a strong starting point for future efforts geared to improve activity.

We aimed to identify the role of DOCK6 in oral squamous cell cancer (OSCC) in this study.DOCK6 expression in OSCC was analyzed using TCGA and GEO datasets and was verified by quantitative real-time PCR, Western blotting, and immunohistochemistry. Statistical analyses were performed to evaluate the relationships between DOCK6 expression and the clinicopathological characteristics of OSCC patients. Wound healing and Transwell assays were performed to assess OSCC cell migration and invasion, respectively. STRING and GO analyses and gene set enrichment analysis were used to identify DOCK6-interacting proteins, their functions and their potential pathways.DOCK6 was significantly upregulated at both the mRNA and protein levels in OSCC tissues (all P 0.05). DOCK6 levels were positively correlated with age (P 0.05), lymph node metastasis status (P 0.001), clinical stage (P 0.001), differentiation (P 0.05), and poor clinical outcome (P 0.05) in OSCC patients. Furthermore, univariate and multivariate analyses revealed that high DOCK6 expression (P 0.01) and clinical stage III-IV (P 0.05) might serve as independent prognostic factors for OSCC patients. Functionally, DOCK6 silencing significantly suppressed OSCC cell migration and invasion (all P 0.05). Ten proteins that interact with DOCK6, more than ten functions related to cancer, and more than six pathways related to DOCK6 in OSCC were identified via bioinformatic methods.DOCK6 is upregulated in OSCC, is associated with a poor prognosis in OSCC patients and increases OSCC cells migration and invasion. These findings suggest that DOCK6 may be a potential therapeutic target with prognostic implication in patients with OSCC.

Probing protein surfaces to accurately predict the binding site and conformation of a small molecule is a challenge currently addressed through mainly two different approaches: blind docking and cavity detection-guided docking. Although cavity detection-guided blind docking has yielded high success rates, it is less practical when a large number of molecules must be screened against many detected binding sites. On the other hand, blind docking allows for simultaneous search of the whole protein surface, which however entails the loss of accuracy and speed. To bridge this gap, in this study, we developed and tested BLinDPyPr, an automated pipeline which uses FTMap and DOCK6 to perform a hybrid blind docking strategy. Through our algorithm, FTMap docked probe clusters are converted into DOCK6 spheres for determining binding regions. Because these spheres are solely derived from FTMap probes, their locations are contained in and specific to multiple potential binding pockets, which become the regions that are simultaneously probed and chosen by the search algorithm based on the properties of each candidate ligand. This method yields pose prediction results (45.2–54.3% success rates) comparable to those of site-specific docking with the classic DOCK6 workflow (49.7–54.3%) and is half as time-consuming as the conventional blind docking method with DOCK6.

Adams-Oliver syndrome (AOS) is a rare, inherited multi-systemic malformation syndrome characterized by a combination of aplasia cutis congenita and transverse terminal limb defects along with variable involvement of the central nervous system, eyes, and cardiovascular system. AOS can be inherited as both autosomal-dominant and recessive traits. Pathogenic variants in theTo report a novel homozygous variant in theCase report.We report a case of a 4-month-old male who presented with microcephaly, global developmental delay, truncal hypotonia, and limb reduction defects. Ophthalmic examination revealed bilateral nystagmus and retinal detachment with mild cataractous changes in addition to retrolental plaque in the left eye. Next generation sequencing analysis identified a novel homozygous frameshift likely pathogenic variant (c.1269_1285dup (p.Arg429Glnfs*32)) in theThe discovery of this new likely pathogenic variant enriches the genotypic spectrum of

First emerged in late December 2019, the outbreak of novel severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) pandemic has instigated public-health emergency around the globe. Although available medications can only alleviate few symptoms like difficulty in breathing, the world is craving to identify specific antiviral agents or vaccines against SARS-CoV-2. Teicoplanin is a glycopeptide class of antibiotic which is regularly used for treating Gram-positive bacterial infections, has shown potential therapeutic efficacy against SARS-CoV-2 in vitro. Therefore, in this study, a mechanistic insight of intermolecular interactions between teicoplanin and SARS-CoV-2 main protease has been scrutinized by employing molecular modelling approaches. Molecular docking study was carried out by three different docking programs including AutoDock4, AutoDock Vina and Dock6. The dynamic and thermodynamics constraints of docked drug in complex with target protein under specific physiological conditions was ascertained by all-atom molecular dynamics (MD) simulation study. Root mean square deviation of carbon α chain exhibited uniform value in the range of 1-1.7 Å while root mean square fluctuations were also recorded below 1.72 Å, justifying the stability of the bound complex in biological environments. Key interacting residues involved in hydrogen bonds include Thr26, His41, Asn142, Ser144, Glu166, and Gln189. Several water bridges and hydrophobic interactions also anchored docked teicoplanin in the inhibitor binding site. These outcomes are supposed to be fruitful in rational design of antiviral drugs against SARS-CoV-2.

Background Adams–Oliver syndrome (AOS [MIM 100300 ]) is a rare, multiple malformation syndrome commonly characterized by scalp aplasia cutis congenita (ACC) and transverse terminal limb defects (TTLD). Brain abnormalities and heart defects are also present in most patients. Both autosomal-dominant and autosomal-recessive inheritance of the disease have been observed. To date, six causative genes have been identified: ARHGAP31, DOCK6, EOGT, RBPJ, NOTCH1, and DLL4. Autosomal-recessive mutations are mostly associated with DOCK6 (MIM: 614219 ) and EOGT (MIM: 615297 ), while mutations in ARHGAP31 (MIM: 100300 ), RBPJ (MIM: 614814 ), NOTCH1 (MIM: 616028 ), and DLL4 (MIM: 616589 ) have been linked to autosomal-dominant inheritance. Case We report a case of AOS caused by DOCK6 mutations (c.3190_3191del and c.4491 + 1G > T), showing no signs of scalp ACC or TTLD, but with bilateral ventricular dilation and ophthalmic abnormalities. Results of whole-exome high-throughput sequencing were analyzed using a combination of pathogenicity prediction algorithms, query of variant databases, and review of the literature. Candidate gene variation sites were identified for pedigree verification. Conclusions The correlation between the genotype and phenotype of AOS has great variability, and the specific pathogenesis of AOS remains to be further studied.

DOCK6 is a RAC1/CDC42 guanine nucleotide exchange factor, however, little is known about its function and sub-cellular localization. DOCK6 regulates the balance between RAC1 and RHOA activity during cell adhesion and is important for CDC42-dependent mitotic chromosome alignment. Surprisingly, a cell intrinsic adaptation mechanism compensates for errors in these DOCK6 functions that arise as a consequence of prolonged DOCK6 depletion or complete removal in DOCK6 knockout cells. Down-regulation of the ubiquitin-like modifier ISG15 accounts for this adaptation. Strikingly, although most other DOCK family proteins are deployed on the plasma membrane, here we show that DOCK6 localizes to the endoplasmic reticulum (ER) in dependence of its DHR-1 domain. ER localization of DOCK6 opens up new insights into its functions.

Structure-based virtual screening usually involves docking of a library of chemical compounds onto the functional pocket of the target receptor so as to discover novel classes of ligands. However, the overall success rate remains low and screening a large library is computationally intensive. An alternative to this "ab initio" approach is virtual screening by binding homology search. In this approach, potential ligands are predicted based on similar interaction pairs (similarity in receptors and ligands). SPOT-Ligand is an approach that integrates ligand similarity by Tanimoto coefficient and receptor similarity by protein structure alignment program SPalign. The method was found to yield a consistent performance in DUD and DUD-E docking benchmarks even if model structures were employed. It improves over docking methods (DOCK6 and AUTODOCK Vina) and has a performance comparable to or better than other binding-homology methods (FINDsite and PoLi) with higher computational efficiency. The server is available at http://sparks-lab.org. © 2016 Wiley Periodicals, Inc.

Adams-Oliver syndrome (AOS) is a rare congenital disease characterized by aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It shows significant genetic heterogeneity and can be transmitted by autosomal dominant or recessive inheritance. Recessive inheritance is associated with mutations of DOCK6 or EOGT; however, only few cases have been published so far. We present two families with EOGT-associated AOS. Due to pseudodominance in one family, the recognition of the recessive inheritance pattern was difficult. We identified two novel AOS-causing mutations (c.404G>A/p.Cys135Tyr and c.311+1G>T). The phenotype in the presented families was dominated by large ACC, whereas TTLD were mostly subtle or even absent and no major malformations occured. Our observations along with the previously published cases indicate that the two types of recessive AOS (EOGT- vs. DOCK6-associated) differ significanty regarding the frequency of neurologic or ocular deficits.

International audience; Background: Drepanocytosis is a genetic blood disorder characterized by red blood cells that assume an abnormal, rigid, sickle shape. In the pathogenesis of vaso-occlusive crises of sickle cell disease, red blood cells bind to the endothelium and promote vaso-occlusion. At the surface of these sickle red blood cells, the overexpressed protein Lutheran strongly interacts with the Laminin 511/521. The aim of this study is to identify a PPI inhibitor with a high probability of binding to Lutheran for the inhibition of the Lutheran-Laminin 511/521 interaction. Methods: A virtual screening was performed with 395 601 compounds that target Lutheran. Prior validation of a robust docking and scoring protocol was considered on the protein CD80 because this protein has a binding site with similar topological and physico-chemical characteristics and it also has a series of ligands with known affinity constants. This protocol consisted of multiple filtering steps based on docked scores, molecular dynamics simulations, post-screening scores, and molecular properties. Results: A robust docking and scoring protocol was validated on the protein CD80 with the docking program DOCK6 and the secondary scoring function XSCORE. We identified four molecules for Lutheran that have good structural and physico-chemical properties. Conclusion: We took advantage of the similarities between the binding site of Lutheran and that of the protein CD80 to set up a robust docking and scoring protocol. Our protocol for primary scoring filtering, molecular dynamics simulation filtering, secondary scoring filtering, and molecular property filtering allows discarding most of the ligands with four compounds that are promising candidates for inhibiting the Lutheran-Laminin 511/521 interaction.

Adams–Oliver syndrome (AOS) is characterized by a combination of congenital scalp defects (aplasia cutis congenita) and terminal transverse limb malformations of variable severity. When neurological findings are present, patients are reported as AOS variants. We describe a child with compound heterozygosity of the DOCK6 gene, aplasia cutis, terminal transverse limb defects, cardiovascular impairment, intellectual disability, and brain malformations with intracranial calcifications. He suffers from a severe refractory epileptic encephalopathy characterized by polymorphic seizures with prolonged periods of electroencephalogram (EEG), continuous epileptiform activity related to clinical inactivity, and closure of eyes with an “ON-OFF” behavior.

cGMP-binding cGMP-specific PDE, PDE5 plays a key role in the hydrolysis of cyclic guanidine monophosphate. Because cGMP mediates vascular functions, a PDE5 inhibitor that elevates cGMP level is an attractive means for vasodilatation and treatment of erectile dysfunction. In this paper we report the elucidation of the common pharmacophore hypothesis of different classes of PDE5 inhibitors. Using LigandScout program, pharmacophore modelling studies were performed on prior reported potent PDE5 inhibitors with a variety of scaffolds in order to identify one common set of critical chemical features of these PDE5 inhibitors 1-52. The best pharmacophore model, model-1, characterized by four chemical features: one aromatic ring, one hydrophobe, one hydrogen acceptors and one hydrogen donor. Using Dock6 program, docking studies were performed in order to investigate the mode of binding of these compounds. The molecular docking study allowed confirming the preferential binding mode of different classes of PDE5 inhibitors inside the active site. The obtained binding mode was as same as that of vardenafil, X-ray ligand with different orientation with varied PDE5 inhibitors׳ scaffold.

Contains fulltext : 152973.pdf (Publisher’s version ) (Closed access) Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

There is a pressing need to discover and develop novel drugs against cancer. With the new era of bioinformatics, which integrates different aspects, drug development has been tremendously improved. Recently, extensive research was directed towards the rational modification of steroid molecules against different disease especially cancer. Moreover, heterocyclic steroid derivatives have shown a lot of different biological activities such as antimicrobial, anti-inflammatory, and anti-cancer activities. Molecular docking methods can be used to explore how the steroid derivatives conformations can adopt within the binding sites of specific macromolecular targets involved in cancer progression. We conducted this study to investigate the accuracy of different molecular docking calculations using different steroidal molecular targets, and to define the most accurate one to study the mode of action of steroid derivatives as potential anti-cancer drugs. Our results revealed that the Dock6, PLANTS, AutoDock, GLIDE (SP and XP), and GOLD (ASP, Chemscore, and PLP) software were able to maintain the binding mode of the co-crystallized ligands inside their proteins by achieving RMSD values lower than two. Moreover, molecular docking study revealed that compound 4, and 5 are promising steroidal derivatives as anti-cancer drugs. Further on, the cytotoxic activity of the selected steroidal derivatives were tested against leukemia cell line using MTT assay. The results revealed that compound 4, and 5 were potential cytotoxic agents against THP-1 cells (IC50s were 44.67 µM, and 46.77 µM, respectively), these results are in agreement with the molecular docking study.

RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

The recent advances in genome-engineering by technologies such as CRISPR/Cas9 now enable an unprecedented level of genome manipulation to support the analysis of phenotypes arising from gene disrup...

Genome-editing technologies allow systematic inactivation of human genes. Whether knockout phenotypes always reflect gene functions as determined by acute RNAi is an important question. Here we show how the acute knockdown of the Adams-Oliver syndrome (AOS) gene DOCK6, coding for a RAC1/CDC42 guanine nucleotide exchange factor, results in strikingly different phenotypes to those generated by genomic DOCK6 disruption. Cell-intrinsic adaptation compensates for loss of DOCK6 function. Prolonged DOCK6 loss impacts upon the MRTF-A/SRF transcription factor, reducing levels of the ubiquitin-like modifier ISG15. Reduced ISGylation of the IQGAP1 protein increases levels of active CDC42 and RAC1 to compensate for DOCK6 disruption. Similar downregulation of ISG15 in cells from DOCK6 AOS patients indicates that such adaptation can compensate for genetic defects during development. Thus, phenotypes of gene inactivation are critically dependent on the timescale, as acute knockdown reflects a transient state of adjustment to a new equilibrium that is attained following compensation.

Angiopoietin-like proteins (ANGPTLs) play major roles in the trafficking and metabolism of lipids. Inactivation of ANGPTL3 , a gene located in an intron of DOCK7 , results in very low levels of LDL-cholesterol (C), HDL-C and triglyceride (TAG). We identified another ANGPTL family member, ANGPTL8 , which is located in the corresponding intron of DOCK6 . A variant in this family member (rs2278426, R59W) was associated with lower plasma LDL-C and HDL-C levels in three populations. ANGPTL8 is expressed in liver and adipose tissue, and circulates in plasma of humans. Expression of ANGPTL8 was reduced by fasting and increased by refeeding in both mice and humans. To examine the functional relationship between the two ANGPTL family members, we expressed ANGPTL3 at physiological levels alone or together with ANGPTL8 in livers of mice. Plasma TAG level did not change in mice expressing ANGPTL3 alone, whereas coexpression with ANGPTL8 resulted in hypertriglyceridemia, despite a reduction in circulating ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in plasma of these mice. In cultured hepatocytes, ANGPTL8 expression increased the appearance of N-terminal ANGPTL3 in the medium, suggesting ANGPTL8 may activate ANGPTL3. Consistent with this scenario, expression of ANGPTL8 in Angptl3 −/− mice failed to promote hypertriglyceridemia. Thus, ANGPTL8 , a paralog of ANGPTL3 that arose through duplication of an ancestral DOCK gene, regulates postprandial TAG and fatty acid metabolism by controlling activation of its progenitor, and perhaps other ANGPTLs. Inhibition of ANGPTL8 provides a new therapeutic strategy for reducing plasma lipoprotein levels.

Rho guanine nucleotide exchange factors (RhoGEFs) integrate cell signaling inputs into morphological and functional responses. However, little is known about the endothelial repertoire of RhoGEFs and their regulation. Thus, we assessed the expression of 81 RhoGEFs (70 homologous to Dbl and 11 of the DOCK family) in endothelial cells. Further, in the case of DH-RhoGEFs, we also determined their responses to VEGF exposure in vitro and in the context of tumors. A phylogenetic analysis revealed the existence of four groups of DH-RhoGEFs and two of the DOCK family. Among them, we found that the most abundant endothelial RhoGEFs were: Tuba, FGD5, Farp1, ARHGEF17, TRIO, P-Rex1, ARHGEF15, ARHGEF11, ABR, Farp2, ARHGEF40, ALS, DOCK1, DOCK7 and DOCK6. Expression of RASGRF2 and PREX2 increased significantly in response to VEGF, but most other RhoGEFs were unaffected. Interestingly murine endothelial cells isolated from tumors showed that all four phylogenetic subgroups of DH-RhoGEFs were altered when compared to non-tumor endothelial cells. In summary, our results provide a detailed assessment of RhoGEFs expression profiles in the endothelium and set the basis to systematically address their regulation in vascular signaling.

Unravelling the role of cytoskeleton regulators may be complicated by adaptations to experimental manipulations. In this issue of Developmental Cell, Cerikan et al. (2016) reveal how acute effects of DOCK6 RhoGEF depletion on RAC1 and CDC42 activation are reversed over time by compensatory mechanisms that re-establish cellular homeostasis.

© 2014 The American Society of Human Genetics Notch signaling determines and reinforces cell fate in multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

68 Background: The prognosis of advanced gastric cancer (GC) remains poor, and the key players in molecular pathogenesis are predominantly unknown at present. Methods: In order to identify novel prognostic factor of GC, we compared the data of iTRAQ proteomics from 6 GC samples with that of Oncomine database from 86 GC samples to intersect 122 high-expression proteins in gastric cancer tissue. The candidate biomarkers were further characterized in fresh GC tumor tissues compared with adjacent non-tumor tissue by using Q-RT-PCR, immunohistochemical staining and western blot. GC cells with depletion of DOCK6 were examined to identify downstream molecules and establish their effects on cell motility, invasion and gastric cancer stem cell (CSC) markers. The possible signaling pathway of DOCK6 were also investigated. Results: DOCK6 is identified as one of the potential GC biomarkers in iTRAQ proteomics. Our data confirm the overexpression of DOCK6 in GC tumor tissue compared with adjacent non-tumor tissue. In addition, the clinical data shows higher DOCK6 expression was positive correlated to depth of invasion, lymph node metastasis, vascular invasion and pathological stage. Furthermore, higher DOCK6 expression represented significantly shorter cumulative survival both in univariate and multivariate analysis for survival outcome. Depletion of DOCK6 repressed the cell migration, proliferation, sphere formation, anchorage-independent growth and tumorigenicity as well as gastric cancer stem cell (CSC) markers. In order to explore the downstream effector of DOCK6, we correlated thousands of gene with DOCK6 in three GC dataset published in the literature. The DOCK6 positive-correlated genes were subsequently analyzed by Metacore. They were significantly involved in WNT/β-catenin signaling pathway. Our result demonstrates nuclear b-catenin, as well as CD44, was decreased accompanied by depleting DOCK6. Moreover, the interaction between DOCK6 and β-catenin was observed in GC cells. Conclusions: Our studies unveil the role of DOCK6 in the self-renewal of CSC and propose DOCK6 as an independent marker of tumor progression.

Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.