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Introduction: Neuropathic pain (NP) conditions arising from injuries to the nervous system due to trauma, disease, or neurotoxins are chronic, severe, debilitating, and exceedingly difficult to treat. However, the mechanisms of NP are not yet clear. Here we explored the role of Dock4, an atypical Rac1 GEF, in the development of NP. Methods: Mechanical allodynia was assessed as paw withdrawal threshold by a dynamic plantar aesthesiometer. Immunofluorescence staining was conducted to investigate the expression and localization of Dock4, Rac1 and GluN2B. Quantitative analysis of Dock4, Rac1 and GluN2B were determined by qRT-PCR and Western blot assay. Spontaneous excitatory and inhibitory postsynaptic currents in spinal cord slices were examined using whole cell patch clam. Dendritic spine remodeling and synaptogenesis were detected in cultured dorsal spinal neurons. Results and discussion: We found that SNL caused markedly mechanical allodynia accompanied by increase of Dock4, GTP-Rac1and GluN2B, which was prevented by knockdown of Dock4. Electrophysiological tests showed that SNL facilitated excitatory synaptic transmission, however, this was also inhibited by Dock RNAi-LV. Moreover, knockdown of Dock4 prevented dendritic growth and synaptogenesis. Conclusion: In summary, our data indicated that Dock4 facilitated excitatory synaptic transmission by promoting the expression of GluN2B at the synaptic site and synaptogenesis, leading to the occurrence of NP. [ABSTRACT FROM AUTHOR]

The scavenger receptor BI (SR-BI) facilitates the transport of both HDL and LDL through endothelial cells. Its two splice variants, SR-BIvar1 and SR-BIvar2, differ in their carboxy terminal domains. Only SR-BIvar1 contains the putative binding sites for the adapter proteins PDZ domain containing protein 1 (PDZK1) and dedicator of cytokinesis 4 (DOCK4), which limit the cell surface abundance and internalization of the receptor. To investigate the cellular localization of the SR-BI variants and their interaction with lipoproteins in endothelial cells, EA.hy926 cells were stably transfected with vectors encoding untagged, GFP- or mCherry-tagged constructs of the two SR-BI variants. Additionally, the cells were transfected with shRNAs against PDZK1 or DOCK4. Microscopy investigation showed that SR-BIvar1 was predominantly localized on the cell surface together with clathrin whereas SR-BIvar2 was absent from the cell surface but retrieved in endosomes and lysosomes. Accordingly, only SR-BIvar1 increased lipoprotein binding to endothelial while HDL and LDL uptake were enhanced by both variants. Silencing of PDZK1 or DOCK4 only reduced HDL association in SR-BIvar2 overexpressing cells while LDL association was reduced both in WT and SR-BIvar2 overexpressing cells. In conclusion, either SR-BI variant facilitates the uptake of HDL and LDL into endothelial cells, however by different mechanisms and trafficking routes. This dual role may explain why the loss of DOCK4 or PDZK1 differently affects the uptake of HDL and LDL in different endothelial cells.

Abstract Background We aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data. Methods A total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using “DESeq2” package in R language. Functional enrichment analysis was conducted using the “clusterProfiler” package, and the cis- and trans-regulatory relationships between lncRNA and target mRNA were analyzed by the lncTar software. A protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through the MCODE plugin in Cytoscape. Results We identified 897 differentially expressed lncRNAs (DELs) and 1366 differentially expressed genes (DEGs) between normal and OP samples. After co-expression network analysis and cis-trans regulatory genes analysis, we identified 69 candidate genes regulated by lncRNAs. Then we further screened 7 genes after PPI analysis. The target gene DOCK4, trans-regulated by two lncRNAs, was found to be significantly upregulated in OP samples. Additionally, 4 miRNAs were identified as potential regulators of DOCK4. The potential diagnostic value of DOCK4 and its two trans-regulatory lncRNAs was supported by ROC analysis, indicating their potential as biomarkers for OP. Conclusion DOCK4 is a potential biomarker for elderly osteoporosis diagnostic. It is identified to be regulated by two lncRNAs and four miRNAs.

Background: We aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data. Methods: A total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using "DESeq2" package in R language. Functional enrichment analysis was conducted using the "clusterProfiler" package, and the cis- and trans-regulatory relationships between lncRNA and target mRNA were analyzed by the lncTar software. A protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through the MCODE plugin in Cytoscape. Results: We identified 897 differentially expressed lncRNAs (DELs) and 1366 differentially expressed genes (DEGs) between normal and OP samples. After co-expression network analysis and cis-trans regulatory genes analysis, we identified 69 candidate genes regulated by lncRNAs. Then we further screened 7 genes after PPI analysis. The target gene DOCK4, trans-regulated by two lncRNAs, was found to be significantly upregulated in OP samples. Additionally, 4 miRNAs were identified as potential regulators of DOCK4. The potential diagnostic value of DOCK4 and its two trans-regulatory lncRNAs was supported by ROC analysis, indicating their potential as biomarkers for OP. Conclusion: DOCK4 is a potential biomarker for elderly osteoporosis diagnostic. It is identified to be regulated by two lncRNAs and four miRNAs. [ABSTRACT FROM AUTHOR]

Auditory hair cells (HCs) are the mechanosensory receptors of the cochlea, and HC loss or malfunction can result from genetic defects. Dock4, a member of the Dock180-related protein superfamily, is a guanine nucleotide exchange factor for Rac1, and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder, myelodysplastic syndromes, and tumorigenesis. Here, we found that Dock4 is highly expressed in the cochlear HCs of mice. However, the role of Dock4 in the inner ear has not yet been investigated. Taking advantage of the piggyBac transposon system, Dock4 knockdown (KD) mice were established to explore the role of Dock4 in the cochlea. Compared to wild-type controls, Dock4 KD mice showed significant hearing impairment from postnatal day 60. Dock4 KD mice showed hair bundle deficits and increased oxidative stress, which eventually led to HC apoptosis, late-onset HC loss, and progressive hearing loss. Furthermore, molecular mechanism studies showed that Rac1/β-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs. Overall, our work demonstrates that the Dock4/Rac1/β-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Abstract The miRNA-181 (miR-181) family regulates neuronal persistence during cerebral ischemia/reperfusion injury (CI/RI). Since the effect of miR-181d on CI/RI has never been studied, the current work sought to determine the involvement of miR-181d in neuronal apoptosis after brain I/R injury. To replicate in vivo and in vitro CI/RI, a transient middle cerebral artery occlusion (tMCAO) model in rats and an oxygen-glucose deficiency/reoxygenation (OGD/R) model in neuro 2A cells were developed. In both in vivo and in vitro stroke models, the expression of miR-181d was considerably higher. miR-181d suppression reduced apoptosis and oxidative stress in OGD/R-treated neuroblastoma cells, but miR-181d overexpression increased both. Furthermore, it was observed that miR-181d has a direct target in dedicator of cytokinesis 4 (DOCK4). The overexpression of DOCK4 partially overcame cell apoptosis and oxidative stress induced by miR-181d upregulation and OGD/R injury. Furthermore, the DOCK4 rs2074130 mutation was related to lower DOCK4 levels in ischemic stroke (IS) peripheral blood and higher susceptibility to IS. These findings suggest that downregulating miR-181d protects neurons from ischemic damage by targeting DOCK4, implying that the miR-181d/DOCK4 axis might be a novel therapeutic target for IS. Graphical Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that lasts lifelong and causes noticeably higher premature mortality. Although the core symptoms and other behavioral deficits of ASD can persist or be deteriorated from early development to old age, how aging affects the behaviors and brain anatomy in ASD is largely unknown. DOCK4 is an ASD risk gene highly expressed in the hippocampus, and Dock4 knockout (KO) mice display ASD-like behaviors in adulthood (4- to 6-month-old). In this study, we evaluated the behavioral and hippocampal pathological changes of late-middle-aged (15- to 17-month-old) Dock4 male KO mice. Aged Dock4 KO mice continuously showed similar social deficit, elevated anxiety, and disrupted object location memory as observed in the adulthood, when compared to their wild-type (WT) littermates. Notably, Dock4 KO mice displayed an age-related decline of hippocampal dependent spatial memory, showing decreased spatial memory in Barnes maze than their WT littermates at late middle age. Morphological analysis from WT and Dock4 KO littermates revealed that Dock4 deficiency led to decreased mature neurons and oligodendrocytes but increased astrocytes in the hippocampus of late-middle-aged mice. Together, we report that ASD-like behaviors mostly persist into late-middle age in Dock4 KO mice, with specific alterations of spatial memory and hippocampal anatomy by age, thus providing new evidence for understanding age differences in behavioral deficits of ASD. [ABSTRACT FROM AUTHOR]

The miRNA-181 (miR-181) family regulates neuronal persistence during cerebral ischemia/reperfusion injury (CI/RI). Since the effect of miR-181d on CI/RI has never been studied, the current work sought to determine the involvement of miR-181d in neuronal apoptosis after brain I/R injury. To replicate in vivo and in vitro CI/RI, a transient middle cerebral artery occlusion (tMCAO) model in rats and an oxygen-glucose deficiency/reoxygenation (OGD/R) model in neuro 2A cells were developed. In both in vivo and in vitro stroke models, the expression of miR-181d was considerably higher. miR-181d suppression reduced apoptosis and oxidative stress in OGD/R-treated neuroblastoma cells, but miR-181d overexpression increased both. Furthermore, it was observed that miR-181d has a direct target in dedicator of cytokinesis 4 (DOCK4). The overexpression of DOCK4 partially overcame cell apoptosis and oxidative stress induced by miR-181d upregulation and OGD/R injury. Furthermore, the DOCK4 rs2074130 mutation was related to lower DOCK4 levels in ischemic stroke (IS) peripheral blood and higher susceptibility to IS. These findings suggest that downregulating miR-181d protects neurons from ischemic damage by targeting DOCK4, implying that the miR-181d/DOCK4 axis might be a novel therapeutic target for IS. [ABSTRACT FROM AUTHOR]

Yi Lu,1 Jiaxi Yu,1 Qiuping Dong,2 Yan Du,3 Zheng Liang1 1Department of Otorhinolaryngology, Tianjin Medical University General Hospital, Tianjin, People’s Republic of China; 2Department of Cancer Cell Biology, Tianjin’s Key Laboratory of Cancer Prevention and Therapy, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, People’s Republic of China; 3Department of Operating Theatre, Tianjin Medical University General Hospital, Tianjin, People’s Republic of ChinaCorrespondence: Zheng Liang, Department of Otorhinolaryngology, Tianjin Medical University General Hospital, Tianjin, People’s Republic of China, Tel +8618802230827, Email liangzheng01@tmu.edu.cnmailtoPurpose: The involvement of dedicator for cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor for Rac1, in immune infiltration in stomach adenocarcinoma (STAD) remains unclear.Methods: The UALCAN database was used to analyze the expression of the DOCK family. The Kaplan–Meier method and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to assess the prognostic value of the DOCK family in STAD. Furthermore, the correlation between expression of DOCK4 as well as other immune-related marker genes and tumor immune infiltration in STAD was explored using the TIMER and GEPIA websites. Subsequently, the relationship between DOCK4 expression and clinical characteristics was verified using the UALCAN database. Finally, DOCK4 mutation was analyzed via the TIMER2.0 and cBioPortal databases and the DOCK4 protein-protein interaction networks were constructed using the GeneMANIA and STRING websites.Results: DOCK4 was found to be a new prognostic biomarker in STAD. DOCK4 expression in tumors was thoroughly evaluated relative to paracancerous tissues; overexpression of DOCK4 had a negative impact on the prognosis of patients with STAD. DOCK4 was found to be significantly associated with tumor immune infiltration in STAD.Conclusion: In summary, DOCK4 is a potential regulator of the recruitment and regulation of immune-infiltrating cells, thus serving as a valuable prognostic biomarker in STAD.Keywords: DOCK4, stomach adenocarcinoma, immune infiltration, prognostic biomarker, TME

Despite that androgen-deprivation therapy results in long-lasting responses, the disease inevitably progresses to metastatic castration-resistant prostate cancer. In this study, we identified miR-33b-3p as a tumor suppressor in prostate cancer. miR-33b-3p was significantly reduced in prostate cancer tissues, and the low expression of miR-33b-3p was correlated with poor overall survival of prostate cancer patients. Overexpression of miR-33b-3p inhibited both migration and invasion of highly metastatic prostate cancer cells whereas inhibition of miR-33b-3p promoted those processes in lowly metastatic cells. The in vivo results demonstrate that miR-33b-3p suppresses metastasis of tail vein inoculated prostate cancer cells to lung and lymph nodes in mice. DOCK4 was validated as the direct target of miR-33b-3p. miR-33b-3p decreased the expression of DOCK4 and restoration of DOCK4 could rescue miR-33b-3p inhibition on cell migration and invasion. Moreover, downregulation of miR-33b-3p was induced by bortezomib, the clinically used proteasome inhibitor, and overexpression of miR-33b-3p enhanced the insufficient inhibition of bortezomib on migration and invasion as well as metastasis of prostate cancer cells. In summary, our findings demonstrate that miR-33b-3p suppresses metastasis by targeting DOCK4 in prostate cancer. Our results suggest that enhancing miR-33b-3p expression may provide a promising therapeutic strategy for overcoming that proteasome inhibitor's poor efficacy against metastatic prostate cancer. [ABSTRACT FROM AUTHOR]

Despite that androgen-deprivation therapy results in long-lasting responses, the disease inevitably progresses to metastatic castration-resistant prostate cancer. In this study, we identified miR-33b-3p as a tumor suppressor in prostate cancer. miR-33b-3p was significantly reduced in prostate cancer tissues, and the low expression of miR-33b-3p was correlated with poor overall survival of prostate cancer patients. Overexpression of miR-33b-3p inhibited both migration and invasion of highly metastatic prostate cancer cells whereas inhibition of miR-33b-3p promoted those processes in lowly metastatic cells. The in vivo results demonstrate that miR-33b-3p suppresses metastasis of tail vein inoculated prostate cancer cells to lung and lymph nodes in mice. DOCK4 was validated as the direct target of miR-33b-3p. miR-33b-3p decreased the expression of DOCK4 and restoration of DOCK4 could rescue miR-33b-3p inhibition on cell migration and invasion. Moreover, downregulation of miR-33b-3p was induced by bortezomib, the clinically used proteasome inhibitor, and overexpression of miR-33b-3p enhanced the insufficient inhibition of bortezomib on migration and invasion as well as metastasis of prostate cancer cells. In summary, our findings demonstrate that miR-33b-3p suppresses metastasis by targeting DOCK4 in prostate cancer. Our results suggest that enhancing miR-33b-3p expression may provide a promising therapeutic strategy for overcoming that proteasome inhibitor’s poor efficacy against metastatic prostate cancer.

The intestinal mucosa is in continuous contact with milliard of microorganisms, thus intestinal epithelial barrier is a critical component in the arsenal of defense mechanisms required to prevent infection and inflammation. Mucin 2 (MUC2), which is produced by the goblet cells, forms the skeleton of the intestinal mucus and protects the intestinal tract from self‐digestion and numerous microorganisms. Dedicator of cytokinesis 4 (DOCK4) is a member of the DOCK‐B subfamily of the DOCK family of guanine nucleotide exchange factors. It is reported that DOCK4 plays a critical role in the repair of the barrier function of the intestinal epithelium after chemical damage. In this study, the role of DOCK4 in the goblet cell differentiation and MUC2 production is explored. Disordered intestinal epithelium and shortage of goblet cells were observed in DOCK4 gene knockout mice. Furthermore, DOCK4 deletion contributed to the low expression of MUC2 and the goblet cell differentiation/maturation factors including growth factor independent 1 (Gfi1) and SAM pointed domain epithelial‐specific transcription factor (Spdef) in mouse ileums and colons. Overexpression of DOCK4 caused a marked increase in Gfi1, Spdef, and MUC2, while siRNA knockdown of endogenous DOCK4 significantly decreased Gfi1, Spdef, and MUC2 in HT‐29 cells. In addition, MUC2, DOCK4, and the goblet cell differentiation/maturation factors mRNA levels were decreased in colorectal cancer samples compared with normal colons. A significant positive correlation was found between MUC2 and DOCK4. In conclusion, DOCK4 may serve as a critical regulator of goblet cell differentiation and MUC2 production in the intestine. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) and dyslexia are both neurodevelopmental disorders with high prevalence in children. Both disorders have strong genetic basis, and share similar social communication deficits co-occurring with impairments of reading or language. However, whether these two disorders share common genetic risks remain elusive. DOCK4 (dedicator for cytokinesis 4), a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1, is one of few genes that are associated with both ASD and dyslexia. Dock4 is important for neuronal development and social behaviors. Two DOCK4 variations, Exon27-52 deletion (protein product: Dock4-945VS) and a missense mutation at rs2074130 (protein product: Dock4-R853H), are associated with dyslexia and/or ASD with reading difficulties. The present study explores the molecular and cellular functions of these two DOCK4 variants on neuronal development, by comparing them with the wild-type Dock4 protein. Notably, it is revealed that both mutants of Dock4 showed decreased ability to activate not only Rac1, but also another small GTPase Rap1. Consistently, both mutants were dysfunctional for regulation of cell morphology and cytoskeleton. Using Neuro-2a cells and hippocampus neurons as models, we found that both mutants had compromised function in promoting neurite outgrowth and dendritic spine formation. Electrophysiological recordings further showed that R853H partially lost the ability to promote excitatory synaptic transmission, whereas 945VS totally lost the ability. Together, we identified R853 as a previously uncharacterized site for the regulation of the integrity of Dock4 function, and provides insights in understanding the common molecular pathophysiology of ASD and dyslexia.

Autism spectrum disorder (ASD) and dyslexia are both neurodevelopmental disorders with high prevalence in children. Both disorders have strong genetic basis, and share similar social communication deficits co-occurring with impairments of reading or language. However, whether these two disorders share common genetic risks remain elusive. DOCK4 (dedicator for cytokinesis 4), a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1, is one of few genes that are associated with both ASD and dyslexia. Dock4 is important for neuronal development and social behaviors. Two DOCK4 variations, Exon27-52 deletion (protein product: Dock4-945VS) and a missense mutation at rs2074130 (protein product: Dock4-R853H), are associated with dyslexia and/or ASD with reading difficulties. The present study explores the molecular and cellular functions of these two DOCK4 variants on neuronal development, by comparing them with the wild-type Dock4 protein. Notably, it is revealed that both mutants of Dock4 showed decreased ability to activate not only Rac1, but also another small GTPase Rap1. Consistently, both mutants were dysfunctional for regulation of cell morphology and cytoskeleton. Using Neuro-2a cells and hippocampus neurons as models, we found that both mutants had compromised function in promoting neurite outgrowth and dendritic spine formation. Electrophysiological recordings further showed that R853H partially lost the ability to promote excitatory synaptic transmission, whereas 945VS totally lost the ability. Together, we identified R853 as a previously uncharacterized site for the regulation of the integrity of Dock4 function, and provides insights in understanding the common molecular pathophysiology of ASD and dyslexia. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is the most prevalent malignant cancer in the world, is the leading cause of cancer‐related death female. Recently, there is accumulating evidence that long noncoding RNAs (lncRNAs) might as an important role in the progression of BC. (epithelial‐mesenchymal transition (EMT) is considered to play a vital role in tumor cells migration and invasion. Nevertheless, the entire biological mechanisms and functions of lncRNAs in tumor migration, invasion, and EMT remain uncertain. In the present research, we observed that the expression of lncRNA AC073284.4 was downregulated in BC paclitaxel‐resistant (PR) cells (MCF‐7/PR) and tissues. Bioinformatics analysis predicted that miR‐18b‐5p was a direct target of AC073284.4, which has been validated by dual‐luciferase reporter gene assay. We further proved that AC073284.4 could directly bind to miR‐18b‐5p and relieve the suppression for dedicator of cytokinesis protein 4 (DOCK4). Furthermore, the underlying functional experiments demonstrated that AC073284.4 might sponge miR‐18b‐5p to attenuate the invasion, metastasis, and EMT of BC cell through upregulating DOCK4 expression. In summary, AC073284.4 might serve as a competing endogenous RNA (ceRNA) in BC progression via modulating miR‐18b‐5p/DOCK4 axis, which weakens EMT and migration of BC. These results suggesting that AC073284.4 might function as a potential novel diagnostic biomarker in the progression of BC. [ABSTRACT FROM AUTHOR]

Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture‐mass spectrometry; SILAC‐MS) to compare protein expression in a bone‐homing variant (BM1) of the human breast cancer cell line MDA‐MB‐231 with parental non‐bone‐homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC‐MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone‐homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4‐shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06–4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176–3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4‐expressing tumours. High DOCK4 expression was not associated with metastasis to non‐skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death.

Background: Dedicator for cytokinesis 4 (DOCK4) is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. However, the functions of DOCK4 concerning the tumor microenvironment (TME) in colon adenocarcinoma (COAD) remain uncertain., Methods: The TIMER and GEPIA databases were used to analyze the DOCK4 expression between COAD tissues and adjunct normal tissues. The PrognoScan database was used to assess the prognosis of DOCK4 expression in COAD. The co-expression networks of DOCK4 in COAD were constructed by the LinkedOmics website. Furthermore, the correlation between DOCK4 expression and TME of COAD was explored using TIMER and TISIDB databases. Finally, the clone formation assay was used to further verify the function of DOCK4 in COAD. The Western blotting assay was used to confirm the mechanism related to DOCK4 in COAD., Results: The DOCK4 expression was different significantly in COAD tissues and paracancerous tissues. The DOCK4 was found to play a poor role in the prognosis of patients with COAD. The DOCK4 was found to participate in the TME by promoting immune evasion of COAD. The reduction of DOCK4 expression inhibited the clone formation and Ras-associated protein 1A (Rap1A) expression of HCT116 cells., Conclusions: DOCK4 potentially plays an important role in the regulation of TME in COAD. DOCK4 facilitates the development through the Rap1A pathway, thus becoming a novel prognostic biomarker in COAD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background Multiple sclerosis (MS) is a multi-factorial disease of the Central Nervous System (CNS) affecting young adults leading to significant disabilities over time. MS is now believed to be prevalent in Arabian Gulf area with high incidence due to environmental factors and unknown genetic variations. The objectives of this study was to detect up-regulated potential genes that might be involved in neuroinflammatory process in MS patients in Bahrain and to measure the protein levels of the expressed genes. Methods A microarray was used to investigate mRNA expression from 12 MS patients and 12 control subjects in Bahrain where the mRNA came from peripheral blood leukocytes. Also, 80 MS patients and 80 control subjects were analyzed to measure serum protein levels of the expressed genes by ELISA. Results The data showed 15,480 genes expressed from over 47,000 transcripts and variants. Only 5 genes were significantly up-regulated in MS patients vs control subjects; namely TNF-AIP6, IL-1RA, OASL, CLC and DOCK4 (p < 0.05). Conversely, KIAA0125 gene was significantly down-regulated (p < 0.0003). Analysis of the effector molecules of the up-regulated genes revealed that 83 MS patients had positive serum level of OASL, 87 MS patients had positive serum levels of IL-1RA, and none of the 88 MS patients showed detectable serum levels of TNF-AIP6, CLC or DOCK4. Conclusions OASL and IL-1RA genes were strongly expressed in MS patients and that their effector molecules may be considered as biomarkers associated with the inflammatory process of the disease and possibly treatment response. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that the DOCK4 gene increases susceptibility to schizophrenia. However, no study has hitherto repeated this association in Chinese, and further investigated the relationship between DOCK4 and clinical symptoms in schizophrenic patients using clinical scales and functional magnetic resonance imaging (fMRI). In this study, we genotyped three single nucleotide polymorphisms (SNPs) (rs2074127, rs2217262, and rs2074130) within the DOCK4 gene using a case-control design (including 1289 healthy controls and 1351 patients with schizophrenia). 55 first-episode schizophrenia (FES) patients and 59 healthy participants were divided by the genotypes of rs2074130 into CC and CT+TT groups. We further investigated the association with clinical symptoms and neural characteristics (brain activation/connectivity and nodal network metrics). Our results showed significant associations between all selected SNPs and schizophrenia (all P < 0.05). In patients, letter fluency and motor speed scores of T allele carriers were significantly higher than the CC group (all P < 0.05). Interestingly, greater brain activity, functional connectivity, and betweenness centrality (BC) in language processing and motor coordination were also observed in the corresponding brain zones in patients with the T allele based on a two-way ANCOVA model. Moreover, a potential positive correlation was found between brain activity/connectivity of these brain regions and verbal fluency and motor speed. Our findings suggest that the DOCK4 gene may contribute to the onset of schizophrenia and lead to language processing and motor coordination dysfunction in this patient population from China. [ABSTRACT FROM AUTHOR]

Abnormal regulation of neural migration and neurite growth is thought to be an important feature of developmental dyslexia (DD). We investigated 16 genetic variants, selected by bioinformatics analyses, in six key genes in the neuronal migration and neurite outgrowth network in a Chinese population. We first observed that KIAA0319L rs28366021, KIAA0319 rs4504469, and DOCK4 rs2074130 were significantly associated with DD risk after false discovery rate (FDR) adjustment for multiple comparisons (odds ratio (OR) = 0.672, 95 % confidence interval (CI) = 0.505-0.894, P = 0.006; OR = 1.608, 95 % CI = 1.174-2.203, P = 0.003; OR = 1.681, 95 % CI = 1.203-2.348, P = 0.002). The following classification and regression tree (CART) analysis revealed a prediction value of gene-gene interactions among DOCK4 rs2074130, KIAA0319 rs4504469, DCDC2 rs2274305, and KIAA0319L rs28366021 variants. Compared with the lowest risk carriers of the combination of rs2074130 CC, rs4504469 CC, and rs2274305 GG genotype, individuals carrying the combined genotypes of rs2074130 CC, rs4504469 CT or TT, and rs28366021 GG had a significantly increased risk for DD (OR = 2.492, 95 % CI = 1.447-4.290, P = 0.001); individuals with the combination of rs2074130 CT or TT and rs28366021 GG genotype exhibited the highest risk for DD (OR = 2.770, 95 % CI = 2.265-6.276, P = 0.000). A significant dose effect was observed among these four variants ( P for trend = 0.000). In summary, this study supports the importance of single- and multiple-risk variants in this network in DD susceptibility in China. [ABSTRACT FROM AUTHOR]

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia. [ABSTRACT FROM AUTHOR]

Background Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-type specific mutation spectrum. Methods We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. Results Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximity ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. Conclusion Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies. [ABSTRACT FROM AUTHOR]

Dock family proteins are evolutionary conserved exchange factors for the Rho GTPases Rac and Cdc42. There are 11 Dock proteins in mammals, named Dock1 (or Dock180) to Dock11 that play different cellular functions. In particular, Dock proteins regulate actin cytoskeleton, cell adhesion and migration. Not surprisingly, members of the Dock family have been involved in various pathologies, including cancer and defects in the central nervous and immune systems. This review proposes an update of the recent findings regarding the function of Dock proteins, focusing on their role in the control of cell migration and invasion and the consequences in human diseases. [ABSTRACT FROM AUTHOR]

Abstract: Dock4 is a member of the Dock180 family of proteins that mediates cancer cell migration through activation of Rac. However, the regulatory mechanism of Dock4 remains unclear. In this study, we show that the C-terminal proline-rich region of Dock4 is essential for the Dock4 mediated promotion of cell migration in MDA-MB-231 breast cancer cells. We found that a phosphoinositide-binding protein SH3YL1 interacted with the C-terminal proline-rich region of Dock4. Interaction of SH3YL1 with Dock4 promoted Dock4-mediated Rac1 activation and cell migration. Mutations in the phosphoinositide-binding domain disrupted the ability of SH3YL1 to promote Dock4-mediated cell migration. In addition, depletion of SH3YL1 in MDA-MB-231 cells suppressed cell migration. Taken together, these results provide evidence for a novel and functionally important interaction between Dock4 and SH3YL1 to promote cancer cell migration by regulating Rac1 activity. [Copyright &y& Elsevier]

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It is well accepted that schizophrenia has a strong genetic component. Several genome-wide association studies (GWASs) of schizophrenia have been published in recent years; most of them population based with a case-control design. Nevertheless, identifying the specific genetic variants which contribute to susceptibility to the disorder remains a challenging task. A family-based GWAS strategy may be helpful in the identification of schizophrenia susceptibility genes since it is protected against population stratification, enables better accounting for genotyping errors and is more sensitive for identification of rare variants which have a very low frequency in the general population. In this project we implemented a family-based GWAS of schizophrenia in a sample of 107 Jewish-Israeli families. We found one genome-wide significant association in the intron of the DOCK4 gene (rs2074127, p value=1.134×10−7) and six additional nominally significant association signals with p<1×10−5. One of the top single nucleotide polymorphisms (p<1×10−5) which is located in the predicted intron of the CEACAM21 gene was significantly replicated in independent family-based sample of Arab-Israeli origin (rs4803480: p value=0.002; combined p value=9.61×10−8), surviving correction for multiple testing. Both DOCK4 and CEACAM21 are biologically reasonable candidate genes for schizophrenia although generalizability of the association of DOCK4 with schizophrenia should be investigated in further studies. In addition, gene-wide significant associations were found within three schizophrenia candidate genes: PGBD1, RELN and PRODH, replicating previously reported associations. By application of a family-based strategy to GWAS, our study revealed new schizophrenia susceptibility loci in the Jewish-Israeli population. [ABSTRACT FROM AUTHOR]

Microdeletion and microduplication copy number variations are found in patients with autism spectrum disorder and in a number of cases they include genes that are involved in the canonical Wnt signaling pathway (for example, FZD9, BCL9 or CDH8). Association studies investigating WNT2, DISC1, MET, DOCK4 or AHI1 also provide evidence that the canonical Wnt pathway might be affected in autism. Prenatal medication with sodium-valproate or antidepressant drugs increases autism risk. In animal studies, it has been found that these medications promote Wnt signaling, including among others an increase in Wnt2 gene expression. Notably, the available genetic information indicates that not only canonical Wnt pathway activation, but also inhibition seems to increase autism risk. The canonical Wnt pathway plays a role in dendrite growth and suboptimal activity negatively affects the dendritic arbor. In principle, this provides a logical explanation as to why both hypo- and hyperactivity may generate a similar set of behavioral and cognitive symptoms. However, without a validated biomarker to stratify for deviant canonical Wnt pathway activity, it is probably too dangerous to treat patients with compounds that modify pathway activity. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral deficits and complex genetic etiology. A recent study of 517 ASD families implicated DOCK4 by single nucleotide polymorphism (SNP) association and a microdeletion in an affected sibling pair. Methods: The DOCK4 microdeletion on 7q31.1 was further characterized in this family using QuantiSNP analysis of 1M SNP array data and reverse transcription polymerase chain reaction. Extended family members were tested by polymerase chain reaction amplification of junction fragments. DOCK4 dosage was measured in additional samples using SNP arrays. Since QuantiSNP analysis identified a novel CNTNAP5 microdeletion in the same affected sibling pair, this gene was sequenced in 143 additional ASD families. Further polymerase chain reaction-restriction fragment length polymorphism analysis included 380 ASD cases and suitable control subjects. Results: The maternally inherited microdeletion encompassed chr7:110,663,978-111,257,682 and led to a DOCK4-IMMP2L fusion transcript. It was also detected in five extended family members with no ASD. However, six of nine individuals with this microdeletion had poor reading ability, which prompted us to screen 606 other dyslexia cases. This led to the identification of a second DOCK4 microdeletion co-segregating with dyslexia. Assessment of genomic background in the original ASD family detected a paternal 2q14.3 microdeletion disrupting CNTNAP5 that was also transmitted to both affected siblings. Analysis of other ASD cohorts revealed four additional rare missense changes in CNTNAP5. No exonic deletions of DOCK4 or CNTNAP5 were seen in 2091 control subjects. Conclusions: This study highlights two new risk factors for ASD and dyslexia and demonstrates the importance of performing a high-resolution assessment of genomic background, even after detection of a rare and likely damaging microdeletion using a targeted approach. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of sporadic breast tumors (referred to Mehrad Hospital in Tehran) were screened using PCR-SSCP method and the number of observed variations compared with the control group (100 normal women). Mutation detection for coding exons of RAP1A gene and the 500 bp upstream of transcription initiation site as promoters of both DOCK4 and RAP1A were carried out and compared with the control group. The promoter region of DOCK4 showed a heterozygous mutation with G>A transition at nucleotide -303 in a fibroadenoma case. With regard to RAP1A we found a heterozygous mutation, G>A transition in an adenoid cystic carcinoma case, and another heterozygous mutation, G>T transversion in an intraductal papilloma case both at nucleotide +45. A homozygous variation, T>A transversion was also found at nucleotide +29 of a fibroadenoma case. The differences in the frequency of variations mentioned above were not statistically significant. However Fisher's exact showed significant difference for T>A transversion. Although, the higher frequency of these mutations and variations may be related to the disease, a larger sample size is needed for the confirmation of our findings. [ABSTRACT FROM AUTHOR]

The canonical Wnt/β-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating β-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/β-catenin pathway. DOCK4 expression regulates cellular β-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the β-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3β (GSK3β). This molecular interaction enhances β-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3β, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/β-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/β-catenin pathway.Oncogene (2008) 27, 5845–5855; doi:10.1038/onc.2008.202; published online 21 July 2008 [ABSTRACT FROM AUTHOR]

DOCK4, a member of DOCK180 family proteins, was originally identified as a product of a gene deleted during tumor progression. Although its tumor suppression properties have been reported, the regulation mechanism of this protein has not been fully elucidated. DOCK4 shares two conserved domains called as DHR-1 and DHR-2 domain as other members including DOCK180. Although DHR-1 in DOCK180 is reported to bind to PIP3, whether that of DOCK4 exhibits similar function has yet not been examined. In a search for novel PIP3 binding proteins by the PIP3 analog beads binding assay, we found that DOCK4 and its novel splicing variant, whose exon1 and exon52 are different from the known one, bind to PIP3. Binding assay using deletion mutants of DOCK4 revealed that the binding region falls into the DHR-1 domain. These results raise the possibility that DOCK4 may be regulated by PIP3 to exert its function. © 2008 IUBMB IUBMB Life, 60(7): 467–472, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Cell migration is essential for normal development and many pathological processes including tumor metastasis. Rho family GTPases play important roles in this event. In particular, Rac is required for lamellipodia formation at the leading edge during migration. Dock4 is a member of the Dock180 family proteins, and Dock4 mutations are present in a subset of human cancer cell lines. However, the function and the regulatory mechanism of Dock4 remain unclear. Here we show that Dock4 is regulated by the small GTPase RhoG and its effector ELMO and promotes cell migration by activating Rac1. Dock4 formed a complex with ELMO, and expression of active RhoG induced translocation of the Dock4–ELMO complex from the cytoplasm to the plasma membrane and enhanced the Dock4- and ELMO-dependent Rac1 activation and cell migration. On the other hand, RNA interference-mediated knockdown of Dock4 in NIH3T3 cells reduced cell migration. Taken together, these results suggest that Dock4 plays an important role in the regulation of cell migration through activation of Rac1, and that RhoG is a key upstream regulator for Dock4. [Copyright &y& Elsevier]

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia. [Copyright &y& Elsevier]

Conventionally, Rho guanine nucleotide exchange factors (GEFs) are known activators of Rho guanosine triphosphatases (GTPases) that promote tumorigenesis. However, the role of Rho GEFs in non-small cell lung cancer (NSCLC) remains largely unknown. Through the screening of 81 Rho GEFs for their expression profiles and correlations with survival, four of them were identified with strong significance for predicting the prognosis of NSCLC patients. The four Rho GEFs, namely ABR, PREX1, DOCK2 and DOCK4, were downregulated in NSCLC tissues compared to normal tissues. The downregulation of ABR, PREX1, DOCK2 and DOCK4, which can be attributfed to promoter methylation, is correlated with poor prognosis. The underexpression of the four key Rho GEFs might be related to the upregulation of MYC signaling and DNA repair pathways, leading to carcinogenesis and poor prognosis. Moreover, overexpression of ABR was shown to have a tumor-suppressive effect in PC9 and H1703 cells. In conclusion, the data reveal the unprecedented role of ABR as tumor suppressor in NSCLC. The previously unnoticed functions of Rho GEFs in NSCLC will inspire researchers to investigate the distinct roles of Rho GEFs in cancers, in order to provide critical strategies in clinical practice.