DVL1 Expression Research Articles

Role of MicroRNA-214 in Dishevelled1-Modulated β-catenin Signalling in Non-Small Cell Lung Cancer Progression.

Background: The mortality of patients with non-small cell lung cancer (NSCLC) is rather high. This is largely because of the lack of specific targets and understanding of the molecular mechanism for early diagnosis. Dishevelled (Dvl) dysregulation leads to malignant progression. We confirmed that Dvl1 expression is associated with a poor prognosis of patients with NSCLC. However, how Dvl1 transmits signals through the Wnt/β-catenin pathway remains unknown. Methods: In this study, the expression levels of Dvl1 and β-catenin in resected NSCLC samples were immunohistochemically analysed. Dvl1 cDNA and small interfering RNA against β-catenin were transfected into NSCLC cells, and their effects on canonical Wnt signalling and biological behaviour of NSCLC cells were analysed. Using bioinformatics analyses, an interaction between microRNA (miR)-214 and β-catenin was identified; miR-214 expression was determined in NSCLC tissues using quantitative real-time polymerase chain reaction. An exogenous miR-214 (mimic) was used to analyse the biological behaviour of NSCLC cells and the effect of Dvl1 on canonical Wnt activation. Results: Dvl1 overexpression in NSCLC tissues as well as Dvl1 and β-catenin nuclear coexpression were significantly associated with poor prognosis of NSCLC (P < 0.05). Additionally, Dvl1 promoted Wnt/β-catenin signalling to enhance the malignant phenotype of NSCLC cells. Moreover, miR-214 directly targeted the 3' untranslated region of β-catenin to inhibit the activation of canonical Wnt signalling induced by Dvl1. Conclusions: Our results suggest that Dvl1 is a potential therapeutic target for NSCLC and that miR-214 plays an inhibitory role in Dvl1-mediated activation of Wnt/β-catenin signalling in NSCLC cells, which could affect NSCLC progression.

Restoration of RNA helicase DDX5 suppresses hepatitis B virus (HBV) biosynthesis and Wnt signaling in HBV-related hepatocellular carcinoma.

Rationale: RNA helicase DDX5 is downregulated during hepatitis B virus (HBV) replication, and poor prognosis HBV-related hepatocellular carcinoma (HCC). The aim of this study is to determine the mechanism and significance of DDX5 downregulation for HBV-driven HCC, and identify biologics to prevent DDX5 downregulation.Methods: Molecular approaches including immunoblotting, qRT-PCR, luciferase transfections, hepatosphere assays, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), and RNA-seq were used with cellular models of HBV replication, HBV infection, and HBV-related liver tumors, as well as bioinformatic analyses of liver cancer cells from two independent cohorts.Results: We demonstrate that HBV infection induces expression of the proto-oncogenic miR17~92 and miR106b~25 clusters which target the downregulation of DDX5. Increased expression of these miRNAs is also detected in HBV-driven HCCs exhibiting reduced DDX5 mRNA. Stable DDX5 knockdown (DDX5KD) in HBV replicating hepatocytes increased viral replication, and resulted in hepatosphere formation, drug resistance, Wnt activation, and pluripotency gene expression. ATAC-seq of DDX5KD compared to DDX5 wild-type (WT) cells identified accessible chromatin regions enriched in regulation of Wnt signaling genes. RNA-seq analysis comparing WT versus DDX5KD cells identified enhanced expression of multiple genes involved in Wnt pathway. Additionally, expression of Disheveled, DVL1, a key regulator of Wnt pathway activation, was significantly higher in liver cancer cells with low DDX5 expression, from two independent cohorts. Importantly, inhibitors (antagomirs) to miR17~92 and miR106b~25 restored DDX5 levels, reduced DVL1 expression, and suppressed both Wnt activation and viral replication.Conclusion: DDX5 is a negative regulator of Wnt signaling and hepatocyte reprogramming in HCCs. Restoration of DDX5 levels by miR17~92 / miR106b~25 antagomirs in HBV-infected patients can be explored as both antitumor and antiviral strategy.

Different behaviour of DVL1, DVL2, DVL3 in astrocytoma malignancy grades and their association to TCF1 and LEF1 upregulation.

Key regulators of the Wnt signalling, DVL1, DVL2 and DVL3, in astrocytomas of different malignancy grades were investigated. Markers for DVL1,DVL2 and DVL3 were used to detect microsatellite instability (MSI) and gross deletions (LOH), while immunohistochemistry and immunoreactivity score were used to determine the signal strengths of the three DVL proteins and transcription factors of the pathway, TCF1 and LEF1. Our findings demonstrated that MSI at all three DVL loci was constantly found across tumour grades with the highest number in grade II (P = 0.008). Collectively, LOHs were more frequent in high‐grade tumours than in low grade ones. LOHs of DVL3 gene were significantly associated with grade IV tumours (P = 0.007). The results on protein expressions indicated that high‐grade tumours expressed less DVL1 protein as compared with low grade ones. A significant negative correlation was established between DVL1 expression and malignancy grades (P < 0.001). The expression of DVL2 protein was found similar across grades, while DVL3 expression significantly increased with malignancy grades (P < 0.001). The signal strengths of expressed DVL1 and DVL3 were negatively correlated (P = 0.002). However, TCF1 and LEF1 were both significantly upregulated and increasing with astrocytoma grades (P = 0.001). A positive correlation was established between DVL3 and both TCF1 (P = 0.020) and LEF1 (P = 0.006) suggesting their joint involvement in malignant progression. Our findings suggest that DVL1 and DVL2 may be involved during early stages of the disease, while DVL3 may have a role in later phases and together with TCF1 and LEF1 promotes the activation of Wnt signalling.

CtBP2 promotes proliferation and reduces drug sensitivity in non-small cell lung cancer via the Wnt/β-catenin pathway.

The C-terminal binding protein 2 (CtBP2) is crucial for the activation of the Wnt/β-catenin pathway and regulates significant cellular processes in multiple cancer cells. However, the role of CtBP2 in non-small cell lung cancer (NSCLC) remains uncertain. Our western blotting and immunohistochemistry assays revealed that CtBP2 expression was obviously increased in NSCLC tissues and cells. In addition, the chi-square test and Kaplan-Meier analysis showed that over-expression of CtBP2 correlates with more invasive tumor phenotype and poor prognosis. In vitro studies with serum starvation-refeeding and CtBP2-shRNA transfection assay demonstrated that CtBP2 expression facilitates NSCLC cell proliferation and reduces sensitivity to cis-diamminedichloroplatinum (CDDP). The possible signaling transduction pathways were investigated, and the immunoprecipitation assay revealed that CtBP2 interacts directly with DvL1. Depletion of CtBP2 resulted in inhibited DvL1 expression and decreased expression of downstream genes. Moreover, our study showed that CtBP2 knockdown enhanced NSCLC cell sensitivity to CDDP through inhibition of the Wnt/β-catenin pathway. These results suggest that CtBP2 plays a crucial role in NSCLC progression and CDDP sensitivity, and that CtBP2 depletion can provide a new target for NSCLC treatment.

MARK2/Par1b Insufficiency Attenuates DVL Gene Transcription via Histone Deacetylation in Lumbosacral Spina Bifida.

Dishevelled (DVL/Dvl) genes play roles in canonical and noncanonical Wnt signaling, both of which are essential in neural tube closing and are involved in balancing neural progenitor growth and differentiation, or neuroepithelial cell polarity, respectively. In mouse Dvl haploinsufficiency leads to neural tube defects (NTDs), which represent the second most common birth defects. However, DVL genes' genetic contributions in human NTDs are modest. We sought to explore the molecular impact on such genes in human NTDs in a Han Chinese cohort. In 47 cases with NTDs and 61 matched controls, in brain tissues, the DVL1/2 mRNA levels were correlated with the levels of a serine/threonine protein kinase MARK2, and in 20 cases with lumbosacral spina bifida, the mRNA levels of DVL1 and MARK2 were significantly decreased; by contrast, only an intronic rare variant was found. Moreover, in an extended population, we found merely three novel rare missense variants in 1% of individuals with NTDs. In cell-based assays, Mark2 depletion indeed reduces Dvl gene expression and interrupts neural stem cell (NSCs) growth and differentiation, which are likely to be mediated through a decrease in class IIa HDAC phosphorylation and reduced H3K4ac and H3K27ac occupancies at the Dvl1/2 promoters. Finally, the detections of folate concentration in human brain tissue and NSCs and MEF cells indicates that folate deficiency contributes to the observed decreases in Mark2 and Dvl1 expression. Our present study raises a potential common pathogenicity mechanism in human lumbosacral spina bifida about DVL genes rather than their genetic pathogenic role.

Expression of DDR1 and DVL1 in invasive ductal and lobular breast carcinoma does not correlate with histological type, grade and hormone receptor status.

Invasive ductal (IDC) and lobular (ILC) carcinomas are the common histological types of breast carcinoma which are difficult to distinguish when poorly differentiated. Discoidin domain receptor (DDR1) and Drosophila dishevelled protein (DVL1) were recently suggested to differentiate IDC from ILC. To assess the expression of DDR1 and DVL1 and their association with histological type, grading and hormonal status of IDC and ILC. This cross sectional study was conducted on IDC and ILC breast tumours. Tumours were immunohistochemically stained for (DDR1) and (DVL1) as well as estrogen receptor (ER), progesterone receptor (PR) and C-erbB2 receptor. Demographic data including age and ethnicity were obtained from patient records. A total of 51 cases (30 IDCs and 21 ILCs) were assessed. DDR1 and DVL1 expression was not significantly associated with histological type (p=0.57 and p=0.66 respectively). There was no association between DDR1 and DVL1 expression and tumour grade (p=0.32 and p=1.00 respectively), ER (p=0.62 and 0.50 respectively), PR (p=0.38 and p=0.63 respectively) and C-erbB2 expression (p=0.19 and p=0.33 respectively) in IDC. There was no association between DDR1 and DVL1 expression and tumour grade (p=0.52 and p=0.33 respectively), ER (p=0.06 and p=0.76 respectively), PR (p=0.61 and p=0.43 respectively) and C-erbB2 expression (p=0.58 and p=0.76 respectively) in ILC. This study revealed that DDR1 and DVL1 are present in both IDC and ILC regardless of the tumour differentiation. More studies are needed to assess the potential of these two proteins in distinguishing IDC from ILC in breast tumours.

Aliskiren Modifies Canonical Wnt/β‐Catenin Signaling Pathway Associated to (Pro)renin Receptor on Heart and Kidney in 5/6 Nephrectomy‐Induced Hypertension in Rats

The Wnt/frizzled receptor signaling pathways are important in morphogenesis and development of essential organs. (Pro)renin receptor (PRR) has been characterized as a multifunctional protein associated with vacuolar H+‐ATPase (v‐H+‐ATPase) (independently of renin) and is required for canonical Wnt/β‐catenin signaling activation. Aliskiren, a direct renin inhibitor, has been used to treat cardiovascular disorders. The aim of this work was to study Aliskiren effects upon PRR and canonical Wnt/β‐catenin pathway in cardiac and renal tissue during 5/6 nephrectomy (5/6 Nx)‐induced hypertension. Male Wistar rats underwent 5/6 Nx or sham operation (S), and were treated with Aliskiren, 10 mg/kg/day/7 days. Control groups received vehicle. 24‐hour urine was collected and blood pressure was measured at the end of experiment. PRR, β‐catenin and dishevelled (Dvl‐1) expression was determined by immunoblot. Our results showed that, compared to sham animals, 5/6 Nx rats treated with Aliskiren had lowered blood pressure and improved seric creatinine, proteinuria and creatinine clearance. We found in nephrectomized rats that PRR expression increased in heart and kidney seven days after surgery, and aliskiren reduced PRR, β‐catenin and Dvl‐1 expression in both tissues. These results confirm the antihypertensive and renoprotective effects of aliskiren and interestingly, showed that aliskiren modifies PRR expression as well as canonical Wnt/β‐catenin signaling expression. Supported by SIP20140636 grant

Significant Overexpression of DVL1 in Taiwanese Colorectal Cancer Patients with Liver Metastasis

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I–III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588–12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469–9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.

Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.

DVL genes play a role in the coordination of socket cell recruitment and differentiation

Specialized plant cells arise from undifferentiated cells through a series of developmental steps. The decision to enter into a certain differentiation pathway depends in many cases on signals from neighbouring cells. The ability of cells to engage in short-range intercellular communication permits the coordination of cell actions necessary in many developmental processes. Overexpression of genes from the DEVIL/ROTUNDIFOLIA (DVL/ROT) family results in severe developmental alterations, but very little is known about their mechanism of action. This work presents evidence that suggests a role for these genes in local signalling, specifically in the coordination of socket cell recruitment and differentiation. Overexpression of different DVL genes results in protuberances at the base of the trichomes surrounded by several rows of elongated epidermal cells, morphologically similar to socket cells. Localized overexpression of DVL4 in trichomes and socket cells during early developmental stages activates expression of socket cell markers in additional cells, farther away from the trichome. The same phenomenon is observed in an activation tagged line of DVL1, which also shows an increase in the number of socket cells in contact with the trichome. The roles of individual DVL genes have been difficult to discover since their overexpression phenotypes are quite similar. In gl1 leaves that lack trichomes and socket cells DVL1 expression shows a 69% reduction, suggesting that this gene could be involved in the coordination of socket cell development in wild-type plants.

BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) superfamily, plays a key role in the induction of the differentiation of mesenchymal cells into chondrocytes to form cartilage tissue. However, it is not clear whether BMP2 regulates the proliferation of chondrocytes. In the present study, the effect of BMP2 on the proliferation of chondrocytes and its underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without BMP2 for 48h. Cell viability was analyzed using the MTT assay. mRNA and protein expression levels of β-catenin, GSK-3β, Dvl1 and Cyclin D1 were detected using real-time RT-PCR and Western blotting, respectively. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. BMP2 stimulation was found to significantly increase cell viability. In addition, following BMP2 treatment, β-catenin, Cyclin D1 and Dvl1 expression was significantly increased, whereas GSK-3β expression was significantly decreased. Moreover, the percentage proportion of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the Sphase was significantly increased. The results indicate that BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway.

Dishevelled family proteins are expressed in non-small cell lung cancer and function differentially on tumor progression

Background Dishevelled (Dvl) family proteins are cytoplasmic mediators of the Wnt/β-catenin signaling pathway and have recently been linked to cancers. However, the roles of individual Dvls and their expression in human cancers are poorly defined. This work aimed to characterize the expression of Dvls and their correlation to clinicopathological factors and β-catenin expression in non-small cell lung cancer (NSCLC). Methods We used immunohistochemistry to assess the presence of the three Dvl family proteins in 113 individual NSCLC specimens. Thirty-nine of the 113 cases were examined further for Dvl and β-catenin protein expression in matched primary growths and autologous nodal metastases. We also examined the effect of Dvl-1 and Dvl-3 overexpression on β-catenin expression and the invasive ability of A549 and QG56 lung cancer cells. Results The positive expression rate in primary tumors was 53.1% (60/113) for total Dvl, 36.3% (41/113) for Dvl-1, 36.3% (41/113) for Dvl-2 and 41.6% (47/113) for Dvl-3, while normal adult bronchial and alveolar epithelia showed negative expression of all these proteins. The expression levels of all three Dvl proteins were significantly higher in adenocarcinomas than in squamous carcinomas, and were associated with poor tumor differentiation. The positive expression of Dvl-1 and Dvl-2 proteins was correlated to advanced pTNM stages (III–IV vs. I–II). In addition, the expression levels of Dvl-1 and Dvl-3 were significantly higher in nodal metastases than in primary growths, with the Dvl-1 expression correlating to β-catenin expression in the metastases. Exogenous expression of Dvl-1 and Dvl-3 both enhanced the invasive ability of A549 and QG56 cells, but had differential effects on β-catenin protein expression in either cell line, without influencing β-catenin mRNA levels. Conclusions Expression of Dvl family proteins, Dvl-1, Dvl-2 and Dvl-3, is common in NSCLCs. They may contribute to the progression of NSCLCs, but Dvl-1 and Dvl-3 may function on this process through different signaling pathways.

Upregulation and Overexpression of DVL1, the Human Counterpart of the Drosophila Dishevelled Gene, in Prostate Cancer

The Wnt/beta-catenin signaling pathway is one of the main carcinogenic mechanisms in human malignancies including prostate cancer. Recently, the DVL1 gene was identified as a middle molecule of the Wnt/beta-catenin signaling pathway. In addition, alterations of the DVL1 gene have been reported in breast and cervical cancer. The abnormality of beta-catenin in prostate cancer has been well studied, so the examination of the DVL1 gene in prostate cancer is appealing. We investigated DVL1 messenger RNA alterations by semiquantitative PCR (SQ-PCR) in 20 primary prostate cancers and assessed the protein expression by immunohistochemical analysis in the same samples. In addition, DVL1 and beta-catenin protein expression was evaluated with a new validated set of 20 prostate cancers. SQ-PCR revealed significant overexpression of DVL1 in prostate cancer (65%). Upregulation of the DVL1 gene product in prostate cancer was confirmed by immunostaining. With SQ-PCR and immunostaining, none of the cases showed underexpression or downregulation of DVL1. In addition, the data showed correlations between DVL1 mRNA and protein expression. Interestingly, the expression level of DVL1 increased with worsening histological grade. In addition, a correlation between DVL1 expression and beta-catenin expression was confirmed. DVL1 was overexpressed in prostate cancer and its overexpression might be related to prostate cancer progression through the Wnt/beta-catenin pathway.

796. DVL-1 Expression in Differentiated N2A Cells Triggers Cytoskeletal Remodeling of Growth Cone to a Bipolar Phenotype

796. DVL-1 Expression in Differentiated N2A Cells Triggers Cytoskeletal Remodeling of Growth Cone to a Bipolar Phenotype

Isolation and Characterization of a Mouse Homolog of the Drosophila Segment Polarity Gene dishevelled

In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2) . Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.