Cadherin Pathways Research Articles

Adiponectin receptor 1 regulates endometrial receptivity via the adenosine monophosphate‑activated protein kinase/E‑cadherin pathway.

Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL95‑2 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL95‑2 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcription‑quantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the mid‑secretory phase compared with that in the proliferative phase and in receptive RL95‑2 cells compared with that in non‑receptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced E‑cadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMP‑activated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5‑aminoimidazole‑4‑carboxamide ribonucleotide affected E‑cadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/E‑cadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.

High-throughput methylome analysis reveals differential methylation for early and late onset preeclampsia for mothers and their children.

Preeclampsia is a hypertensive disorder of pregnancy that affects ∼2%-5% of all pregnancies, contributes to 4 of the top 10 causes of pregnancy-related deaths, and remains a long-term risk factor for cardiometabolic diseases. Yet, little is still known about the molecular mechanisms that lead to this disease. There is evidence that some cases have a genetic cause. However, it is well appreciated that harmful factors in the environment, such as poor nutrition, stress, and toxins, may lead to epigenetics changes that can contribute to this disease. DNA methylation is one of the epigenetic modifications known to be fairly stable and impact gene expression. Using DNA from buccal swabs, we analyzed global DNA methylation among three groups of individuals: mothers who experienced 1) early-stage preeclampsia (<32 wk), 2) late-stage preeclampsia (>37 wk), or 3) no complications during their pregnancies, as well as the children from these three groups. We found significant differentially methylated regions (DMRs) between mothers who experienced preeclampsia compared with those with no complications adjacent or within genes that are important for placentation, embryonic development, cell adhesion, and inflammation (e.g., the cadherin pathway). A significant portion of DMR genes showed expression in tissues relevant to preeclampsia (i.e., the brain, heart, kidney, uterus, ovaries, and placenta). As this study was performed on DNA extracted from cheek swabs, this opens the way to future studies in different tissues, aimed at identifying possible biomarkers of risk and early detection, developing targeted interventions, and reducing the progression of this life-threatening disease.NEW & NOTEWORTHY Preeclampsia is a life-threatening hypertensive disorder, affecting 2%-5% of pregnancies, that remains poorly understood. This study analyzed DNA methylation from buccal swabs from mothers who experienced early and late-stage preeclampsia and those with uncomplicated pregnancies, along with their children. Differentially methylated regions were found near and within genes crucial for placental function, embryonic development, and inflammation. Many of these genes are expressed in preeclampsia-related tissues, offering hope for future biomarker development for this condition.

TCF1/LEF1 triggers Wnt-dependent chemokine/cytokine-induced inflammation and cadherin pathways to drive T-ALL cell migration

T-cell acute lymphoblastic leukemia (T-ALL) is a type of aggressive hematologic malignancy. It progresses quickly and it is likely to be fatal within a few months without treatment. Despite the limitations of current clinical therapies, there is an urgent need for novel and targeted therapies. To explore potential targeted therapies, molecular genetic mechanisms of T-ALL metastasis must be uncovered. However, the genes and mechanisms that mediate T-ALL metastasis are largely unknown. Recent insights into T-ALL biology have identified several genes that can be grouped into several targetable signaling pathways. The Wnt/β-catenin signaling pathway is one of the most important pathways. Our work investigated the functions of TCF1 and LEF1 in cell growth and migration mediated by the Wnt signaling pathway. We found that TCF1 and LEF1 knockdown weakly repressed T-ALL cell proliferation but distinctly impaired cell migration. T-ALL metastasis is dependent on cell migration and invasion. Our results displayed that TCF1 and LEF1 regulated T-ALL cell migration by the Wnt-dependent chemokine and cytokine-induced inflammation and cadherin signaling pathways. By transcriptionally regulating these pathways-associated genes, TCF1 and LEF1 inhibited cell adhesion and promoted cell migration and invasion.

Fibroblast Growth Factor 9 Inhibited Apoptosis in Random Flap via the ERK1/2-Nrf2 Pathway to Improve Tissue Survival.

The application of random pattern skin flaps is limited in plastic surgery reconstruction due to necrosis. Fibroblast growth factor 9 (FGF9) was reported to exert a protective effect against myocardial damage and cerebral ischemia injury, but the impact of FGF9 in random flap survival is still unclear. In this study, we used a mouse model of random flaps to verify that FGF9 can directly increase flap survival area and blood flow intensity by promoting angiogenesis. In total, 84 male C57BL/6 mice weighing between 22 and 25 g were randomly divided into three groups (n = 28 each group). After skin flap operation, one group served as a control, a treatment group received FGF9, and a treatment group received FGF9+U0126. All flap samples were incised on postoperative day 7. Our results showed that flap survival was significantly increased in the FGF9 group compared with that in the control group. This protective function was restrained by U0126. The results of histopathology, laser Doppler, and fluorescent staining all showed significant increases in capillary count, collagen deposition, and angiogenesis. FGF9 also significantly increased the expression of antioxidant stress proteins SOD1, eNOS, HO-1, vascular marker proteins CD31, VE cadherin, and pericyte marker protein PDGFRβ. Western blot showed that the phosphorylation degree of ERK1/2 increased after FGF9 treatment, and the expression of Nrf2, a downstream factor, was u-regulated. Western blot and immunofluorescence results of apoptosis-related proteins cleaved caspase-3, BAX, and Bcl2 showed that FGF9 inhibited apoptosis. ERK inhibitor U01926 reduced the beneficial effects of FGF9 on skin flap survival, including promoting angiogenesis, and showing antiapoptosis and antioxidative stress activities. Exogenous FGF9 stimulates angiogenesis of random flap and survival of tissue. the impact of FGF9 is closely linked to the prevention of oxidative stress mediated by ERK1/2-Nrf2. In the function of FGF9 in promoting effective angiogenesis, there may be a close interaction in the FGF9-FGFR-PDGFR-ERK-VE cadherin pathway. In particular, PDGFR and VE cadherin may interact.

In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

Meta-Analysis and Bioinformatics Detection of Susceptibility Genes in Diabetic Nephropathy.

The latest meta-analysis of genome-wide linkage studies (GWLS) identified nine cytogenetic locations suggestive of a linkage with diabetic nephropathy (DN) due to type 1 diabetes mellitus (T1DM) and seven locations due to type 2 diabetes mellitus (T2DM). In order to gain biological insight about the functional role of the genes located in these regions and to prioritize the most significant genetic loci for further research, we conducted a gene ontology analysis with an over representation test for the functional annotation of the protein coding genes. Protein analysis through evolutionary relationships (PANTHER) version 16.0 software and Cytoscape with the relevant plugins were used for the gene ontology analysis, and the overrepresentation test and STRING database were used for the construction of the protein network. The findings of the over-representation test highlight the contribution of immune related molecules like immunoglobulins, cytokines, and chemokines with regard to the most overrepresented protein classes, whereas the most enriched signaling pathways include the VEGF signaling pathway, the Cadherin pathway, the Wnt pathway, the angiogenesis pathway, the p38 MAPK pathway, and the EGF receptor signaling pathway. The common section of T1DM and T2DM results include the significant over representation of immune related molecules, and the Cadherin and Wnt signaling pathways that could constitute potential therapeutic targets for the treatment of DN, irrespective of the type of diabetes.

Genome-wide CNV investigation suggests a role for cadherin, Wnt, and p53 pathways in primary open-angle glaucoma

BackgroundTo investigate whether copy number variations (CNVs) are implicated in molecular mechanisms underlying primary open-angle glaucoma (POAG), we used genotype data of POAG individuals and healthy controls from two case-control studies, AGS (n = 278) and GLGS-UGLI (n = 1292). PennCNV, QuantiSNP, and cnvPartition programs were used to detect CNV. Stringent quality controls at both sample and marker levels were applied. The identified CNVs were intersected in CNV region (CNVR). After, we performed burden analysis, CNV-genome-wide association analysis, gene set overrepresentation and pathway analysis. In addition, in human eye tissues we assessed the expression of the genes lying within significant CNVRs.ResultsWe reported a statistically significant greater burden of CNVs in POAG cases compared to controls (p-value = 0,007). In common between the two cohorts, CNV-association analysis identified statistically significant CNVRs associated with POAG that span 11 genes (APC, BRCA2, COL3A1, HLA-DRB1, HLA-DRB5, HLA-DRB6, MFSD8, NIPBL, SCN1A, SDHB, and ZDHHC11). Functional annotation and pathway analysis suggested the involvement of cadherin, Wnt signalling, and p53 pathways.ConclusionsOur data suggest that CNVs may have a role in the susceptibility of POAG and they can reveal more information on the mechanism behind this disease. Additional genetic and functional studies are warranted to ascertain the contribution of CNVs in POAG.

Level of Murine DDX3 RNA Helicase Determines Phenotype Changes of Hepatocytes In Vitro and In Vivo.

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo—deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60–65%) showed discordant results in vitro and in vivo—similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors.

Identified a disintegrin and metalloproteinase with thrombospondin motifs 6 serve as a novel gastric cancer prognostic biomarker by bioinformatics analysis.

Objective: We aimed to explore the prognostic value of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes in gastric cancer (GC). Methods: The RNA-sequencing (RNA-seq) expression data for 351 GC patients and other relevant clinical data were acquired from The Cancer Genome Atlas (TCGA). Survival analysis and a genome-wide gene set enrichment analysis (GSEA) were performed to define the underlying molecular value of the ADAMTS genes in GC development. Besides, qRT-PCR and immunohistochemistry were all employed to validate the relationship between the expression of these genes and GC patient prognosis. Results: The Log rank test with both Cox regression and Kaplan–Meier survival analyses showed that ADAMTS6 expression profile correlated with the GC patients clinical outcome. Patients with a high expression of ADAMTS6 were associated with poor overall survival (OS). Comprehensive survival analysis of the ADAMTS genes suggests that ADAMTS6 might be an independent predictive factor for the OS in patients with GC. Besides, GSEA demonstrated that ADAMTS6 might be involved in multiple biological processes and pathways, such as the vascular endothelial growth factor A (VEGFA), kirsten rat sarcoma viral oncogene (KRAS), tumor protein P53, c-Jun N-terminal kinase (JNK), cadherin (CDH1) or tumor necrosis factor (TNF) pathways. It was also confirmed by immunohistochemistry and qRT-PCR that ADAMTS6 is highly expressed in GC, which may be related to the prognosis of GC patients. Conclusion: In summary, our study demonstrated that ADAMTS6 gene could be used as a potential molecular marker for GC prognosis.

Deregulation of the Epithelial-to-Mesenchymal Transition Process Underlies Zic2-Linked Holoprosencephaly

Mutations in the Zic2 gene cause holoprosencephaly type 5, but the mechanisms that translate Zic2 mutations into this devastating pathology remain unclear. Here, we report that Zic2 is expressed in a few epiblast cells during gastrulation to become transiently upregulated in the primitive streak. Later, during neurulation, Zic2 is expressed in neural crest cells and downregulated as they delaminate from the neural tube. In combination with transcriptomic data from mutant embryos, chromatin occupancy profiles in gastrula and neural crest cells reveal that Zic2 regulates a large number of genes associated with the Wnt, cadherin and TGF-β pathways. Functional experiments in chick and mice demonstrate that Zic2 blocks the activation of the Wnt canonical pathway at the time that induces the TGF-β pathway to trigger cell delamination. All these experiments together demonstrate that Zic2 initiates epithelial-to-mesenchymal transition at early developmental stages.

Associations between genetic variants of KIF5B, FMN1, and MGAT3 in the cadherin pathway and pancreatic cancer risk.

Because the cadherin‐mediated signaling pathway promotes cancer progression, we assessed associations between genetic variants in 109 cadherin‐related genes and risk of pancreatic cancer (PanC) by using genotyping data from publically available genome‐wide association studies (GWAS) datasets comprising 15,423 individuals of European ancestry. After initial single‐locus analyses and subsequent meta‐analysis with multiple testing correction for 29,963 single‐nucleotide polymorphisms (SNPs), 11 SNPs remained statistically significant (p < 0.05). In the stepwise logistic regression analysis, three independent PanC risk‐associated SNPs (KIF5B rs211304 C > G, FMN1 rs117648907 C > T, and MGAT3 rs34943118 T > C) remained statistically significant (p < 0.05), with odds ratios of 0.89 (95% confidence interval = 0.82–0.95 and p = 6.93 × 10−4), 1.33 (1.13–1.56 and 2.11 × 10−4), and 1.11 (1.05–1.17 and 8.10 × 10−5), respectively. Combined analysis of unfavorable genotypes of these three independent SNPs showed an upward trend in the genotype‐risk association (p trend < 0.001). Expression quantitative trait loci analyses indicated that the rs211304 G and rs34943118 C alleles were associated with increased mRNA expression levels of KIF5B and MGAT3, respectively (all p < 0.05). Additional bioinformatics prediction suggested that these three SNPs may affect enhancer histone marks that likely have an epigenetic effect on the genes. Our findings provide biological clues for these PanC risk‐associated SNPs in cadherin‐related genes in European ancestry populations, possibly by regulating the expression of the affected genes. However, our findings need to be validated in additional population, molecular and mechanistic investigations.

Bioinformatics analysis reveals TSPAN1 as a candidate biomarker of progression and prognosis in pancreatic cancer.

Pancreatic cancer (PCC) is a common malignant tumor of the digestive system that is resistant to traditional treatments and has an overall 5-year survival rate of <7%. Transcriptomics research provides reliable biomarkers for diagnosis, prognosis, and clinical precision treatment, as well as the identification of molecular targets for the development of drugs to improve patient survival. We sought to identify new biomarkers for PCC by combining transcriptomics and clinical data with current knowledge regarding molecular mechanisms. Consequently, we employed weighted gene co-expression network analysis and differentially expressed gene analysis to evaluate genes co-expressed in tumor versus normal tissues using pancreatic adenocarcinoma data from The Cancer Genome Atlas and dataset GSE16515 from the Gene Expression Omnibus. Twenty-one overlapping genes were identified, with enrichment of key Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways, including epidermal growth factor receptor signaling, cadherin, cell adhesion, ubiquinone, and glycosphingolipid biosynthesis pathways, and retinol metabolism. Protein-protein interaction analysis highlighted 10 hub genes, according to Maximal Clique Centrality. Univariate and multivariate COX analyses indicated that TSPAN1 serves as an independent prognostic factor for PCC patients. Survival analysis distinguished TSPAN1 as an independent prognostic factor among hub genes in PCC. Finally, immunohistochemical staining results suggested that the TSPAN1 protein levels in the Human Protein Atlas were significantly higher in tumor tissue than in normal tissue. Therefore, TSPAN1 may be involved in PCC development and act as a critical biomarker for diagnosing and predicting PCC patient survival.

Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.

DNA methylation pattern changes following a short-term hypocaloric diet in women with obesity.

We aimed to investigate the effects of short-term hypocaloric diet-induced weight loss on DNA methylation profile in leukocytes from women with severe obesity. Eleven women with morbid obesity (age: 36.9 ± 10.3 years; BMI: 58.5 ± 10.5 kg/m2) were assessed before and after 6 weeks of a hypocaloric dietary intervention. The participants were compared with women of average weight and the same age (age: 36.9 ± 11.8 years; BMI: 22.5 ± 1.6 kg/m2). Genome-wide DNA methylation analysis was performed in DNA extracted from peripheral blood leukocytes using the Infinium Human Methylation 450 BeadChip assay. Changes (Δβ) in the methylation level of each CpGs were calculated. A threshold with a minimum value of 10%, p < 0.001, for the significant CpG sites based on Δβ and a false discovery rate of <0.05 was set. Dietary intervention changed the methylation levels at 16,064 CpG sites. These CpGs sites were related to cancer, cell cycle-related, MAPK, Rap1, and Ras signaling pathways. However, regardless of hypocaloric intervention, a group of 878 CpGs (related to 649 genes) remained significantly altered in obese women when compared with normal-weight women. Pathway enrichment analysis identified genes related to the cadherin and Wnt pathway, angiogenesis signaling, and p53 pathways by glucose deprivation. A short-term hypocaloric intervention in patients with severe obesity partially restored the obesity-related DNA methylation pattern. Thus, the full change of obesity-related DNA methylation patterns could be proportional to the weight-loss rate in these patients after dietary interventions.

Sulforaphene induces apoptosis and inhibits the invasion of esophageal cancer cells through MSK2/CREB/Bcl-2 and cadherin pathway in vivo and in vitro

BackgroundAs a novel type of isothiocyanate derived from radish seeds from cruciferous vegetables, sulforaphene (SFE, 4-methylsufinyl-3-butenyl isothiocyanate) has various important biological effects, such as anti-oxidative and anti-bacterial effects. Recently, sulforaphene has attracted increasing attention for its anti-tumor effects and its ability to suppress the development of multiple tumors through different regulatory mechanisms. However, it has not yet been widely investigated for the treatment of esophageal cancer.MethodsWe observed an increased apoptosis in esophageal cancer cells on sulforaphene treatment through flow cytometry (FCM) analysis and transmission electron microscopy (TEM). Through mass spectrometry (MS) analysis, we further detected global changes in the proteomes and phosphoproteomes of esophageal cancer cells on sulforaphene treatment. The molecular mechanism of sulforaphene was verified by western blot,the effect and mechanism of SFE on esophageal cancer was further verified by patient-derived xenograft mouse model.ResultsWe identified multiple cellular processes that were changed after sulforaphene treatment by proteomics. We found that sulforaphene could repress the phosphorylation of CREB through MSK2, leading to suppression of Bcl-2 and further promoted cell apoptosis. Additionally, we confirmed that sulforaphene induces tumor cell apoptosis in mice. Interestingly, we also observed the obvious inhibition of cell migration and invasion caused by sulforaphene treatment by inhibiting the expression of cadherin, indicating the complex effects of sulforaphene on the development of esophageal cancer.ConclusionsOur data demonstrated that sulforaphene induced cell apoptosis and inhibits the invasion of esophageal cancer through a mechanism involving the inhibition of the MSK2–CREB–Bcl2 and cadherin pathway. Sulforaphene could therefore serve as a promising anti-tumor drug for the treatment of esophageal cancer.

Unsupervised discovery of phenotype-specific multi-omics networks.

Complex diseases often involve a wide spectrum of phenotypic traits. Better understanding of the biological mechanisms relevant to each trait promotes understanding of the etiology of the disease and the potential for targeted and effective treatment plans. There have been many efforts towards omics data integration and network reconstruction, but limited work has examined the incorporation of relevant (quantitative) phenotypic traits. We propose a novel technique, sparse multiple canonical correlation network analysis (SmCCNet), for integrating multiple omics data types along with a quantitative phenotype of interest, and for constructing multi-omics networks that are specific to the phenotype. As a case study, we focus on miRNA-mRNA networks. Through simulations, we demonstrate that SmCCNet has better overall prediction performance compared to popular gene expression network construction and integration approaches under realistic settings. Applying SmCCNet to studies on chronic obstructive pulmonary disease (COPD) and breast cancer, we found enrichment of known relevant pathways (e.g. the Cadherin pathway for COPD and the interferon-gamma signaling pathway for breast cancer) as well as less known omics features that may be important to the diseases. Although those applications focus on miRNA-mRNA co-expression networks, SmCCNet is applicable to a variety of omics and other data types. It can also be easily generalized to incorporate multiple quantitative phenotype simultaneously. The versatility of SmCCNet suggests great potential of the approach in many areas. The SmCCNet algorithm is written in R, and is freely available on the web at https://cran.r-project.org/web/packages/SmCCNet/index.html. Supplementary data are available at Bioinformatics online.

Partially overlapping guidance pathways focus the activity of UNC-40/DCC along the anteroposterior axis of polarizing neuroblasts.

Directional migration of neurons and neuronal precursor cells is a central process in nervous system development. In the nematode Caenorhabditis elegans, the two Q neuroblasts polarize and migrate in opposite directions along the anteroposterior body axis. Several key regulators of Q cell polarization have been identified, including MIG-21, DPY-19/DPY19L1, the netrin receptor UNC-40/DCC, the Fat-like cadherin CDH-4 and CDH-3/Fat, which we describe in this study. How these different transmembrane proteins act together to direct Q neuroblast polarization and migration is still largely unknown. Here, we demonstrate that MIG-21 and DPY-19, CDH-3 and CDH-4, and UNC-40 define three distinct pathways that have partially redundant roles in protrusion formation, but also separate functions in regulating protrusion direction. Moreover, we show that the MIG-21, DPY-19 and Fat-like cadherin pathways control the localization and clustering of UNC-40 at the leading edge of the polarizing Q neuroblast, and that this is independent of the UNC-40 ligands UNC-6/netrin and MADD-4. Our results provide insight into a novel mechanism for ligand-independent localization of UNC-40 that directs the activity of UNC-40 along the anteroposterior axis.

Bacillus thuringiensis Cry1A toxins exert toxicity by multiple pathways in insects

Adoption of biotech crops engineered to express insecticidal toxins from Bacillus thuringiensis (Bt) has revolutionized insect pest control in agriculture. For continuing effective application and development of the environmentally friendly Bt biotechnology, it is fundamental to understand pathways of toxicity of Bt toxins in insects. In this study, mutations were introduced in the midgut cadherin gene in the cabbage looper, Trichoplusia ni, by CRISPR/Cas9 mutagenesis. T. ni strains with mutations in the genes of two major receptors for Bt toxins, the midgut cadherin and ABCC2, and three Cry1A toxins with shared and differential midgut binding sites were used as an experimental system to dissect the roles of the cadherin and ABCC2 in the pathways of toxicity of Bt toxins. Results from assays of responses of the T. ni strains to different Bt toxins revealed that the cadherin and ABCC2 play independent roles in the mode of action of Cry1A toxins and that Bt toxins exert insecticidal activity through multiple redundant pathways of toxicity in insects. Besides the cadherin and ABCC2 pathways, there exists an additional major pathway of toxicity to be identified for Cry1Aa. The results also confirmed that the toxicity of Cry2Ab involves neither the cadherin nor the ABCC2 protein. The multiple pathway model for Bt toxins clarified from this study provided new insights into the molecular modes of action of Bt toxins and mechanisms of insect resistance to Bt toxins.

The function of FAK/CCDC80/E-cadherin pathway in the regulation of B16F10 cell migration.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in the development and progression of cancer. However, the regulatory role of FAK in cell migration remains unclear. The aim of the present study was to investigate the mechanism underlying the regulation of melanoma cell migration by FAK. The effect of FAK knockdown on gene expression in B16F10 cells was examined by gene chip analysis. The expression levels of coiled-coil domain containing 80 (CCDC80) and epithelial (E)-cadherin were analyzed by reverse transcription quantitative polymerase chain reaction and western blotting. Wound healing and transwell assays were used to monitor B16F10 cell migration. It was identified that the knockdown of FAK increased the expression levels of CCDC80 and E-cadherin, while the overexpression of CCDC80 elevated E-cadherin expression. Concurrently, upregulation of CCDC80 inhibited the migration of B16F10 cells, and downregulation of CCDC80 promoted the migration of B16F10 cells. The clinical data from the Oncomine database also revealed that the mRNA level of FAK was increased while the mRNA levels of CCDC80 and E-cadherin were decreased in patients with melanoma compared with normal controls. Taken together, the results of the present study suggest that the regulation of B16F10 melanoma cell migration by FAK is potentially mediated by CCDC80.

Stable silencing of dll4 gene suppresses the growth and metastasis of esophagus cancer cells by attenuating Akt phosphorylation.

δ‑Like ligand4 (DLL4) has recently been reported to be involved in the process of cancer angiogenesis and considered to play a vital role in vascular endothelial growth factor (VEGF) signaling, while the role of DLL4 in cancer metastasis and growth has not been systematically studied. In the present study, the esophagus cancer cell line Eca109 was infected invitro with a lentiviral vector loaded with dll4‑shRNA to obtain a stable cell line of DLL4 expression which was downregulated through puromycin screening. The migration and invasion ability of the Eca109 dll4‑shRNA cells were evaluated by scratch and Transwell assays, respectively. The underlying signaling pathway was further explored by western blotting. Subsequently, to explore the role of dll4 in the development of esophagus cancer cells invivo, a xenograft model was established by intraperitoneal injection with Eca109 dll4‑shRNA cells containing luciferase activity in nude mice. Then, small animal imaging system was used to evaluate the volume and metastatic potential of the tumors. Additionally, the overall survival rate of the nude mice was also recorded. Following infection with lentivirus, the expression of DLL4 in the Eca109 cells could be stably silenced through screening with puromycin, which was confirmed by western blotting. The scratch and Transwell assays demonstrated that downregulated DLL4 significantly diminished the aggressive invasion and migration properties of the Eca109 cells. The underlying mechanisms may be attributed to the inactivation of the PI3K/Akt/E‑cadherin pathway by western blotting. Finally, the results from the invivo study indicated that the tumor growth rate in the Eca109 dll4‑shRNA group, as displayed by the tumor volume and the weak staining of the proliferating cell nuclear antigen (PCNA), was significantly slower than the control group, and the metastasis ability of the Eca109 dll4‑shRNA cells was also dramatically abolished invivo. It was also observed that downregulated DLL4 led to the formation of less pulmonary nodules in mice lungs and to a prolonged survival rate of nude mice. In summary, this study revealed that DLL4 has pathophysiological roles on the progression of esophagus cancer cells, including migration, invasion and apoptosis, which indicated that DLL4 may be considered as a potent therapeutic target for the treatment of malignant esophageal cancer.