Netrin-1-induced activation of Notch signaling mediates glioblastoma cell invasion.

Glioblastoma multiforme is an aggressively invasive human brain cancer, which lacks effective treatment. The axonal guidance protein, netrin-1, is overexpressed in glioblastoma tumor biopsies. In Matrigel invasion assays we observed that experimental overexpression of netrin-1 increased cell invasiveness and its downregulation decreased invasiveness. Using tandem affinity purification and mass spectrometry protein identification we found that netrin-1 forms a complex with both Notch2 and Jagged1. Recombinant netrin-1 colocalized with Jagged1 and Notch2 at the cell surface and was also present in the intracellular vesicles with Jagged1, but not with Notch2. Netrin-1 activated Notch signaling and subsequent glioblastoma cell invasion. Interestingly, the recombinant central domain of netrin-1 counteracted the effects of the full-length netrin-1: it inhibited glioblastoma cell invasion and Notch activation by retaining the Notch signaling complex at the cell surface. This finding may give rise to therapeutic applications. These results reveal a new mechanism leading to glioblastoma cell invasion, in which netrin-1 activates Notch signaling.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture.

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination-termed placode-that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.

Ephrin-As, Eph receptors and integrin α3 interact and colocalise at membrane protrusions of U251MG glioblastoma cells.

Glioblastoma is the most common brain cancer. Ephrins and their Eph receptors play important roles in the development of central nervous system and the regulation of cancer cell migration and invasion. In a search for the Eph receptor complexes, we used tandem affinity purification based interaction screening with tagged ephrins A1, A3 and A4 combined with protein identification by mass-spectrometry in U251MG glioblastoma cells. Ephrins bound to Eph receptors, mainly to EphA2 in these cells. Integrin α3 was identified in protein complexes with ephrin-As. Soluble ephrin-A1 colocalised with integrin α3 at the cell surface, and was rapidly endocytosed by the cells. However, integrin α3 did not colocalise with internalised ephrin-A1, whereas EphA2 receptor did. In U251MG cells, integrin α3 colocalised with EphA2 receptor at the cell edges and protrusions. Sites of EphA2-integrin α3 colocalisation were positive for vinculin, focal adhesion kinase and phosphotyrosine, that is, markers for cell adhesion and active signalling. The interaction between ephrin-As, Eph receptors and integrin α3 is plausibly important for the crosstalk between Eph and integrin signalling pathways at the membrane protrusions and in the migration of brain cancer cells.

Genetic determinants of serum 25-hydroxyvitamin D concentration during pregnancy and type 1 diabetes in the child.

OBJECTIVE: The in utero environment plays an important role in shaping development and later life health of the fetus. It has been shown that maternal genetic factors in the metabolic pathway of vitamin D associate with type 1 diabetes in the child. In this study we analyzed the genetic determinants of serum 25-hydroxyvitamin D (25OHD) concentration during pregnancy in mothers whose children later developed type 1 diabetes and in control mothers. STUDY DESIGN: 474 mothers of type 1 diabetic children and 348 mothers of non-diabetic children were included in the study. We previously selected 7 single nucleotide polymorphisms (SNPs) in four genes in the metabolic pathway of vitamin D vitamin based on our previously published data demonstrating an association between genotype and serum 25OHD concentration. In this re-analysis, possible differences in strength in the association between the SNPs and serum 25OHD concentration in mothers of type 1 diabetic and non-diabetic children were investigated. Serum 25OHD concentrations were previously shown to be similar between the mothers of type 1 diabetic and non-diabetic children and vitamin D deficiency prevalent in both groups. RESULTS: Associations between serum 25OHD concentration and 2 SNPs, one in the vitamin D receptor (VDR) gene (rs4516035) and one in the group-specific component (GC) gene (rs12512631), were stronger during pregnancy in mothers whose children later developed type 1 diabetes than in mothers whose children did not (pinteraction = 0.03, 0.02, respectively). CONCLUSIONS: We show for the first time that there are differences in the strength of genetic determinants of serum 25OHD concentration during pregnancy between the mothers of type 1 diabetic and non-diabetic children. Our results emphasize that the in utero environment including maternal vitamin D metabolism should be important lines of investigation when searching for factors that lead to early programming of type 1 diabetes.

Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma.

Malignant mesothelioma originates from mesothelial cells and is a cancer type that aggressively invades into the surrounding tissue, has poor prognosis and no effective treatment. Gremlin-1 is a cysteine knot protein that functions by inhibiting BMP-pathway activity during development. BMP-independent functions have also been described for gremlin-1. We have previously shown high gremlin-1 expression in mesothelioma tumor tissue. Here, we investigated the functions of gremlin-1 in mesothelioma cell migration and invasive growth. Gremlin-1 promoted mesothelioma cell sprouting and invasion into three dimensional collagen and Matrigel matrices. The expression level of gremlin-1 was linked to changes in the expression of SNAI2, integrins, matrix metalloproteinases (MMP) and TGF-ß family signaling - all previously associated with a mesenchymal invasive phenotype. Small molecule inhibitors of MMPs completely blocked mesothelioma cell invasive growth. In addition, inhibitors of TGF-ß receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-ß pathway dependent and independent mechanisms of action. Results of in vivo mesothelioma xenograft experiments indicated that gremlin-1 overexpressing tumors were more vascular and had a tendency to send metastases. This suggests that by inducing a mesenchymal invasive cell phenotype together with enhanced tumor vascularization, gremlin-1 drives mesothelioma invasion and metastasis. These data identify gremlin-1 as a potential therapeutic target in mesothelioma.

Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness.

BACKGROUND: Glioblastoma is an untreatable brain cancer. The tumors contain a population of stem-like cells which are highly invasive and resistant to therapies. These cells are the main reason for the lethality of glioblastoma. Extracellular guidance molecule netrin-1 promotes the invasiveness and survival of various cancer cell types. We have previously found that netrin-1 activates Notch signaling, and Notch signaling associates with cell stemness. Therefore, we have here investigated the effects of netrin-1 on glioblastoma pathogenesis and glioblastoma cell stemness. METHODS: Glioma tissue microarrays were stained with immunohistochemistry and the results were used to evaluate the association between netrin-1 and survival of glioma patients. The localization of netrin-1 was analyzed utilizing fresh frozen glioblastoma tissues. The glioma cell invasion was investigated using ex vivo glioma tissue cultures and newly established primary cell cultures in 3D in vitro invasion assays. Intracranial mouse xenograft models were utilized to investigate the effects of netrin-1 on glioblastoma growth and invasion in vivo. RESULTS: Netrin-1 expression associated with poor patient prognosis in grade II-III gliomas. In addition, its expression correlated with the stem-like cell marker nestin. Netrin-1 overexpression in cultured cells led to increased formation of stem-like cell spheroids. In glioblastoma tumor biopsies netrin-1 localized to hypoxic tumor areas known to be rich in the stem-like cells. In xenograft mouse models netrin-1 expression altered the phenotype of non-invasive glioblastoma cells into diffusively invading and increased the expression of glioma stem-like cell markers. Furthermore, a distinct invasion pattern where netrin-1 positive cells were following the invasive stem-like cells was detected both in mouse models and ex vivo human glioblastoma tissue cultures. Inhibition of netrin-1 signaling targeted especially the stem-like cells and inhibited their infiltrative growth. CONCLUSIONS: Our findings describe netrin-1 as an important regulator of glioblastoma cell stemness and motility. Netrin-1 activates Notch signaling in glioblastoma cells resulting in subsequent gain of stemness and enhanced invasiveness of these cells. Moreover, inhibition of netrin-1 signaling may offer a way to target stem-like cells.

Netrin-1: A regulator of cancer cell motility?

Netrins form a family of secreted and membrane-associated proteins, netrin-1 being the prototype and most investigated member of the family. The major physiological functions of netrin-1 lie in the regulation of axonal development as well as morphogenesis of different branched organs, by promoting the polarity of migratory/invasive front of the cell. On the other hand, netrin-1 acts as a factor preventing cell apoptosis. These events are mediated via a range of different receptors, including UNC5 and DCC-families. Cancer cells often employ developmental pathways to gain survival and motility advantage. Within recent years, there has been increasing number of observations of upregulation of netrin-1 expression in different forms of cancer, and the increased expression of netrin-1 has been linked to its functions as a survival and invasion promoting factor. We review here recent advances in the netrin-1 related developmental processes that may be of special interest in tumor biology, in addition to the known functions of netrin-1 in tumor biology with special focus on cancer cell migration.

NETRIN-4 protects glioblastoma cells FROM temozolomide induced senescence.

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.

Netrin-4 promotes glioblastoma cell proliferation through integrin ß4 signaling.

Netrin-4 is a laminin-related secreted molecule originally found to have roles in neuronal axon migration. Recent studies have indicated that netrin-4 also participates in the development of nonneural tissues and modulates tumor cell proliferation and tumor metastasis. Here we have explored the functions and molecular mechanisms of netrin-4 in glioblastoma multiforme. The suppression of netrin-4 expression in glioblastoma cell lines significantly reduced cell proliferation and motility and increased serum deprivation-induced apoptosis. Using tandem affinity purification combined with protein identification by mass spectrometry, we found that integrin ß(4) interacts with netrin-4 and that it mediates mitogenic effects as well as AKT and mammalian target of rapamycin phosphorylation induced by netrin-4. Interestingly, netrin-4 acted as an inhibitor of cell proliferation in integrin ß(4)-silenced glioblastoma cells, and high concentrations of netrin-4 reduced cell proliferation. The negative effects of netrin-4 on proliferation were mediated by UNC5B. Analysis of more than 400 primary tumors from The Cancer Genome Atlas repository revealed that the expression of netrin-4 is significantly downregulated in glioblastoma and that the reduced expression is linked to poor patient survival time. The expression of integrin ß(4) is increased in glioblastoma, and it predicts poor patient survival time. Current results illustrate a novel mechanism for glioma progression, where glioma cells reduce netrin-4 expression to decrease its inhibitory effects. In parallel, the expression of integrin ß(4) is upregulated to sensitize the cells to low concentrations of netrin-4 for maintaining cell proliferation.