Irene Ylivinkka

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

Summary 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.

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

BackgroundGlioblastoma 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.MethodsGlioma 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.ResultsNetrin-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.ConclusionsOur 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-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-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.

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-β 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-β receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-β 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.

Abstract 1894: Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma

The purpose of this study was to clarify the role of BMP inhibitor gremlin 1 in invasion and migration in mesothelioma. Primary mesothelioma cells isolated from patient pleural fluid as well as mesothelioma cell lines were used for in vitro studies. Cells were transfected with siRNAs or transduced with lentiviral expression vectors. Invasive growth was analyzed in 3D Matrigel or collagen I matrices. mRNA expressions were analyzed using a commercial PCR array and quantitative RT-PCR. Migration assays were performed using scratch wound assay or transwell migration assay with fibronectin or collagen coating. TGF-β and BMP signaling activity was measured with luciferase reporter assays. For in vivo mouse xenograft experiment cells were additionally transduced to express a luciferase marker. Subcutaneous cell injection with Matrigel matrix was performed in the flank of nude mice. Malignant mesothelioma is an aggressive cancer that develops from mesothelial cells, most often in the pleural lining of the lung. We have previously shown that the BMP inhibitor protein gremlin 1 is highly expressed in mesothelioma and induces a mesenchymal and chemoresistant phenotype in mesothelioma cells. Since mesothelioma tumors are locally very invasive and has poor prognosis, we analyzed the role of gremlin 1 in mesothelioma cell migration and invasive growth. We found that mesothelioma cells expressing gremlin 1 showed invasive sprouting when tumor cell spheroids were imbedded into 3D collagen matrix. Silencing of gremlin 1 expression significantly reduced invasive growth. In addition, cells overexpressing gremlin 1 gained more invasive phenotype. This was associated with increased mRNA expression levels of Slug and matrix metalloproteinases (MMP) as well as reduced expression of E-cadherin. The cells were more migratory and exhibited increased expression of certain integrins, especially the αv subunit. Gremlin 1 induced invasive growth was dependent on MMP activity and associated with increased TGF-β activity. Intrapleural injection of mesothelioma cells overexpressing gremlin 1 isolated from a patient with epithelioid mesothelioma, produced tumors in 2/4 mice over 5 months after injection. Mice with cells transduced with vector only did not develop tumors (0/4). When cells were injected subcutaneously together with Matrigel gremlin 1 overexpressing tumors appeared more slowly, but exhibited comparable luciferase signal 2.5 months after injection. However, gremlin 1 tumors showed more vascularization and in contrast to control tumors some also developed metastases (2/6 mice). As conclusion, we identified gremlin 1 as an important regulator of mesothelioma invasive growth and chemoresistance. Blocking gremlin 1 function may overcome drug resistance and reduce invasive behavior of mesothelioma. Citation Format: Miao Yin, Mira Tissari, Emmi I. Joensuu, Jenni Tamminen, Irene Ylivinkka, Mikko Ronty, Kaisa Lehti, Marko Hyytiainen, Marjukka Myllarniemi, Katri Koli. Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2017-1894

Abstract 3348: Netrin-4/Integrin beta-4 interaction promotes glioblastoma cell proliferation and protects from temozolomide induced cellular senescence via activating PI3K/AKT pathway

Netrin-4 (NTN4) is a secreted laminin-related protein, which was originally observed to guide neuronal axons during neuronal development. Recently, it has been found to be expressed in many other tissues and tumor types, and to contribute to the regulation of cell adhesion, migration, proliferation, and apoptosis. Glioblastoma multiforme (GBM) is the most common primary tumor of central nervous system (CNS). Although there is no remedy for this fatal disease, the efficacy of temozolomide (TMZ) -an orally taken alkylating agent- has been demonstrated in the treatment of glioblastoma. However, the sensitivity of GBM cells to TMZ is interfered by many factors such as MGMT expression, AKT activation, etc. Here we have explored the functions and molecular mechanisms of NTN4 in GBM. The suppression of NTN4 expression in GBM cell lines significantly reduced cell proliferation and motility and increased serum deprivation-induced apoptosis. By using tandem affinity purification and mass spectrometric analysis, we identified the physical interaction between integrin beta-4 (ITGB4) and NTN4. This interaction mediates AKT phosphorylation and concomitant mitogenic effects. Furthermore, silencing either NTN4 or ITGB4 in GBM cell induced cellular senescence. Temozolomide induces cellular senescence in MGMT devoid glioma cell lines. Addition of exogenous recombinant NTN4 protein rescued TMZ induced senescence and AKT dephosphorylation in GBM cells in an ITGB4 dependent manner. Current data suggest that the NTN4-ITGB4 interaction promotes GBM cell proliferation and protects GBM cell from TMZ triggered cellular senescence via activating PI3K/AKT pathway. Citation Format: Yizhou Hu, Irene Ylivinkka, Li Li, Ping Chen, Sampsa Hautaniemi, Tuula A. Nyman, Jorma Keski-Oja Keski-Oja, Marko Hyytiainen. Netrin-4/Integrin beta-4 interaction promotes glioblastoma cell proliferation and protects from temozolomide induced cellular senescence via activating PI3K/AKT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3348. doi:10.1158/1538-7445.AM2014-3348

Abstract 4104: Netrin-1 promotes the invasiveness and survival of human glioblastoma cells.

Glioblastoma multiforme (GBM) is the most severe human brain cancer, which has no efficient treatment. The main reason for the lethality of GBM is its resistance to all current therapies and highly invasive. According to microarray data on Oncomine database netrin-1 is upregulated in glioblastoma tumors. Netrin-1 is a secreted laminin related extracellular matrix protein. Its main function is to regulate the axon guidance during embryogenesis. Recently it has been observed to act as a survival factor for different forms of cancer such as aggressive neuroblastoma, metastatic breast cancer and non small cell lung cancer. It also promotes tumorigenesis of colorectal cancer. Based on these observations we explored the possibility whether netrin-1 plays a role in human GBM. We analyzed the effects of netrin-1 expression on human GBM cells. According to Matrigel invasion assays overexpression of netrin-1 increased the invasiveness whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness of human GBM cells. On the other hand, complete knock-down of netrin-1 led to apoptotic death of those cells. To further analyze netrin-1 signaling in human GBM we performed a mass spectrometry screen to identify binding partners of netrin-1. We identified Notch as a novel receptor for netrin-1. In addition, we observed that netrin-1 overexpression increased and knockdown decreased Notch activation. The role of Notch signaling regarding the aggressiveness of various cancers has been controversial. Our results confirm that increased Notch signaling increases glioblastoma cell invasiveness, and that netrin-1 increases Notch signaling. Further, we identified here a recombinant netrin-1 fragment that inhibits Notch signaling by blocking the Notch signaling complex to the cell surface. Current studies provide new mechanistic information on the tumorigenesis of GBM. Activation of Notch signaling pathway has been linked to the initiation of EMT which is essential for cancer cell invasion. Our results suggest that netrin-1 regulates GBM invasion by activating Notch signaling. The inhibition of netrin-1 signaling may prove out to be a treatment option for human glioblastoma. Citation Format: Jorma K. Keski-Oja, Irene Ylivinkka, Yizhou Hu, Tuula Nyman, Marko Hyytiainen. Netrin-1 promotes the invasiveness and survival of human glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4104. doi:10.1158/1538-7445.AM2013-4104