Netrins in glioma biology : regulators of tumor cell proliferation, motility and stemness

Glioblastoma is the most malignant brain cancer. Currently no cure has been established. The lethality of glioblastoma is a consequence of its extremely invasive nature: it rarely metastasizes outside the nervous system but effectively spreads throughout the brain parenchyma. This property, in addition to its challenging location makes it impossible to remove surgically. Furthermore, the tumors are heterogeneous and contain cells that are resistant to radiation therapy as well as to the only currently approved chemotherapy, temozolomide. Netrins are secreted extracellular matrix proteins. They were initially identified as proteins essential for the correct axonal wiring of both vertebrates and invertebrates. Later, they were observed to regulate the branching morphogenesis of various organs including mammary gland, lungs and pancreas. During recent years increasing number of studies have linked netrins to various forms of cancer. For example, netrin-1 induces the invasion of pancreatic, colorectal and hepatocytic cancers and medulloblastoma and promotes the survival of breast and lung cancer and neuroblastoma. Netrin-4 can modulate tumor growth, angiogenesis and metastasis. In the current work, we analyzed how netrins contribute to glioblastoma growth. We discovered that both netrin-1 and -4 regulate the malignancy of glioblastoma via independent pathways. Netrin-1 expression was upregulated in glioblastoma whereas netrin-4 was downregulated. However, both were associated with poor patient prognosis. The signaling pathways mediating the effects of these proteins were systematically explored. First, our results revealed a new mechanism where netrin-4 controls glioma cell proliferation via UNC5B and ITGB4 receptors. During normal cell culture conditions netrin-4 is abundantly expressed, and it binds to both UNC5B and ITGB4 receptors that counteract each other and keep glioma cell proliferation in balance. However, during glioma progression netrin-4 expression is decreased and the signaling is shifted towards ITGB4. This led to increased cell proliferation and tumor growth. Second, we discovered a mechanism through which netrin-1 promotes cell invasion. Netrin-1 expression associated with astrocytomas which are invasive glioma subtype. In glioblastoma cells it interacted with Notch2 and Jagged1 and facilitated the activation of the signaling. Subsequently, this led to an increase in cell invasion in vitro and in vivo. Furthermore, a unique invasion pattern was characterized where netrin-1 expressing cells were promoting the motility and stemness of the invasion leading stem-like cells. Third, we designed and engineered a recombinant peptide that had the capacity to inhibit netrin-1 signaling. This peptide was able to overcome the effects of the full-length netrin-1 and specifically inhibited the invasiveness of the stem-like glioblastoma cells in vitro and in vivo. This peptide may prove out to be of therapeutic value in GBM treatment.Glioomat ovat glia- eli hermotukisoluista alkunsa saavia aivokasvaimia. Glioblastooma on näistä syöpäkasvaimista pahanlaatuisin. Siihen ei toistaiseksi ole kehitetty hoitoa, ja potilaiden keskimääräinen elinaika on 15 kuukautta diagnosoinnin jälkeen. Glioblastooman pahanlaatuisuus johtuu kasvaimen syöpäsolujen aggressiivisesta leviämisestä terveen aivokudoksen sekaan. Tästä syystä se on lähes mahdoton poistaa kirurgisesti. Nämä syöpäsolut ovat myös sädehoidolle sekä toistaiseksi ainoalle hyväksytylle lääkehoidolle, temozolomidille, vastustuskykyisiä. Netriinit ovat erittyviä soluväliaineen proteiineja. Niiden on alun perin havaittu säätelevän aksonien kehitystä yksilönkehityksen aikana sekä selkärankaisissa että selkärangattomissa. Myöhemmin niiden on osoitettu säätelevän myös muiden haaroittuneiden elinten, kuten rintarauhasen ja keuhkojen, muodostumista. Viime vuosien aikana yhä useammat tutkimukset ovat osoittaneet netriinien olevan tärkeässä osassa myös syöpäbiologiassa: netriini-1 lisää useiden eri syöpätyyppien leviämistä sekä syöpäsolujen selviytymistä, ja netriini-4 puolestaan on yhdistetty syöpäkasvainten kykyyn kasvattaa verisuonia sekä niiden kykyyn lähettää etäpesäkkeitä. Tässä väitöskirjatyössä on tutkittu netriinien vaikutuksia glioblastoomaan. Tutkimuksissa osoitettiin, että lisääntynyt netriini-1:n tuotto ja vastakohtaisesti vähentynyt netriini-4:n tuotto ovat yhteydessä potilaiden huonompaan ennusteeseen. Signalointireittejä, joiden kautta nämä proteiinit vaikuttavat syöpäsoluihin, seulottiin systemaattisesti. Tutkimuksessa havaittiin, että kumpikin näistä proteiineista vaikuttaa glioblastoomasoluihin itsenäisesti, eri signaloittireittien välityksellä. Netriini-4:n osoitettiin olevan tärkeä glioblastoomasolujen jakautumisen säätelijä ja sen tuoton vähentyminen mahdollisti kontrolloimattoman solujen jakautumisen ja täten kasvaimen kasvun. Netriini-1:n puolestaan osoitettiin olevan merkittävä glioblastoomasolujen liikkuvuuden säätelijä. Sen lisääntynyt tuotto sai aikaan syöpäsolujen muuttumisen kantasolumaisemmiksi ja aggressiivisemmin terveen aivokudoksen sekaan leviäviksi. Lisäksi tässä työssä suunniteltiin ja rakennettiin peptidi, joka pystyi estämään netriini-1:n vaikutukset glioblastoomasoluissa. Koska tämän peptidin tuotto vähensi glioblastoomasolujen kantasolumaisuutta sekä niiden kykyä levitä terveen aivokudoksen sekaan, voi sillä tulevaisuudessa olla potentiaalia glioblastooman lääkehoidon kehityksessä

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. (C) 2016 Elsevier GmbH. All rights reserved.Peer reviewe

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

Peer reviewe

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Peer reviewe

La formación docente en momentos de cambios: ¿Qué nos dicen los profesores principiantes universiatrios?

Este estudio cualitativo con una metodología de estudios de casos múltiple examina la opinión de los profesores principiantes de la Universidad de Barcelona sobre las consecuencias del proceso de implementación del Espacio Europeo de Educación Superior (EEES). Indaga sobre la valoración que realiza el profesorado sobre la formación recibida en el postgrado de 'Iniciación a la docencia universitaria' y sobre los aprendizajes que les generó la experiencia formativa. La muestra de la investigación ha estado constituida por diez profesores principiantes de diferentes ámbitos de conocimiento de la Universidad de Barcelona y los datos proceden de la realización de entrevistas en profundidad. Entre los hallazgos más importantes, cabe destacar su percepción sobre la implementación del EEES y las propuestas que dan sobre los cambios que debería realizar la Universidad para una verdadera reforma de la formación de los docente

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.Peer reviewe

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 (p(interaction) = 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.Peer reviewe

Netrin-4 Promotes Glioblastoma Cell Proliferation through Integrin β4 Signaling12

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 β4 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 β4-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 β4 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 β4 is upregulated to sensitize the cells to low concentrations of netrin-4 for maintaining cell proliferation

NTN4 partially rescues AKT and ERK phosphorylation in TMZ treated U251MG cells, but not in TMZ treated U87MG cells.

<p>U251MG and U87MG cells were treated with TMZ [100μM] for 3 h, and then cultured in medium containing 10% FCS supplemented with recombinant NTN4 protein [100 ng/ml]. Recombinant NTN4 protein was resupplied every 24 h. The cells were lysed for immunoblotting before (0 h) or after TMZ treatment (3 h), and every 24 h after TMZ treatment (24 h, 48 h, 72 h), total mTOR, total AKT, total ERK, and tubulin were used as loading control. In both U251MG and U87MG cell lines, the levels of p-AKT increased immediately after TMZ treatment and were significantly reduced 72 h later, while p-ERK (marked by arrowhead) was continuously inhibited after TMZ treatment. 72 h after TMZ treatment, NTN4 partially rescued AKT and ERK phosphorylation in U251MG cells (A), but not in U87MG cells (B).</p

NTN4 partially prevents TMZ induced cell senescence of U251MG, but not of U87MG cells.

<p>U251MG and U87MG cells were treated with TMZ [100μM] for 3 h, and then cultured in medium containing 10% FCS and recombinant NTN4 protein [100 ng/ml] as indicated. After 4 d (U251MG) or 2 d (U87MG) the cells were subjected to beta-gal staining. NTN4 reduced the senescence rate of TMZ treated U251MG cells (A), but had no significant effects on U87MG cells (B). Both U87MG and U251MG cells were cultured to about 50% confluence, and then lysed for western blotting (WB) analysis. ITGB4 protein was observed in U251MG cells, but was barely detectable in U87MG cells. Tubulin was used as a loading control (C). Mean ± SE, <i>n</i> ≥ 3, ** p-value < 0.01.</p