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.