Abstract
Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.
Highlights
Cell migration is essential for normal embryonic development, wound healing, and immunity but can be devastating in tumor invasion and metastasis
Glioblastoma cells express netrin and netrin receptors To determine if netrins regulate glioblastoma cell migration, we first characterized netrin and netrin receptor expression in human astrocytoma cell lines U87, U343, and U373, and in cultures of astrocytes isolated from newborn rat cortex (Fig. 1A)
Ectopic Deleted in Colorectal Cancer (DCC) expression conferred on U343 and U373 cells the capacity to respond to a gradient of netrin-1
Summary
Cell migration is essential for normal embryonic development, wound healing, and immunity but can be devastating in tumor invasion and metastasis. Netrin-1 and netrin receptors DCC, the DCC paralogue neogenin, and UNC5 proteins, are expressed in many adult tissues [2,3,4,5,6,7,8,9], but their function in mature tissues is poorly understood. Reduced expression of netrin-1 has been documented in brain tumors, including glioblastoma [4], a role for netrins regulating brain tumor cell migration has not been established. Dcc expression is reduced in many cancers, including most high-grade gliomas [12] [13]and loss of DCC correlates with the development of highly invasive glioblastoma multiformae [13]. Unc homologue netrin receptors signal chemorepulsion, and co-expression of DCC often facilitates UNC5 function (reviewed by [1]). Altered expression of UNC5A, B, C, and D has been detected in various cancers and tumor cell lines [6,18][19]
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