Over the last few years, the expression of chemotropic axon guidance molecules in a variety of cancer cell lines and tumors has been thoroughly
investigated . These studies revealed that many of these molecules are either up- or down-regulated in tumor cells.
Axon Guidance Molecules Highly Expressed in Cancer Cells
SEMA3C was the first secreted semaphorin proposed to be involved in tumorigenesis, as it is overexpressed in non-MDR (multidrug resistance) drug resistant ovarian and lung cancer cell lines [15, 16]. Several glioma cell lines also express high levels of SEMA3C. Likewise, SEMA3E expression was correlated positively with tumor progression in mouse mammary carcinoma and is over-expressed in metastatic human lung adenocarcinoma cell. SEMA3A, SEMA3F are also overexpressed several cancer cell lines and tumors . Last, SLIT2 is strongly expressed in many tumor cell lines (melanoma, neuroblastoma, colon adenocarcinoma ...) and primary tumors, while SLIT1-3 expression and ROBO1 expression are upregulated in prostate tumors and colorectal cancer respectively [18, 19]. Although the function of the overexpressed semaphorins and slits in cancer cell lines is unclear, they could via an autocrine/paracrine action modify cell survival and migration, inhibit the immune response , but also regulate tumor vasculature.
Down-Regulation of Axon Guidance Molecules in
Cancer Cells: Putative Tumor Suppressors?
Most studies point to a down-regulation of the expression of secreted axon guidance proteins in cancers. Thus, the netrin-1 receptor DCC was first characterized as a gene of frequently DCCs and its expression is also down-regulated in many tumor cell lines . DCC is at 18q21.2, a locus with frequent loss of heterozygosity (LOH) in gastrointestinal cancers, suggesting that DCC is a tumor suppressor gene. Interestingly, the UNC5 genes, that encode the other netrin-1 receptors, are frequently down-regulated in many primary tumors in association with significant LOH .
SEMA3B and SEMA3F, were mapped to the 3p21.3 locus, a region that is thought to contain putative tumor suppressor genes . Many studies suggest that SEMA3B may be a candidate tumor suppressor. First, SEMA3B is down-regulated in lung cancer cells and is also often mutated, suggesting that it may play a suppressive role in tumorigenesis. Morevover, SEMA3B promoter is hypermethylated in multiple cancer cell lines, or in tumor samples and there is a significant LOH and hypermethylation of its promoter . Ovarian adenocarcinoma cells also express 25-fold less SEMA3B than in normal human ovary and have decreased tumorigenic properties in xenograft model . SEMA3F expression is also down-regulated in several cancer cell lines and tumors and its promoter methylated . As mentioned above, deletions and heterozygous loss in regions 3p12, 3p14 and 3p21 occur frequently in lung cancer. Interestingly, ROBO1/Dutt1 was mapped within the deletion and its promoter region is hypermethylated in primary lung, renal breast and cervical cancer . Although, no somatic point mutation of ROBO1 (or of its ligands slits) was reported in tumors, ROBO1 may be a tumor suppressor gene . Likewise, SLIT2 is mapped to 4p15.2 a region associated with frequent LOH in many tumors. Accordingly, the inactivation of SLIT2 in tumors was shown to be epigenetic and caused by the hypermethylation of the promoter region. SLIT1-3 expression is decreased in breast, lung cancer cell lines, gliomas or tumors and epigenetic inactivation of slit genes was demonstrated [18, 24]. The observation that exogenous SLIT2 suppresses colony growth in breast cancer cell lines further supports a possible tumor suppressor function of SLIT2.
The development and growth of tumors require the simultaneous formation and sprouting of new blood vessels from pre-existing capillaries and veins . Surprisingly, mounting evidence suggests that diffusible axon guidance molecules are very potent angiogenic or anti-angiogenic factors  and that blood vessels that irrigate tumors express receptors for several secreted axon guidance proteins, in particular ROBO1, neuropilin-1, plexins-D1 and UNC5B.
The first direct evidence for a link between axon guidance molecules and angiogenesis came from studies focused on neuropilin receptors . Binding experiments revealed that in addition to binding most class 3 semaphorins, neuropilin-1 is a receptor for VEGF-A (the VEGF165 but not the VEGF121 isoform), VEGF-B, VEGF-E and placental-derived growth factor-2. Neuropilin-1 is expressed by tumor cells and endothelial cells, where it is a co-receptor for VEGFR-2 mediating VEGF function in angiogenesis . The analysis of neuropilin-1 knockout mice has confirmed that neuropilin-1/VEGF interaction is required for normal development of the vasculature . A soluble neuropilin-1 isoform was identified and found to have anti-tumor activity. Recently, two other soluble forms of neuropilin-1, sIIINRP1 and sIVNRP1, generated by alternative splicing, were discovered and both are expressed in human cancerous tissue. These soluble neuropilins also bind VEGF165 and SEMA3A. Likewise, neu-ropilin-2 is a receptor for VEGF165, VEGF145 and placental-derived growth factor-2. Interestingly, SEMA3A binding to neuropilin-1 and SEMA3F binding to neuropilin-2 block the migration of endothelial cells . SEMA3F also inhibits endothelial cell survival . Several class 3 semaphorins are also expressed by endothelial cells and could have an autocrine action. Accordingly, SEMA3A seems to exert a permissive role on angiogenesis by inhibiting integrin-mediated adhesion of endothelial cells allowing their deadhesion. The ratio of SEMA3A/VEGF165 expression in patients with multiple myeloma was also proposed to be critical to the angiogenic potential of bone marrow endothelial cells . More recent studies have shown that in contrast with other secreted semaphorins, SEMA3E directly binds and signals via the plexin-D1 receptor . Interestingly, plexin-D1 controls angiogenesis during development and both molecules could exert a similar function in tumors.
As mentioned above, many tumors express high levels of SLIT2 and several studies suggest that SLIT2 and its receptors have a potent angiogenic activity. ROBO1 is expressed on human umbilical vein endothelial cells and SLIT2 increases their migration . This chemotactic activity of SLIT2 requires phosphatidylinositol-3 kinase (PI-3K) activation and can be inhibited by recombinant ectodomain (RoboN). There is also an in vivo evidence for a role of slit/robo in angiogenesis.
Recent studies suggested that netrins could also regulate angiogenesis. Thus, endothelial cells express UNC5B and A2b receptors, respond to netrin-1 and vascular defects were detected in UNC5B knockouts . However, contradictory data have just appeared . Thereby, the exact mechanism of action of netrin-1 in endothelial cells, and its pro and anti-angiogenic activity are still debated.
Overall these studies suggest that in tumors, some axon guidance proteins (SLIT2, netrin-1) are up-regulated and may increase angiogenesis upon binding their receptors on endothelial cells. Other axon guidance molecules (SEMA3E, SEMA3A and SEMA3C ...), may act as inhibitors of angiogenesis in normal condition for instance by interfering with VEGF function.
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