br Materials and methods br Results br Discussion We

Materials and methods
Results
Discussion We demonstrate reduced angiogenic activity in NDRG1 overexpressing malignant glioma leading to reduced glioma growth. This antiangiogenic phenotype is paralleled by a significant upregulation of the antiangiogenic gene TNFSF15. TNFSF15 upregulation is associated with NDRG1 induced activation of the NF-κB and AP-1 response elements within the TNFSF15 promoter region. Consequently, NDRG1 overexpressing gliomas are demonstrated to be intrinsically resistant to targeted antiangiogenic therapy. NDRG1 prognosticates the natural course of disease in WHO grade II glioma, where patients with high NDRG1 expression show an improved survival [8], [22]. This improved survival may be the result of reduced tumor cell proliferation, reduced tumor cell invasion and angiogenesis in response to NDRG1 alteration [8], [23]. Angiogenesis is driven by various angiogenic molecules, the foremost being VEGF[24]. With NDRG1 overexpression we did not observe alterations in the expression of proangiogenic molecules. The only proangiogenic effector upregulated was CCL2[25]. However, we did observe an antiangiogenic phenotype in our tumors, therefore CCL2 expression exerted only minor effects in our experiments. Instead, we identified a highly significant alteration of the endogenous TNFSF15. This phenomenon suggests that TNFSF15 may be a key molecule responsible for the antiangiogenic phenotype. TNFSF15 was initially described as a specific autocrine inhibitor expressed by endothelial SB1518 [26]. Gonsky et al. demonstrated that monocytes also express TNFSF15[17]. Moreover, luminal, glandular and deep gland epithelial cells of the bovine uterus stained positive for TNFSF15, showing that TNFSF15 expression is not confined to endothelial cells [27]. In this scenario, it does not seem surprising that glioma cells, which are known for diverse gene mutations and regulation, also express TNFSF15. Accordingly, TNFSF15 expression is demonstrated in GBM patients with a highly diverse expression and DNA copy number profile (Supplementary Fig. S1). Three isoforms of TNFSF15 have been discovered: VEGI-174, VEGI-192 and VEGI-251[28]. The only isoform including a secretion signal peptide and found to be secreted by endothelial and cancer cells was VEGI-251[29]. The supernatant of NDRG1 overexpressing glioma cells exerts negative effects on HUVEC proliferation, migration and tube formation similar to findings in experiments with VEGI-251 overexpressing mammary carcinoma cells [29]. Thus, it seems likely that NDRG1 overexpressing gliomas secrete the splice variant VEGI-251. In this context, TNF-alpha and Il1b were described to induce TNFSF15 secretion [30], [31]. However, in our study these mediators were not changed by NDRG1 overexpression excluding TNF-alpha and Il1b as inducers of TNFSF15 in our experiments. Secreted TNFSF15 inhibits vasculogenesis by downregulating membrane bound VEGF receptor 1 (mFlt1) and by increasing soluble VEGF receptor 1 (sFlt1), thereby interfering with vasculogenesis [32]. Moreover, TNFSF15 has been shown to exert antiangiogenic effects by inducing an early G1 arrest in quiescent endothelial cells and by stimulating apoptosis in proliferating endothelial cells [33]. In different tumor models, the application of TNFSF15 has produced tumor growth arrest due to antiangiogenic properties underlining the results of our study [33], [34], [35]. The antiangiogenic effect of NDRG1 contradicts the general concept that hypoxia is one of the main inducers of neoangiogenesis. Interestingly, in pancreatic cancer NDRG1 suppresses tumor growth and angiogenesis due to reduced VEGF activity [36]. Especially in glioma, this finding highlights the diverse effects that hypoxia may have on the angiogenic system. In this context it may be hypothesized that NDRG1 induced upregulation of TNFSF15 may occur in the necrotic core of malignant glioma, which is characterized by massive hypoxia and reduced angiogenesis [37], [38]. The contrary may occur in the less hypoxic but proangiogenic infiltration zone of malignant glioma [39]. In this scenario, TNFSF15 expression is regulated via different pathways, the most important being the NF-κB and AP-1 pathways [18]. The TNFSF15 promoter region underlies a complex regulation mechanism consisting of different, cell type-dependent activating and inhibiting response elements [17], [18]. TNFSF15 in endothelial cells is regulated in an autocrine fashion as the critical control of its expression resides in a promoter segment containing a NF-κB binding site [40], [41]. In our study, TNFSF15 promoter activity was significantly reduced by mutations in the NF-κB and AP-1 promoter region, suggesting both elements as dependent positive regulators of TNFSF15 expression in glioma. This regulatory mechanism has been previously demonstrated in human microvascular endothelial cells [18]. In human gastric cancer cells, knock-down of AP-1 results in downregulation of VEGF, CXC chemokines and matrix-metalloproteinases, thereby altering angiogenesis [21], [42]. Moreover, NDRG1 is differently regulated by mTOR signaling as AKT acts inhibitory on NDRG1 while mTOR mediated SGK induction activates NDRG1[42], [43]. These data underline the complex regulation of NDRG1 activity explaining the diverse cellular and tissue-specific effects of NDRG1 (Fig. 5).