miRNA Mediated Noise Making of 3′UTR Mutations in Cancer

Somatic mutations in 3′-untranslated regions (3′UTR) do not alter amino acids and are considered to be silent in cancers. We found that such mutations can promote tumor progression by altering microRNA (miRNA) targeting efficiency and consequently affecting miRNA⁻mRNA interactions. We identified 67,159 somatic mutations located in the 3′UTRs of messenger RNAs (mRNAs) which can alter miRNA⁻mRNA interactions (functional somatic mutations, funcMutations), and 69.3% of these funcMutations (the degree of energy change > 12 kcal/mol) were identified to significantly promote loss of miRNA-mRNA binding. By integrating mRNA expression profiles of 21 cancer types, we found that the expression of target genes was positively correlated with the loss of absolute affinity level and negatively correlated with the gain of absolute affinity level. Functional enrichment analysis revealed that genes carrying funcMutations were significantly enriched in the MAPK and WNT signaling pathways, and analysis of regulatory modules identified eighteen miRNA modules involved with similar cellular functions. Our findings elucidate a complex relationship between miRNA, mRNA, and mutations, and suggest that 3′UTR mutations may play an important role in tumor development

The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

Background: Glioblastoma (GBM) is a highly migratory, invasive, and angiogenic brain tumor. Like vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF) promotes GBM angiogenesis. VEGF-A is a ligand for both VEGF receptor-1 (VEGFR-1) and VEGFR-2, while PlGF interacts exclusively with VEGFR-1. We recently generated the novel anti-VEGFR-1 monoclonal antibody (mAb) D16F7 that diminishes VEGFR-1 homodimerization/activation without affecting VEGF-A and PlGF binding. Methods: In the present study, we evaluated the expression of VEGFR-1 in human GBM tissue samples (n = 42) by immunohistochemistry, in cell lines (n = 6) and GBM stem cells (GSCs) (n = 18) by qRT-PCR and/or western blot analysis. In VEGFR-1 positive GBM or GSCs we also analyzed the ability of D16F7 to inhibit GBM invasiveness in response to VEGF-A and PlGF. Results: Most of GBM specimens stained positively for VEGFR-1 and all but one GBM cell lines expressed VEGFR-1. On the other hand, in GSCs the expression of the receptor was heterogeneous. D16F7 reduced migration and invasion of VEGFR-1 positive GBM cell lines and patient-derived GSCs in response to VEGF-A and PlGF. Interestingly, this effect was also observed in VEGFR-1 positive GSCs transfected to over-express wild-type EGFR (EGFRwt+) or mutant EGFR (ligand binding domain-deficient EGFRvIII+). Furthermore, D16F7 suppressed intracellular signal transduction in VEGFR-1 over-expressing GBM cells by reducing receptor auto-phosphorylation at tyrosine 1213 and downstream Erk1/2 activation induced by receptor ligands. Conclusion: The results from this study suggest that VEGFR-1 is a relevant target for GBM therapy and that D16F7-derived humanized mAbs warrant further investigation

Additional file 4: Figure S4. of The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

Inhibition of ECM invasion by D16F7 cells in a spheroid assay with P3 cells. Representative pictures of spheroids taken at 24, 48 and 72 h after embedding P3 cells in matrigel (40× magnification) and referring to the experiment described in Fig. 3c legend. (PDF 255 kb

The angiogenic switch leads to a metabolic shift in human glioblastoma

Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic interventio

Additional file 6: Figure S6. of The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

Inhibition of ECM invasion by D16F7 cells in a spheroid assay with EGFRvIII​+ cells. Representative pictures of spheroids taken at 24, 48 and 72 h after embedding EGFRvIII+ cells in matrigel (40× magnification) and referring to the experiment described in Fig. 3c legend. (PDF 198 kb

Additional file 1: Figure S1. of The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

Immunohistochemical analysis of VEGFR-1 expression in GBM tissue sections. Representative images are presented (25× magnification). VEGFR-1 immunostaining was scored as described in Table 1: score 1 (<10%); score 2 (11–25%); score 3 (26–50%); score 4 (51–75%); 5 (>75%). (PDF 484 kb