Targeting TRAF3IP2 inhibits angiogenesis in glioblastoma

Abstract

Increased vascularization, also known as neoangiogenesis, plays a major role in many cancers, including glioblastoma multiforme (GBM), by contributing to their aggressive growth and metastasis. Although anti-angiogenic therapies provide some clinical improvement, they fail to significantly improve the overall survival of GBM patients. Since various pro-angiogenic mediators drive GBM, we hypothesized that identifying targetable genes that broadly inhibit multiple pro-angiogenic mediators will significantly promote favorable outcomes. Here, we identified TRAF3IP2 (TRAF3-interacting protein 2) as a critical regulator of angiogenesis in GBM. We demonstrated that knockdown of TRAF3IP2 in an intracranial model of GBM significantly reduces vascularization. Targeting TRAF3IP2 significantly downregulated VEGF, IL6, ANGPT2, IL8, FZGF2, PGF, IL1β, EGF, PDGFRB, and VEGFR2 expression in residual tumors. Our data also indicate that exogenous addition of VEGF partially restores angiogenesis by TRAF3IP2-silenced cells, suggesting that TRAF3IP2 promotes angiogenesis through VEGF- and non-VEGF-dependent mechanisms. These results indicate the anti-angiogenic and anti-tumorigenic potential of targeting TRAF3IP2 in GBM, a deadly cancer with limited treatment options.

Keywords: Glioblastoma multiforme; TRAF3IP2; angiogenesis; inflammation; tumor microenvironment.

Figure 1

TRAF3IP2 is expressed at the highest levels at the infiltrating edge of the tumor. RNA sequencing data were mined from the IVY GBM Atlas project, accessed through https://glioblastoma.alleninstitute.org. Significance was calculated by ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Immunohistochemical analysis demonstrating that knockdown of TRAF3IP2 in GBM reduces angiogenic markers. The U87 and U118 cells were transduced with anti-TRAF3IP2-shRNA (U87_TRAF3IP2KD and U118_TRAF3IP2KD, respectively) to silence TRAF3IP2 or Scrambled-shRNA (U87_{Control shRNA} and U118_{Control shRNA}, respectively) as a control. The cells (U87_TRAF3IP2KD or U87_{control shRNA} or U118_TRAF3IP2KD or U118_{Control shRNA}) were used to induce intracranial tumors to generate an orthotopic mouse GBM mouse model. The cells (3 × 10⁵ cells) were injected into the left somatosensory cortex (SSCx) of NSG mice for tumor induction. The animals were euthanized 28 days post-tumor induction. Histology and immunohistochemistry revealed that U87_TRAF3IP2KD and U118_TRAF3IP2KD compared to control had significantly less TRAF3IP2 expression, which correlated with decreased CD31 and CD34 (endothelial vascular markers) and VEGF. Higher magnification of selected areas is shown (scale bar is 100 μm) (A). Quantification of vascular-like structures (B). Analysis using GEPIA revealed that GBM tumors express higher levels of CD34 (C) and significantly higher levels of CD31 (p < 0.05) (D), compared to normal brain controls. Targeting TRAF3IP2 in GBM cell lines decreases protein levels of CD31 (E). *P<0.05, ****P<0.0001.

Figure 3

Tube formation assays demonstrating that knockdown of TRAF3IP2 in GBM reduces angiogenesis. HBMEC (human brain microvascular endothelial cells) were cultured in the following conditions: normal media (EBM), conditioned media (CM) from U87_{Control shRNA} or U118 _{Control shRNA} cells, or CM from U87_TRAF3IP2KD or U118_TRAF3IP2KD cells (Figure 3AI, 3BI). The addition of exogenous VEGF at a concentration of 40 ng/ml was also performed (bottom rows, AI and 3BI). Effects of CM from U87_TRAF3IP2KD (A) or U118_TRAF3IP2KD (B) with or without VEGF on angiogenesis parameters including total tube [measured in pixels (px)], total branching points, and total loops are shown (I-IV). U87_TRAF3IP2KD and U118_TRAF3IP2KD compared to U87_{Control shRNA} and U118_{Control shRNA} secreted significantly lower amounts VEGF in the medium by enzyme-linked immunosorbent assay (ELISA) (C). Significance was calculated by ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

Targeting TRAF3IP2 in GBM decreases pro-angiogenic factors and receptors. Gene expression fold changes (U87_TRAF3IP2KD vs. U87_{Control shRNA}) are shown. *p < 0.05, ***p < 0.001, ****p < 0.0001 (A). Protein levels of key angiogenic factors in U87_TRAF3IP2KD vs. U87_Control (B) and quantification (C). Levels of key angiogenic factors in GBM vs. normal tissue: clinical GBM tumor samples and data (red bars) are from The Cancer Genome Atlas (TCGA) red bars and normal controls (gray bars) are from Genotype-Tissue Expression (GTEx) accessed through GEPIA (http://gepia.cancer-pku.cn), p < 0.01 (D–O).

Figure 5

Ingenuity pathway analysis (IPA) reveals that targeting TRAF3IP2 coordinately inhibits multiple pro-angiogenic pathways. IPA reveals that targeting TRAF3IP2 coordinately inhibits multiple pro-angiogenic pathways. Gene expression analysis revealed a differentially expressed gene list between the U87_TRAF3IP2KD and U87_{control shRNA} cells. IPA^® canonical pathway analysis and molecular network analysis revealed HIF-1α signaling, tumor microenvironment signaling, and neuroinflammation pathways as top regulated pathways.

Figure 6

Forest plots demonstrate hazard ratios and 95% confidence intervals (CI) for angiogenic genes significantly changed by targeting TRAF3IP2. Data are from The Cancer Genome Atlas (TCGA), accessed through GEPIA (http://gepia.cancer-pku.cn). Group cutoff for high expression vs. low expression of the gene of interest is the median. Hazard ratio (HR) is calculated based on the COX proportional hazard model. Hazard ratios are for the “high expression” group for each gene and refer to overall survival (OS) Panel (A) or disease-free survival (DFS) Panel (B).