Upregulation of TRAF3IP2 and NAMPT Inversely Correlated with Decreased Overall Survival in Glioblastoma

Analysis of the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases demonstrated upregulation of both NAMPT and TRAF3IP2 in glioblastoma compared to normal tissues (Fig. 1A). When stratified by TRAF3IP2 expression, patients with tumors expressing high levels of TRAF3IP2 demonstrated decreased overall survival (OS) (Fig. 1B). In vitro, shRNA-mediated knockdown of TRAF3IP2 (TRAF3IP2KD) in U87 and U118 glioblastoma cells led to decreased NAMPT and SIRT1 expression (Fig. 1C), suggesting a correlation between TRAF3IP2 and NAD metabolism via NAMPT.

Fig. 1

Inhibition of TRAF3IP2 reduces NAMPT expression in glioblastoma. Analysis of glioblastoma samples from TCGA (red bars) compared to non-malignant brain samples (blue bars) from GTEx shows an increase in the expression of TRAF3IP2 and NAMPT mRNA, accessed through GEPIA 2 (http://gepia2.cancer-pku.cn) (A). Kaplan-Meier curve analysis of overall survival from TCGA sample analysis using the Xena platform (https://xena.ucsc.edu/) reveals an inverse association between TRAF3IP2 gene expression and length of survival in glioblastoma patients (B). Levels of mRNA expression, measured through qRT PCR, show reduced TRAF3IP2, NAMPT and SIRT1 expression in U87TRAF3IP2KD and U118TRAF3IP2KD cells, compared to U87SCR and U118TRAF3IP2KD, respectively (p < 0.05) (C). Silencing TRAF3IP2 reduces relative NAD levels in glioblastoma cell lines, compared to scrambled controls (SCR). The level of NAD was measured in U87TRAF3IP2KD and U87SCR in presence or absence of NAMPT inhibitor “FK866” (DMSO was used as solvent for FK866) (D). Silencing TRAF3IP2 decreases ATP production mechanism, measured through reduced oxygen consumption rate (OCR) in Agilent Seahorse XF Cell Mito Stress Test kit, in glioblastoma cell line KNS (Compared to SCR controls) (E). Triplicate experiments, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Silencing TRAF3IP2 Depletes NAD and Energetic Intermediates in Glioblastoma Cells

To understand the impact of TRAF3IP2 on cellular energetics in glioblastoma, we assessed the metabolomic profile in glioblastoma cells. Through colorimetric analysis, the NAD in U87TRAF3IP2KD cells was quantified and compared to control U87 cells (U87SCR). Additionally, we treated U87 cells with NAMPT inhibitor FK866, creating two conditions: U87SCR cells treated with FK866 (U87SCR+FK866) and U87TRAF3IP2KD cells treated with FK866 (U87TRAF3IP2KD+FK866). Results demonstrate significant decrease in NAD + levels in U87TRAF3IP2KD cells compared to U87SCR cells (Fig. 1D), illustrating the role of TRAF3IP2 in maintaining NAD homeostasis. Furthermore, analysis of mitochondrial stress in KNS cells revealed a reduction in ATP production, indicated by reduced oxygen consumption rate (OCR) following knockdown of TRAF3IP2 (Fig. 1E) suggesting that silencing TRAF3IP2 significantly perturbs cellular energetics in glioblastoma cells.

Targeting TRAF3IP2 Alters Protein Expression of NAMPT and NAD-Dependent Markers

Western blot analysis was conducted to elucidate the effect of targeting TRAF3IP2 on key metabolic and apoptotic regulators in glioblastoma cells. Silencing TRAF3IP2 significantly reduced the expression of NAMPT and SIRT1, while simultaneously increasing total, phosphorylated, and acetylated p53 compared with SCR control cells (Fig. 2A-C). These results indicate a direct correlation between TRAF3IP2 activity, and the expression levels of key markers involved in glioblastoma cellular metabolism and tumor suppression. The decrease in NAMPT expression following TRAF3IP2 knockdown leads to a subsequent reduction in SIRT1 levels, a NAD-dependent deacetylase involved in cellular stress responses and longevity. The reduction in SIRT1 correlates with an increase in both total, phosphorylated, and acetylated forms of p53 resulting in enhanced apoptosis. The increase in acetylated-p53 suggests enhanced activation and stability of p53, contributing to potential anti-tumor effects in glioblastoma cells. Furthermore, cleaved caspase-3 was markedly elevated in TRAF3IP2KD cells, confirming activation of the intrinsic apoptotic pathway, and Annexin V staining (Fig. 4) demonstrated a significant rise in early apoptotic populations, providing complementary evidence of apoptosis.

Fig. 2

Targeting TRAF3IP2 alters protein levels of NAD salvage pathway markers in glioblastoma. Protein levels of TRAF3IP2 and NAMPT in U87, U118, and KNS cell lines (A). Protein levels of SIRT1, total p53, Acetyl-p53[K382] (a-p53), and Phosphorylated-p53[S392] (p-p53) in U87, U118, and KNS cell lines (B). Imagej analysis of band intensity in western blots, relative to β-actin (TRAF3IP2KD vs. SCR) (C). Immunohistochemical analysis of the tumor tissues demonstrating that U87TRAF3IP2KD -derived tumors had reduced TRAF3IP2 and NAMPT expression, compared to SCR control tumor tissues (Size bar = 50 µM) (D). Relative fluorometric intensity of ROS in U87TRAF3IP2KD, compared to U87SCR showed that silencing TRA3IP2 increases ROS levels (E). Protein expression of LKB1, p-LKB1, AMPK, p-AMPK in transduced U87, U118, and KNS glioblastoma cell lines (F). Triplicate experiments, (*P < 0.05)

Fig. 4

TRAF3IP2 inhibition is associated with increased ROS presence and apoptosis. Gene expression analysis showed elevated levels of survivin in TRAF3IP2KD cells, compared to SCR controls (Triplicate experiments/cell lines, p < 0.05) (A). Western blot analysis of showed levels of survivin, total caspase-3 and cleaved caspase-3 protein in U87 and U118 cells (B). Scatterplot of glioblastoma cell groups and treatment types (U87SCR+DMSO (i); U87SCR+FK866 (ii); U87TRAF3IP2KD+DMSO (iii); U87TRAF3IP2KD+FK866 (iv)), with bar graph representation of Annexin V and PI staining of U87 (v) (C). *P < 0.05

To elucidate the in vivo implications of targeting TRAF3IP2 on NAMPT expression, we induced tumors using U87TRAF3IP2KD and U87SCR cells. As previously reported, compared to U87SCR, U87TRAF3IP2KD induced significantly smaller tumors Immunohistochemistry of resulting tumors showed a notable decrease in TRAF3IP2 and NAMPT levels in tumors derived from U87TRAF3IP2KD cells, in comparison to tumors of U87SCR cells (Fig. 2D). These findings support the inverse relationship between TRAF3IP2 and NAMPT expressions in vivo.

Given the importance of reactive oxygen species (ROS) in glioblastoma biology, we measured intracellular ROS levels using fluorometric assay. Our results show that targeting TRAF3IP2 led to further elevation of ROS in U87TRAF3IP2KD compared to SCR control cells, potentially overcoming the elevated ROS resistance in glioblastoma cells (Fig. 2E). This suggests that TRAF3IP2 plays a critical role in regulating oxidative stress and maintaining redox balance in glioblastoma cells.

TRAF3IP2 Inhibition Increases AMPK-Associated Expression

To assess whether decline in cellular energetics from silencing TRAF3IP2 in glioblastoma cells affects nutrient-sensing pathways, LKB1 and AMPK protein expression were examined. Results show that silencing TRAF3IP2 in glioblastoma cells increased expression and phosphorylation in LKB1 and AMPK (Fig. 2F). These results indicate that targeting TRAF3IP2 disrupts signaling pathways that are crucial for glioblastoma growth and proliferation. Specifically, TRAF3IP2KD cells, when compared to control cells, demonstrated enhanced expression of phosphorylated LKB1 and AMPK, which are pivotal components of the nutrient-sensing signaling pathway. These results suggest that inhibiting TRAF3IP2 in glioblastoma cells is associated with disruption of metabolomic signaling pathways, thereby potentially impairing tumor growth and proliferation mechanisms that are dependent on these signaling networks.

Silencing TRAF3IP2 is Associated with a Decrease in mTORC Activity

The increased activity of mTOR is a characteristic feature of glioblastoma cells, contributing significantly to their proliferation and survival. We next studied the effects of targeting TRAF3IP2 on mTOR activity. The silencing TRAF3IP2 in glioblastoma cells led to significant decreases in RNA expression of mTOR complex such as Raptor (mTORC1) and Rictor (mTORC2) (Fig. 3A). In addition, western blot analysis shows a decreased expression of phosphorylated both Raptor and Rictor proteins (Fig. 3B). These results show that targeting TRAF3IP2 in glioblastoma is associated with a reduction in mTOR complex expression.

Fig. 3

Targeting TRAF3IP2 reduces mTOR associated cell growth markers and glycolysis and glioblastoma cells. Fold change difference in mTOR and mTORC associated markers Raptor and Rictor in TRAF3IP2KD cells, compared to SCR controls, in U87, U118 and KNS cell lines (Triplicate experiments/cell lines, p < 0.05) (A). Western blot of Raptor, p-Raptor, Rictor, and p-Rictor protein expression in U87SCR and U87TRAF3IP2KD cells (B). Agilent Seahorse XF Glycolytic stress test measuring glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification through assessment of real-time ECAR levels, normalized to total µg protein in cell culture wells, of glioblastoma cell lines U87 (C) and KNS (D)

Inhibition of TRAF3IP2 Reduces Glycolysis in Glioblastoma Cells

Glioblastoma cells predominantly rely on glycolysis for ATP production, a metabolic pathway that supports glioblastoma cell rapid proliferation and survival under hypoxic conditions typical to tumor microenvironment. Analysis of glycolytic stress in glioblastoma cell lines (U87TRAF3IP2KD vs. U87SCR, and KNSTRAF3IP2KD vs. KNSSCR,) shows a significant decline in both glycolysis and glycolytic capacity upon TRAF3IP2 knockdown (Fig. 3C-D). This result indicates the efficacy of targeting TRAF3IP2 in diminishing glycolytic function in glioblastoma cells.

Targeting TRAF3IP2 Progresses Apoptotic Signaling and Leads Toward Cell Death

Further analysis underscores the effects of targeting TRAF3IP2 in suppression of survivin, a marker of cell proliferation, in glioblastoma cells (Fig. 4A). The reduction of surviving in RNA levels was also shown at the protein level, with western blot analysis displaying a significant decrease in survivin protein expression in U87TRAF3IP2KD and U118TRAF3IP2KD cells (Fig. 4B). Moreover, an increase in cleaved caspase-3 was observed, indicating an upregulation of apoptotic activity (Fig. 4B). These results collectively show that inhibiting TRAF3IP2 directly impacts survivin expression, leading to initiation of apoptotic activity through increased cleaved caspase-3.

Further exploration into the consequences of altering NAD metabolism through NAMPT inhibition revealed its impact on cell viability. In addition, Annexin V/PI assays post-FK866 treatment showed a significant elevation in both early and late apoptotic populations in TRAF3IP2KD cells (Fig. 4C), further corroborating enhanced apoptotic signaling. These results indicate that silencing TRAF3IP2 not only disrupts NAD metabolism but also potentiate apoptotic progression beyond the effects of NAMPT inhibition alone.