FAM135B suppresses glioblastoma angiogenesis via stabilizing the IKK complex and inactivating the NF-κB/IL-6...

Glioblastoma multiforme (GBM), the most aggressive primary central nervous system malignancy in adults, is hallmarked by robust angiogenesis and pronounced heterogeneity, with the mesenchymal subtype linked to the poorest prognosis. The median survival of GBM patients is merely ~ 14 months, and approved anti-angiogenic agents like bevacizumab only yield short-term benefits without improving overall survival, highlighting the urgent need for novel anti-angiogenic targets. FAM135B, a brain-enriched protein-coding gene, is implicated in tumorigenesis and chemoresistance in other cancers, but its role and underlying mechanism in GBM angiogenesis remain unelucidated.

Bioinformatic analysis: Gene Set Variation Analysis (GSVA), single-cell and spatial transcriptomic analyses were performed on GBM datasets from TCGA, CGGA and other databases to screen angiogenesis-related genes and explore the correlation between FAM135B expression and clinical prognosis. In vitro experiments: FAM135B-overexpressing/knockdown GBM cell models were constructed. Tube formation, Transwell migration and colony formation assays were used to detect the angiogenic potential of human umbilical vein endothelial cells (HUVECs) treated with GBM cell-conditioned medium. Co-immunoprecipitation, mass spectrometry, Western blot, RT-qPCR and ELISA were applied to verify protein-protein interactions and detect the expression of cytokines and signaling pathway molecules. Dual-luciferase reporter assays confirmed the transcriptional regulation of FAM135B. In vivo experiments: Nude mouse orthotopic and subcutaneous GBM xenograft models were established to assess the effect of FAM135B on tumor growth, angiogenesis and mouse survival. Rescue experiments with NF-κB inhibitor PDTC and recombinant IL-6 were conducted to validate the key role of the NF-κB/IL-6 axis. Clinical sample validation: Immunohistochemistry and magnetic resonance imaging were used to analyze the correlation between FAM135B expression, microvessel density and prognosis in 72 GBM clinical samples.

SPSS 19.0 was used for data analysis with Kaplan-Meier, t-tests and Log-rank tests; the significance level was set at P < 0.05.

FAM135B is a novel key regulator of GBM angiogenesis, with significantly downregulated expression in GBM tissues (especially the mesenchymal subtype). Low FAM135B expression correlates with enhanced endothelial cell communication, high angiogenic potential and poor progression-free/overall survival in GBM patients. FAM135B inhibits GBM angiogenesis in vitro and in vivo: its overexpression suppresses HUVEC tube formation, migration and colony formation, while knockdown exerts the opposite effect. In orthotopic xenografts, FAM135B overexpression slows tumor growth, prolongs mouse survival, and reduces intratumoral microvessel formation and M2 macrophage infiltration. FAM135B downregulates IL-6 expression to inhibit the JAK/STAT signaling pathway: its expression is negatively correlated with pro-angiogenic cytokines (notably IL-6) in GBM. FAM135B overexpression reduces IL-6 transcription and secretion in GBM cells, thereby inhibiting JAK/STAT phosphorylation in HUVECs; high FAM135B expression in GBM tissues is associated with low IL-6 and CD31 levels. FAM135B binds to the IKK complex (IKKα/IKKβ) to stabilize it, inhibit IKKβ activation and P65 phosphorylation, and block the canonical NF-κB signaling pathway, which is the upstream mechanism of IL-6 downregulation. Co-overexpression of IKBKB partially reverses the anti-angiogenic effect of FAM135B, and PDTC rescues NF-κB activation induced by FAM135B knockdown. HNF4A is the upstream positive transcription factor of FAM135B: it directly binds to two key sites in the FAM135B promoter to promote its transcription and protein expression. HNF4A expression is positively correlated with FAM135B and favorable GBM prognosis; its overexpression inhibits GBM angiogenesis, and this effect is abrogated by FAM135B knockdown.

FAM135B acts as a critical negative regulator of GBM angiogenesis, whose low expression contributes to high tumor angiogenic potential and poor patient prognosis. Mechanistically, HNF4A transcriptionally upregulates FAM135B, which then binds to and stabilizes the IKK complex, inactivating the NF-κB signaling pathway and downregulating IL-6 expression, ultimately inhibiting the JAK/STAT-mediated angiogenic process. FAM135B also remodels the GBM tumor microenvironment by reducing M2 macrophage infiltration. Targeting the HNF4A/FAM135B/NF-κB/IL-6 signaling axis provides a novel and promising therapeutic strategy for GBM anti-angiogenic therapy.