The development of glioblastoma (GBM) is typically accompanied by marked changes in lipid metabolism. Oxylipins and their catalyzed enzymes lipoxygenases (LOXs) have been shown to participate in the development of cancers via multiple pathways, while the understanding of LOXs in GBM remains enigmatic. Thus, we aimed to explore the expression and functional roles of LOXs in the development of GBM. Here we showed that ALOXE3 was markedly down-regulated in human GBM. Knockdown of ALOXE3 in GBM cells fostered the orthotopic tumor growth and shortened lifespan in mice. ALOXE3 deficiency rendered GBM cells resistant to p53-SLC7A11 dependent ferroptosis, promoting GBM cell survival. Mechanistically, miR-18a directly targeted ALOXE3 and suppressed its expression and functions in GBM cells. Furthermore, ALOXE3 silencing promoted 12-hydroxyeicosatetraenoic acids (12-HETE) secretion from GBM cells, in turn, 12-HETE enhanced migration of GBM cells by activating G_s-protein-coupled receptor (G_sPCR)-PI3K-Akt pathway in an autocrine manner. Altogether, miR-18a/ALOXE3 axis exerts tumor promoting functions by regulating ferroptosis and migration of GBM cells. Targeting miR-18a/ALOXE3 axis may provide novel therapeutic approaches for GBM treatment.

Background: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. Methods: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). Results: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)⁺ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. Conclusions: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Our previous study illustrated that nuclear factor IX (NFIX) promotes glioblastoma (GBM) progression by inducing migration and proliferation of GBM cells. However, the underlying mechanism of how NFIX regulates GBM cell proliferation remains obscure. In this study, we uncovered that Go-Ichi-Ni-San 1 (GINS1) is upregulated and positively correlated with NFIX in human GBM specimen. NFIX silencing downregulates the expression of GINS1, which is pivotal for cell-cycle progression and proliferation of GBM cells. Replenishment of GINS1 largely rescues the NFIX-null effect on GBM cell proliferation. Mechanistic investigation revealed that NFIX transcriptionally actives GINS1 expression by directly binding to promoter region (-1779 to -1793bp) of the GINS1 gene. Furthermore, knockdown of NFIX sensitizes GBM cells to DNA damage-inducing agents including doxorubicin and temozolomide, in a GINS1-dependent manner. Implications: Our study highlights that targeting NFIX–GINS1 axis could be a novel and potential therapeutic approach for GBM treatment.

S1. Full images of immunoblotting. S2. NFIX knockdown has no effect on p53 and p21 expression. S3. Knockdown of GINS1 by shRNAs in U87 cells. S4. Knockdown of GINS1 represses proliferation of GL261 cells S5. GINS1 rescues the delayed growth of NFIX-null GL261 tumor. S6. NFIX directly up-regulated GINS1 gene by occupying its promoter

Table S1. Primer sequences for mRNA expression analysis. Table S2. Sequences of shRNA. Table S3. Antibodies for immunoblotting.

S1. Full images of immunoblotting. S2. NFIX knockdown has no effect on p53 and p21 expression. S3. Knockdown of GINS1 by shRNAs in U87 cells. S4. Knockdown of GINS1 represses proliferation of GL261 cells S5. GINS1 rescues the delayed growth of NFIX-null GL261 tumor. S6. NFIX directly up-regulated GINS1 gene by occupying its promoter