Abstract
BackgroundGlioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies.MethodsTarget genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence.ResultsHIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway.ConclusionsHIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.
Highlights
Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy
Receptors, and substrates in AKT signaling pathway, and Hypoxia inducible factor subunit alpha (HIF1α) target genes involved in glycolysis, de novo lipogenesis, and angiogenesis, revealed that Platelet-derived growth factor (PDGF) D, PDGFRA, Insulin like growth factor binding protein 2 (IGFBP2), Pyruvate dehydrogenase kinase 1 (PDK1),3, SLC16A1,3/MCT1,4, and lipogenesis genes Stearoyl-CoA desaturase (SCD) and Fatty acid synthase (FASN) were all consistently downregulated in U251 cells following genetic ablation of HIF1α, in comparison to scrambled small Cas9-guided RNA (sgRNA) control (Fig. 1B,C, Table S2, S3)
These data indicate that the expression of PDGFRA and PDGFD in GBM cells depends more on HIF1α compared with the expression of other receptors and growth factors, such as PDGFB
Summary
Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; available inhibitors targeting isolated signaling pathways are minimally effective. Genes encoding epithelial growth factor receptor (EGFR) and PDGFRα were altered by amplification, rearrangements and mutations, resulting in increased receptor tyrosine phosphorylation in GBM [9]. Other genetic lesions, including PDGFRA activating mutations and gene rearrangements, as well as EGFR amplification, often occur concurrently in tumors with PDGF RA amplification [11,12,13,14,15]. Co-overexpression and co-activation of PDGF Rα with EGFR often occur in GBM tumors without amplification of either gene [17,18,19] but with a typical feature of high angiogenesis such as the most common EGFRvIII mutant-overexpressing GBM [20, 21]
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