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Glioblastoma is a highly aggressive brain tumor for which there is no cure. The metabolic enzyme 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is essential for glioblastoma stem-like cell (GSC) survival but its mode of action is unclear. Understanding the role of PFKFB4 in tumor cell survival could allow it to be leveraged in a cancer therapy. Here, we show the importance of PFKFB4 for glioblastoma growth in vivo in an orthotopic patient derived mouse model. In an evaluation of patient tumor samples of different cancer entities, PFKFB4 protein was found to be overexpressed in prostate, lung, colon, mammary and squamous cell carcinoma, with expression level correlating with tumor grade. Gene expression profiling in PFKFB4-silenced GSCs revealed a downregulation of hypoxia related genes and Western blot analysis confirmed a dramatic reduction of HIF (hypoxia inducible factor) protein levels. Through mass spectrometric analysis of immunoprecipitated PFKFB4, we identified the ubiquitin E3 ligase, F-box only protein 28 (FBXO28), as a new interaction partner of PFKFB4. We show that PFKFB4 regulates the ubiquitylation and subsequent proteasomal degradation of HIF-1α, which is mediated by the ubiquitin ligase activity of FBXO28. This newly discovered function of PFKFB4, coupled with its cancer specificity, provides a new strategy for inhibiting HIF-1α in cancer cells. ispartof: ONCOGENESIS vol:11 issue:1 ispartof: location:United States status: published

In a previous study, Protein Kinase C iota (PRKCI) emerged as an important candidate gene for glioblastoma (GBM) stem-like cell (GSC) survival. Here, we show that PKCι is overexpressed and activated in patient derived GSCs compared with normal neural stem cells and normal brain lysate, and that silencing of PRKCI in GSCs causes apoptosis, along with loss of clonogenicity and reduced proliferation. Notably, PRKCI silencing reduces tumor growth in vivo in a xenograft mouse model. PKCι has been intensively studied as a therapeutic target in non-small cell lung cancer, resulting in the identification of an inhibitor, aurothiomalate (ATM), which disrupts the PKCι/ERK signaling axis. However, we show that, although sensitive to pharmacological inhibition via a pseudosubstrate peptide inhibitor, GSCs are much less sensitive to ATM, suggesting that PKCι acts along a different signaling axis in GSCs. Gene expression profiling of PRKCI-silenced GSCs revealed a novel role of the Notch signaling pathway in PKCι mediated GSC survival. A proximity ligation assay showed that Notch1 and PKCι are in close proximity in GSCs. Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM.

In a previous study, Protein Kinase C iota (PRKCI) emerged as an important candidate gene for glioblastoma (GBM) stem-like cell (GSC) survival. Here, we show that PKCι is overexpressed and activated in patient derived GSCs compared with normal neural stem cells and normal brain lysate, and that silencing of PRKCI in GSCs causes apoptosis, along with loss of clonogenicity and reduced proliferation. Notably, PRKCI silencing reduces tumor growth in vivo in a xenograft mouse model. PKCι has been intensively studied as a therapeutic target in non-small cell lung cancer, resulting in the identification of an inhibitor, aurothiomalate (ATM), which disrupts the PKCι/ERK signaling axis. However, we show that, although sensitive to pharmacological inhibition via a pseudosubstrate peptide inhibitor, GSCs are much less sensitive to ATM, suggesting that PKCι acts along a different signaling axis in GSCs. Gene expression profiling of PRKCI-silenced GSCs revealed a novel role of the Notch signaling pathway in PKCι mediated GSC survival. A proximity ligation assay showed that Notch1 and PKCι are in close proximity in GSCs. Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM.

Glioblastoma is one of the most aggressive primary brain tumors in adults, with a dismal median overall survival of only 14 months after diagnosis. Glioblastoma stem-like cells (GSCs) are particularly resistant to current therapies, capable of self-renewal and tumour initiation and are hence thought to be major contributors to patient relapse. A kinome and phosphatome wide screen revealed glycolysis gene 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) as an important candidate gene for GSC survival. Notably, silencing PFKFB4 in an orthotopic xenograft mouse model of glioblastoma completely rid the mice of the tumor. Here we show a brand new function of PFKFB4, independent of its role in glycolysis - namely in the regulation of HIF1α, which is upregulated in GSCs. Gene expression profiling revealed a downregulation in HIF1α target genes in PFKFB4-silenced GSCs, and HIF1α protein levels are also dramatically reduced upon silencing. Mass spectrometric analysis of immunoprecipitated PFKFB4 protein revealed a novel interaction partner, F-box only protein 28 (FBXO28), an E3 ubiqutin ligase. Co-immunoprecipation assays show that FBXO28 forms an SCF multi-protein ubiquitin ligase complex with CUL1 and SKP1. Ubiquitylation studies of HIF1α show that PFKFB4 stabilizes this crucial protein in GSCs by preventing its targeting by FBXO28 for proteasomal degradation. These new findings, coupled with its cancer specific expression in a variety of tumor entities, makes PFKFB4 a compelling target. Citation Format: Emma L. Phillips, Frederic Bethke, Jörg Balss, Stefan Pusch, Stefan Christen, Martina Schnölzer, Antje Habel, David Capper, Andreas Von Deimling, Sarah-Maria Fendt, Peter Lichter, Violaine Goidts. PFKFB4, much more than just a glycolytic gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1906.