Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity.

Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.

PFKFB4 interacts with FBXO28 to promote HIF-1α signaling in glioblastoma.

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.

A novel patient stratification strategy to enhance the therapeutic efficacy of dasatinib in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal, and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, have failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. METHODS: We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. RESULTS: Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen-related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. CONCLUSION: This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

PERK-mediated expression of peptidylglycine α-amidating monooxygenase supports angiogenesis in glioblastoma.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor.

OBJECTIVE: Despite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM's genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton's tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach. METHODS: Twelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models. RESULTS: Kinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA-mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts. CONCLUSIONS: This study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM's resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.

A Dereplication and Bioguided Discovery Approach to Reveal New Compounds from a Marine-Derived Fungus Stilbella fimetaria.

A marine-derived Stilbella fimetaria fungal strain was screened for new bioactive compounds based on two different approaches: (i) bio-guided approach using cytotoxicity and antimicrobial bioassays; and (ii) dereplication based approach using liquid chromatography with both diode array detection and high resolution mass spectrometry. This led to the discovery of several bioactive compound families with different biosynthetic origins, including pimarane-type diterpenoids and hybrid polyketide-non ribosomal peptide derived compounds. Prefractionation before bioassay screening proved to be a great aid in the dereplication process, since separate fractions displaying different bioactivities allowed a quick tentative identification of known antimicrobial compounds and of potential new analogues. A new pimarane-type diterpene, myrocin F, was discovered in trace amounts and displayed cytotoxicity towards various cancer cell lines. Further media optimization led to increased production followed by the purification and bioactivity screening of several new and known pimarane-type diterpenoids. A known broad-spectrum antifungal compound, ilicicolin H, was purified along with two new analogues, hydroxyl-ilicicolin H and ilicicolin I, and their antifungal activity was evaluated.

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2.

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity-dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Targeting atypical protein kinase C iota reduces viability in glioblastoma stem-like cells via a notch signaling mechanism.

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.

Serine/Threonine Kinase MLK4 Determines Mesenchymal Identity in Glioma Stem Cells in an NF-κB-dependent Manner.

Activation of nuclear factor κB (NF-κB) induces mesenchymal (MES) transdifferentiation and radioresistance in glioma stem cells (GSCs), but molecular mechanisms for NF-κB activation in GSCs are currently unknown. Here, we report that mixed lineage kinase 4 (MLK4) is overexpressed in MES but not proneural (PN) GSCs. Silencing MLK4 suppresses self-renewal, motility, tumorigenesis, and radioresistance of MES GSCs via a loss of the MES signature. MLK4 binds and phosphorylates the NF-κB regulator IKKα, leading to activation of NF-κB signaling in GSCs. MLK4 expression is inversely correlated with patient prognosis in MES, but not PN high-grade gliomas. Collectively, our results uncover MLK4 as an upstream regulator of NF-κB signaling and a potential molecular target for the MES subtype of glioblastomas.

Retinoid resistance and multifaceted impairment of retinoic acid synthesis in glioblastoma.

Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies.

Kinome-wide shRNA screen identifies the receptor tyrosine kinase AXL as a key regulator for mesenchymal glioblastoma stem-like cells.

Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs) were recently identified: mesenchymal (MES) and proneural (PN). To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers.

Aberrant self-renewal and quiescence contribute to the aggressiveness of glioblastoma.

Cancer cells with enhanced self-renewal capacity influence tumour growth in glioblastoma. So far, a variety of surrogate markers have been proposed to enrich these cells, emphasizing the need to devise new characterization methods. Here, we screen a large panel of glioblastoma cultures (n = 21) cultivated under stem cell-permissive conditions and identify several cell lines with enhanced self-renewal capacity. These cell lines are capable of matrix-independent growth and form fast-growing, orthotopic tumours in mice. Employing isolation, re-plating, and label-retention techniques, we show that self-renewal potential of individual cells is partitioned asymmetrically between daughter cells in a robust and cell line-specific fashion. This yields populations of fast- and slow-cycling cells, which differ in the expression of cell cycle-associated transcripts. Intriguingly, fast-growing cells keep their slow-cycling counterparts in a reversible state of quiescence associated with high chemoresistance. Our results suggest that two different subpopulations of tumour cells contribute to aberrant growth and tumour recurrence after therapy in glioblastoma.

Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells.

The concept of cancer stem cells has gained considerable interest in the last few decades, partly because of their potential implication in therapy resistance. However, the lack of specific cellular surface markers for these cells has impeded their isolation, making the characterization of this cellular subpopulation technically challenging. Recent studies have indicated that leucine-rich repeat-containing G-protein-coupled receptor 4 and 5 (LGR4 and LGR5) expression in multiple organs may represent a global marker of adult stem cells. This review aims to give an overview of LGR4 and LGR5 as cancer stem cell markers and their function in development.

Functional characterization of ENPP1 reveals a link between cell cycle progression and stem-like phenotype in glioblastoma.

A high-throughput phenotypic screen in glioblastoma stem-like cells (GSCs) identified a novel molecular mechanism in which ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) plays an important role in balancing the pool of nucleotides, thus maintaining GSCs in an undifferentiated proliferative state. This finding highlights the connection between cell cycle length and the stem-like tumor state.

LGR5 is a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells.

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem-like cells (CSCs), characterized by stem cell marker expression, capability of differentiation and self-renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co-immunofluorescence staining demonstrated the co-localization of CD133- and LGR5-positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA-mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs.

The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis.

Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.

Differentiation therapy exerts antitumor effects on stem-like glioma cells.

PURPOSE: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid-induced differentiation to target the stem-like tumor cell pool could have therapeutic implications. EXPERIMENTAL DESIGN: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid-containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model. RESULTS: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo. CONCLUSIONS: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma.