Marco Mineo

Extracellular Vesicles Modulate the Glioblastoma Microenvironment via a Tumor Suppression Signaling Network Directed by miR-1

Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem-like glioblastoma cells. An extracellular vesicle-dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle-derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management.

Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles

We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood‐brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well‐known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain β1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity

A lack of experimental models of tumor heterogeneity limits our knowledge of the complex subpopulation dynamics within the tumor ecosystem. In high-grade gliomas (HGG), distinct hierarchical cell populations arise from different glioma stem-like cell (GSC) subpopulations. Extracellular vesicles (EV) shed by cells may serve as conduits of genetic and signaling communications; however, little is known about how HGG heterogeneity may impact EV content and activity. In this study, we performed a proteomic analysis of EVs isolated from patient-derived GSC of either proneural or mesenchymal subtypes. EV signatures were heterogeneous, but reflected the molecular make-up of the GSC and consistently clustered into the two subtypes. EV-borne protein cargos transferred between proneural and mesenchymal GSC increased protumorigenic behaviors in vitro and in vivo Clinically, analyses of HGG patient data from the The Cancer Genome Atlas database revealed that proneural tumors with mesenchymal EV signatures or mesenchymal tumors with proneural EV signatures were both associated with worse outcomes, suggesting influences by the proportion of tumor cells of varying subtypes in tumors. Collectively, our findings illuminate the heterogeneity among tumor EVs and the complexity of HGG heterogeneity, which these EVs help to maintain. Cancer Res; 76(10); 2876-81. ©2016 AACR.

MicroRNA and extracellular vesicles in glioblastoma: small but powerful

To promote the tumor growth, angiogenesis, metabolism, and invasion, glioblastoma (GBM) cells subvert the surrounding microenvironment by influencing the endogenous activity of other brain cells including endothelial cells, macrophages, astrocytes, and microglia. Large number of studies indicates that the intra-cellular communication between the different cell types of the GBM microenvironment occurs through the functional transfer of oncogenic components such as proteins, non-coding RNAs, DNA and lipids via the release and uptake of extracellular vesicles (EVs). Unlike the communication through the secretion of chemokines and cytokines, the transfer and gene silencing activity of microRNAs through EVs is more complex as the biogenesis and proper packaging of microRNAs is crucial for their uptake by recipient cells. Although the specific mechanism of EV-derived microRNA uptake and processing in recipient cells is largely unknown, the screening, identifying and finally targeting of the EV-associated pro-tumorigenic microRNAs are emerging as new therapeutic strategy to combat the GBM.

MicroRNA Signatures and Molecular Subtypes of Glioblastoma: The Role of Extracellular Transfer

Summary Despite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes.

Abstract 2032: A novel function of WW domain binding protein 2 (WBP2) in regulating cytoskeletal function and cellular division through binding to co-chaperone BAG3

BAG3, a BAG co-chaperone family member, co-localizes with actin microfilaments and influences processes that involve actin function, including adhesion and migration. Because actin function regulates cell division, we hypothesized that the BAG3 WW-domain regulates cellular division through its cytoskeletal association. We show that BAG3 co-localizes with leading lamellipodia exclusively during G2 and mitosis phases. Co-immunoprecipitation of BAG3 and pulldown of GST-tagged BAG3 WW-domain confirmed that BAG3 WW-domain association with actin increased during G2 and mitosis relative to asynchronous cells. Deletion of the BAG3 WW-domain caused an abnormal cellular morphology, degenerate F-actin organization, longer doubling time (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2032. doi:1538-7445.AM2012-2032

Abstract PR04: The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma

Purpose: While multiple protein-coding genes are known to play a crucial role in the formation and progression of glioblastoma multiforme (GBM), the role of long-non-coding RNAs (lncRNAs) in these cascades remains to be fully characterized. Considering the fundamental roles of hypoxia in the cellular and microenvironmental complexity and unexplored function of lncRNAs in GBM pathobiology, the identification of novel lncRNAs and their target pathways that drive the adaptation to hypoxic niche is crucial for better understanding of the development and progression of this highly heterogeneous brain tumor. Transcriptome profiling of GBM have revealed the presence of four clearly distinguishable subtypes, variably expressed in individual cells within a tumor. This finding may have potential clinical implications, including subtype-specific rearrangements of transcriptional programs related to oncogenic signaling, growth and hypoxia. However, the subtype-specific role of lncRNAs in tumorigenic potential of GBM subtypes remains largely unknown. Materials and Methods: The brain tissue samples including GBM tumors and non-pathological tissue adjacent to the tumor were collected. GBM-derived primary stem cells (GSCs) classified by transcriptome analysis as proneural (P) and mesenchymal (M), were collected and exposed to hypoxic conditions. Using custom designed Nanostring platform analysis followed by qPCR, the expression patterns of 70 cancer-related lncRNAs were characterized. The in situ hybridization and qPCR analysis was used to validate sub-cellular localization of selected lncRNA. RNA immunoprecipitation (RIP) and mass-spectroscopy (MS) was performed to map RNA-protein interaction and identified targets were validated by Western blotting. The global GSC transcriptome profiling upon lncRNA de-regulation was performed using Pan Cancer Nanostring platform. The knock-down and overexpression strategies in GSC were used to characterize cellular and molecular phenotypes in vitro and in in vivo intracranial GBM model. Results: We identified lncRNA HIF1A-AS2 (Hypoxia Inducible Factor 1 alpha - antisense 2) as one of the most deregulated in GBM comparing to the matched adjacent tissue. Despite lack of difference in the hypoxia-dependent activation of HIF1A protein between P and M GSC, HIF1A-AS2 was specifically upregulated in M GSC in vitro and in vivo. The deregulation of expression of HIF1A-AS2 had a broad effect on autophagy-related signaling network regulated in response to hypoxia in M but not P GSC. The IGF2BP2 and DHX9 were identified as direct protein partners of this lncRNA. The deregulation of HIF1A-AS2 affected the recruitment of IGF2BP2 to its mRNA targets (e.g. HMG1) and stability of DHX9 protein (but not mRNA), resulting in deregulation of its target genes (e.g FOSL1). Downregulation of HIF1A-AS2 in vivo led to de-regulation of its downstream effectors and resulted in survival benefit of mice bearing M GSC-originated tumors. Conclusion: P and M GSC respond differently to hypoxic stress by induction of autophagy to maintain their homeostasis and viability and this mechanism is controlled by HIF1A-AS2. This abstract is also presented as Poster A16. Citation Format: Marco Mineo, Franz Ricklefs, Shawn S. Lyons, Pavel Ivanov, E. Antonio Chiocca, Jakub Godlewski, Agnieszka Bronisz. The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR04.

Abstract 1929: The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex

Heterogeneous glioblastoma multiforme (GBM) was categorized based on transcriptional signatures into four subtypes (proneural (PN), neural, classical, and mesenchymal (MES)). In order to develop effective targeted therapeutic strategies, understanding the heterogeneous gene expression and molecular features of these subtypes is crucial. De-regulation of microRNA expression and activity has been shown to play an important role in tumor initiation and progression, including gliomagenesis. We have previously reported that expression of microRNA-128 (miR-128) is significantly down regulated in GBM and it diminishes self-renewal of GBM stem-like cells (GSCs) and sensitizes them to irradiation. Proneural-to-mesenchymal transition (PMT) manifested by concomitant up regulation of MES markers and down regulation of PN markers, is associated with increased malignancy, therapy-resistance and worse prognosis, but the underlying causes of PMT have not been convincingly characterized yet. In this study, we have demonstrated that miR-128 can regulate the PMT in GSC subsets. We showed that the expression of miR-128 in PN GSCs was significantly higher compared to MES subtype. As a tumor suppressive microRNA, miR-128 inhibited the MES GSC-specific high expression of Bmi1 and Suz12, two components of Polycomb Repressor Complexes (PRC) 1 and 2, respectively. In both GSC subtypes, miR-128 driven targeting of PRCs suppressed their epigenetic activity measured by ubiquitination of H2AK119 and tri-methylation of H3K27. Stable down regulation of miR-128 in PN GSCs significantly increased the expression of MES-specific gene signature (BCL2A1, CD44, WT-1, LYN, and MET) while its stable up regulation in MES GSCs resulted in the restoration of PN specific gene signature (CD133, SOX2, NES, OLIG2, and NOTCH1). We also showed that stable expression of miR-128 in GSCs could regulate the process of irradiation-induced PMT. Our in vivo studies showed the anti-tumorigenic role of miR-128 in both PN and MES GSC-derived intracranial tumor models. Taken together, we demonstrated that altering levels of miR-128 was sufficient to cause or reverse PMT, most likely by targeting the level/functions of PRCs and their target genes in GBM. Citation Format: Arun K. Rooj, Marco Mineo, Franz Ricklefs, Agnieszka Bronisz, Ennio Chiocca, Jakub Godlewski. The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1929.

Abstract 1000: The long non-coding RNA HIF1A-AS2 regulates mesenchymal glioma stem cell tumorigenicity

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults, which initiation and progression is driven by a subset of self-renewing GBM stem-like cells (GSCs). Long-non coding RNAs (lncRNAs) have been recently shown to play important roles in regulating numerous biological processes both in physiologic and pathologic condition. Identification of functional lncRNAs important for GBM initiation and progression may shed new light on understanding pathophysiology of the disease. We used a custom made lncRNA Nanostring platform to profile the expression of lncRNAs in subtype-characterized collection of patient-derived GSCs. We demonstrated that lncRNA signature may distinguish between GSC subtypes. Out of 73 lncRNAs we found 7 that were overexpressed specifically in the most aggressive mesenchymal (M) GSC subtype. Among them, HIF1A-AS2 was the most differentially expressed lncRNA. HIF1A-AS2 was reported to be overexpressed in many types of cancers; however its biological function and its role in GBM progression are unknown. Knockdown of HIF1A-AS2 in M GSCs resulted in reduced growth, increased cytotoxicity, and it strongly inhibited their neurosphere formation capability. Using more global approach we found out that knockdown of HIF1A-AS2 in M GSCs caused deregulation of several out of 730 cancer-related genes. Functional bioinformatic analysis revealed that these differentially expressed mRNAs are closely related to proliferation, transcriptional regulation and cell division. RNA pull-down assay showed that HIF1A-AS2 may exert its effects through specific binding of RNA helicase DHX9, a multifunctional protein with important roles in transcription, pre-mRNA processing and translation. We also demonstrated that HMGA1, a gene known to be regulated by DHX9, was specifically down-regulated in HIF1A-AS2 knockdown cells both at mRNA and protein level. Finally, we showed that silencing of HIF1A-AS2 blocked M GSC tumor growth in vivo resulting in significant survival benefits. Taken together, our results suggest HIF1A-AS2 as an important lncRNA in pathophysiology of GBM. Citation Format: Marco Mineo, Franz Ricklefs, Arun Rooj, Shawn M. Lyons, Pavel Ivanov, Ennio A. Chiocca, Jakub Godlewski, Agnieszka Bronisz. The long non-coding RNA HIF1A-AS2 regulates mesenchymal glioma stem cell tumorigenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1000.

Abstract 4372: Chronic myeloid leukemia (CML) exosomes promote angiogenesis in a Src-dependent fashion in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL CML is an uncontrolled proliferation of bone marrow myeloid cells driven by the constitutively active fusion product tyrosine kinase BCR/ABL. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is newly recognized as a factor in CML progression. Exosomes, released by a broad spectrum of cells, are microvesicles that play an important role in cell-to-cell communication both in physiological and pathological conditions. The role of exosomes released by CML cells in angiogenesis is emerging; however, little is known about the mechanisms involved in this process. We first isolated and characterized exosomes released by K562 CML cells and we demonstrated their ability to stimulate human vascular endothelial cells (HUVECs) tube differentiation on Matrigel. K562 exosomes induced an increase of the cumulative tube length in a dose-dependent manner, with a maximum effect at 10ug/ml (p=0.003). Next, we evaluated the effect on exosome behavior of imatinib and dasatinib, two tyrosine kinase inhibitors in use in CML treatment. K562 CML cell treatment with either imatinib or dasatinib reduced exosome release by 58% and 56%, respectively (p<0.01). Equivalent exosome concentrations isolated from imatinib or dasatinib treated K562 cells maintained their ability to stimulate HUVEC tube formation, compared to those isolated from untreated cells. Dasatinib treatment of HUVECs strongly reduced exosome-induced vascular differentiation (p=0.0002). On the contrary, little effect was observed following treatment with imatinib. K562 exosomes stimulated vascularization when added to Matrigel plug in vivo; angiogenesis was markedly inhibited by oral administration of dasatinib (p<0.01), but not imatinib. Consistent with the differential effects of dasatinib and imatinib, exosome-induced HUVEC Src and FAK phosphorylation was only inhibited by dasatinib. Confocal images showed that both FAK and Src phosphorylation were increased at points of membrane-matrix contact. Immunoblot analysis confirmed that K562 exosomes induced a dasatinib-sensitive phosphorylation of Src and FAK and their downstream effectors, Erk and Akt. Again, imatinib was minimally active against exosome stimulation of HUVEC cell signaling. Thus, K562 CML exosomes stimulate angiogenesis in vitro and in vivo in a dasatinib-sensitive fashion. This credentials exosomes and angiogenesis as molecular targets in CML via activation of Src both in leukemia and its microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4372. doi:1538-7445.AM2012-4372