Radiation-induced extracellular vesicle (EV) release of miR-603 promotes IGF1-mediated stem cell state in glioblastomas.

BACKGROUND: Recurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. METHODS: microRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. FINDINGS: While nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. INTERPRETATION: Profiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. FUNDING: NIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).

A single-cell translocation and secretion assay (TransSeA).

Understanding biological heterogeneity at the single cell level is required for advancing insights into the complexity of human physiology and diseases. While advances in technological and analytical methods have afforded unprecedented glimpses of this heterogeneity, the information captured to date largely represents one-time "snap" shots of single cell physiology. To address the limits of existing methods and to accelerate discoveries from single cell studies, we developed a single-cell translocation and secretion assay (TransSeA) that supports time lapse analysis, enables molecular cargo analysis of secretions such as extracellular vesicles (EVs) from single cells, allows massively parallel single cell transfer according to user-defined cell selection criteria, and supports tracking of phenotypes between parental and progeny cells derived from single cells. To demonstrate the unique capabilities and efficiencies of the assay, we present unprecedented single cell studies related to cell secretions, EV cargos and cell intrinsic properties. Although used as examples to demonstrate the feasibility and versatility of the technology, the studies already provided insights into key unanswered questions such as the microRNAs carried by EVs, the relationships between EV secretion rates and gene expressions, and the spontaneous, trans-generational phenotypic changes in EV secretion between parental and progeny cells.

A cerebrospinal fluid microRNA signature as biomarker for glioblastoma.

PURPOSE: To develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma. EXPERIMENTAL DESIGN: Glioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArray® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts. RESULTS: miRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor "signature" consisting of nine miRNAs. The "signature" correlated with glioblastoma tumor volume (p=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the 'signature' for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF. CONCLUSION: We report a CSF miRNA signature as a "liquid biopsy" diagnostic platform for glioblastoma.

Differential localization of glioblastoma subtype: implications on glioblastoma pathogenesis.

INTRODUCTION: The subventricular zone (SVZ) has been implicated in the pathogenesis of glioblastoma. Whether molecular subtypes of glioblastoma arise from unique niches of the brain relative to the SVZ remains largely unknown. Here, we tested whether these subtypes of glioblastoma occupy distinct regions of the cerebrum and examined glioblastoma localization in relation to the SVZ. METHODS: Pre-operative MR images from 217 glioblastoma patients from The Cancer Imaging Archive were segmented automatically into contrast enhancing (CE) tumor volumes using Iterative Probabilistic Voxel Labeling (IPVL). Probabilistic maps of tumor location were generated for each subtype and distances were calculated from the centroid of CE tumor volumes to the SVZ. Glioblastomas that arose in a Genetically Modified Murine Model (GEMM) model were also analyzed with regard to SVZ distance and molecular subtype. RESULTS: Classical and mesenchymal glioblastomas were more diffusely distributed and located farther from the SVZ. In contrast, proneural and neural glioblastomas were more likely to be located in closer proximity to the SVZ. Moreover, in a GFAP-CreER; PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/- GEMM model of glioblastoma where tumor can spontaneously arise in different regions of the cerebrum, tumors that arose near the SVZ were more likely to be of proneural subtype (p < 0.0001). CONCLUSIONS: Glioblastoma subtypes occupy different regions of the brain and vary in proximity to the SVZ. These findings harbor implications pertaining to the pathogenesis of glioblastoma subtypes.

Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid.

BACKGROUND: Tumor specific genetic material can be detected in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids of glioblastoma patients. As such, EVs have emerged as a promising platform for biomarker discovery. However, the optimal procedure to transport clinical EV samples remains poorly characterized. METHODS: We examined the stability of EVs isolated from CSF of glioblastoma patients that were stored under different conditions. EV recovery was determined by Nanoparticle tracking analysis, and qRT-PCR was performed to determine the levels of miRNAs. RESULTS: CSF EVs that were lyophilized and stored at room temperature (RT) for seven days exhibited a 37-43% reduction in EV number. This reduction was further associated with decreased abundance of representative miRNAs. In contrast, the EV number and morphology remained largely unchanged if CSF were stored at RT. Total RNA and representative miRNA levels were well-preserved under this condition for up to seven days. A single cycle of freezing and thawing did not significantly alter EV number, morphology, RNA content, or miRNA levels. However, incremental decreases in these parameters were observed after two cycles of freezing and thawing. CONCLUSIONS: These results suggest that EVs in CSF are stable at RT for at least seven days. Repeated cycles of freezing/thawing should be avoided to minimize experimental artifacts.

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF).

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression.

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

miRNA contents of cerebrospinal fluid extracellular vesicles in glioblastoma patients.

Analysis of extracellular vesicles (EVs) derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of EVs in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in EVs derived from the serum and cerebrospinal fluid of glioblastoma patients. EVs were isolated from glioblastoma cell lines as well as the plasma and CSF of glioblastoma patients. The microvesicle subpopulation was isolated by pelleting at 10,000×g for 30 min after cellular debris was cleared by a 2000×g (20 min) spin. The exosome subpopulation was isolated by pelleting the microvesicle supernatant at 120,000×g (120 min). qRT-PCR was performed to examine the distribution of miR-21, miR-103, miR-24, and miR-125. Global miRNA profiling was performed in select glioblastoma CSF samples. In plasma and cell line derived EVs, the relative abundance of miRNAs in exosome and microvesicles were highly variable. In some specimens, the majority of the miRNA species were found in exosomes while in other, they were found in microvesicles. In contrast, CSF exosomes were enriched for miRNAs relative to CSF microvesicles. In CSF, there is an average of one molecule of miRNA per 150-25,000 EVs. Most EVs derived from clinical biofluids are devoid of miRNA content. The relative distribution of miRNA species in plasma exosomes or microvesicles is unpredictable. In contrast, CSF exosomes are the major EV compartment that harbor miRNAs.

RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas.

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

A genome-wide miRNA screen revealed miR-603 as a MGMT-regulating miRNA in glioblastomas.

MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs and characterized the top candidate, miR-603. Transfection of miR-603 suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. Moreover, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents.

MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development.

Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development.

miR-181d: a predictive glioblastoma biomarker that downregulates MGMT expression.

Genome-wide microRNA (miRNA) profiling of 82 glioblastomas demonstrated that miR-181d was inversely associated with patient overall survival after correcting for age, Karnofsky performance status, extent of resection, and temozolomide (TMZ) treatment. This association was validated using the Cancer Genome Atlas (TCGA) dataset (n= 424) and an independent cohort (n= 35). In these independent cohorts, an association of miR-181d with survival was evident in patients who underwent TMZ treatment but was not observed in patients without TMZ therapy. Bioinformatic analysis of potential genes regulated by miR-181d revealed methyl-guanine-methyl-transferase (MGMT) as a downstream target. Indeed, transfection of miR-181d downregulated MGMT mRNA and protein expression. Furthermore, luciferase reporter assays and coprecipitation studies showed a direct interaction between miR-181d and MGMT 3'UTR. The suppressive effect of miR-181d on MGMT expression was rescued by the introduction of an MGMT cDNA. Finally, MGMT expression inversely correlated with miR-181d expression in independent glioblastoma cohorts. Together, these results suggest that miR-181d is a predictive biomarker for TMZ response and that its role is mediated, in part, by posttranscriptional regulation of MGMT.

Regulation of γ-globin gene expression involves signaling through the p38 MAPK/CREB1 pathway.

In response to sodium butyrate and trichostatin A treatment in erythroid cells, p38 mitogen activated protein kinase (MAPK) mediates fetal hemoglobin (HbF) induction by activating cAMP response element binding protein 1 (CREB1). To expand on this observation, we completed studies to determine the role of p38 MAPK in steady-state γ-globin regulation. We propose that p38 signaling regulates Gγ-globin transcription during erythroid maturation through its downstream effector CREB1 which binds the Gγ-globin cAMP response element (G-CRE). We demonstrated that a loss of p38 or CREB1 function by siRNA knockdown resulted in target gene silencing. Moreover, gain of p38 or CREB1 function augments γ-globin transcription. These regulatory effects were conserved under physiological conditions tested in primary erythroid cells. When the G-CRE was mutated in a stable chromatin environment Gγ-globin promoter activity was nearly abolished. Furthermore, introduction of mutations in the G-CRE abolished Gγ-globin activation via p38 MAPK/CREB1 signaling. Chromatin immunoprecipitation assays (ChIP) demonstrated that CREB1 and its binding partner CREB binding protein (CBP) co-localize at the G-CRE region. These data support the role of p38 MAPK/CREB1 signaling in Gγ-globin gene transcription under steady-state conditions.

Post-transcriptional regulation of O(6)-methylguanine-DNA methyltransferase MGMT in glioblastomas.

BACKGROUND: The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) confers therapeutic resistance to DNA alkylating agents, including temozolomide. It is largely believed that MGMT promoter methylation is associated with down regulation of MGMT transcription and corresponding protein expression, thereby predisposing tumor cells to the toxic effect of temozolomide. Here we rigorously examined this underlying assumption. METHODS: We examined the correlation between MGMT promoter methylation, transcription, and protein expression using The Cancer Genome Atlas (TCGA) glioblastoma database as well as an independent collection of glioblastoma specimens. RESULTS: In both analyses, we found that MGMT promoter methylation status correlates well with low MGMT mRNA levels (p = 0.04). On the other hand, glioblastomas with unmethylated MGMT promoters exhibited a wide range of MGMT mRNA expression. Intriguingly, the MGMT mRNA levels correlated poorly with MGMT protein levels by Western blotting (R(2) = 0.04, p = 0.34) or by ImmunoHistoChemical (IHC) stain quantitation (R(2) = 0.02, p = 0.50). To exclude the possibility that the poor correlation was due to substandard specimens, we determined the mRNA and protein levels of Colony Stimulating Factor 1 (CSF1), a gene previously shown to exhibit excellent mRNA/protein correlation. In contrast to MGMT, the mRNA level of CSF1 correlated well with the protein level (R(2) = 0.47, p = 0.001). Importantly, long-term passaged glioblastoma cell lines with comparable MGMT transcript levels differed in MGMT protein levels, suggesting mechanisms of post-transcriptional regulation. Accordingly, the correlation between MGMT promoter methylation and MGMT protein expression was poor (p = 0.27). In silico analysis predicted potential binding sites for several miRNA within the 3'UTR of MGMT, suggesting a mechanism for the post-transcriptional of MGMT. CONCLUSION: Our results suggest mechanisms such as miRNA mediated regulation for post-transcriptional regulation of MGMT. Identification of these mechanisms should enhance the value of MGMT based prognostic or predictive biomarker strategies.

Histone deacetylase 9 activates gamma-globin gene expression in primary erythroid cells.

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of ß-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.

Identification of fetal hemoglobin-inducing agents using the human leukemia KU812 cell line.

Fetal hemoglobin (HbF) ameliorates the clinical severity of sickle cell disease; therefore continued research to identify efficacious HbF-inducing agents is desirable. In this study, we investigated KU812 leukemia cells that express the fetal γ-globin and adult ß-globin genes, as a system for screening and discovery of novel HbF inducers. KU812 cells were analyzed in the presence or absence of fetal bovine serum and then expression levels of the globin genes, cell surface markers and transcription factors were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). For comparison, primary erythroid cells were grown in a two-phase liquid culture system. After drug inductions for 48-72 h, globin mRNA and HbF levels were quantified by RT-qPCR and enzyme-linked immunosorbent assay, respectively. Erythroid markers and transcription factors expression levels in KU812 cells were comparable to days 7-14 erythroid cells. We also tested several drugs including butyrate, trichostatin A, scriptaid, suberoylanilide hydroxamic acid and hydroxyurea, which induced γ-globin in KU812 cells; however, some agents also induced ß-globin. A novel agent STI-571 was studied in the system, which non-selectively induced the globin genes. Additional studies showed comparable globin gene response patterns in KU812 and primary erythroid cells after treatments with the various drug inducers. Mechanisms of drug-mediated γ-globin induction in KU812 cells require signaling through the p38 mitogen-activated protein kinase pathway similar to that previously demonstrated in primary erythroid cells. These data suggest that KU812 cells serve as a good screening system to identify potential HbF inducers for the treatment of ß-hemoglobinopathies.