Mesenchymal Glioma Stem Cells Research Articles

Mesenchymal glioma stem cells trigger vasectasia—distinct neovascularization process stimulated by extracellular vesicles carrying EGFR

Targeting neovascularization in glioblastoma (GBM) is hampered by poor understanding of the underlying mechanisms and unclear linkages to tumour molecular landscapes. Here we report that different molecular subtypes of human glioma stem cells (GSC) trigger distinct endothelial responses involving either angiogenic or circumferential vascular growth (vasectasia). The latter process is selectively triggered by mesenchymal (but not proneural) GSCs and is mediated by a subset of extracellular vesicles (EVs) able to transfer EGFR/EGFRvIII transcript to endothelial cells. Inhibition of the expression and phosphorylation of EGFR in endothelial cells, either pharmacologically (Dacomitinib) or genetically (gene editing), abolishes their EV responses in vitro and disrupts vasectasia in vivo. Therapeutic inhibition of EGFR markedly extends anticancer effects of VEGF blockade in mice, coupled with abrogation of vasectasia and prolonged survival. Thus, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new therapeutic target in a subset of GBMs.

E2F1-regulated USP5 contributes to the tumorigenic capacity of glioma stem cells through the maintenance of OCT4 stability

Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a criticial factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM.

Exploring the Influence of tGLI-1 on Temozolomide Resistance in Glioblastomas: Unraveling Novel Therapeutic Targets

Background/Objective:Temozolomide (TMZ) is a standard chemotherapy treatment for patients with glioblastoma (GBM), but its effectiveness is limited, with only 50% of patients initially responding and developing resistance over time. Glioma stem cells (GSCs) have been implicated in TMZ resistance, particularly the mesenchymal subtype. The truncated form of GLI1, known as tGLI1, is highly expressed in mesenchymal GSCs and has been associated with poor patient outcomes in GBM. However, the role of tGLI1 in TMZ resistance remains unknown.
 Methods:The GBM cell line, U87MG, was utilized for this study. The IC50 of TMZ was determined using a cell viability assay. After successful transfection with vector, GLI1, and tGLI1, the cells were treated with the IC50 of TMZ to assess changes in cell viability between the groups.
 Results:The IC50 of TMZ is 290.1 μM, as averaged between replicate assays. Thus far, the results showed that tGLI1-expressing cells exhibited significantly higher cell viability (average: 39.09%) compared to Vector (average: 26.78%) and GLI1 (average: 27.11%). However, the tGLI1 group displayed higher variability in cell viability results, as evidenced by a larger standard deviation (0.2758) and standard error (0.1592) compared to vector (SD: 0.0325, SE: 0.0188) and GLI1 (SD: 0.1354, SE: 0.0781). The One-Way ANOVA, followed by Tukey's Multiple Comparison Test, results showed no statistically significant differences in cell viability between the groups.
 Conclusion/Impact:The increased cell viability observed in tGLI1-expressing cells suggests a potential association between tGLI1 and TMZ resistance, warranting additional research to fully comprehend its impact on GBM treatment response. Further investigation and replication studies are needed to establish the robustness of these results. This knowledge may contribute to the development oftargeted therapies aimed at inhibiting tGLI1 or its downstream signaling pathways, potentiallyimproving the response to TMZ and patient outcomes.

SPI1 activates TGF-β1/PI3K/Akt signaling through transcriptional upregulation of FKBP12 to support the mesenchymal phenotype of glioma stem cells.

Glioma stem cells (GSCs) exhibit diverse molecular subtypes with the mesenchymal (MES) population representing the most malignant variant. The oncogenic potential of Salmonella pathogenicity island 1 (SPI1), an oncogenic transcription factor, has been established across various human malignancies. In this study, we explored the association between the SPI1 pathway and the MES GSC phenotype. Through comprehensive analysis of the Cancer Genome Atlas and Chinese Glioma Genome Atlas glioma databases, along with patient-derived GSC cultures, we analyzed SPI1 expression. Using genetic knockdown and overexpression techniques, we assessed the functional impact of SPI1 on GSC MES marker expression, invasion, proliferation, self-renewal, and sensitivity to radiation in vitro, as well as its influence on tumor formation in vivo. Additionally, we investigated the downstream signaling cascades activated by SPI1. Our findings revealed a positive correlation between elevated SPI1 expression and the MES phenotype, which in turn, correlated with poor survival. SPI1 enhanced GSC MES differentiation, self-renewal, and radioresistance in vitro, promoting tumorigenicity in vivo. Mechanistically, SPI1 augmented the transcriptional activity of both TGF-β1 and FKBP12 while activating the non-canonical PI3K/Akt pathway. Notably, inhibition of TGF-β1/PI3K/Akt signaling partially attenuated SPI1-induced GSC MES differentiation and its associated malignant phenotype. Collectively, our results underscore SPI1's role in activating TGF-β1/PI3K/Akt signaling through transcriptional upregulation of FKBP12, thereby supporting the aggressive MES phenotype of GSCs. Therefore, SPI1 emerges as a potential therapeutic target in glioma treatment.

SPI1-mediated MIR222HG transcription promotes proneural-to-mesenchymal transition of glioma stem cells and immunosuppressive polarization of macrophages

Background: Glioma stem cells (GSCs) are a key factor in glioblastoma (GBM) development and treatment resistance. GSCs can be divided into the mesenchymal (MES) and proneural (PN) subtypes, and these two subtypes of GSCs can undergo interconversion under certain conditions. MES GSCs have higher malignancy and radioresistance and are closely associated with an immunosuppressive microenvironment. Long noncoding RNAs (lncRNAs) play a broad role in GBM, while the role of GSCs subtype remains unknown. Methods: We performed RNA sequencing to explore the lncRNA expression profile in MES- and PN-subtype GBM tissues. The biological function of a host gene-MIR222HG-in GBM development was confirmed in vitro and in vivo. Specifically, RNA sequencing, RNA pulldown, mass spectrometry, RIP, ChIP, luciferase reporter assays and Co-IP were performed. Results: MIR222HG, the expression of which can be induced by SPI1, has high levels in MES GBM tissues. Functionally, we demonstrated that MIR222HG promotes the MES transition and radioresistance in GSCs in vivo and in vitro. Mechanistically, MIR222HG can bind to the YWHAE/HDAC5 complex to promote the MES transition of GSCs through H4 deacetylation. Moreover, cotranscribed miR221 and miR222 can be delivered to macrophages via exosomes to target SOCS3, causing immunosuppressive polarization. Finally, PLX-4720 sensitivity is associated with SPI1 expression and acts on MES GSCs to enhance radiosensitivity. Conclusions: This study demonstrates that targeting SPI1 to block transcription of the MIR222HG cluster helps to reduce radioresistance and combat the immunosuppressive microenvironment in GBM. PLX-4720 is a potential GBM drug and radiosensitizer.

Cell-Population Dynamics in Diffuse Gliomas during Gliomagenesis and Its Impact on Patient Survival

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations' content in diffuse gliomas of different molecular profiles and grades, considering the cell populations' functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells' proportion and mesenchymal cell populations' appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells' hypoxia response programs' activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs' activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas' pathogenetic tracing up to the practical plane.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.

Mesenchymal and Proneural Subtypes of Glioblastoma Disclose Branching Based on GSC Associated Signature.

Glioblastomas (GBM)—the most common, therapy-resistant, and lethal tumors driven by populations of glioma stem cells (GSCs) are still on the list of the most complicated pathologies. Thus, deeper understanding and characterization of GSCs is indispensable to find suitable targets and develop more effective therapies. In the present study, we applied native glioblastoma cells and GSCs sequencing, screened for GSC-specific targets and checked if the signature is related to GBM patient pathological, clinical data as well as molecular subtypes applying TCGA cohort. Data analysis revealed that tumors of proneural and mesenchymal subtypes are branching in separate clusters based on screened gene expression. Samples of the same subtype revealed significantly different patient survival prognosis as well as recurrence chance between the clusters. Recently, different subpopulations of mesenchymal GSC demonstrating different properties were shown, which indicates possible internal heterogeneity of GBM subtypes as well. Current findings also revealed branching of molecular GBM subtypes that were significantly linked to patient outcome and that might be decided by distinct GSC subpopulations.

TAMI-15. THE EFFECT OF INTERSTITIAL FLUID FLOW AND ASTROCYTES / MICROGLIA ON INVASION, PROLIFERATION AND STEMNESS OF PATIENT-DERIVED GLIOMA STEM CELLS

Abstract SIGNIFICANCE Glioblastoma is a highly infiltrative, malignant, and deadly glioma that can be classified into subtypes based on molecular classification. Treatment resistant glioma stem cells (GSCs) depend on the tumor microenvironment (TME) to drive recurrence. Cellular composition and interstitial fluid flow (IFF) are significant aspects of the TME. IFF and astrocyte and microglia (A+M) presence have independently been shown to mediate invasion. This study’s goal is to expand our knowledge of IFF and A+M effects on invasion to proliferation and stemness. METHODS Seven patient-derived GSC lines were tested in an in vitro 3D model, which consists of GSCs ± A+M resuspended in 0.2% hyaluronan / 0.12% rat tail collagen I gel. The gel was applied to an 8um pore 96-well transwell system. Flow and static conditions were modeled with and without a pressure head above the gel, respectively. Cells beyond the transwell membrane after 18 hrs of incubation were considered invaded. Stemness and proliferation were determined via flow cytometry for CD71 and Ki67, respectively. RESULTS/CONCLUSIONS The three mesenchymal GSC lines tested exhibited the largest IFF fold increases in stemness, proliferation, and invasion with averages of 23.9, 19.1, and 2.1, respectively. CD44+ cell populations, highest in mesenchymal cells, had a strong correlation with proliferation (R=0.8439) and stemness (R=0.7829) under flow. Furthermore, depending on the cell line/subtype, the addition of A+M either amplified, reduced, reversed, mitigated, or kept constant the effect of IFF on invasion and proliferation. Incorporating A+M never amplified the effect of IFF on stemness. Adding A+M had a strong effect on the IFF fold change of at least one parameter in six of the cell lines. This is the first presentation showing that IFF, patient-specific, and context-specific factors contribute to both increased proliferation, and maintenance of stem-like phenotypes in glioma.

Proneural and mesenchymal glioma stem cells display major differences in splicing and lncRNA profiles

Therapy resistance and recurrence in high-grade gliomas are driven by their populations of glioma stem cells (GSCs). Thus, detailed molecular characterization of GSCs is needed to develop more effective therapies. We conducted a study to identify differences in the splicing profile and expression of long non-coding RNAs in proneural and mesenchymal GSC cell lines. Genes related to cell cycle, DNA repair, cilium assembly, and splicing showed the most differences between GSC subgroups. We also identified genes distinctly associated with survival among patients of mesenchymal or proneural subgroups. We determined that multiple long non-coding RNAs with increased expression in mesenchymal GSCs are associated with poor survival of glioblastoma patients. In summary, our study established critical differences between proneural and mesenchymal GSCs in splicing profiles and expression of long non-coding RNA. These splicing isoforms and lncRNA signatures may contribute to the uniqueness of GSC subgroups, thus contributing to cancer phenotypes and explaining differences in therapeutic responses.

Different Cytokine Dependency of Proneural to Mesenchymal Glioma Stem Cell Transition in Tumor Microenvironments

Different Cytokine Dependency of Proneural to Mesenchymal Glioma Stem Cell Transition in Tumor Microenvironments

Molecular subtypes and differentiation programmes of glioma stem cells as determinants of extracellular vesicle profiles and endothelial cell-stimulating activities

ABSTRACTWe have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells.Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting

Abstract 3342: Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44

Abstract The subpopulation of stem-like cells, or glioma stem cells (GSCs), is a key driver of tumor initiation, recurrence, and chemoresistance. Recently, patient-derived GSC lines were identified to contain two mutually exclusive subtypes termed proneural and mesenchymal. Mesenchymal GBM and GSCs were found to be more aggressive and radioresistant; however, the molecular pathways supporting mesenchymal GBM and mesenchymal GSCs are not well understood. To address this knowledge gap, we examined truncated glioma-associated oncogene homolog 1 (tGLI1) for its role in mesenchymal subtypes of GBM and GSCs. Discovered in our laboratory, tGLI1 is a novel alternatively spliced, gain-of-function variant of GLI1 zinc-finger transcription factor with an in-frame deletion of 41 codons retains all of the known functional domains of GLI1, but gains the ability to activate genes not regulated by GLI1. In this study, we report for the first time that tGLI1 is a tumor-specific transcription factor that facilitates GBM growth, enriches in the mesenchymal subtype of GBM and GSCs, and promotes mesenchymal GSCs via transcriptionally activating CD44 expression. Using an orthotopic GBM xenograft mouse model, we observed that tGLI1-overexpressing tumors grew more aggressively, with increased proliferation and angiogenesis, compared to control and GLI1-overexpressing xenografts. IHC analysis using tGLI1- and GLI1-selective antibodies we developed showed that tGLI1 was significantly expressed in GBM specimens but undetectable in normal brains whereas GLI1 was expressed in both tissues. Datamining further showed that tGLI1 activation signature (tGAS) was correlated with glioma grade, tumor angiogenesis, and poor overall survival, and that GBMs with high tGAS were enriched with mesenchymal GBM and GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared to the monolayer culture. Mesenchymal GSCs expressed more tGLI1 than proneural GSCs. Ectopic tGLI1 expression significantly enhanced the ability of GSCs to yield neurospheres in vitro and form aggressive tumors in mouse brains. Finally, we examined the ability of tGLI1 to regulate stemness genes and found that tGLI1 binds to and transactivates the promoter of the CD44 gene, a marker and mediator for mesenchymal GSCs. Collectively, these findings advanced our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSCs. Citation Format: Tadas K. Rimkus. Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44 [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 3342.

Abstract 2004: Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer

Abstract Cancer stem cells (CSCs) are a small population of cancer cells that are capable of self-renewal and tumor initiation. As a result, targeting CSCs is a potential therapeutic strategy for preventing cancer relapse and metastasis. Mixed lineage kinase 4 (MLK4), a serine/threonine kinase, is known to regulate mesenchymal glioma stem cells and to drive tumorigenesis in colorectal cancer. We found that MLK4 was highly expressed in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes according to the database from The Cancer Genome Atlas. Furthermore, multiple datasets indicated that higher expression of MLK4 was associated with poor prognosis in breast cancer. Despite the correlation of MLK4 and clinical outcome, the function of MLK4 in breast cancer is still not known. In this study, we found that knockdown of MLK4 significantly decreased secondary mammosphere formation, the CD44+/CD24- CSC population, invasion and migration in TNBC cell lines. In addition to suppressing CSC phenotypes in vitro, knockdown of MLK4 also inhibited tumor growth in NOD/SCID mice. Secondary transplantation of tumor cells demonstrated that silencing of MLK4 significantly decreased tumor-initiating cell frequency. As accessed by qPCR, knockdown of MLK4 led to decreased expression of mesenchymal genes (CD44 and VIM) and concomitant increased expression of epithelial genes (CDH1 and claudin genes). Immunohistochemical staining revealed that knockdown of MLK4 in xenografts resulted in increased expression of CK19, a luminal-epithelial marker. These studies indicate that MLK4 plays an important role in the function of mesenchymal-like CSCs in TNBC. Silencing of MLK4 in TNBC cell lines can transform CSC in these tumors to a more differentiated epithelial cell state. Together, these studies identify that MLK4 is a potential therapeutic target for CSCs in TNBC. Citation Format: Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Mari Gasparyan, Max Wicha, Duxin Sun. Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer [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 2004.

Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44.

The molecular pathways driving mesenchymal glioblastoma (GBM) are still not well understood. We report here that truncated glioma-associated oncogene homolog 1 (tGLI1) is a tumor-specific transcription factor that facilitates GBM growth, is enriched in the mesenchymal subtype of GBM and glioma stem cells (GSC), and promotes mesenchymal GSC by upregulating transcription of CD44. In an orthotopic GBM xenograft mouse model, tGLI1-overexpressing tumors grew more aggressively with increased proliferation and angiogenesis compared with control and GLI1-overexpressing xenografts. tGLI1 was highly expressed in GBM clinical specimens but undetectable in normal brains, whereas GLI1 was expressed in both tissues. A tGLI1 activation signature (tGAS) correlated with glioma grade, tumor angiogenesis, and poor overall survival, and GBMs with high tGAS were enriched with mesenchymal GBM/GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared with the monolayer culture; mesenchymal GSC expressed more tGLI1 than proneural GSC. Ectopic tGLI1 expression enhanced the ability of mesenchymal GSC to yield neurospheres in vitro and to form tumors in mouse brains. Selective tGLI1 knockdown reduced neurosphere formation of GBM cells. tGLI1 bound to and transactivated the promoter of the CD44 gene, a marker and mediator for mesenchymal GSC, leading to its expression. Collectively, these findings advance our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSC.Significance: These findings highlight the role of a tumor-specific gain-of-function transcription factor tGLI1 in mesenchymal glioma stem cell maintenance and mesenchymal GBM growth. Cancer Res; 78(10); 2589-600. ©2018 AACR.

Targeting glioma stem cells through combined BMI1 and EZH2 inhibition.

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile and hypoxic regions a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress, due to downregulation of the E3 ligase, RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are preferentially sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be necessary to overcome resistance to therapies caused by intratumoral heterogeneity.

Divergent evolution of temozolomide resistance in glioblastoma stem cells is reflected in extracellular vesicles and coupled with radiosensitization.

Glioblastoma (GBM) is almost invariably fatal due to failure of standard therapy. The relapse of GBM following surgery, radiation, and systemic temozolomide (TMZ) is attributed to the ability of glioma stem cells (GSCs) to survive, evolve, and repopulate the tumor mass, events on which therapy exerts a poorly understood influence. Here we explore the molecular and cellular evolution of TMZ resistance as it emerges in vivo (xenograft models) in a series of human GSCs with either proneural (PN) or mesenchymal (MES) molecular characteristics. We observed that the initial response of GSC-initiated intracranial xenografts to TMZ is eventually replaced by refractory growth pattern. Individual tumors derived from the same isogenic GSC line expressed divergent and complex profiles of TMZ resistance markers, with a minor representation of O6-methylguanine DNA methyltransferase (MGMT) upregulation. In several independent TMZ-resistant tumors originating from MES GSCs we observed a consistent diminution of mesenchymal features, which persisted in cell culture and correlated with increased expression of Nestin, decline in transglutaminase 2 and sensitivity to radiation. The corresponding mRNA expression profiles reflective of TMZ resistance and stem cell phenotype were recapitulated in the transcriptome of exosome-like extracellular vesicles (EVs) released by GSCs into the culture medium. Intrinsic changes in the tumor-initiating cell compartment may include loss of subtype characteristics and reciprocal alterations in sensitivity to chemo- and radiation therapy. These observations suggest that exploiting therapy-induced changes in the GSC phenotype and alternating cycles of therapy may be explored to improve GBM outcomes.

The stem cell/cancer stem cell marker ALDH1A3 regulates the expression of the survival factor tissue transglutaminase, in mesenchymal glioma stem cells.

Tissue transglutaminase (tTG), a dual-function enzyme with GTP-binding and acyltransferase activities, has been implicated in the survival and chemotherapy resistance of aggressive cancer cells and cancer stem cells, including glioma stem cells (GSCs). Using a model system comprising two distinct subtypes of GSCs referred to as proneural (PN) and mesenchymal (MES), we find that the phenotypically aggressive and radiation therapy-resistant MES GSCs exclusively express tTG relative to PN GSCs. As such, the self-renewal, proliferation, and survival of these cells was sensitive to treatment with tTG inhibitors, with a benefit being observed when combined with the standard of care for high grade gliomas (i.e. radiation or temozolomide). Efforts to understand the molecular drivers of tTG expression in MES GSCs revealed an unexpected link between tTG and a common marker for stem cells and cancer stem cells, Aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3, as well as other members of the ALDH1 subfamily, can function in cells as a retinaldehyde dehydrogenase to generate retinoic acid (RA) from retinal. We show that the enzymatic activity of ALDH1A3 and its product, RA, are necessary for the observed expression of tTG in MES GSCs. Additionally, the ectopic expression of ALDH1A3 in PN GSCs is sufficient to induce the expression of tTG in these cells, further demonstrating a causal link between ALDH1A3 and tTG. Together, these findings ascribe a novel function for ALDH1A3 in an aggressive GSC phenotype via the up-regulation of tTG, and suggest the potential for a similar role by ALDH1 family members across cancer types.

Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid.

The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest.

MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma.

These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of glioblastoma multiforme. Mol Cancer Res; 14(10); 984-93. ©2016 AACR.