Promising Prognosis Marker Candidates on the Status of Epithelial-Mesenchymal Transition and Glioma Stem Cells in Glioblastoma.
ABSTRACT: Multivariable analyses of global expression profiling are valid indicators of the prognosis of various diseases including brain cancers. To identify the candidates for markers of prognosis of glioblastoma, we performed multivariable analyses on the status of epithelial (EPI)-mesenchymal (MES) transition (EMT), glioma (GLI) stem cells (GSCs), molecular target therapy (MTT), and potential glioma biomarkers (PGBs) using the expression data and clinical information from patients. Random forest survival and Cox proportional hazards regression analyses indicated significant variable values for DSG3, CLDN1, CDH11, FN1, HDAC3/7, PTEN, L1CAM, OLIG2, TIMP4, IGFBP2, and GFAP. The analyses also comprised prognosis prediction formulae that could distinguish between the survival curves of the glioblastoma patients. In addition to the genes mentioned above, HDAC1, FLT1, EGFR, MGMT, PGF, STAT3, SIRT1, and GADD45A constituted complex genetic interaction networks. The calculated status scores obtained by principal component analysis indicated that GLI genes covered the status of EPI, GSC, and MTT-related genes. Moreover, survival tree analyses indicated that MEShigh, MEShighGLIlow, GSChighGLIlow, MEShighMTTlow, and PGBhigh showed poor prognoses and MESmiddle, GSClow, and PGBlow showed good prognoses, suggesting that enhanced EMT and GSC are associated with poor survival and that lower expression of EPI markers and the pre-stages of EMT are relatively less malignant in glioblastoma. These results demonstrate that the assessment of EMT and GSC enables the prediction of the prognosis of glioblastoma that would help develop novel therapeutics and de novo marker candidates for the prognoses of glioblastoma.
Project description:Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 ?M).MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 ?M. Clonogenic GSC surviving 500 ?M TMZ (GSC-500 ?M TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC.The molecular signatures characterized an activation of protective stress responses in GSC-500 ?M TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 ?M TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 ?M TMZ and GSC-parental to 35 ?M TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p?=?0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p?=?0.0489).These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.
Project description:High aggressiveness is a hallmark of glioblastoma and predicts poor prognosis of patients with glioblastoma. The expression level of sortilin has been preliminarily reported to be elevated in high-grade glioma; however, the potential significance of sortilin in glioblastoma progression has not been elucidated. In this study, we investigated the oncogenic effect of sortilin in glioblastoma. Increased levels of sortilin were noted in the mesenchymal subtype of glioblastoma and highly aggressive subtypes of glioblastoma tissues and cell lines. In addition, high levels of sortilin predicted poor prognoses in patients with glioblastoma. Sortilin knockdown or inhibition with AF38469 (an orally bioavailable inhibitor of sortilin) significantly suppressed migration and invasion by inhibiting EMT-like mesenchymal transition in glioblastoma cells. Furthermore, we proved that sortilin promoted cell invasion mainly via Glycogen synthase kinase 3 beta (GSK-3?)/?-catenin/Twist-induced EMT-like mesenchymal transition in glioblastoma. Taken together, our results demonstrate a critical role of sortilin in glioblastoma invasion and EMT-like mesenchymal transition, indicating that sortilin contributes to glioblastoma progression. These data also highlight the dramatic antitumor effects of AF38469 in glioblastoma, suggesting that AF38469 is a potentially powerful antitumor agent for sortilin-overexpressing human glioblastoma.
Project description:Glioblastomas, which contain stem cell-like glioblastoma stem cells (GSCs), are universally lethal cancers. While neural stem cells (NSCs) are usually quiescent, single-cell studies suggest that proliferating glioblastoma cells reside in the GSC population. Interrogating in silico glioma databases for epigenetic regulators that correlate with cell cycle regulation, we identified the chromatin remodeler HELLS as a potential target in glioblastoma. GSCs preferentially expressed HELLS compared with their differentiated tumor progeny and nonmalignant brain cells. Targeting HELLS disrupted GSC proliferation, survival, and self-renewal with induction of replication stress and DNA damage. Investigating potential molecular mechanisms downstream of HELLS revealed that HELLS interacted with the core oncogenic transcription factors, E2F3 and MYC, to regulate gene expression critical to GSC proliferation and maintenance. Supporting the interaction, HELLS expression strongly correlated with targets of E2F3 and MYC transcriptional activity in glioblastoma patients. The potential clinical significance of HELLS was reinforced by improved survival of tumor-bearing mice upon targeting HELLS and poor prognosis of glioma patients with elevated HELLS expression. Collectively, targeting HELLS may permit the functional disruption of the relatively undruggable MYC and E2F3 transcription factors and serve as a novel therapeutic paradigm for glioblastoma.
Project description:Glioblastoma is a highly heterogeneous brain tumor. The presence of cancer cells with stem-like and tumor initiation/propagation properties contributes to poor prognosis. Glioblastoma cancer stem-like cells (GSC) reside in hypoxic and acidic niches favoring cell quiescence and drug resistance. A high throughput screening recently identified the laxative Bisacodyl as a cytotoxic compound targeting quiescent GSC placed in acidic microenvironments. Bisacodyl activity requires its hydrolysis into DDPM, its pharmacologically active derivative. Bisacodyl was further shown to induce tumor shrinking and increase survival in <i>in vivo</i> glioblastoma models. Here we explored the cellular mechanism underlying Bisacodyl cytotoxic effects using quiescent GSC in an acidic microenvironment and GSC-derived 3D macro-spheres. These spheres mimic many aspects of glioblastoma tumors <i>in vivo</i>, including hypoxic/acidic areas containing quiescent cells. Phosphokinase protein arrays combined with pharmacological and genetic modulation of signaling pathways point to the WNK1 serine/threonine protein kinase as a mediator of Bisacodyl cytotoxic effect in both cell models. WNK1 partners including the Akt and SGK1 protein kinases and NBC-family Na<sup>+</sup>/HCO3<sup>-</sup> cotransporters were shown to participate in the compound's effect on GSC. Overall, our findings uncover novel potential therapeutic targets for combatting glioblastoma which is presently an incurable disease.
Project description:Esophageal adenocarcinoma (EAC) accounts for the most esophageal cancer cases in the US, and is notoriously aggressive. This study examines the role of Sonic Hedgehog (SHh)/Gli signaling in the regulation of epithelial-mesenchymal transition (EMT), a process tied to invasion and metastasis, in EAC. Gli/EMT protein expression levels were examined by western blot in paired EAC patient tissues (n = 24) and cell lines (OE19, OE33). Functional analyses were performed (siRNA, treatment with Gli-inhibitor, AKT-inhibitor, and N-Shh recombinant proteins) to investigate SHh/Gli signaling and EMT, cell cycle, and prognostic markers in EAC cell lines. MTS, luciferase reporter, qRT-PCR, western blot, wound healing, and transwell assays were executed to analyze pathway activity, cell migration, and invasion. Aberrant Gli1/2 expression was found in EAC patient tissues, and was significantly associated with increased EMT and AKT pathway activity. Stimulation of SHh/Gli resulted in EMT signaling, including expression of E-cadherin, N-cadherin, Vimentin, ?-catenin, Snail, and Slug, as well as cell cycle progression at mRNA and protein levels in EAC cell lines. Gli inhibition via small molecule administration and siRNA significantly reduced EMT, decreasing cell mobility and invasion. Both Gli and AKT inhibition rescued E-cadherin expression and suppressed AKT phosphorylation. This study provides evidence for a strong association between aberrant Gli1/2 expression and AKT/EMT markers in EAC; activated SHh/Gli signaling may be a critical component in promoting cell survival, metastases, and resistance to chemotherapy, and represents a promising avenue to target tumor proliferation and mobility.
Project description:Glioma stem-like cells (GSCs) are a subset of tumor cells that initiate malignant growth and promote the therapeutic resistance of glioblastoma, the most lethal primary brain tumor. Ribosome biogenesis is an essential cellular process to maintain cell growth, but its regulatory mechanism in GSCs remains largely unknown. Here, we show that WD repeat domain 12 (WDR12), a component of the Pes1-Bop1 complex (PeBoW), is required for ribosome biogenesis in GSCs. WDR12 is preferentially expressed in GSCs compared to non-stem tumor cells and normal brain cells. High levels of WDR12 are associated with glioblastoma progression and poor prognosis. Silencing WDR12 results in the degradation of PeBoW complex components and prevents the maturation of 28S rRNA, thereby inhibiting ribosome biogenesis in GSCs. Subsequently, WDR12 depletion compromises GSC proliferation, inhibits GSC-derived orthotopic tumor growth, and extends animal survival. Together, our results suggest that WDR12 is crucial for ribosome biogenesis in GSCs, and is thus a potential target for GSC-directed therapy of glioblastoma.
Project description:<h4>Background</h4>Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43<sub>266-283</sub> reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43<sub>266-283</sub>.<h4>Methods</h4>Metabolic impairment induced by the c-Src inhibitor TAT-Cx43<sub>266-283</sub> in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence.<h4>Findings</h4>TAT-Cx43<sub>266-283</sub> decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43<sub>266-283</sub> impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43<sub>266-283</sub> showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3.<h4>Interpretation</h4>The reduced ability of TAT-Cx43<sub>266-283</sub>-treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43<sub>266-283</sub>.<h4>Funding</h4>Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).
Project description:Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.
Project description:Low levels of reactive oxygen species (ROS) are crucial for maintaining cancer stem cells (CSCs) and their ability to resist therapy, but the ROS regulatory mechanisms in CSCs remains to be explored. Here, we discover that prohibitin (PHB) specifically regulates mitochondrial ROS production in glioma stem-like cells (GSCs) and facilitates GSC radiotherapeutic resistance. We find that PHB is upregulated in GSCs and is associated with malignant gliomas progression and poor prognosis. PHB binds to peroxiredoxin3 (PRDX3), a mitochondrion-specific peroxidase, and stabilizes PRDX3 protein through the ubiquitin-proteasome pathway. Knockout of PHB dramatically elevates ROS levels, thereby inhibiting GSC self-renewal. Importantly, deletion or pharmacological inhibition of PHB potently slows tumor growth and sensitizes tumors to radiotherapy, thus providing significant survival benefits in GSC-derived orthotopic tumors and glioblastoma patient-derived xenografts. These results reveal a selective role of PHB in mitochondrial ROS regulation in GSCs and suggest that targeting PHB improves radiotherapeutic efficacy in glioblastoma.