Glioblastoma Tumor-initiating Cells Research Articles

867 Postoperative Metformin is Associated With Improved Survival of Glioblastoma Patients

INTRODUCTION: Metformin may improve clinical outcomes for glioblastoma (GBM) patients as metformin showed in vitro tumor suppressor functions, such as reducing the proliferation of GBM tumor-initiating cells by reversing Akt kinase activation. METHODS: A single-centre retrospective study of 288 patients who underwent biopsy or surgery for GBM between 2014-2018 was conducted. Postoperative metformin treatment includes patients who received metformin as part of an adjuvant therapy with temozolomide and/or hyper-fractionated radiotherapy. Kaplan-Meier curves and log-rank p test were used for univariate analysis. Cox-proportional hazards model was used to generate adjusted hazard ratios with 95% CI for multivariate analysis. RESULTS: Metformin use impacted OS of patients with MGMT-methylated (MGMTm+) tumors (n = 71 no metformin (Met-), n = 35 metformin use (Met +); median OS = 442 vs. 662 days; p = 0.13) but not in patients with unmethylated MGMT tumors (n = 104 Met-, n = 44 Met+; median OS = 279 vs 295 days; p = 0.674). Postoperative metformin treatment of MGMTm+ tumor patients improved OS when compared to no or only preoperative metformin treatment (n = 66 no or preoperative metformin use, n = 40 postoperative metformin use; median OS = 443 vs 595; p = 0.012). Further, postoperative metformin treatment of nondiabetic patients with MGMTm+ tumors improved OS (n = 70 Met-, n = 18 postoperative Met+; median OS = 422 vs 582; p = 0.017). A multivariable model considering age, Karnofsky Performance Status (KPS), and extent of resection (EOR), demonstrated that postoperative metformin improves OS of MGMTm+ patients (aHR 0.500, 0.287-0.872, p = 0.015). Comparing postoperative with no or preoperative use of metformin produced a similar effect on OS (aHR 0.411, 0.177-0.954, p = 0.038). Postoperative treatment of nondiabetic patients with MGMTm+ tumors demonstrated improved OS (aHR 0.367, 0.156-0.864, p = 0.022). CONCLUSIONS: Postoperative metformin treatment of patients with MGMTm+ tumors is associated with improved OS, irrespective of age, diabetic status, KPS, or EOR.

332 Tumor collection and establishment of tumor-initiating cell cultures as antigen source for AV-GBM-1 dendritic cell vaccines for a phase II trial in patients with newly diagnosed glioblastoma

BackgroundDespite standard aggressive therapy (maximum safe surgical resection, concurrent radiation therapy and temozolomide chemotherapy (RT/TMZ), then maintenance TMZ), 2-year survival is only about 25% for patients with newly diagnosed primary glioblastoma (GBM). Adding AV-GBM-1, a vaccine consisting of autologous dendritic cells (DC) pulsed with autologous tumor antigens (ATA) may improve survival. One objective of a multi-center phase II clinical trial was to determine the feasibility of collecting fresh GBM and establishing short-term cell cultures of GBM tumor-initiating cells (TIC) to serve as ATA source.MethodsKey eligibility criteria for tumor collection were (1) clinical suspicion of new primary GBM, (2) age 18 to 70 years (3) tentative agreement to undergo a leukapheresis procedure after recovery from surgery, and (4) tentative plans for RT/TMZ. Fresh tumor was placed in media and shipped in a transport kit by overnight courier to AIVITA where a cell suspension was placed in culture and incubated in serum-free medium with factors that favor survival and proliferation of TICS (stem cells and early progenitor cells). The intent was to produce a patient-specific DC-ATA vaccine by incubating a lysate of irradiated TICs with autologous DC for subsequent subcutaneous injection.ResultsPatients were enrolled from five sites in California, one in Kentucky and one in New Jersey. Tumors were collected between August 2018 and January 2020. 106 patients consented for tumor collection, but 15 were not GBM, 4 had insufficient tissue to send, 2 patients withdrew consent, 4 were ineligible because of age, and 1 was ineligible because of autoimmune disease. Of the 80 GBM tumors that were placed into culture, 7 were discontinued because of patient withdrawal. 71/73 (97%) resulted in a successful cell culture; two were unsuccessful because of contamination. 60/71 subsequently consented for intent-to-treat ; 46/60 (77%) had cells in culture for 28 days or less, 11 were in culture for 30 to 35 days, and the remaining 3 were cultured 46, 54, and 55 days. The average number of cells per culture at the time of irradiation was 14.0 million (range 0.78 to 63.3 million). 58/60 (97%) yielded more than 1 million TICs for irradiation for the tumor cell lysate; 36/60 (60%) had more than 10 million cells irradiated. 57 patients were subsequently treated with AV-GBM-1 after recovery from RT/TMZ.ConclusionsSelf-renewing GBM TIC cultures can be reliably and rapidly established for use as the antigen source for personal DC-ATA vaccines.Trial RegistrationClinicaltrialsgov NCT03400917Ethics ApprovalThis study was approved by the Western IRB, approval number 20182582; all participants gave written informed consent before taking part

Suppression of LIM Kinase 1 and LIM Kinase 2 Limits Glioblastoma Invasion.

The aggressive brain tumor glioblastoma (GBM) is characterized by rapid cellular infiltration of brain tissue, raising the possibility that disease progression could potentially be slowed by disrupting the machinery of cell migration. The LIM kinase isoforms LIMK1 and LIMK2 (LIMK1/2) play important roles in cell polarization, migration, and invasion and are markedly upregulated in GBM and many other infiltrative cancers. Yet, it remains unclear whether LIMK suppression could serve as a viable basis for combating GBM infiltration. In this study, we investigated effects of LIMK1/2 suppression on GBM invasion by combining GBM culture models, engineered invasion paradigms, and mouse xenograft models. While knockdown of either LIMK1 or LIMK2 only minimally influenced invasion in culture, simultaneous knockdown of both isoforms strongly reduced the invasive motility of continuous culture models and human GBM tumor-initiating cells (TIC) in both Boyden chamber and 3D hyaluronic acid spheroid invasion assays. Furthermore, LIMK1/2 functionally regulated cell invasiveness, in part, by disrupting polarized cell motility under confinement and cell chemotaxis. In an orthotopic xenograft model, TICs stably transduced with LIMK1/2 shRNA were implanted intracranially in immunocompromised mice. Tumors derived from LIMK1/2 knockdown TICs were substantially smaller and showed delayed growth kinetics and more distinct margins than tumors derived from control TICs. Overall, LIMK1/2 suppression increased mean survival time by 30%. These findings indicate that LIMK1/2 strongly regulate GBM invasive motility and tumor progression and support further exploration of LIMK1/2 as druggable targets. SIGNIFICANCE: Targeting the actin-binding proteins LIMK1 and LIMK2 significantly diminishes glioblastoma invasion and spread, suggesting the potential value of these proteins as therapeutic targets.

Abstract 2456: NetZen: A comprehensive network-based pathway and target discovery platform

Abstract BACKGROUND: Success in precision medicine depends on the ability to accurately and rapidly identify master regulators of critical gene subnetworks. To achieve this goal requires a seamless computational target discovery platform that integrates global network visualization with identification of master regulatory subnetworks (MRS) and their key influencers. METHODS: We have created NetZen - a computer cluster-capable computational suite allowing for 1) robust network generation with million edges; 2) network-based MRS identification; 3) hierarchical analysis with automated annotations; and 4) whole-network 3D visualization of all 20,000 genes. To identify cancer MRS, we applied NetZen to expression datasets of tumors and normal tissues, tumor-initiating cells (TICs), non-TIC cancer cells, and normal stem and non-stem cells from TCGA, GEO and internal sources. For validation, we introduced these subnetworks in normal cells to recapitulate TIC phenotypes in vivo or disrupted them in patients-derived TICs and mouse xenografts (PDX). To identify MRS of mortality, we applied NetZen to survival data in TCGA. RESULTS: Networks of 28 human cancers were generated, integrating transcriptomics, microRNAs, proteomics, and clinical data. For cancers with TIC datasets, we identified TIC-specific MRS with clear-cut hierarchy controlling immune surveillance, EMT and developmental functions. Interestingly, in some cancers, the immune subnetwork including immune checkpoints was entirely encompassed in the developmental subnetwork. We present representative validation on glioblastoma TIC (GIC) MRS with features common in other cancers’ TICs. Exogenous introduction of this subnetwork in normal astrocytes forced them to become GICs and form tumors in mice. Disrupting this subnetwork in 10 independent PDXs profoundly blunted tumor growth and prolonged survival. Lastly, we present detailed MRS of mortality analysis of multiple cancer types and identify common pathways directly influencing outcomes. DISCUSSION: NetZen provides a complete solution to big data analysis by effortlessly processing million-edge hierarchical networks with biological insights through automated annotations. The findings that the immune and developmental subnetworks were intimately linked suggest that immune escape by TICs is intrinsic of cellular transformation rather than a product of selection. Lastly, detailed network hierarchy provides a logical roadmap for cancer therapeutic development. Citation Format: Son Le, Alberto Riva, Changwang Deng, David D. Tran. NetZen: A comprehensive network-based pathway and target discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2456.

Transcriptomic analysis reveals that BMP4 sensitizes glioblastoma tumor-initiating cells to mechanical cues

The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.

Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells with a Cell-Friendly Culture System

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

Tumor-initiating cell frequency is relevant for glioblastoma aggressiveness

Glioblastoma (GBM) is maintained by a small subpopulation of tumor-initiating cells (TICs). The arduous assessment of TIC frequencies challenges the prognostic role of TICs in predicting the clinical outcome in GBM patients. We estimated the TIC frequency in human GBM injecting intracerebrally in mice dissociated cells without any passage in culture.All GBMs contained rare TICsand were tumorigenic in vivo but only 54% of them grew in vitro as neurospheres. We demonstrated that neurosphere formation in vitro did not foretell tumorigenic ability in vivo and frequencies calculated in vitro overestimated the TIC content.Our findings assert the pathological significance of GBM TICs. TIC number correlated positively with tumor incidence and inversely with survival of tumor-bearing mice. Stratification of GBM patients according to TIC content revealed that patients with low TIC frequency experienced a trend towards a longer progression free survival. The expression of either putative stem-cell markers or markers associated with different GBM molecular subtypes did not associate with either TIC content or neurosphere formation underlying the limitations of TIC identification based on the expression of some putative stem cell-markers.

Scalable Production of Glioblastoma Tumor-initiating Cells in 3 Dimension Thermoreversible Hydrogels.

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods, however, cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (~50 days, 10 passages tested, accumulative ~>1010-fold expansion) with both high growth rate (~20-fold expansion/7 days) and high volumetric yield (~2.0 × 107 cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient, affordable glioblastoma TICs for drug discovery.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

In This Issue

In This Issue

Chromosomal Instability Affects the Tumorigenicity of Glioblastoma Tumor-Initiating Cells.

Tumors are dynamic organs that evolve during disease progression with genetic, epigenetic, and environmental differences among tumor cells serving as the foundation for selection and evolution in tumors. Tumor-initiating cells (TIC) that are responsible for tumorigenesis are a source of functional cellular heterogeneity, whereas chromosomal instability (CIN) is a source of karyotypic genetic diversity. However, the extent that CIN contributes to TIC genetic diversity and its relationship to TIC function remains unclear. Here, we demonstrate that glioblastoma TICs display CIN with lagging chromosomes at anaphase and extensive nonclonal chromosome copy-number variations. Elevating the basal chromosome missegregation rate in TICs decreases both proliferation and the stem-like phenotype of TICs in vitro Consequently, tumor formation is abolished in an orthotopic mouse model. These results demonstrate that TICs generate genetic heterogeneity within tumors, but that TIC function is impaired if the rate of genetic change is elevated above a tolerable threshold. Genetic heterogeneity among TICs may produce advantageous karyotypes that lead to therapy resistance and relapse; however, we found that TICs have an upper tolerable limit for CIN. Thus, increasing the chromosome missegregation rate offers a new therapeutic strategy to eliminate TICs from tumors. Cancer Discov; 6(5); 532-45. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 461.

CDC20 maintains tumor initiating cells.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21CIP1/WAF1, a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21CIP1/WAF1, resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes-FOXM1 and p21CIP1/WAF1-elucidating a potential point for therapeutic intervention.

Constitutive activation of myosin-dependent contractility sensitizes glioma tumor-initiating cells to mechanical inputs and reduces tissue invasion.

Tumor-initiating cells (TIC) perpetuate tumor growth, enable therapeutic resistance, and drive initiation of successive tumors. Virtually nothing is known about the role of mechanotransductive signaling in controlling TIC tumorigenesis, despite the recognized importance of altered mechanics in tissue dysplasia and the common observation that extracellular matrix (ECM) stiffness strongly regulates cell behavior. To address this open question, we cultured primary human glioblastoma (GBM) TICs on laminin-functionalized ECMs spanning a range of stiffnesses. Surprisingly, we found that these cells were largely insensitive to ECM stiffness cues, evading the inhibition of spreading, migration, and proliferation typically imposed by compliant ECMs. We hypothesized that this insensitivity may result from insufficient generation of myosin-dependent contractile force. Indeed, we found that both pharmacologic and genetic activation of cell contractility through RhoA GTPase, Rho-associated kinase, or myosin light chain kinase restored stiffness-dependent spreading and motility, with TICs adopting the expected rounded and nonmotile phenotype on soft ECMs. Moreover, constitutive activation of RhoA restricted three-dimensional invasion in both spheroid implantation and Transwell paradigms. Orthotopic xenotransplantation studies revealed that control TICs formed tumors with classical GBM histopathology including diffuse infiltration and secondary foci, whereas TICs expressing a constitutively active mutant of RhoA produced circumscribed masses and yielded a 30% enhancement in mean survival time. This is the first direct evidence that manipulation of mechanotransductive signaling can alter the tumor-initiating capacity of GBM TICs, supporting further exploration of these signals as potential therapeutic targets and predictors of tumor-initiating capacity within heterogeneous tumor cell populations.

ONC201, a small molecule Foxo3a activator, activity against patient-derived glioblastoma tumor-initiating cells.

e13022 Background: ONC201 is a novel small molecule that inactivates MEK and Akt to induce Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) tumor suppressor expression in a Foxo3a-dependent manner and is able to cross the blood-brain barrier. Previous studies have verified preclinical cytotoxic activity in numerous cancer models including glioma. We sought to determine activity of ONC201 against patient-derived glioma tumor initiating cell (TIC) models in vitro and in orthotopic xenograft. Methods: We tested 5 TIC lines derived using the neurosphere system from untreated (MGG4, MGG8, MGG18) and recurrent (MGG67R and MGG152) glioblastomas. Several lines are known to have variable sensitivity to secreted TRAIL, including one (MGG4) that was reported to be resistant to secreted TRAIL-mediated cell death. We performed cell viability assays using ONC201 and IC50 values were calculated. SCID mouse brains were stereotactically implanted with TICs and mice were treated with ONC201 by oral gavage fo...

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Glioblastoma Tumor Initiating Cells: Therapeutic Strategies Targeting Apoptosis and MicroRNA Pathways

Glioblastoma (GBM) is a highly malignant primary brain tumor known for its invasiveness and aggressive resistance to standard treatment. It is currently the most common primary brain tumor which is associated with a high mortality rate. Tumor initiating cells (TICs) are a subpopulation of GBM stem cells which are capable of self-renewal and apoptotic resistance, and are thought to account for GBMs aggressive nature. Recent efforts have focused on therapies which target key intracellular apoptotic pathways which may confer tumor resistance, such as Akt, p53, Bcl-2 family proteins, caspase family proteases, and more recently microRNAs. Research into microRNA's role in GBM has shown that microRNAs play a key regulatory role in the GBM apoptotic pathway, making it a potential therapeutic target. In this review we summarized the molecular mechanisms involved in the signaling pathways of human GBM TIC apoptosis and microRNAs, the contemporary treatments involving different members of the signaling cascade, and the future direction of GBM treatment strategies.

Glioblastoma Tumor Initiating Cells: Therapeutic Strategies Targeting Apoptosis and MicroRNA Pathways

Glioblastoma (GBM) is a highly malignant primary brain tumor known for its invasiveness and aggressive resistance to standard treatment. It is currently the most common primary brain tumor which is associated with a high mortality rate. Tumor initiating cells (TICs) are a subpopulation of GBM stem cells which are capable of self-renewal and apoptotic resistance, and are thought to account for GBMs aggressive nature. Recent efforts have focused on therapies which target key intracellular apoptotic pathways which may confer tumor resistance, such as Akt, p53, Bcl-2 family proteins, caspase family proteases, and more recently microRNAs. Research into microRNA's role in GBM has shown that microRNAs play a key regulatory role in the GBM apoptotic pathway, making it a potential therapeutic target. In this review we summarized the molecular mechanisms involved in the signaling pathways of human GBM TIC apoptosis and microRNAs, the contemporary treatments involving different members of the signaling cascade, and the future direction of GBM treatment strategies.

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.

Metformin selectively affects human glioblastoma tumor-initiating cell viability

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.