Brain Tumor-initiating Cells Growth Research Articles

STEM-26. BLOOD-TUMOR BARRIER IS COMPOSED OF MECHANOSENSING TUMOR CELLS THAT MASK THERAPEUTIC VULNERABILITY

Abstract Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB), which restricts delivery of most therapeutic agents, and the quiescent brain tumor-initiating cells (BTICs), which evade cell cycle-targeting chemotherapy. Mechanosensation, the transduction of mechanical cues into cellular signaling, underlies physiological processes such as touch, pain, proprioception, hearing, respiration, epithelial homeostasis, and vascular and lymphatic development. We report that medulloblastoma (MB) BTICs are mechanosensing, a property conferred by force-activated ion channel Piezo2. In contrast to the prevailing view that astrocytes function as a physical barrier in BTB, BTICs project endfeet to ensheathe capillaries. MB develops a tissue stiffness gradient as a function of distance to capillaries. Piezo2 senses substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion at BTIC growth cones, which allow BTICs to mechanically interact with their substrate and sequester β-Catenin to prevent WNT/β-Catenin signaling in BTICs. Our single cell analysis uncovers a two-branched BTIC trajectory that progresses from a deep quiescent state to two cycling states. Tumor cell-specific Piezo2 knockout reverses the off-on WNT/β-Catenin signaling states in BTICs and endothelial cells, collapses the BTB, reduces quiescence depth of BTICs, and markedly enhances MB response to chemotherapy. Our study reveals that BTICs co-opt astrocytic mechanism to contribute to the BTB and provides the first evidence that BTB depends on mechanochemical signaling to mask tumor chemosensitivity. Targeting Piezo2 addresses BTB and BTIC properties that underlie therapy failures in brain cancer.

Demeclocycline Reduces the Growth of Human Brain Tumor-Initiating Cells: Direct Activity and Through Monocytes.

Myeloid cells that infiltrate into brain tumors are deactivated or exploited by the tumor cells. We previously demonstrated that compromised microglia, monocytes, and macrophages in malignant gliomas could be reactivated by amphotericin-B to contain the growth of brain tumorinitiating cells (BTICs). We identified meclocycline as another activator of microglia, so we sought to test whether its better-tolerated derivative, demeclocycline, also stimulates monocytes to restrict BTIC growth. Monocytes were selected for study as they would be exposed to demeclocycline in the circulation prior to entry into brain tumors to become macrophages. We found that demeclocycline increased the activity of monocytes in culture, as determined by tumor necrosis factor-α production and chemotactic capacity. The conditioned medium of demeclocycline-stimulated monocytes attenuated the growth of BTICs generated from human glioblastoma resections, as evaluated using neurosphere and alamarBlue assays, and cell counts. Demeclocycline also had direct effects in reducing BTIC growth. A global gene expression screen identified several genes, such as DNA damage inducible transcript 4, frizzled class receptor 5 and reactive oxygen species modulator 1, as potential regulators of demeclocycline-mediated BTIC growth reduction. Amongst several tetracycline derivatives, only demeclocycline directly reduced BTIC growth. In summary, we have identified demeclocycline as a novel inhibitor of the growth of BTICs, through direct effect and through indirect stimulation of monocytes. Demeclocycline is a candidate to reactivate compromised immune cells to improve the prognosis of patients with gliomas.

Microglia induces Gas1 expression in human brain tumor-initiating cells to reduce tumorigenecity

We reported previously that microglia decreased the growth of human brain tumor-initiating cells (BTICs). Through microarray analyses of BTICs exposed in vitro to microglia, we found the induction of several genes ascribed to have roles in cell cycle arrest, reduced cell proliferation and differentiation. Herein, we tested the hypothesis that one of these genes, growth arrest specific 1 (Gas1), is a novel growth reduction factor that is induced in BTICs by microglia. We found that microglia increased the expression of Gas1 transcript and protein in glioblastoma patient-derived BTIC lines. Using neurosphere assay we show that RNAi-induced reduction of Gas1 expression in BTICs blunted the microglia-mediated BTIC growth reduction. The role of Gas1 in mediating BTIC growth arrest was further validated using orthotopic brain xenografts in mice. When microglia-induced Gas1-expressing BTIC cells (mGas1-BTICs) were implanted intra-cranially in mice, tumor growth was markedly decreased; this was mirrored in the remarkable increase in survival of mGas1-BT025 and mGas1-BT048 implanted mice, compared to mice implanted with non-microglia-exposed BTIC cells. In conclusion, this study has identified Gas1 as a novel factor and mechanism through which microglia arrest the growth of BTICs for anti-tumor property.

MYC-Regulated Mevalonate Metabolism Maintains Brain Tumor-Initiating Cells.

Metabolic dysregulation drives tumor initiation in a subset of glioblastomas harboring isocitrate dehydrogenase (IDH) mutations, but metabolic alterations in glioblastomas with wild-type IDH are poorly understood. MYC promotes metabolic reprogramming in cancer, but targeting MYC has proven notoriously challenging. Here, we link metabolic dysregulation in patient-derived brain tumor-initiating cells (BTIC) to a nexus between MYC and mevalonate signaling, which can be inhibited by statin or 6-fluoromevalonate treatment. BTICs preferentially express mevalonate pathway enzymes, which we find regulated by novel MYC-binding sites, validating an additional transcriptional activation role of MYC in cancer metabolism. Targeting mevalonate activity attenuated RAS-ERK-dependent BTIC growth and self-renewal. In turn, mevalonate created a positive feed-forward loop to activate MYC signaling via induction of miR-33b. Collectively, our results argue that MYC mediates its oncogenic effects in part by altering mevalonate metabolism in glioma cells, suggesting a therapeutic strategy in this setting. Cancer Res; 77(18); 4947-60. ©2017 AACR.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Activation of NOTCH Signaling by Tenascin-C Promotes Growth of Human Brain Tumor-Initiating Cells.

Oncogenic signaling by NOTCH is elevated in brain tumor-initiating cells (BTIC) in malignant glioma, but the mechanism of its activation is unknown. Here we provide evidence that tenascin-C (TNC), an extracellular matrix protein prominent in malignant glioma, increases NOTCH activity in BTIC to promote their growth. We demonstrate the proximal localization of TNC and BTIC in human glioblastoma specimens and in orthotopic murine xenografts of human BTIC implanted intracranially. In tissue culture, TNC was superior amongst several extracellular matrix proteins in enhancing the sphere-forming capacity of glioma patient-derived BTIC. Exogenously applied or autocrine TNC increased BTIC growth through an α2β1 integrin-mediated mechanism that elevated NOTCH ligand Jagged1 (JAG1). Microarray analyses and confirmatory PCR and Western analyses in BTIC determined that NOTCH signaling components including JAG1, ADAMTS15, and NICD1/2 were elevated in BITC after TNC exposure. Inhibition of γ-secretase and metalloproteinase proteolysis in the NOTCH pathway, or silencing of α2β1 integrin or JAG1, reduced the proliferative effect of TNC on BTIC. Collectively, our findings identified TNC as a pivotal initiator of elevated NOTCH signaling in BTIC and define the establishment of a TN-α2β1-JAG1-NOTCH signaling axis as a candidate therapeutic target in glioma patients. Cancer Res; 77(12); 3231-43. ©2017 AACR.

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma.

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Neurotrophin receptors TrkB and TrkC promote Brain Tumor Initiating Cells growth and are novel targets for glioma therapy. (P4.242)

Neurotrophin receptors TrkB and TrkC promote Brain Tumor Initiating Cells growth and are novel targets for glioma therapy. (P4.242)

Neurotrophin Signaling via TrkB and TrkC Receptors Promotes the Growth of Brain Tumor-initiating Cells

Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma.

CS-14 * NEUROTROPHIN RECEPTORS TrkB AND TrkC ARE REQUIRED FOR SURVIVAL OF BRAIN TUMOR INITIATING CELLS

Neurotrophins (NGF, BDNF and NT3) and their receptors (TrkA, TrkB, TrkC and p75NTR) are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75NTR is required for glioma invasion and proliferation. However, the role of other Trk receptors has not been examined. In this study, we investigated the importance of neurotrophin receptors TrkB and TrkC in survival of brain tumor initiating cells (BTICs). We used human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas and examined the expression of neurotrophin receptors and studied their role in survival/growth these cells in vitro, we have also examined the mechanism underlie. Here, we show that human malignant glioma tissues and tumor-initiating cells express the neurotrophin receptors TrkB and TrkC, not TrkA and they also express neurotrophins NGF, BDNF and NT3. Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 stimulates Trk phosphorylation, activates Erk and Akt and enhances tumor-initiating cell viability and growth. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition using inhibitors of Trk, Erk and Akt, decreases neurotrophin-dependent Trk phosphorylation, Erk and Akt activation and BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling and further neurotrophin signaling is required for maintains long-term BTIC growth in the absence of EGF and FGF, and this effect is attenuated with Trk, Erk and Akt inhibitors. In vivo experiments are under progress. Our results highlight a novel role for neurotrophin signaling in malignant glioma and suggest that Trks could be a target for combinatorial treatment.

Therapeutic activation of macrophages and microglia to suppress brain tumor-initiating cells

Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient-derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.

Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) due to preferential BTIC survival and adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3 and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, TICs may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may instruct the tumor hierarchy and portend poor prognosis.