Dimitris Placantonakis

Sox2 antagonizes the Hippo pathway to maintain stemness in cancer cells

The repressive Hippo pathway has a profound tumour suppressive role in cancer by restraining the growth-promoting function of the transcriptional coactivator, YAP. We previously showed that the stem cell transcription factor Sox2 maintains cancer stem cells (CSCs) in osteosarcomas. We now report that in these tumours, Sox2 antagonizes the Hippo pathway by direct repression of two Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), leading to exaggerated YAP function. Repression of Nf2, WWC1 and high YAP expression marks the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression. YAP depletion sharply reduces CSCs and tumorigenicity of osteosarcomas. Thus, Sox2 interferes with the tumour-suppressive Hippo pathway to maintain CSCs in osteosarcomas. This Sox2-Hippo axis is conserved in other Sox2-dependent cancers such as glioblastomas. Disruption of YAP transcriptional activity could be a therapeutic strategy for Sox2-dependent tumours.

Two distinct oscillatory states determined by the NMDA receptor in rat inferior olive

1 The effects of N‐methyl‐d‐aspartate (NMDA) receptor activation and blockade on subthreshold membrane potential oscillations of inferior olivary neurones were studied in brainstem slices from 12‐ to 21‐day‐old rats. 2 Dizocilpine (MK‐801), a non‐competitive NMDA antagonist, at 1‐45 μm abolished spontaneous subthreshold oscillations, without affecting membrane potential, input resistance, or the low‐threshold calcium current, IT. Ketamine (100 μm), a non‐competitive NMDA antagonist, and L‐689,560 (20 μm), an antagonist at the glycine site of the NMDA receptor, also abolished the oscillations, while the competitive non‐NMDA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 20‐50 μm) had no effect. 3 NMDA (100 μm) induced 4.1 Hz subthreshold oscillations and reversibly depolarized olivary neurones by 13.7 mV. In contrast, 10 μmα‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and 20 μm kainic acid depolarized the membrane equivalently but did not induce oscillations. 4 Both NMDA‐induced and spontaneous subthreshold oscillations were unaffected by 1 μm tetrodotoxin and were prevented by substituting extracellular calcium with cobalt. 5 Removing magnesium from the perfusate did not affect spontaneous subthreshold oscillations but did prevent NMDA‐induced oscillations. 6 NMDA‐induced oscillations were resistant to 50 μm mibefradil, an IT blocker, in contrast to spontaneous oscillations. Both oscillations were inhibited by 20 μm nifedipine, an L‐type calcium channel antagonist, and 200 nmω‐agatoxin IVA, a P‐type calcium channel blocker. Bay K 8644 (10 μm), an L‐type Ca2+ agonist, significantly enhanced the amplitude of both spontaneous and NMDA‐induced oscillations. 7 The data indicate that NMDA receptor activation induces olivary neurones to manifest high amplitude membrane potential oscillations in part mediated by L‐ and P‐ but not T‐type calcium currents. Moreover, the data demonstrate that NMDA receptor currents are necessary for generation of spontaneous subthreshold oscillations in the inferior olive.

Serotonin suppresses subthreshold and suprathreshold oscillatory activity of rat inferior olivary neurones in vitro

1 The effect of serotonin on membrane potential oscillations of inferior olivary neurones was studied in brainstem slices from 10‐ to 19‐day‐old rats. 2 Serotonin at 50 and 5 μM induced a mean depolarization of 9.4 and 7.7 mV, respectively, that was preceded by a reversible suppression of subthreshold membrane potential oscillations. These effects were not changed by 1 μM tetrodotoxin and the suppression of subthreshold oscillations persisted after current‐mediated restoration of resting potential. 3 In spontaneously active neurones, serotonin abolished the rhythmicity of action potential firing without affecting spike frequency. 4 Serotonin reduced the slope of the calcium‐mediated rebound spike and both the duration and amplitude of the subsequent afterhyperpolarization. Serotonin also shifted the voltage dependence of the rebound spike to more negative values. 5 Hyperpolarizing current pulses (200 ms) revealed that serotonin increased the pre‐rectification and steady‐state components of membrane resistance by 37 and 38 %, respectively, in 66 % of neurones, but decreased these parameters by 14 and 20 % in the remaining cells. 6 The serotonin effects were antagonized by 5 μM methysergide or 1–5 μM ketanserin and were mimicked by 10–20 μM dimethoxy‐4‐iodoamphetamine but not 10 μM 8‐hydroxy‐2‐(di‐N‐propylamino)‐tetralin. 7 The data indicate that serotonin suppresses the rhythmic activity of olivary neurones via 5‐HT2 receptors by inhibition of the T‐type calcium current in combination with membrane depolarization due to activation of a cation current (Ih) and block of a resting K+ current (fast IK(ir)). This modulatory action of serotonin may account for the differential propensity of olivary neurones to fire rhythmically during different behavioural states in vivo.

Posterior stabilization strategies following resection of cervicothoracic junction tumors: review of 90 consecutive cases

OBJECT In this retrospective analysis the authors describe the assessment and outcomes of 90 patients who underwent placement of posterior instrumentation at the cervicothoracic junction following the resection of a primary or metastatic tumor during a 10-year period. METHODS All patients underwent a posterolateral laminectomy including uni- or bilateral facetectomy, and 44 patients additionally required vertebral body resection and reconstruction. In patients who underwent C-6 or C-7 decompression, the posterior instrumentation strategies changed from the use of lateral mass plate systems (LMPSs) to lateral mass screw/rod systems (LMSRSs). Similarly, for T1-3 tumor decompression, the strategy shifted from sublaminar hook/rod systems (SHRSs) to the use of pedicle screw systems (PSSs) in which the surgeon used either a 6.25-mm rod or dual-diameter rods with or without a connector. RESULTS The overall surgical complication rate was 19% including fixation failure in 11 patients (12%), 6 of whom required reoperation. Fixation failure rates for cervical decompression decreased from 2 (29%) of 7 patients in the LMPS group to 0 (0%) of 8 in the LMSRS group (p = 0.2). The fixation failure rates for thoracic decompression were 7 (15%) of 48 patients in the SHRS group, and there was a decrease to 2 (7%) of 27 in the PSS group (p = 0.48). Neurological and functional outcomes including American Spinal Injury Association, Eastern Cooperative Oncology Group, and Medical Research Council muscle strength and pain scores remained stable or improved in 94, 96, 100, and 96% of patients, respectively. CONCLUSIONS Current posterior instrumentation strategies involving LMSRSs and PSSs provide excellent and safe stabilization of the cervicothoracic junction following resection of primary or metastatic tumors.

Glioblastoma stem cells: Molecular characteristics and therapeutic implications

Glioblastoma Multiforme (GBM) is a grade IV astrocytoma, with a median survival of 14.6 mo. Within GBM, stem-like cells, namely glioblastoma stem cells (GSCs), have the ability to self-renew, differentiate into distinct lineages within the tumor and initiate tumor xenografts in immunocompromised animal models. More importantly, GSCs utilize cell-autonomous and tumor microenvironment-mediated mechanisms to overcome current therapeutic approaches. They are, therefore, very important therapeutic targets. Although the functional criteria defining GSCs are well defined, their molecular characteristics, the mechanisms whereby they establish the cellular hierarchy within tumors, and their contribution to tumor heterogeneity are not well understood. This review is aimed at summarizing current findings about GSCs and their therapeutic importance from a molecular and cellular point of view. A better characterization of GSCs is crucial for designing effective GSC-targeted therapies.

Brain stem cells as the cell of origin in glioma.

Glioma incidence rates in the United States are near 20000 new cases per year, with a median survival time of 14.6 mo for high-grade gliomas due to limited therapeutic options. The origins of these tumors and their many subtypes remain a matter of investigation. Evidence from mouse models of glioma and human clinical data have provided clues about the cell types and initiating oncogenic mutations that drive gliomagenesis, a topic we review here. There has been mixed evidence as to whether or not the cells of origin are neural stem cells, progenitor cells or differentiated progeny. Many of the existing murine models target cell populations defined by lineage-specific promoters or employ lineage-tracing methods to track the potential cells of origin. Our ability to target specific cell populations will likely increase concurrently with the knowledge gleaned from an understanding of neurogenesis in the adult brain. The cell of origin is one variable in tumorigenesis, as oncogenes or tumor suppressor genes may differentially transform the neuroglial cell types. Knowledge of key driver mutations and susceptible cell types will allow us to understand cancer biology from a developmental standpoint and enable early interventional strategies and biomarker discovery.

A panoramic view of the skull base: systematic review of open and endoscopic endonasal approaches to four tumors.

Endoscopic endonasal surgery has been established as the safest approach to pituitary tumors, yet its role in other common skull base lesions has not been established. To answer this question, we carried out a systematic review of reported series of open and endoscopic endonasal approaches to four major skull base tumors: olfactory groove meningiomas (OGM), tuberculum sellae meningiomas (TSM), craniopharyngiomas (CRA), and clival chordomas (CHO). Data from 162 studies containing 5,701 patients were combined and compared for differences in perioperative mortality, gross total resection (GTR), cerebrospinal fluid (CSF) leak, neurological morbidity, post-operative visual function, post-operative anosmia, post-operative diabetes insipidus (DI), and post-operative obesity/hyperphagia. Weighted average rates for each outcome were calculated using relative study size. Our findings indicate similar rates of GTR and perioperative mortality between open and endoscopic approaches for all tumor types. CSF leak was increased after endoscopic surgery. Visual function symptoms were more likely to improve after endoscopic surgery for TSM, CRA, and CHO. Post-operative DI and obesity/hyperphagia were significantly increased after open resection in CRA. Recurrence rates per 1,000 patient-years of follow-up were higher in endoscopy for OGM, TSM, and CHO. Trends for open and endoscopic surgery suggested modest improvement in all outcomes over time. Our observations suggest that endonasal endoscopy is a safe alternative to craniotomy and may be preferred for certain tumor types. However, endoscopic surgery is associated with higher rates of CSF leak, and possibly increased recurrence rates. Prospective study with long-term follow-up is required to verify these preliminary observations.

Selective Lentiviral Gene Delivery to CD133-Expressing Human Glioblastoma Stem Cells

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.

Notch signaling regulates metabolic heterogeneity in glioblastoma stem cells

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.

GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.