Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis.

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.

A Supplemented High-Fat Low-Carbohydrate Diet for the Treatment of Glioblastoma.

PURPOSE: Dysregulated energetics coupled with uncontrolled proliferation has become a hallmark of cancer, leading to increased interest in metabolic therapies. Glioblastoma (GB) is highly malignant, very metabolically active, and typically resistant to current therapies. Dietary treatment options based on glucose deprivation have been explored using a restrictive ketogenic diet (KD), with positive anticancer reports. However, negative side effects and a lack of palatability make the KD difficult to implement in an adult population. Hence, we developed a less stringent, supplemented high-fat low-carbohydrate (sHFLC) diet that mimics the metabolic and antitumor effects of the KD, maintains a stable nutritional profile, and presents an alternative clinical option for diverse patient populations. EXPERIMENTAL DESIGN: The dietary paradigm was tested in vitro and in vivo, utilizing multiple patient-derived gliomasphere lines. Cellular proliferation, clonogenic frequency, and tumor stem cell population effects were determined in vitro using the neurosphere assay (NSA). Antitumor efficacy was tested in vivo in preclinical xenograft models and mechanistic regulation via the mTOR pathway was explored. RESULTS: Reducing glucose in vitro to physiologic levels, coupled with ketone supplementation, inhibits proliferation of GB cells and reduces tumor stem cell expansion. In vivo, while maintaining animal health, the sHFLC diet significantly reduces the growth of tumor cells in a subcutaneous model of tumor progression and increases survival in an orthotopic xenograft model. Dietary-mediated anticancer effects correlate with the reduction of mTOR effector expression. CONCLUSIONS: We demonstrate that the sHFLC diet is a viable treatment alternative to the KD, and should be considered for clinical testing. Clin Cancer Res; 22(10); 2482-95. ©2015 AACR.

Differential connexin function enhances self-renewal in glioblastoma.

The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

Macrokinetic analysis of blockade of NMDA-gated currents by substituted alcohols, alkanes and ethers.

Volatile hydrocarbon based CNS depressants including short chain alcohols and anesthetics act, in part, by inhibition of the excitatory effect of glutamate at the NMDA receptor. While effects of several of these volatile agents on NMDA-gated currents have been demonstrated, there has been no direct comparison of different chemical classes of CNS depressant drugs on NMDA-gated currents. Here, whole-cell voltage clamp measurements of currents gated by 100 microM NMDA from cultured cerebrocortical neurons were examined in the presence of varying concentrations of the alcohols ethanol and hexanol, the halogenated alcohol trichloroethanol, the halogenated alkane halothane and the halogenated ethers isoflurane and sevoflurane. All drugs tested showed concentration-dependent inhibition of NMDA-gated currents with anesthetic concentrations of each agent producing approximately 30% inhibition of the NMDA-gated current. A rapid-translation perfusion system was used to study the onset and offset kinetics of each of the volatile agents. Onset kinetics for the CNS depressants was similar with tau values near 100 ms. Offset kinetics was more variable with tau ranging from 88.2 ms for ethanol to 221.4 ms for trichloroethanol. These data indicate that a wide variety of volatile hydrocarbon based CNS depressants produce a similar inhibition of NMDA-gated currents and that the kinetics for these agents are inconsistent with an open channel block.

Comparison of effect of ethanol on N-methyl-D-aspartate- and GABA-gated currents from acutely dissociated neurons: absence of regional differences in sensitivity to ethanol.

In vivo, ethanol alters the effect of N-methyl-D-aspartate (NMDA) and GABA in some brain regions but is without effect in others. To determine whether these regional differences were due to differences in the effect of ethanol on postsynaptic NMDA or GABAA receptors, we examined the effect of ethanol on NMDA- and GABA-gated currents from neurons acutely dissociated from the lateral septal nucleus, substantia nigra, thalamus, hippocampus, and cerebellum. Ethanol decreased the effect of NMDA similarly in all brain areas tested and had similar effects on Chinese hamster ovary cells expressing NR2A or NR2B subunits with an NR1-1a subunit. However, ifenprodil reduced the inhibition by ethanol of NMDA-gated currents from neurons isolated from the lateral septum without affecting neurons from the substantia nigra. In contrast to the robust effect of ethanol on NMDA-gated currents, ethanol (25-300 mM) was without effect on GABA-gated currents at all brain sites tested or on Ltk- cells stably expressing the alpha1, beta2, and gamma2L or gamma2S subunits. The neuroactive steroid alphaxalone profoundly enhanced GABA-gated currents in all brain areas and cell types tested, indicating a similar sensitivity to allosteric modulation; however, there was no interaction of alphaxalone with ethanol at any site tested. These data suggest that the regional differences in the effect of ethanol observed in vivo are not due to a differential action of ethanol at the postsynaptic NMDA or GABAA receptor subtypes.

Changes in the effect of isoflurane on N-methyl-D-aspartic acid-gated currents in cultured cerebral cortical neurons with time in culture: evidence for subunit specificity.

BACKGROUND: Developmental changes in NR1 splice variants and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor have been associated with changes in the sensitivity of NMDA receptors to agonists, antagonists, and pharmacologic modulators. The authors have investigated changes in the effect of isoflurane on NMDA-gated currents from cultured cortical neurons with time in culture and related these changes to the subunit composition of the NMDA receptors. METHODS: N-methyl-D-aspartate-gated currents were measured using whole-cell voltage clamp recording in cortical neurons cultured for 1-4 weeks and HEK 293 cells transiently expressing NR1-1a + NR2A or NR1-1a + NR2B subunit-containing receptors. NMDA alone or NMDA with treatment agents (isoflurane or ifenprodil) was applied to cells using a U tube. RESULTS: The effect of isoflurane and the NR2B selective antagonist ifenprodil on NMDA-gated currents from cortical neurons decreased significantly with time in culture. NMDA-gated currents mediated by NR2A-containing receptors were less sensitive to isoflurane than those mediated by NR2B-containing receptors. Tachyphylaxis to repeated application of isoflurane was found in cortical neurons and HEK 293 cells with recombinant NMDA receptors. Hooked tail currents were induced by isoflurane in cultured cortical neurons and HEK 293 cells with expressed NMDA receptors. CONCLUSIONS: Isoflurane inhibits NMDA-gated currents at concentrations well below 1 minimum alveolar concentration (MAC). This effect of isoflurane was subunit dependent with the NR2B-containing receptors more sensitive to isoflurane than the NR2A-containing receptors. A potent tachyphylaxis occurred after brief exposure to isoflurane.