EAG2 potassium channel with evolutionarily conserved function as a brain tumor target
Ontology highlight
ABSTRACT: Characterizing the impact of pharmacological and shRNA-mediated silencing of EAG2 in medulloblastoma. A medulloblastoma (MB) cell line was untreated, mock(DMSO)-treated and treated to inhibit EAG2 (both pharmacologically and via shRNA).
Project description:Characterizing the impact of pharmacological and shRNA-mediated silencing of EAG2 in medulloblastoma. Medulloblastoma (MB) is the most common pediatric CNS malignancy. Previously, we demonstrated that overexpression of the ion channel EAG2, identified in a subset of histological and molecular subtypes of this disease, functionally contributes to tumor progression (PMID: 22855790). Here, we demonstrate the evolutionarily conserved function of EAG2 potassium channel in promoting brain tumor growth and metastasis, delineate downstream pathways and uncover a co-option mechanism for different potassium channels to regulate mitotic cell volume and tumor progression. We show that EAG2 potassium channel is enriched at the trailing edge of migrating MB cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identify the FDA- approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings thus illustrate the potential of targeting ion channels in cancer treatment.
Project description:Microarray profiling of amplified total RNA isolated from FACS-sorted Drosophila neuroblasts 2 samples per group, RNA isolated and amplified. Hybridized as a one color experiment.
Project description:Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.
Project description:Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl- (PAC) currents (I Cl,H). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl- channel family in physiological and pathological processes associated with acidic pH.
Project description:Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac I(Ks) current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50-60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K(+) current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.
Project description:Induction of the transcription factor Sox2 from a doxycycline-inducible promoter in iSox2-DAOY medulloblastoma cells. Affymetrix microarrays were used to characterize the gene expression profile of iSox2-DAOY cells in the absence and presence of doxycycline. Human DAOY medulloblastoma cells were engineered to express epitope tagged Sox2 under the control of a doxycycline inducible promoter, to produce iSox2-DAOY cells. iSox2-DAOY cells were FACS sorted into CD133 low and high populations. The CD133 low population was cultured in the absence and presence of 0.5 µg/mL doxycycline for 24 hours. RNA was extracted from cells cultured without and with doxycycline, and was used for microarray analysis.
Project description:Sequencing cancer genomes is predicted to uncover therapeutic tumor vulnerabilities. This has been complicated by the abundance of genetic alterations which are either non-functional, or only important in tumor initiation or progression. Distinguishing tumor maintenance genes from initiation, progression, and passenger genes is critical for developing effective cancer therapy. We employed a functional genomic approach using the Lazy Piggy transposon to identify tumor maintenance genes in vivo, and apply this to SHH medulloblastoma (MB). Combining Lazy Piggy screening in mice and transcriptomic profiling of human MB, we discovered candidate maintenance genes including the voltage-gated potassium channel Kcnb2. Genetic knockout of Kcnb2 impairs MB growth. Kcnb2 mediates potassium efflux in MB-propagating cells to govern cell volume. Loss of Kcnb2 leads to chronic osmotic swelling and decreased plasma membrane tension, which elevates endocytosis to dampen Egfr signaling, thereby mitigating proliferation of MB-propagating cells. Kcnb2 is largely dispensable for mouse development and synergizes with anti-SHH therapy in treating MB. These results demonstrate the utility of the Lazy Piggy functional genomic approach in identifying cancer maintenance genes, and elucidate a mechanism by which a potassium channel integrates biomechanical and biochemical signaling to promote MB aggression.
Project description:Sequencing cancer genomes is predicted to uncover therapeutic tumor vulnerabilities. This has been complicated by the abundance of genetic alterations which are either non-functional, or only important in tumor initiation or progression. Distinguishing tumor maintenance genes from initiation, progression, and passenger genes is critical for developing effective cancer therapy. We employed a functional genomic approach using the Lazy Piggy transposon to identify tumor maintenance genes in vivo, and apply this to SHH medulloblastoma (MB). Combining Lazy Piggy screening in mice and transcriptomic profiling of human MB, we discovered candidate maintenance genes including the voltage-gated potassium channel Kcnb2. Genetic knockout of Kcnb2 impairs MB growth. Kcnb2 mediates potassium efflux in MB-propagating cells to govern cell volume. Loss of Kcnb2 leads to chronic osmotic swelling and decreased plasma membrane tension, which elevates endocytosis to dampen Egfr signaling, thereby mitigating proliferation of MB-propagating cells. Kcnb2 is largely dispensable for mouse development and synergizes with anti-SHH therapy in treating MB. These results demonstrate the utility of the Lazy Piggy functional genomic approach in identifying cancer maintenance genes, and elucidate a mechanism by which a potassium channel integrates biomechanical and biochemical signaling to promote MB aggression.
Project description:Ever since they were first observed in Purkinje fibers of the heart, funny channels have had close connections to potassium channels. Indeed, funny channels were initially thought to produce a potassium current in the heart called I K2. However, funny channels are completely unlike potassium channels in ways that make their contributions to the physiology of cells unique. An important difference is the greater ability for sodium to permeate funny channels. Although it does not flow through the funny channel as easily as does potassium, sodium does permeate well enough to allow for depolarization of cells following a strong hyperpolarization. This is critical for the function of funny channels in places like the heart and brain. Computational analyses using recent structures of the funny channels have provided a possible mechanism for their unusual permeation properties.