The authors acknowledge support from the Thomas E. &#38; Pamela M. Mischell Family Foundation to S.S. and the Harold C. Schott Foundation funding of the Harold C. Schott Endowed Chair, UC College of Medicine, to S.S.

Pharmacological Targeting of Ion Channels to Treat Solid Tumors

<ol>
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D.A.P.K. is a co-founder, president, and CEO of Amlal Pharmaceuticals Inc. S.S. is a co-founder of Amlal Pharmaceuticals Inc. and sits on the Drug Safety Monitoring Board of Bexion Pharmaceuticals, Inc.

Changes in ion channel function alter intracellular signaling cascades, which can impact the overall functioning of a cell. Over the past decade, it has become increasingly clear that ion channels are important to cancer cell growth and metastasis. Importantly, many ion channels are primary targets for approved therapeutics targeting a broad range of disorders24. Investigators have probed whether ion channels could be anti-cancer targets, and the initial results are promising2</s...

Membrane transport proteins are critical for communication between cells, as well as for maintaining cellular homeostasis. Amongst the membrane transport proteins, ion channels serve to drive the growth and development of cells and to maintain the state of cells in challenging and changing environments. Ion channels have also been reported to drive and support the development of solid tumors, both systemically and in the central nervous system (CNS)1,2. For example, KCa3.1 channels are responsible for regulating membrane potential and controlling cell volume, which is important in cell-cycle regulation. Defective KCa3.1 channels have been reported to contribute to the abnormal proliferation of tumor cells3. Further, ion channels may contribute to the metastatic dissemination of cancers. Transient receptor potential (TRP) channels, for example, are involved in Ca2+ and Mg2+ influx; this influx activates several kinases and heat shock proteins that function to regulate the extracellular matrix surrounding a tumor, which is, in turn, important for initiating cancer metastasis4.
Since ion channels can contribute to the development of cancers, they may also be targets for drug-related cancer treatment. For example, resistance to treatment modalities, including chemotherapy and novel immunotherapy, is related to ion channel function dysregulation5,6,7. In addition, ion channels are emerging as important drug targets to impede the growth and development of cancers, with repurposed small molecule (FDA-approved) drugs being examined, as well as biopolymers, including monoclonal antibodies1,2,8,9. While there has been much progress on this front, ion channel cancer drug discovery remains underdeveloped. This is partly due to the unique challenges of studying ion channels in cancer cells. For example, there are technical limitations in setting up electrophysiology assays for slow-acting compounds and temporal differences in channel activation and drug action. Further, the solubility of compounds can also impede progress, as most of the automated electrophysiology systems commonly in use today utilize hydrophobic substrates, which may contribute to artifacts as a result of compound adsorption. In addition, large bioorganic molecular therapeutics such as natural products, peptides, and monoclonal antibodies are technically challenging to screen using conventional electrophysiology assays10. Finally, the bioelectrical properties of cancer cells remain poorly understood11.
Meanwhile, the immunofluorescence staining of ion channels is often challenging. This is due, in part, to the complexity of their structures and their context in the membrane, which impact the ability to both generate and employ antibodies for microscopy studies. It is especially important that the antibodies used to stain ion channels are validated for specificity, affinity, and reproducibility. Commercial antibodies for ion channels should be considered based on their validation strategy and publication record. Experiments should include negative controls to demonstrate the lack of nonspecific binding by either knockdown or knockout of the target protein. Alternatively, cell lines in which the target protein is absent or in low abundance based on mRNA or protein determinations may serve as negative controls. For example, this study shows the localization of the (GABA) receptor&#160;subunit Gabra5 in a medulloblastoma cell line (D283). D283 cells with an siRNA knockdown and Daoy&#160;cells, another cerebellar medulloblastoma cell line, were stained for Gabra5 and showed no appreciatable staining (data not shown).
Here, methods are presented to analyze and assay ion channel function, as well as the effect of ion channel modulators on cancer cells. Protocols are provided for (1) staining cells for an ion channel, (2) testing the polarized state of mitochondria, (3) establishing ion channel function using electrophysiology, and (4) in vitro drug validation. These protocols emphasize studies of the type A gamma-aminobutyric acid (GABAA) receptor2,12,13,14,15,16, a chloride anion channel and major inhibitory neurotransmitter receptor. However, the methods presented here apply to studying many other cancer cells and ion channels.

<table><tbody><tr><td>ABS SpectraMax Plate Reader</td><td>Molecular Devices</td><td>ABS</td><td></td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>00-4555-56</td><td></td></tr><tr><td>Alexa Flor 488</td><td>Invitrogen</td><td>A32723</td><td>Goat Anti-Rabbit</td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td>100x</td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>12587-010</td><td>Lacks vitamin A</td></tr><tr><td>Biosafety Cabinet</td><td>LABCONCO</td><td>302381101</td><td>Class II, Type A2</td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP1606-100</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>13-998-211</td><td>Heracell VIOS 160i</td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>C7902</td><td>Dihydrate</td></tr><tr><td>Cell Culture Dishes, 150 mm</td><td>Fisher Scientific</td><td>12-600-004</td><td>Cell culture treated</td></tr><tr><td>Cell Culture Flasks, 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Fisher Scientific</td><td>430641U</td><td>Cell culture treated</td></tr><tr><td>Cell Culture Plates, 6 well</td><td>Fisher Scientific</td><td>353046</td><td>Cell culture treated</td></tr><tr><td>Cell Culture Plates, 96 well</td><td>Fisher Scientific</td><td>353072</td><td>Cell culture treated</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>EP-5804R</td><td>Refrigerated</td></tr><tr><td>Corning CoolCell</td><td>Fisher Scientific</td><td>07-210-0006</td><td></td></tr><tr><td>Coverslips, 22 x 22 mm</td><td>Fisher Scientific</td><td>12-553-450</td><td>Corning brand</td></tr><tr><td>D283 Med</td><td>ATCC</td><td>HTB-185</td><td></td></tr><tr><td>DABCO Mounting Media</td><td>EMS</td><td>17989-97</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma Life Sciences</td><td>D9434</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich</td><td>D2650</td><td>Cell culture grade</td></tr><tr><td>DMEM/F12, base media</td><td>Fisher Scientific</td><td>11330-032</td><td>With phenol red</td></tr><tr><td>DMEM/F12, phenol red free</td><td>Fisher Scientific</td><td>21041-025</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>STEMCELL</td><td>78006.1</td><td></td></tr><tr><td>FCCP</td><td>Abcam</td><td>AB120081</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>Fibroblast Growth Factor, Basic</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>GARBA5 Antibody</td><td>Aviva</td><td>ARP30687_P050</td><td>Rabbit Polyclonal</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Glycerol Mounting Medium</td><td>EMS</td><td>17989-60</td><td>With DAPI+DABCO</td></tr><tr><td>Hemocytometer</td><td>Millipore Sigma</td><td></td><td></td></tr><tr><td>Heparin</td><td>STEMCELL</td><td>7980</td><td></td></tr><tr><td>HEPES</td><td>HyClone</td><td>SH3023701</td><td>Solution</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td>Solid</td></tr><tr><td>ImageJ</td><td>Open platform</td><td></td><td>With Fiji plugins</td></tr><tr><td>Immuno Mount DAPI</td><td>EMS</td><td>17989-97</td><td></td></tr><tr><td>KRM-II-08</td><td></td><td></td><td>experimental compounds not available from a commercial source</td></tr><tr><td>Leica Application Suite X</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Novus</td><td>N276314100U</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma Aldrich</td><td>M9272</td><td>Hexahydrate</td></tr><tr><td>Microscope, Confocal</td><td>Leica</td><td>SP8</td><td></td></tr><tr><td>Microscope, Light</td><td>VWR</td><td>76382-982</td><td>DMiL Inverted</td></tr><tr><td>MTS - Promega One Step</td><td>Promega</td><td>G3581</td><td></td></tr><tr><td>Multi-channel pipette, 0.5-10 &amp;micro;L</td><td>Eppendorf</td><td>Z683914</td><td></td></tr><tr><td>Multi-channel pipette, 10-100 &amp;micro;L</td><td>Eppendorf</td><td>Z683930</td><td></td></tr><tr><td>Multi-channel pipette, 30-300 &amp;micro;L</td><td>Eppendorf</td><td>Z683957</td><td></td></tr><tr><td>Nest-O-Patch</td><td>Heka</td><td></td><td></td></tr><tr><td>Neurobasal-A Medium</td><td>Gibco</td><td>10888022</td><td>Without vitamin A</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibco</td><td>12348-017</td><td>Phenol red free</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>NOR-QH-II-66</td><td></td><td></td><td>experimental compounds not available from a commercial source</td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>50-998-944</td><td>4 inch width</td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>RT-15710</td><td></td></tr><tr><td>PATHCHMASTER</td><td>Heka</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Perfusion System</td><td>Nanion</td><td>4000120</td><td></td></tr><tr><td>PFA</td><td>EMS</td><td>RT-15710</td><td></td></tr><tr><td>Phosphate Bufered Saline</td><td>Fisher Scientific</td><td>AAJ75889K2</td><td>Reagent grade</td></tr><tr><td>Poly-D-Lysine</td><td>Fisher Scientific</td><td>A3890401</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma Life Sciences</td><td>P4707</td><td></td></tr><tr><td>Port-a-Patch</td><td>Nanion</td><td>21000072</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Life Sciences</td><td>P5405</td><td></td></tr><tr><td>Primary Antibody</td><td>Invitrogen</td><td>MA5-34653</td><td>Rabbit Monoclonal</td></tr><tr><td>Prism</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Propofol</td><td>Fisher Scientific</td><td>NC0758676</td><td>1 mL ampule</td></tr><tr><td>QH-II-66</td><td></td><td></td><td>experimental compounds not available from a commercial source</td></tr><tr><td>Reagent Reservoirs</td><td>VWR</td><td>89094-664</td><td>Sterile</td></tr><tr><td>Slides, 75 x 25 mm</td><td>Fisher Scientific</td><td>12-544-7</td><td>Frosted one side</td></tr><tr><td>Sodium Bicarbonate</td><td>Corning</td><td>25-035-Cl</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-3</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Synth-a-Freeze Medium</td><td>Gibco</td><td>R00550</td><td>Cryopreservation</td></tr><tr><td>TMRE</td><td>Fisher Scientific</td><td>50-196-4741</td><td>Reagent</td></tr><tr><td>TMRE Kit</td><td>Abcam</td><td>AB113852</td><td>Kit</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>NC0704309</td><td></td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15-250-061</td><td>Solution, 0.4%</td></tr><tr><td>Trypsin/EDTA</td><td>Gibco</td><td>25200-072</td><td>Solution, 0.25%</td></tr><tr><td>Vortex Mixer</td><td>VWR</td><td>97043-562</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Fisher Scientific</td><td>09-927-841</td><td></td></tr></tbody></table>

screening ion channels in cancer cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Above are select procedures that can be employed to characterize ion channels in cancerous cells. The first protocol highlights the staining of an ion channel. As detailed, there are many challenges when staining an ion channel or, for that matter, any protein that is present in the extracellular membrane. Shown in Figure 1 is the staining for a subunit of the pentameric GABAA receptor. The second protocol highlights the results of testing the polarized state of mitochondria in ca...

Watch this Scientific Journal Video about Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches at JoVE.com

Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches 

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

Screening Ion Channels in Cancer Cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

Research

JoVE Journal

Cancer Research

2.5K Views.  University of Cincinnati College of Medicine. De functie van ionkanalen is cruciaal in de celbiologie en heeft een aanzienlijke invloed op de groei van kanker. Ons team is gericht op het richten van ionkanalen om kankerbehandelingen te ondersteunen. Het veld heeft onlangs een beter begrip gekregen van de micro-omgeving van de tumor en hoe kankercellen zich vastgrijpen in gezond weefsel, wat het belang van ionkanalen bij kanker benadrukt.Een van de grootste uitdagingen in het huidige experiment is de beschikbaarheid van immuun-comp...

Video: Auteur in de schijnwerpers: onderzoek naar de rol van ionenkanalen bij kanker: karakterisering en mogelijke behandelingsbenaderingen 

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Biochemistry

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Genetics

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Fluorescent Assay for Detection of Novel Iron Chelators

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Biology

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Screening Ion Channels in Cancer Cells

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