Christian Beaudry

Inhibition of Rho-Kinase Affects Astrocytoma Morphology, Motility, and Invasion through Activation of Rac1

Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell systems has implicated a role for the Rho-GTPases in cell motility and invasion. Here, we examine how the inhibition or activation of Rho-kinase (ROCK) affects astrocytoma morphology, motility, and invasion. ROCK was inhibited in astrocytoma cells by using 5 to 100 mumol/L of Y27632 or by expressing the dominant-negative ROCK mutant, RB/PH TT. ROCK activation was achieved by expressing a constitutively active mutant, CAT. ROCK inhibition led to morphologic and cytoskeletal alterations characterized by an increase in the number and length of cell processes, increased membrane ruffling, and collapse of actin stress fibers. Using two-dimensional radial migration and Boyden chamber assays, we show that astrocytoma migration and invasion were increased at least 2-fold by ROCK inhibition. On the contrary, ROCK activation significantly inhibited migration and invasion of astrocytoma cells. Furthermore, using a Rac-GTP pull-down assay, we show that Rac1 is activated as a consequence of ROCK inhibition. Finally, we show that treatment of astrocytoma cells with small interfering RNA duplexes specific for Rac1-reversed stellation, prevented membrane ruffling formation and abrogated the increased motility observed following treatment with Y27632. Our data show that Rac1 plays a major role in astrocytoma morphology, motility, and invasion. These findings warrant further investigation to determine precisely how the modulation of Rac1 and ROCK can be exploited to inhibit glioma invasion.

Migrating glioma cells activate the PI3-K pathway and display decreased susceptibility to apoptosis

Glioma cells that migrate out of the main tumor mass into normal brain tissue contribute to the failure of most gliomas to respond to treatment. Treatments that target migratory glioma cells may enhance the therapeutic response. Multiple lines of evidence suggest that suppression of apoptosis accompanies activation of the migratory phenotype. Here, we determine whether migration and apoptosis are consistently linked in glioma cells and whether manipulation of migration influences cytotoxic therapy-induced apoptosis. Camptothecin and Trail-induced apoptosis were decreased 2-5-fold in actively migrating glioma cells relative to migration-restricted cells. Consistent with a mechanistic link between migration and apoptosis, the dose-response for stimulation of migration on laminin was inversely proportional to apoptosis induction. Treatment of glioma cells with migration inhibitors alone had little effect on basal rates of apoptosis and had little effect on Trail-induced or camptothecin-induced apoptosis in migration-restricted cells. By contrast, migration inhibitors increased camptothecin and Trail-induced apoptosis in actively migrating glioma cells. Migrating glioma cells have increased amounts of phosphorylated Akt and its downstream substrate glycogen synthase kinase-3 relative to migration restricted cells. Treatment of migrating cells with a specific inhibitor of phosphoinositide 3-kinase (PI3-K), LY294002, blocked the phosphorylation of Akt and increased the sensitivity to apoptosis. LY294002 had no effect on the migration of restricted cells. This suggests that migrating glioma cells activate the PI3-K survival pathway, protecting migrating cells from apoptosis. Taken together, these data provide support for a link between migration and apoptosis in glioma cells. In addition, evidence indicates that treatment with migration inhibitors, while not affecting apoptosis-induction in migration-restricted cells, can sensitize migrating glioma cells to cytotoxic agents.

MAP-ing glioma invasion: Mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy. [Mol Cancer Ther 2007;6(4):1212–22]

Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

BackgroundGlioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death.To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis.ResultsGene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells.ConclusionGene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation.

BackgroundNeurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimers disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.ResultsWe report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways.ConclusionsThese findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.

Detailed Molecular Fingerprinting of Four New Anaplastic Thyroid Carcinoma Cell Lines and Their Use for Verification of RhoB as a Molecular Therapeutic Target

CONTEXT Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma

BackgroundEwings sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewings sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewings sarcoma cells.ResultsFour Ewings sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewings sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewings sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewings sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis.ConclusionIn summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewings sarcoma.

A high-content RNAi-screening assay to identify modulators of cholesterol accumulation in Niemann-Pick type C cells.

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder characterized by a defect in intracellular trafficking of exogenous cholesterol and glycosphingolipids. A goal for therapeutic treatment of NPC is to decrease/normalize cholesterol accumulation. We developed a functional genomics-based assay, combining high-throughput RNA interference (HT-RNAi) screening with high-content fluorescence imaging to identify specific genes in NPC cells that will result in more normal cholesterol levels in the diseased cells. Conditions for siRNA tranfections were optimized for 2 NPC fibroblast cell lines (GM03123, GM18453) and a normal fibroblast cell line (GM05659). RNAi screening was done using a focused-set siRNA library targeting 40 cholesterol trafficking-associated genes, knowledge mined from the existing literature on NPC disease, and/or their association with NPC1/NPC2 genes. We utilized filipin staining as a measure of cholesterol accumulation in fixed NPC cells. Data analysis of these screens confirmed several genes including LDLR and RAB9A that reduced cholesterol content in NPC cells. Nine genes were validated using filipin staining to detect unesterified cholesterol as well as cholesteryl BODIPY esters to study lipid trafficking. Gene silencing was also confirmed using qRT-PCR. Our results show that this technology can be applied to larger screens to identify genes responsible for lipid accumulation and/or trafficking in NPC disease, which could be instrumental in developing innovative therapies for individuals afflicted with NPC disease.

Chemogenomic analysis identifies Macbecin II as a compound specific for SMAD4-negative colon cancer cells.

The tumor suppressor gene, SMAD4, is mutated in approximately 30% of colon cancers. To identify compounds with enhanced potency on cells with a SMAD4‐negative context, we combined genomic and cheminformatic analyses of publicly available data relating to the colon cancer cell lines within the NCI60 panel. Two groups of cell lines were identified with either wild‐type or negative SMAD4 status. A cheminformatic analysis of the NCI60 screening data was carried out, which led to the identification of 14 compounds that preferentially inhibited cell growth of the SMAD4‐negative cell lines. Using cell viability assays, the effect of these compounds was validated on four colon cancer cell lines: HCT‐116 and HCT‐15 (SMAD4‐expressing), and HT‐29 and COLO‐205 (SMAD4‐negative). Our data identified Macbecin II, a hydroquinone ansamycin antibiotic, as having increased potency in the SMAD4‐negative cells compared to SMAD4 wild‐type cells. In addition, we showed that silencing of SMAD4 using siRNA in HCT‐116 enhanced Macbecin II potency. Our results demonstrate that Macbecin II is specifically active in colon cancer cells having a SMAD4‐negative background and thus is a potential candidate for further investigation in a drug discovery perspective.

P2-141: Unraveling the process of tau hyperphosphorylation one gene at a time

RiLee H. Robeson, Gillian R. Brautigam, Danielle Frost, David Azorsa, Chad Dickey, Christian Beaudry, Gargi Basu, David Holz, Joseph Hernandez, Kristen Bisanz, Leslie Gwinn, Bessie Meechoovet, Andrew Grover, Joe Rogers, Eric M. Reiman, Dietrich A. Stephan, Spyro Mousses, Travis Dunckley, Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA; Pharmaceutical Genomics Division, Translational Genomics Research Institute, Scottsdale, AZ, USA; Department of Pharmacology, University of South Florida, Tampa, FL, USA; Center for Alzheimer’s Research, Sun Health Research Institute, Phoenix, AZ, USA; Banner Alzheimer’s Disease Institute, Banner Good Samaeritan Medical Center, Phoenix, AZ, USA. Contact e-mail: rherbert@tgen.org