Alain Charest

Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy

Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell–derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.

Selective ablation of αv integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death

Mouse embryos genetically null for all αv integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating αv expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of αv expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of αv integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that αv integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for αv integrins expressed on axons in the postnatal central nervous system.

Fusion of FIG to the receptor tyrosine kinase ROS in a glioblastoma with an interstitial del(6)(q21q21).

The transmembrane proto‐oncogene receptor tyrosine kinase (RTK) ROS is an orphan receptor that is aberrantly expressed in neoplasms of the central nervous system. Here, we report the fusion of its carboxy‐terminal kinase domain to the amino‐terminal portion of a protein called FIG (Fused in Glioblastoma) in a human glioblastoma multiforme (GBM). By characterizing both FIG and ROS genes in normal and in U118MG GBM cells, we determined that an intra‐chromosomal homozygous deletion of 240 kilobases on 6q21 is responsible for the formation of the FIG‐ROS locus. The FIG‐ROS transcript is encoded by 7 FIG exons and 9 ROS‐derived exons. We also demonstrate that the FIG‐ROS locus encodes for an in‐frame fusion protein with a constitutively active kinase activity, suggesting that FIG‐ROS may act as an oncogene. This is the first example of a fusion RTK protein that results from an intra‐chromosomal deletion, and it represents the first fusion RTK protein isolated from a human astrocytoma.

Functional delivery of siRNA in mice using dendriworms.

Small interfering RNAs (siRNAs) mediate cleavage of specific, complementary mRNA sequences and thus regulate gene expression. Not surprisingly, their use for treatment of diseases that are rooted in aberrant gene expression, such as cancer, has become a paradigm that has gained wide interest. Here, we report the development of dendrimer-conjugated magnetofluorescent nanoworms that we call “dendriworms” as a modular platform for siRNA delivery in vivo. This platform maximizes endosomal escape to robustly produce protein target knockdown in vivo, and is tolerated well in mouse brain. We demonstrate that siRNA-carrying dendriworms can be readily internalized by cells and enable endosomal escape across a wide range of loading doses, whereas dendrimers or nanoworms alone are inefficient. Further, we show that dendriworms carrying siRNA against the epidermal growth factor receptor (EGFR) reduce protein levels of EGFR in human glioblastoma cells by 70−80%, 2.5-fold more efficiently than commercial cationic lipids. Dendriworms were well-tolerated after 7-days of convection-enhanced delivery to the mouse brain and in an EGFR-driven transgenic model of glioblastoma, anti- EGFR dendriworms led to specific and significant suppression of EGFR expression. Collectively, these data establish dendriworms as a multimodal platform that enables fluorescent tracking of siRNA delivery in vivo, cellular entry, endosomal escape, and knockdown of target proteins.

Cloning and chromosomal mapping of an orphan chemokine receptor: mouse RDC1

Abstract Degenerate RT-PCR was used to identify a new seven-transmembrane-spanning receptor expressed in astrocytes. A receptor, termed RDC1, displaying the characteristic structural features of a chemokine receptor was cloned. The predicted 362-amino-acid sequence displayed 92% and 91% similarity to the human and dog orphan receptor RDC1, respectively. In addition, RDC1 shares 43% amino acid similarity to rabbit and mouse CXCR2. Transcripts of RDC1 were found in astrocytes, heart, kidney, the mesangial tumor line MES-13, spleen, and neutrophils by means of northern blot. Using linkage analysis of interspecies backcross mice, we localized to chromosome 1 the genes for mouse CXCR2, CXCR4, and RDC1. Mouse RDC1 is linked to and lies between the genes for the mouse CXC chemokine receptors CXCR2 and CXCR4. The combined data of chromosomal location and sequence similarity suggest that RDC1 is an orphan CXC chemokine receptor.

Genome complexity reduction for SNP genotyping analysis

Efficient single nucleotide polymorphism (SNP) genotyping methods are necessary to accomplish many current gene discovery goals. A crucial element in large-scale SNP genotyping is the number of individual biochemical reactions that must be performed. An efficient method that can be used to simultaneously amplify a set of genetic loci across a genome with high reliability can provide a valuable tool for large-scale SNP genotyping studies. In this paper we describe and characterize a method that addresses this goal. We have developed a strategy for reducing genome complexity by using degenerate oligonucleotide primer (DOP)-PCR and applied this strategy to SNP genotyping in three complex eukaryotic genomes; human, mouse, and Arabidopsis thaliana. Using a single DOP-PCR primer, SNP loci spread throughout a genome can be amplified and accurately genotyped directly from a DOP-PCR product mixture. DOP-PCRs are extremely reproducible. The DOP-PCR method is transferable to many species of interest. Finally, we describe an in silico approach that can effectively predict the SNP loci amplified in a given DOP-PCR, permitting the design of an efficient set of reactions for large-scale, genome-wide SNP studies.

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.

Coupling of the murine protein tyrosine phosphatase PEST to the epidermal growth factor (EGF) receptor through a Src homology 3 (SH3) domain-mediated association with Grb2.

The involvement of murine protein tyrosine phosphatase-PEST (MPTP – PEST) in signal transduction pathways is suggested by its ability to dephosphorylate phosphotyrosine residues, its interaction with the adaptor protein SHC and by the presence of five proline-rich stretches in its non-catalytic carboxyl terminus. Proline-rich sequences have been identified as binding sites for Src homology 3 (SH3) domains found in proteins associated with signal transduction events. The ability of these sequences to act as SH3 domain recognition motifs was investigated using bacterially expressed SH3 domains derived from several different signalling proteins. In vitro binding assays indicate that four of these proline-rich sequences constitute specific binding sites for both SH3 domains of the adaptor molecule Grb2. Wild type Grb2, but not Grb2 proteins corresponding to loss-of-function mutants in the Caenorhabditis elegans sem-5 protein, associate with MPTP – PEST in vivo. Experiments in EGF receptor expressing cells show that the interaction between MPTP – PEST and Grb2 results in the binding of this complex to activated EGF receptors. In addition, identification of putative substrate(s) of MPTP – PEST have revealed a candidate protein of ∼120 kDa which is tyrosine phosphorylated upon EGF stimulation. Together, these results describe a novel SH3 domain-dependent recruitment of a protein tyrosine phosphatase to an activated receptor tyrosine kinase and establish a potential role for MPTP – PEST in signalling pathways at the molecular level.

Acquired MET expression confers resistance to EGFR inhibition in a mouse model of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type epidermal growth factor receptor (EGFR) and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that an important component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.

Inhibition of EGFR Induces a c-MET-Driven Stem Cell Population in Glioblastoma

Glioblastoma multiforme (GBM) is the most lethal form of primary brain tumors, characterized by highly invasive and aggressive tumors that are resistant to all current therapeutic options. GBMs are highly heterogeneous in nature and contain a small but highly tumorigenic and self‐renewing population of stem or initiating cells (glioblastoma stem cells or GSCs). GSCs have been shown to contribute to tumor propagation and resistance to current therapeutic modalities. Recent studies of human GBMs have elucidated the genetic alterations common in these tumors, but much remains unknown about specific signaling pathways that regulate GSCs. Here we identify a distinct fraction of cells in a genetically engineered mouse model of EGFR‐driven GBM that respond to anti‐EGFR therapy by inducing high levels of c‐MET expression. The MET‐positive cells displayed clonogenic potential and long‐term self‐renewal ability in vitro and are capable of differentiating into multiple lineages. The MET‐positive GBM cells are resistant to radiation and highly tumorigenic in vivo. Activation of MET signaling led to an increase in expression of the stemness transcriptional regulators Oct4, Nanog, and Klf4. Pharmacological inhibition of MET activity in GSCs prevented the activation of Oct4, Nanog, and Klf4 and potently abrogated stemness. Finally, the MET expressing cells were preferentially localized in perivascular regions of mouse tumors consistent with their function as GSCs. Together, our findings indicate that EGFR inhibition in GBM induces MET activation in GSCs, which is a functional requisite for GSCs activity and thus represents a promising therapeutic target. Stem Cells 2014;32:338–348