Glial Cell Adhesion Molecule (GlialCAM) Determines Proliferative versus Invasive Cell States in Glioblastoma.

The malignant brain cancer glioblastoma (GBM) contains groups of highly invasive cells that drive tumor progression as well as recurrence after surgery and chemotherapy. The molecular mechanisms that enable these GBM cells to exit the primary mass and disperse throughout the brain remain largely unknown. Here we report using human tumor specimens and primary spheroids from male and female patients that glial cell adhesion molecule (GlialCAM), which has normal roles in brain astrocytes and is mutated in the developmental brain disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC), is differentially expressed in subpopulations of GBM cells.

MMP9 Clears the Way for Metastatic Cell Penetration Across the Blood-Brain Barrier.

Although brain metastases are 10-fold more prevalent than primary brain cancers, relatively little is understood about the genes and pathways that promote metastatic cell entry, growth, and survival in the brain. Hence, determining how metastatic tumors colonize the brain and thrive within the neural microenvironment is a topic of both fundamental importance and direct clinical relevance. In this issue, a report by Karreman and colleagues explores pathways that are exploited by metastatic tumor cells to arrest in the circulation, cross the endothelial blood-brain barrier (BBB), and thrive in the brain microenvironment.

The alpha7 integrin subunit in astrocytes promotes endothelial blood-brain barrier integrity.

The blood-brain barrier (BBB) is a vascular endothelial cell boundary that partitions the circulation from the central nervous system to promote normal brain health. We have a limited understanding of how the BBB is formed during development and maintained in adulthood. We used quantitative transcriptional profiling to investigate whether specific adhesion molecules are involved in BBB functions, with an emphasis on understanding how astrocytes interact with endothelial cells.

The β8 integrin cytoplasmic domain activates extracellular matrix adhesion to promote brain neurovascular development.

In the developing mammalian brain, neuroepithelial cells interact with blood vessels to regulate angiogenesis, blood-brain barrier maturation and other key neurovascular functions. Genetic studies in mice have shown that neurovascular development is controlled, in part, by Itgb8, which encodes the neuroepithelial cell-expressed integrin β8 subunit. However, these studies have involved complete loss-of-function Itgb8 mutations, and have not discerned the relative roles for the β8 integrin extracellular matrix (ECM) binding region versus the intracellular signaling tail.

Mlc1-Expressing Perivascular Astrocytes Promote Blood-Brain Barrier Integrity.

In the mammalian brain, perivascular astrocytes (PAs) closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). Deciphering specific functions for PAs in BBB biology, however, has been limited by the ability to distinguish these cells from other astrocyte populations. In order to characterize selective roles for PAs , a new mouse model has been generated in which the endogenous megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) gene drives expression of Cre fused to a mutated estrogen ligand-binding domain (Mlc1-T2A-CreERT2).

Megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1) promotes glioblastoma cell invasion in the brain microenvironment.

Glioblastoma (GBM), or grade IV astrocytoma, is a malignant brain cancer that contains subpopulations of proliferative and invasive cells that coordinately drive primary tumor growth, progression, and recurrence after therapy. Here, we have analyzed functions for megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1), an eight-transmembrane protein normally expressed in perivascular brain astrocyte end feet that is essential for neurovascular development and physiology, in the pathogenesis of GBM. We show that Mlc1 is expressed in human stem-like GBM cells (GSCs) and is linked to the development of primary and recurrent GBM.

Isolation and transcriptional characterization of mouse perivascular astrocytes.

In the post-natal mammalian brain perivascular astrocytes (PAs) ensheath blood vessels to regulate their unique permeability properties known as the blood-brain barrier (BBB). Very little is known about PA-expressed genes and signaling pathways that mediate contact and communication with endothelial cells (ECs) to regulate BBB physiology. This is due, in part, to lack of suitable models to distinguish PAs from other astrocyte sub-populations in the brain.

The vascular endothelial cell-expressed prion protein doppel promotes angiogenesis and blood-brain barrier development.

The central nervous system (CNS) contains a complex network of blood vessels that promote normal tissue development and physiology. Abnormal control of blood vessel morphogenesis and maturation is linked to the pathogenesis of various neurodevelopmental diseases. The CNS-specific genes that regulate blood vessel morphogenesis in development and disease remain largely unknown.

α integrins in Schwann cells promote attachment to axons, but are dispensable in vivo.

In the developing peripheral nervous system, Schwann cells (SCs) extend their processes to contact, sort, and myelinate axons. The mechanisms that contribute to the interaction between SCs and axons are just beginning to be elucidated. Using a SC-neuron coculture system, we demonstrate that Arg-Gly-Asp (RGD) peptides that inhibit α -containing integrins delay the extension of SCs elongating on axons.

αvβ8 integrin adhesion and signaling pathways in development, physiology and disease.

Cells must interpret a complex milieu of extracellular cues to modulate intracellular signaling events linked to proliferation, differentiation, migration and other cellular processes. Integrins are heterodimeric transmembrane proteins that link the extracellular matrix (ECM) to the cytoskeleton and control intracellular signaling events. A great deal is known about the structural and functional properties for most integrins; however, the adhesion and signaling pathways controlled by αvβ8 integrin, which was discovered nearly 30 years ago, have only recently been characterized.

Metastatic Brain Tumors Disrupt the Blood-Brain Barrier and Alter Lipid Metabolism by Inhibiting Expression of the Endothelial Cell Fatty Acid Transporter Mfsd2a.

Disruption of the blood-brain barrier (BBB) by cancer cells is linked to metastatic tumor initiation and progression; however, the pathways that drive these events remain poorly understood. Here, we have developed novel patient-derived xenograft (PDX) models of brain metastases that recapitulate pathological growth features found in original patient samples, thus allowing for analysis of BBB disruption by tumor cells. We report that the BBB is selectively disrupted in brain metastases, in part, via inhibition of the endothelial cell-expressed docosahexaenoic acid (DHA) transporter, major facilitator superfamily domain 2a (Mfsd2a).

PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells.

Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary mass, escaping surgical resection and invariably giving rise to lethal recurrent lesions. Here we report that PTP-PEST, a cytoplasmic protein tyrosine phosphatase, controls GBM cell invasion by physically bridging the focal adhesion protein Crk-associated substrate (Cas) to valosin-containing protein (Vcp), an ATP-dependent protein segregase that selectively extracts ubiquitinated proteins from multiprotein complexes and targets them for degradation via the ubiquitin proteasome system. Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) in Vcp impacting affinity for Cas in focal adhesions and controlling ubiquitination levels and protein stability.

Integrins in Vascular Development and Pathology.

During vascular development, endothelial cells (ECs) and neighboring stromal cells interact and communicate through autocrine and paracrine signaling mechanisms involving extracellular matrix (ECM) proteins and their cell surface integrin adhesion receptors. Integrin-mediated adhesion and signaling pathways are crucial for normal vascular development and physiology, and alterations in integrin expression and/or function drive several vascular-related pathologies including thrombosis, autoimmune disorders, and cancer. The purpose of this chapter is to discuss integrin adhesion and signaling pathways important for EC growth, survival, and migration.

Neuropilin-1 modulates TGFβ signaling to drive glioblastoma growth and recurrence after anti-angiogenic therapy.

Glioblastoma (GBM) is a rapidly progressive brain cancer that exploits the neural microenvironment, and particularly blood vessels, for selective growth and survival. Anti-angiogenic agents such as the vascular endothelial growth factor-A (VEGF-A) blocking antibody bevacizumab yield short-term benefits to patients due to blood vessel regression and stabilization of vascular permeability. However, tumor recurrence is common, and this is associated with acquired resistance to bevacizumab.

Selective expression of eGFP in mouse perivascular astrocytes by modification of the Mlc1 gene using T2A-based ribosome skipping.

Perivascular astrocyte end feet closely juxtapose cerebral blood vessels to regulate important developmental and physiological processes including endothelial cell proliferation and sprouting as well as the formation of the blood-brain barrier (BBB). The mechanisms underlying these events remain largely unknown due to a lack of experimental models for identifying perivascular astrocytes and distinguishing these cell types from other astroglial populations. Megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) is a transmembrane protein that is expressed in perivascular astrocyte end feet where it controls BBB development and homeostasis.

RICE CRISPR: Rapidly increased cut ends by an exonuclease Cas9 fusion in zebrafish.

Application of CRISPR-Cas9 technology in diverse organisms has resulted in an explosion of genome modification efforts. To expand the toolbox of applications, we have created an E. coli Exonuclease I (sbcB)-Cas9 fusion that has altered enzymatic activity in zebrafish embryos.

The Cytoskeletal Adapter Protein Spinophilin Regulates Invadopodia Dynamics and Tumor Cell Invasion in Glioblastoma.

Unlabelled: Glioblastoma is a primary brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to invasive cancer cells that disperse from the main tumor site, escape surgical resection, and contribute to recurrent secondary lesions. The adhesion and signaling mechanisms that drive glioblastoma cell invasion remain enigmatic, and as a result there are no effective anti-invasive clinical therapies.

Neuropilin 1 balances β8 integrin-activated TGFβ signaling to control sprouting angiogenesis in the brain.

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells.

Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth.

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells' adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth.

Protein tyrosine phosphatase-PEST and β8 integrin regulate spatiotemporal patterns of RhoGDI1 activation in migrating cells.

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1).

Inducible gene deletion in glial cells to study angiogenesis in the central nervous system.

Most organs and tissues of the vertebrate body harbor elaborate network of blood vessels with diverse functions that are determined, in part, by cues within the local environment (Warren and Iruela-Arispe, Curr Opin Hematol 17:213-218, 2010). How vascular endothelial cells decipher these cues to promote normal blood vessel development and physiology remains largely uncharacterized. In this review, we will focus on genetic strategies to analyze glial regulation of blood vessel growth and sprouting within the microenvironment of the retina, a component of the central nervous system (CNS) that contains a complex web of blood vessels with many unique features, including a blood-retinal barrier (Abbott et al.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency.

The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity.

Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear.

Glioblastoma resistance to anti-VEGF therapy: has the challenge been MET?

In glioblastoma cells the receptor tyrosine kinase c-Met is upregulated in response to bevacizumab and plays an important role in promoting invasion and tumor recurrence. These data support novel links between VEGF-A and hepatocyte growth factor and suggest that c-Met and its signaling effectors may be effective targets for anti-invasive therapies.

αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion.

The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion.