Perspectives on Vascular Regulation of Mechanisms Controlling Selective Immune Cell Function in the Tumor Immune Response.

The vasculature plays a major role in regulating the tumor immune cell response although the underlying mechanisms explaining such effects remain poorly understood. This review discusses current knowledge on known vascular functions with a viewpoint on how they may yield distinct immune responses. The vasculature might directly influence selective immune cell infiltration into tumors by its cell surface expression of cell adhesion molecules, expression of cytokines, cell junction properties, focal adhesions, cytoskeleton and functional capacity. This will alter the tumor microenvironment and unleash a plethora of responses that will influence the tumor's immune status. Despite our current knowledge of numerous mechanisms operating, the field is underexplored in that few functions providing a high degree of specificity have yet been provided in relation to the enormous divergence of responses apparent in human cancers. Further exploration of this field is much warranted.

An AKT2-specific nanobody that targets the hydrophobic motif induces cell cycle arrest, autophagy and loss of focal adhesions in MDA-MB-231 cells.

The AKT kinase family is a high-profile target for cancer therapy. Despite their high degree of homology the three AKT isoforms (AKT1, AKT2 and AKT3) are non-redundant and can even have opposing functions. Small-molecule AKT inhibitors affect all three isoforms which severely limits their usefulness as research tool or therapeutic. Using AKT2-specific nanobodies we examined the function of endogenous AKT2 in breast cancer cells. Two AKT2 nanobodies (Nb8 and Nb9) modulate AKT2 and reduce MDA-MB-231 cell viability/proliferation. Nb8 binds the AKT2 hydrophobic motif and reduces IGF-1-induced phosphorylation of this site. This nanobody also affects the phosphorylation and/or expression levels of a wide range of proteins downstream of AKT, resulting in a G0/G1 cell cycle arrest, the induction of autophagy, a reduction in focal adhesion count and loss of stress fibers. While cell cycle progression is likely to be regulated by more than one isoform, our results indicate that both the effects on autophagy and the cytoskeleton are specific to AKT2. By using an isoform-specific nanobody we were able to map a part of the AKT2 pathway. Our results confirm AKT2 and the hydrophobic motif as targets for cancer therapy. Nb8 can be used as a research tool to study AKT2 signalling events and aid in the design of an AKT2-specific inhibitor.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

The Ly49Q receptor plays a crucial role in neutrophil polarization and migration by regulating raft trafficking.

Neutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.