PI3Kß is selectively required for growth factor-stimulated macropinocytosis.

Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kß (containing the p110ß catalytic subunit encoded by PIK3CB). Loss of PI3Kß expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kß is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kß.

PI3Kß links integrin activation and PI(3,4)P2 production during invadopodial maturation.

The invasion of tumor cells from the primary tumor is mediated by invadopodia, actin-rich protrusive organelles that secrete matrix metalloproteases and degrade the extracellular matrix. This coupling between protrusive activity and matrix degradation facilitates tumor invasion. We previously reported that the PI3Kß isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and G protein-coupled receptors, is required for invasion and gelatin degradation in breast cancer cells. We have now defined the mechanism by which PI3Kß regulates invadopodia. We find that PI3Kß is specifically activated downstream from integrins, and is required for integrin-stimulated spreading and haptotaxis as well as integrin-stimulated invadopodia formation. Surprisingly, these integrin-stimulated and PI3Kß-dependent responses require the production of PI(3,4)P2 by the phosphoinositide 5'-phosphatase SHIP2. Thus, integrin activation of PI3Kß is coupled to the SHIP2-dependent production of PI(3,4)P2, which regulates the recruitment of PH domain-containing scaffolds such as lamellipodin to invadopodia. These findings provide novel mechanistic insight into the role of PI3Kß in the regulation of invadopodia in breast cancer cells.

A single discrete Rab5-binding site in phosphoinositide 3-kinase ß is required for tumor cell invasion.

Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.

Rac1-stimulated macropinocytosis enhances Gßγ activation of PI3Kß.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kß, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gßγ subunits to the C2-helical domain linker of p110ß, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110ß. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gßγ led to an additive PI3Kß-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kß that cannot bind Gßγ, Gßγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kß was blocked. Surprisingly, PI3Kß-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110ß that cannot bind Rac1. Instead of directly binding to PI3Kß, CA-Rac1 acts by enhancing Gßγ coupling to PI3Kß, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gßγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kß by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gßγ coupling to PI3Kß.

In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability.

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.

Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue.

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.

There are four dynamically and functionally distinct populations of E-cadherin in cell junctions.

E-cadherin is a trans-membrane tumor suppressor responsible for epithelial cell adhesion. E-cadherin forms adhesive clusters through combined extra-cellular cis- and trans-interactions and intracellular interaction with the actin cytoskeleton. Here we identify four populations of E-cadherin within cell junctions based on the molecular interactions which determine their mobility and adhesive properties. Adhesive and non-adhesive populations of E-cadherin each consist of mobile and immobile fractions. Up to half of the E-cadherin immobilized in cell junctions is non-adhesive. Incorporation of E-cadherin into functional adhesions require all three adhesive interactions, with deletion of any one resulting in loss of effective cell-cell adhesion. Interestingly, the only interaction which could independently slow the diffusion of E-cadherin was the tail-mediated intra-cellular interaction. The adhesive and non-adhesive mobile fractions of E-cadherin can be distinguished by their sensitivity to chemical cross-linking with adhesive clusters. Our data define the size, mobility, and adhesive properties of four distinct populations of E-cadherin within cell junctions, and support association with the actin cytoskeleton as the first step in adhesion formation.

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic. Furthermore, interaction with other cell types, including stromal fibroblasts and immune cells, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo. Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo. As such, they can serve an important role in reducing the need for experiments on living animals.