The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a longrange gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Iglike and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ϳ45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3 H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed ␥-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)-and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.motor-protein driven transport ͉ clathrin adaptor AP-1 ͉ endosomal vesicles ͉ recycling T he endosomal system comprises a mosaic of dynamically interconnected organelles that fulfills a variety of important cell physiological functions ranging from the uptake, recycling, and degradation of nutrients, signaling molecules and cell surface receptors to the regulation of cell migration, differentiation, and morphogenesis (1-3). The endocytic pathway also intersects with the biosynthetic delivery of lysosomal enzymes at several stations, most notably at the trans-Golgi network (TGN)/ endosomal boundary (1). Endosomes and TGN-or endosomederived vesicles exhibit characteristic distribution patterns with Rab4-positive sorting endosomal vesicles (EVs) and tubular recycling endosomes (REs) typically concentrated in the pericentrosomal area (4). Early endosomes, by contrast, appear dispersed throughout the cytoplasm (5). Function and dynamics of endosomes and EVs requires a so far ill-defined interplay between organellar sorting adaptors, the cytoskeleton (6) and molecular motors (7). Here we show that ␥-BAR, a recently described accessory factor of the clathrin adaptor complex AP-1 at the TGN/endosomal interface (8), modulates the dynamics of transferrin (Tf)-and Rab4-positive EVs by directly associating with AP-1 and kinesin KIF5. Because ␥-BAR does not harbor a curvature-sensing BIN/amphiphysin/Rvs167 (BAR) domain, we refer to this protein as Gadkin, for ␥1-adaptin and kinesin interactor. The AP-1/Gadkin/KIF5 complex identified here provides a hitherto unknown molecular link between TGN-derived EVs and the microtubule-based cytoskeleton. Our work also suggests a surprising complexity of endosomal membrane dynamics and its integration with cargo sorting. Results Gadkin Localizes to the TGN and to Perinuclear EVs and Regulates EVPositioning. Gadkin has originally been identified as an AP-1 binding protein localized to the T...