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SLIT2/ROBO1-Signaling Inhibits Macropinocytosis by Opposing Cortical Cytoskeletal Remodeling

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SLIT2/ROBO1-Signaling Inhibits Macropinocytosis by Opposing Cortical Cytoskeletal Remodeling ARTICLE https://doi.org/10.1038/s41467-020-17651-1 OPEN SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling Vikrant K. Bhosle 1, Tapas Mukherjee 2, Yi-Wei Huang 1, Sajedabanu Patel 1, Bo Wen (Frank) Pang1,3,14, Guang-Ying Liu1, Michael Glogauer4,5,6, Jane Y. Wu7,8,9, Dana J. Philpott2, Sergio Grinstein 1,10,11 & ✉ Lisa A. Robinson 1,3,12,13 Macropinocytosis is essential for myeloid cells to survey their environment and for growth of 1234567890():,; RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa. 1 Program in Cell Biology, The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto, ON M5G 0A4, Canada. 2 Department of Immunology, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. 3 Institute of Medical Science, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. 4 Faculty of Dentistry, University of Toronto, 101 Elm Street, Toronto, ON M5G 2L3, Canada. 5 Department of Dental Oncology and Maxillofacial Prosthetics, University Health Network, Princess Margaret Cancer Centre, 610 University Avenue, Toronto, ON M5G 2C1, Canada. 6 Centre for Advanced Dental Research and Care, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada. 7 Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. 8 Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. 9 Lurie Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. 10 Department of Biochemistry, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. 11 Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 290 Victoria Street, Toronto, ON M5C 1N8, Canada. 12 Division of Nephrology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada. 13 Department of Paediatrics, Faculty of Medicine, University of Toronto, 555 University Avenue, Toronto, ON M5G 1X8, Canada. 14Present address: ✉ BenchSci, Suite 201, 559 College Street, Toronto, ON M6G 1A9, Canada. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:4112 | https://doi.org/10.1038/s41467-020-17651-1 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17651-1 acrophages, which are specialized cells of the innate the actions of SLIT2 on macrophage functions have been largely immune system, perform diverse functions, including unexplored. M 1 fl host defense against pathogens . Pro-in ammatory The role played by Slit-Robo signaling in tumor progression vs. macrophages use various tools to combat invading microorgan- suppression also remains a topic of active investigation and isms, including secretion of inflammatory mediators, phagocy- appears to be context dependent36,37. Nonetheless, SLIT2 has tosis of the pathogen, and production of reactive oxygen species. been shown to be silenced in several invasive tumors and in These cellular processes are carefully regulated by dynamic cancer cell lines, including PDAC cells, and conversely, high changes in the macrophage cytoskeleton2. During immune sur- levels of SLIT2 mRNA are associated with suppressed tumor veillance, macrophages, and immature dendritic cells (iDCs) growth in vivo38,39. Although SLIT2-induced tumor suppression constitutively sample their extracellular surroundings via mac- has been presumed to be due inhibition of cancer cell migration, ropinocytosis, a phenomenon brought about by active plasma its effects on cancer cell growth have not been carefully membrane ruffling induced by remodeling of the cortical elucidated. cytoskeleton3,4. These cells also internalize bacterial pathogen- We report here that SLIT2 prevents macrophage spreading not associated molecular patterns (PAMPs), such as muramyl by inhibiting Rac activation, but rather, by activating RhoA. We dipeptide (MDP), via macropinocytosis, thereby initiating an further demonstrate that SLIT2 can inhibit macropinocytic immune response5. Intracellular pathogens, including some uptake of bacterial PAMPs and subsequent upregulation of viruses, exploit macropinocytosis to facilitate their entry into the inflammatory chemokines in vivo, thereby modulating macro- host cells6,7. Several growth factors, including colony stimulating phage immune responses. We show that SLIT2, in a ROBO1- factor 1 (CSF1), secreted by the host, as well as some pathogen- dependent manner, negatively impacts the survival of KRAS- derived proteins such as IpaC from Shigella are known to induce transformed cancer cells by inhibiting macropinocytosis, thus macropinocytosis3,8,9. It is estimated that macrophages can con- limiting protein uptake in a Glut-deprived state similar to the sume liquid equal to their cell volume every hour10. This would tumor microenvironment found in vivo. Our work identifies an leave them particularly vulnerable to the entry of soluble PAMPs endogenous inhibitor of macropinocytosis with potent effects and viruses, via macropinocytosis, under specific conditions. both in vitro and in vivo. Macropinocytosis is a near-universal feature of cancers driven by mutations in the RAS family of oncogenes11–13. RAS- transformed tumors account for approximately one third of all Results malignancies in humans14. Among the RAS family members, NSlit2 induces actin remodeling in macrophages in a ROBO1- KRAS mutations are the most common in cancer, especially in dependent manner.Wefirst investigated which Slit-binding adenocarcinomas such as pancreatic adenocarcinoma (PDAC)15, Robo receptors are expressed in macrophages and found that and they are associated with poorer response to the conventional Robo1, but not Robo2, messenger RNA (mRNA) is expressed in therapies and worse prognosis16. KRAS-transformed cancer cells the RAW264.7 macrophage cell line and in primary murine bone use macropinocytosis to internalize large proteins, such as albu- marrow-derived macrophages (BMDM) (Fig. 1a). Using immu- min, from their extracellular environment, to subsequently break noblotting, ROBO1 protein was detected in RAW264.7 cells as them down into amino acids, which enter the cell metabolism to well as in primary macrophages derived from human peripheral promote cancer cell survival11. This might be particularly relevant blood mononuclear (MDM) cells (Supplementary Fig. 1a). To in the tumor core, which contains low concentrations of amino investigate the effects of SLIT2 on macrophage cytoskeleton, we acids, including glutamine (Glut)17. Inhibition of macro- incubated cells with control vehicle, or with recombinant bioac- pinocytosis could be of therapeutic benefit in this context11. tive NSlit2 (the amino-terminal fragment of SLIT2), or with other However, no physiological factor that can inhibit macro- recombinant SLIT2 proteins, CSlit2 and Slit2ΔD2, which do not pinocytosis has been identified. bind to the Robo receptors (Fig. 1b)22,30,32. The endotoxin levels Spatiotemporal regulation of activity of Rho-family of small in all recombinant Slit protein preparations were below 0.05 EU/ GTPases is essential to bring about localized and reversible ml (Supplementary Fig. 1b; Supplementary Table 1). Treatment changes in the cellular actin cytoskeleton18. Accordingly, mul- with NSlit2 significantly reduced macrophage spreading, as seen tiple steps during macropinocytosis are regulated by small Rho by reduction in their total three-dimensional surface area (Fig. 1c, GTPases, Rac1 and Cdc423,19. The conserved Slit family of d and Supplementary Fig. 1c). To quantify the circularity of cells, neuronal guidance cues, together with their transmembrane we calculated the ‘shape factor’ using Volocity 6.3 software40. Roundabout (Robo) receptors, act as repellents during develop- Shape factor values range from 0 to 1, the latter defining a per- ment of the central nervous system by regulating actin cytos- fectly circular shape (see “Methods” for mathematical calcula- keletal rearrangements in migrating neurons and projecting tion). Only NSlit2 treatment significantly increased rounding in axons20. Slit proteins (SLIT1-3) undergo proteolytic cleavage human and murine macrophages (Fig. 1c, e; Supplementary in vivo, with N-terminal fragments binding to Robo receptors to Fig. 1d). To determine whether these effects of NSlit2 were induce signaling21. A growing number of
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