A roundabout way to cancer

Abstract

The Slit family of secreted proteins and their transmembrane receptor, Robo, were originally identified in the nervous system where they function as axon guidance cues and branching factors during development. Since their discovery, a great number of additional roles have been attributed to Slit/Robo signaling, including regulating the critical processes of cell proliferation and cell motility in a variety of cell and tissue types. These processes are often deregulated during cancer progression, allowing tumor cells to bypass safeguarding mechanisms in the cell and the environment in order to grow and escape to new tissues. In the past decade, it has been shown that the expression of Slit and Robo is altered in a wide variety of cancer types, identifying them as potential therapeutic targets. Further, studies have demonstrated dual roles for Slits and Robos in cancer, acting as both oncogenes and tumor suppressors. This bifunctionality is also observed in their roles as axon guidance cues in the developing nervous system, where they both attract and repel neuronal migration. The fact that this signaling axis can have opposite functions depending on the cellular circumstance make its actions challenging to define. Here, we summarize our current understanding of the dual roles that Slit/Robo signaling play in development, epithelial tumor progression, and tumor angiogenesis.

Figure 1. Structural Representation of Slits, Robos and Their Interaction

(A) At their N-terminus, vertebrate and invertebrate Slits consist of four Leucine-Rich Repeats (LRRs), termed D1 – D4. These LRRs are followed by seven-nine epidermal growth factor (EGF)-like domains, a laminin G-like domain (ALPS), and a C-terminal cysteine-rich knot. Slits are proteolytically cleaved between two EGF-like domains. (B) Vertebrates have four Robos (Robo1–4), while fly, chick and zebrafish have three (Robo1–3). Robo1, 2 and 3 contain five immunoglobulin (Ig) domains and 3 fibronectin type 3 (FN3) domains. Robo4 contains only two Ig domains and two FN3 domains. Zebrafish Robo4 is unique in that it contains three Ig domains instead of two. In their cytoplasmic tail, Robos contain between two and four conserved proline-rich domains (CC0-CC3). (C) The Slit/Robo signaling pair can be stabilized via heparan sulfate glycosaminoglycans (GAGs) that are present either in the extracellular matrix or attached to membrane-associated proteins such as the heparan sulfate proteoglycan (HSPG) syndecan.

Figure 2. Slit/Robo Signaling Regulates Cell Proliferation and Cell-Cell Contacts by Controlling the Localization of β-catenin In the Cell

(A) As illustrated by red arrows, binding of Slit to Robo inhibits phosphatidylinositol kinase (PI3K)-induced Akt activity. Glycogen synthase kinase-3beta (GSK3β) is consequently left in its non-phosphorylated, active form, and targets β-catenin for phosphorylation, excluding it from the nucleus and thus preventing its transcriptional activity. Cytoplasmic β-catenin either becomes ubiquitinated through the GSK3β- adenomatous polyposis coli (APC)-Axin complex or transferred to the membrane where it interacts with E-cadherin, stabilizing cell-cell contacts and preventing cell migration. As illustrated by green lines, Slit2/Robo1 signaling blocks Snail expression by preventing the translocation of β-catenin to the nucleus, thus relieving the repression of E-cadherin expression and enhancing cell-cell contacts. Slit/Robo signaling can also function to decrease cell-cell contacts and increase proliferation (B, C). (B) Slit/Robo signaling recruits the Abelson tyrosine kinase (Abl), which binds to the adaptor protein Cables. Cables links the Robo/Abl complex to the N-Cadherin/β-catenin complex, thus enabling Abl to phosphorylate β-catenin on Y489, causing its translocation to the nucleus where it activates transcription of cell proliferation genes. (C) In another context, Slit/Robo signaling drives cell migration by recruiting the ubiquitin ligase Hakai to E-cadherin and of β-catenin. This results proteasomal degradation of β-catenin and Hakai-mediated lysosomal degradation of E-cadherin, causing decreased cell-cell contacts and enhanced cell migration.

Figure 3. Slit/Robo Signaling Regulates Cell Migration by Controlling the Activation State of Actin Cytoskeleton Modulators

(A) Slit/Robo signaling regulates actin polymerization, and thus cell migration, by controlling the activity level of Rho GTPases. Slit/Robo signaling prevents cell migration by recruiting Slit/Robo (s/r)GAPs to the CC1 and CC2 domains, which inactivate the small Rho GTPases RhoA and Cdc42, and Vilse/CrGAP to the CC0 domain, which exchanges RacGTP for RacGDP. In other contexts, Slit/Robo signaling drives actin polymerization by recruiting Dock, which in turn recruits son of sevenless (SoS) GEF and PAK p21-activated kinase. SoS GEF activates the small Rho GTPase Rac by exchanging GDP for GTP, leading to actin polymerization. RacGTP in turn activates PAK, which also drives actin polymerization. (B) The anti-migratory function of Slit/Robo signaling is regulated by the Abl, which attenuates Robo signaling via phosphorylation of Robo at Y1073 near CC0, possibly preventing substrate binding, and by directly targeting the Robo effector protein Enabled (Ena). In the absence of Abl, Ena binds to CC0 and functions to inhibit cell migration by preventing actin polymerization.

Figure 4. Slit/Robo Signaling Regulates the Process of Tumor Angiogenesis

(A) Slit2 binds to Robo4/proteoglycan complex and signals to block pro-angiogenic signaling downstream of VEGF/VEGFR. (B) Slit promotes angiogenesis by binding to a Robo1/Robo4 heterodimer and driving endothelial migration. (C) Robo4 binds UNC5B, and signals to block pro-angiogenic signaling downstream of VEGF/VEGFR.