The SLIT-ROBO pathway: a regulator of cell function with implications for the reproductive system

Abstract

The secreted SLIT glycoproteins and their Roundabout (ROBO) receptors were originally identified as important axon guidance molecules. They function as a repulsive cue with an evolutionarily conserved role in preventing axons from migrating to inappropriate locations during the assembly of the nervous system. In addition the SLIT-ROBO interaction is involved in the regulation of cell migration, cell death and angiogenesis and, as such, has a pivotal role during the development of other tissues such as the lung, kidney, liver and breast. The cellular functions that the SLIT/ROBO pathway controls during tissue morphogenesis are processes that are dysregulated during cancer development. Therefore inactivation of certain SLITs and ROBOs is associated with advanced tumour formation and progression in disparate tissues. Recent research has indicated that the SLIT/ROBO pathway could also have important functions in the reproductive system. The fetal ovary expresses most members of the SLIT and ROBO families. The SLITs and ROBOs also appear to be regulated by steroid hormones and regulate physiological cell functions in adult reproductive tissues such as the ovary and endometrium. Furthermore several SLITs and ROBOs are aberrantly expressed during the development of ovarian, endometrial, cervical and prostate cancer. This review will examine the roles this pathway could have in the development, physiology and pathology of the reproductive system and highlight areas for future research that could further dissect the influence of the SLIT/ROBO pathway in reproduction.

Figure 1. The domain structure of the vertebrate SLIT and ROBO proteins

All three vertebrate SLIT proteins have the same structure. From the N-terminus: a putative signal peptide (SS); four tandem leucine rich repeat domains (LRR); six epidermal growth factor (EGF) like domains; a laminin G (LG) like domain; a further three EGF-like domains and a C terminal cysteine knot (C) domain. A putative proteolytic cleavage site has been mapped to EGF5 and part of EGF6. Four ROBO receptors have been identified in vertebrates. ROBO1, ROBO2 and ROBO3 share a common extracellular domain structure of five immunoglobulin-like (Ig) domains and three fibronectin type 3 (FNIII) repeats. The intracellular domains of ROBO1 and ROBO2 share the same conserved cytoplasmic motifs (CC0, CC1, CC2 and CC3). The CC1 motif is absent in ROBO3. ROBO4, which has the lowest homology with other ROBO family members, contains only two Ig and FNIII domains along with one CC motif, CC2. The second LRR domain of SLIT along with IG1 and IG2 of ROBO are essential for the ligand-receptor interaction. The LRR domain is circled to highlight its importance in the SLIT-ROBO interaction. SLIT has the same affinity for ROBO1-3 receptors. However whether ROBO4 is receptor for SLIT is debatable.

Figure 2. Localisation of SLIT2 and ROBO1 in the endometrium and Fallopian tube

Immunohistochemistry for SLIT2 and ROBO1 (brown) using protocols and pre-absorption controls using the techniques of reported in Dickinson et al., 2008 and Dickinson et al., 2009a. a) SLIT2 in human endometrium with insert showing negative control and b) ROBO1 in human endometrium. The epithelial glandular staining is highlighted by the arrows. c) SLIT2 in human Fallopian tube with insert showing negative control and d) ROBO1 in human Fallopian tube. The luminal epithelial staining is highlighted by the arrows. Scale bar represents 50 μm.

Figure 3. The SLIT-ROBO interaction and their regulation of cell function

SLIT can inhibit invasion and promote a cell cycle arrest by blocking Wnt, HGF and SDF-1 signalling. The SLIT-ROBO can also prevent invasion and stimulate a cell cycle arrest directly by negatively regulating cdc42 activity. SLIT binding to ROBO also relieves inhibition of DCC by Netrin-1. This allows the activation of pro-apoptotic pathways through Caspase 3 and 9. SLIT can also bind and sequester Netrin-1 preventing its interaction with DCC and inhibitory role in apoptosis. Depending on the particular cellular environment, the SLIT-ROBO interaction can also promote and inhibit adhesion. The SLIT-ROBO interaction promotes adhesion in breast tumour cells and during mammary gland development, possibly by enhancing the association between E-cadherin and β-catenin at cell borders. However during formation of the heart lumen, SLIT-ROBO signalling antagonises E-cadherin/β-catenin mediated cell-cell adhesion. During neural development SLIT binding promotes an interaction between ROBO and N-cadherin. Subsequently β-catenin becomes disassociated from the complex and there is a reduction in cadherin mediated cell-cell adhesion. Black arrows represent promoting an activity while red arrows depict inhibiting an action.