Abstract
BackgroundThe neuronal guidance molecule Slit2 plays suppressive role in tumorigenesis and progression. We previously showed that Slit2-Robo1 inhibit cell migration in colorectal cancer (CRC). However, little is known about its downstream effectors in CRC. This study tries to identify whether the Slit-Robo Rho GTPase activating protein 1 (srGAP1) could mediate the inhibitory effect of Slit2-Robo1 on CRC cell migration.MethodsThe protein expression of srGAP1 in clinical CRC tissues was tested by immunohistochemistry staining. Conditioned medium was prepared from HEK293 cells stably expressing Slit2-myc, Robo1-HA or RoboN (a soluble extracellular domain of Robo1). Immunoprecipitation (IP) was applied to check the interaction between Robo1 and srGAP1, and immunofluorescence (IF) was used to observe the subcellular localization of Robo1 and srGAP1. Small GTPase pull-down assay was used to determine the activity of Cdc42. A modified wound healing assay was performed to detect cell migration.ResultsThe protein expression of srGAP1 was remarkably decreased in 47.5% of CRC tissues compared with adjacent noncancerous tissues, and the decreased srGAP1 expression was associated with lymphatic invasion, poor tumor differentiation, high TNM stage, and poor survival (P < 0.05). IP and IF assays revealed that srGAP1 was a Robo1-interacting protein and exhibited similar dynamic subcellular distribution after Slit2 treatment in CRC cells. Small GTPase pull-down assay and migration assay indicated that Slit2-Robo1 signaling inhibited Cdc42 activity and CRC cell motility through srGAP1.ConclusionDownregulation of srGAP1 in CRC was associated with tumor progression and poor prognosis. srGAP1 is an important downstream molecule of Slit2 signalling in CRC, and mediates the anti-migration function of Slit2 by inhibiting Cdc42.
Highlights
The neuronal guidance molecule Slit2 plays suppressive role in tumorigenesis and progression
Results Slit-Robo Rho GTPase activating protein 1 (srGAP1) is downregulated in colorectal cancer (CRC) and is associated with poor survival Little was known about the expression and clinical significance of srGAP1 in human cancers, including CRC, so we measured the protein levels of srGAP1 in 156 paired CRC and noncancerous tissues (NCT) tissues by IHC staining
SrGAP1 expression in CRCs was significantly correlated with tumor differentiation (Spearman r = −0.162, P = 0.044), lymphatic invasion (Spearman r = −0.194, P = 0.015) and TNM stage (Spearman r = −0.202, P = 0.011), suggesting that srGAP1 expression was associated with tumor progression
Summary
The neuronal guidance molecule Slit plays suppressive role in tumorigenesis and progression. Previous studies have showed that Slit-Robo signaling affects cell motility by controlling the activity of several proteins involved in reorganizing the actin cytoskeleton, including the Rho GTPases family (Rac, Cdc, and RhoA) [3]. As the key neuronal guidance molecules, Slits were originally identified to regulate axonal guidance and neuronal migration in the central nervous system by binding to Roundabout receptor family (Robo1-Robo4) [4, 5]. In addition to their functions in nervous system, recent data revealed that
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