These data were in concordance with its role in mediating the inhibitory function of Slit2 signaling on the CRC cell motility and metastasis [7]. Conclusion In summary, our data revealed that the downregulation of srGAP1 in CRC tissues is associated with tumor progression and poor prognosis, and srGAP1-Cdc42 regulatory axis is a key Deferasirox downstream target of Slit2-Robo1 pathway in CRC. survival (valuevalue Rabbit polyclonal to A4GNT of less than 0.05 was considered Deferasirox statistically significant. The Graphpad prism 5.0 software (GraphPad Software, USA) and SPSS 20.0 package (IBM, USA) were used for the statistical analyses and scientific graphing, respectively. Results srGAP1 is downregulated in 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 NCT tissues by IHC staining. The srGAP1 expression results were not available in 34 NCT samples due to technical difficulty in the tissue microarray construction and IHC staining. As shown in Fig.?1a, srGAP1 was expressed mainly in cytoplasm and was significantly downregulated in 47.5% (58 of 122) of CRCs compared with NCTs (Fig.?1b). To evaluate the clinical significance of srGAP1 in CRC, the potential relationships between srGAP1 expression and patients clinicophathological characteristics were analyzed (Table?1). Noticeably, srGAP1 expression in CRCs was significantly correlated with tumor differentiation (Spearman hazard ration, 95%, 95% confidence interval Slit2, Robo1 and srGAP1 expression in CRC cell lines The mRNA levels of Slit2, Robo1 and srGAP1 were detected in CRC cell lines using RT-PCR. The results revealed that both Slit2 and Robo1 were expressed in HT29 and LoVo cells, whereas srGAP1 mRNA expressed in all these six CRC cell lines with lowest expression in HT29 cells (Fig.?2a). Western blot results confirmed the Robo1 expression in HT29 and LoVo cells, whereas no detectable srGAP1 protein was observed in HT29 cells (Fig.?2b). Based on these results, we focused on LoVo cell line for the subsequent experiments. Since no satisfactory anti-Slit2 antibody could be used to detect the endogenous Slit2 protein expression based on our preliminary study, the anti-myc antibody was used to detect Deferasirox the secretary Slit2 protein in the CM from HEK293 cells stably expressing Slit2-myc (Fig.?2c). Open in a separate window Fig. 2 Slit2 inhibits CRC cell migration in a Robo dependent manner. a The mRNA expression of components in the Slit/Robo signaling in CRC cell lines. b The protein expressions of Robo1 and srGAP1 in CRC cell lines were detected by western blot, with beta-actin as a loading control. c Detection of Slit2-myc in slit2 conditional medium (CM). d Mutation at srGAP1 locus was analyzed based on data obtained from the Stand Up to Cancer cBio portal (http://cbio.mskcc.org/su2c-portal/) To investigate potential role of srGAP1 in CRC, we analyzed its alteration frequency in CRC using an online database tool (http://www.cbioportal.org/public-portal/index.do). Based Deferasirox Deferasirox on the data from 3 different CRC cohorts, srGAP1 locus was found to be mutated in 4%?~?7% CRCs (Fig.?1d), whereas no deletion or amplification was observed. srGAP1 is a Robo1-interacting protein in CRC Previous studies have revealed that srGAP1 could interact with Robo1 in neuron [10, 12]. To check potential interaction between Robo1 and srGAP1 in CRC cells, we performed CoIP assay. Plasmids of Robo1-HA, srGAP1-GFP or empty vector were transfected into LoVo cells. After IP using an anti-HA antibody, srGAP1 was detected in the immunoprecipitates of the cells expressing both Robo1-HA and srGAP1-GFP proteins, but not that of the control cells (Fig.?3a). To further confirm it, we performed CoIP using an anti-Flag antibody in LoVo and HCT116 cells cotranfected with Robo1-HA, srGAP1-Flag and/or USP33-GFP (also a Robo1-interacting protein). CoIP results showed that Robo1 and USP33 could be detected in the immunoprecipitates of LoVo cells expressing endogenous Robo1 but not in those of HCT116 cells without endogenous Robo1 expression (Fig.?3b). These results confirmed that Robo1 could interact with srGAP1 and USP33 in CRC cells, and srGAP1 could not interact with USP33 directly. Open in a separate window Fig. 3 srGAP1 is a Robo1-interacting protein in CRC. a srGAP1 interacted with Robo1. Coimmunoprecipitation was performed to detect the interaction of Robo1-HA with srGAP1-GFP in LoVo cells transfected with Robo1-HA, srGAP1-GFP. Immunoprecipitates and total cell lysates (TCL) were immunoblotted with antibodies as indicated. b srGAP1 could indirectly interact with USP33 through Robo1. Coimmunoprecipitation was performed to detect the interaction of srGAP1-GFP with Robo1, GFP-USP33 in LoVo and HCT116 cells. srGAP1 could interact with USP33 in LoVo cells expressing endogenetic Robo1 but not in HCT116 cells without endogenetic.