DAB2IP Suppresses Tumor Malignancy By Inhibiting GRP75-driven Wild-type P53 Ubiquitination In Colorectal Cancer

Objectives: To explore the correlation between DAB2 IP and p53,and to evaluate the correlation between DAB2 IP expression and clinical prognosis of patients in colorectal cancer.Methods: Publicly available CRC expression profiles in the TCGA-COAD and GSE21510 datasets were used for the analysis of the correlation between DAB2 IP expression level and p53 signaling.Gene expression correlation analysis was performed to analyze the correlation between DAB2 IP,TP53 and the downstream genes of TP53.Colorectal cancer tissue microarrays were used to evaluate the protein levels of DAB2 IP and p53 in human colorectal cancer specimens.DAB2 IP,non-specific p53 and mutant p53 were detected by immunohistochemical(IHC)staining.Taking advantage of clinical databases,the correlation between DAB2 IP expression and clinical prognosis of p53 mutant and wild-type colorectal cancer patients was explored.Results: Analysis of the expression profile datasets from publicly available databases revealed that the expression level of DAB2 IP was positively correlated with p53 signaling pathway.The expression of DAB2 IP and TP53 downstream genes(CDKN1A,GDF15,PHLDA3)were also positively correlated whereas the expression of DAB2 IP and TP53 exhibited no significant association.The IHC of colorectal cancer tissue microarrays showed that in p53 wild-type colorectal cancer,the protein levels of DAB2 IP and p53 were positively correlated.Kaplan Meier overall survival analysis based on DAB2 IP expression in 419 colorectal cancer patients from the TCGA-COAD dataset revealed that high DAB2 IP expression was related to better prognosis,but only in p53 wild-type subgroup.Conclusions: The expression of DAB2 IP is positively correlated with p53 signaling and wild-type p53 protein level in colorectal cancer,and is positively correlated with the clinical prognosis of p53 wild-type colorectal cancer patients.Objectives: To explore the tumor-suppressive effects of DAB2 IP in p53 wild-type colorectal cancer and to confirm the regulation effect of DAB2 IP on wild-type p53.Methods: Cancer Cell Line Encyclopedia(CCLE)database and Western blot were employed to identify p53 wild-type colorectal cancer cell lines with adequate DAB2 IP expression for further studies.Transwell cell migration assay,plate colony formation assay and FACS cell apoptosis assay were performed under the manipulation of DAB2 IP to evaluate the effects of DAB2 IP on migration,proliferation and apoptosis of p53 wild-type colorectal cancer cells.Meanwhile,Western blot was used to detect the regulation effects of DAB2 IP on wild-type p53 under normal and stressed conditions.In vitro and in vivo reverse experiments were conducted to confirm whether wild-type p53 was involved in the tumor suppressive effects of DAB2 IP on colorectal cancer cells.Results: The p53 wild-type colorectal cell lines HCT116 and SW48 were selected by examing the p53 mutation patterns of colorectal cell lines from CCLE database for further studies,Western blot indicated that both cell lines expressed adequate DAB2 IP protein.The two tumor cells exhibited stronger migration ability and attenuated 5-FU induced apoptosis after DAB2 IP knockdown by transient si RNA transfection.HCT116 and SW48 showed higher proportions of apoptotic cells when DAB2 IP was overexpressed.The colony formation ability of both cell lines significantly reduced when DAB2 IP was stably downregulated.Western blot analysis revealed that DAB2 IP knockdown reduced the protein level of p53 and its downstream targets in HCT116 and SW48 cells.Correspondingly,overexpression of DAB2 IP led to increased protein levels of p53 and its downstream targets.Flow cytometry analysis showed that p53 knockdown significantly reduced DAB2 IPinduced cell apoptosis.Additionally,the overexpression of p53 significantly reduced the induction of cell migration by si DAB2 IP in both SW48 and HCT116 cells.The subcutaneous xenograft experiment of HCT116 cells showed that knockdown of DAB2 IP significantly promoted tumor growth,whereas knocking down DAB2 IP could not markedly improve tumor growth in mice with wild-type p53 overexpressing by the intratumoral injection of p53-adenovirus.Conclusion: DAB2 IP exerts tumor-suppressive effects by regulating wild-type p53 protein levels in colorectal cancer cells.Objective: To elucidate the mechanism of DAB2 IP regulating wild-type p53 in colorectal cancer cells.Methods: Western blot and q PCR were conducted to detect the protein and m RNA level of p53 under DAB2 IP manipulation to preliminarily confirm whether DAB2 IP transcriptionally or post-transcriptionally regulated wild-type p53.Protein synthesis inhibitor CHX and proteasome inhibitor MG132 were used to confirm whether DAB2 IP affected the the synthesis or degradation of wild-type p53.Co-immunoprecipitation(CoIP)assay was conducted to verify the interaction of DAB2 IP and wild-type p53 in colorectal cancer cell lines.Co-IP and mass spectrometry were combined to screen the potential proteins that might mediate DAB2IP-regulated ubiquitination of wild-type p53.The interaction was reconfirmed by Co-IP assay.A series of in vitro and in vivo experiments were performed to validate whether the selected protein mediated the biological process of DAB2IP-regulated p53 ubiquitination and the corresponding tumor-suppressive effects.CoIP was used to evaluate the amount of GRP75 binding to p53 under different DAB2 IP expression levels.The truncated and domain-deletion DAB2 IP plasmids were used to examine which domain of DAB2 IP was crucial for the binding with GRP75.Functional experiments were performed to validate whether the domain-deleted DAB2 IP could still affect the ubiquitination level of wild-type p53.Results: Knocking down DAB2 IP in SW48 cells,Western blot and q PCR showed that the protein level of p53 was significantly reduced,while the m RNA level of TP53 exhibited no significant change.In SW48 and HCT116 cells,after MG132 treatment,the DAB2 IP knockdown group showed accelerated p53 synthesis efficiency at certain time points.After CHX treatment,the half life of p53 in the DAB2 IP knockdown group significantly decreased.In normal and stressed SW48 cells,Co-IP results showed an increased ubiquitination level of endogenous p53 after DAB2 IP knockdown.Similarly,the overexpression of DAB2 IP significantly inhibited exogenous p53 ubiquitination in HEK293 T cells.Co-IP assay showed that DAB2 IP couldn’t directly bind with wild-type p53.Immunoprecipitation and mass spectrometry were used to purify and identify DAB2 IPand p53-binding proteins in SW48 cell line.The E3 ligase-related protein GRP75 was identified as the target molecule for further study.Co-IP assay revealed that GRP75 could bind with DAB2 IP and wild-type p53.The quantitive co-immunoprecipitation showed that the amount of GRP75 protein interacting with p53 increased with the depletion of DAB2 IP.Similarily,DAB2 IP overexpression led to a decreased GRP75 protein level bound to p53.Functional experiments revealed that GRP75 promoted the ubiquitination and degradation of wild-type p53 and mediated the regulation of p53 ubiquitination by DAB2 IP.The Co-IP experiment declared that the Ras-GAP domain at N-terminal of DAB2 IP was crucial for the interaction with GRP75.The expression of Ras-GAP-deleted DAB2 IP could neither reduce the ubiquitination level of wild-type p53 nor increase the protein level of wild-type p53 and its downstream genes.Conclusion: DAB2 IP competitively binds to the ubiquitin ligase-related protein GRP75 through its Ras-GAP domain,reducing the ubiquitination level of wild-type p53 protein to maintain its stability.