DAB2IP Suppresses Colon Cancer Tumor-repopulating Cells Via Negative Regulation Of C-Myc

Objective Colorectal cancer has become a worldwide medical burden due to its high incidence and high fatality rate.The presence of cancer stem cells(CSCs)is one of the important causes of tumor metastasis,recurrence,and drug resistance.Our previous work showed that downregulation of DAB2 IP enhanced tumor stem cell phenotypes,and the mechanisms of DAB2 IP involved in regulating colon CSCs required further investigation.It has been reported that the core transcription factor c-Myc is critical for maintaining the stemness and self-renewal characteristics of CSCs,but its regulatory mechanisms were not fully clear.In this study,a subgroup of colon cancer cells with stem-like characteristic(defined as tumor-repopulating cells,TRCs)were enriched by three-dimensional soft fibrin gels and used to study whether DAB2 IP could influence the formation and growth of TRC spheroids from colon cancer via regulating c-Myc,and the specific regulatory mechanisms were further explored.Methods The colon TRCs were isolated from HCT116 and HT29 cells via 3D soft fibrin gels culture.The stemness and self-renewal characteristics were determined by immunofluorescence,western blot,sphere formation assay,and serial passage assay.Subsequently,RT-PCR,western blot,immunofluorescence,RNA interference technology,and colony formation assay were used to explore the roles and mechanisms of DAB2 IP in TRC formation and growth via regulation of c-Myc.Cycloheximide and proteasome inhibitor MG132 were used to detected whether DAB2 IP regulated the turnover of c-Myc via the ubiquitin-proteasome pathway.The interaction between DAB2 IP and c-Myc were detected by immunoprecipitation and western blot.Finally,the expression pattern of DAB2 IP and c-Myc was verified in human colorectal cancer specimens.The relationship between DAB2 IP expression and clinical pathological features of colorectal cancer were analyzed based on Gene Expression Omnibus(GEO)database.Results To enrich colon TRCs,colon cancer cells were cultured in soft fibrin gels.The stem cell markers were increased in colon TRCs,compared with the normal differentiated colon cancer cell lines.Colon TRCs displayed self-renewal and longterm repopulating capabilities.Knockdown of c-Myc formed fewer spheroids with smaller size than the control group.The protein expressions of Nanog,CD133,and CD44 were attenuated following c-Myc knockdown.DAB2 IP overexpression significantly reduced c-Myc protein levels in HCT116 and HT29 cells.In contrast,DAB2 IP knockdown displayed the opposite effect.Overexpression of c-Myc significantly promoted the TRC spheroid formation and growth in DAB2 IPoverexpressing cells.However,neither c-Myc overexpression nor knockdown modulated the expression of DAB2 IP in HCT116 and HT29 cells.DAB2 IP transient overexpression significantly accelerated the turnover rate of c-Myc compared with control cells.The proteasome inhibitor MG132 treatment largely blocked the DAB2 IPinduced reduction of c-Myc protein,indicating that DAB2 IP modulated c-Myc protein stability.Overexpression of DAB2 IP enhanced ubiquitination of c-Myc in HCT116 and HT29 cells.Specifically,DAB2 IP disrupted c-Myc stability through the GSK3β/PP2AB56α-induced phosphorylation/dephosphorylation cascade.Furthermore,a coimmunoprecipitation analysis was undertaken and confirmed that endogenous DAB2 IP interacted with endogenous c-Myc in HCT116 cells.DAB2 IP protein levels were downregulated and c-Myc was upregulated in tumor tissues compared with adjacent normal tissues.DAB2 IP was negatively correlated with c-Myc in colorectal cancer specimens.In addition,colorectal cancer patients with high DAB2 IP expression responded well to 5-fluorouracil-based adjuvant chemotherapy.Low DAB2 IP expression was significantly associated with distant metastasis in CRC patients.Conclusions The c-Myc protein was critical for the TRC maintenance.DAB2 IP promoted the ubiquitination of c-Myc to disrupt its protein stability through the GSK3β/PP2A-B56α-dependent phosphorylation-dephosphorylation cascade,which inhibited the formation and growth of TRC spheroids.DAB2 IP protein levels was negatively correlated with c-Myc in colorectal cancer specimens.Low DAB2 IP expression was associated with distant metastasis and poor prognosis of colorectal cancer,indicating that DAB2 IP might be used as a diagnostic and prognostic biomarker.