The Roles And Molecular Mechanisms Of CUL4B In Regulating Colorectal Cancer Stem Cells

CUL4B acts as a scaffold protein to form Cullin 4B-RING ubiquitin ligases(CRL4Bs).CRL4B catalyzes either polyubiquitination for proteosomal degradation or H2AK119 monoubiquitination for epigenetic modification.Through these mechanisms,CUL4B participate in a broad variety of physiological and developmentally controlled processes such as cell cycle regulation,signal transduction,DNA damage and repair,chromatin remodeling,and embryonic development.On the other hand,CUL4B is frequently overexpressed in a variety of solid tumors such as breast cancer,stomach cancer,prostate cancer,liver cancer and bladder cancer,and plays an important role in tumorigenesis through transcriptionally repressing various tumor suppressors.However,the role and mechanism of CUL4B in regulating colorectal cancer stem cells(CCSCs)are still unclear.Colorectal cancer is the main casue of cancer-related death,as a result of metastasis and chemoresistance,which is related to cancer stem cells(Cancer stem cells,CSCs).CSCs are a subgroup of cells within the tumor that can self-renew and give rise to various differentiated cell types constituting the tumor.Colorectal cancer stem cells(CCSCs)are highly tumorigenic,invasive and metastatic,and are more resistant to chemo-and radiotherapy.However,its regulatory mechanisms have not been fully characterized.In recent years,the advent of patient-derived organoids(PDOs)has revolutionized in vitro studies of tumor biology.PDOs are capable of maintaining the functionality,molecular and cellular heterogeneity of the originating tumors,and widely used to various aspects of cancer research,such as drug screening,drug toxicity testing,and personalized medicine.In addition,organoids are stem cell-originated.Therefore,colorectal cancer organoids is a good model for CCSCs.In this study,we aim to investigate the roles and mechanisms of CUL4B in CCSCs using colorectal cancer cells,colorectal cancer organoids and clinical tissue samples.Part Ⅰ CUL4B promotes sphere formation,self-renewal and metastasis of CCSCsOur previous studies showed that CUL4B is highly expressed in a variety of solid tumors and promotes the proliferation,metastasis and invasion of tumor cells.However,whether CUL4B promote the malignant behavior of colorectal cancer by regulating CCSCs remains to be determined.In this section we analyzed the role of CUL4B in colorectal cancer and obtains the following results:(1)We first examined CUL4B expression by immunohistochemistry in tissue microarrays comprising tumor tissues and adjacent tissues from 75 cases of CRCs,and found that the expression level of CUL4B was significantly higher in tumor tissues than that in adjacent tissues.(2)We next examined the correlation between CUL4B expression and patients’survival status,and found that the expression level of CUL4B was negatively correlated with the survival time of tumor patients,that is,the higher the CUL4B expression,the shorter the survival time of the patients,and the lower the CUL4B expression,the longer the survival time of the patients.We further explore whether the expression level of CUL4B is related to other clinicopathological features such as tumor size,stage classification and metastasis.The results showed that the age,gender,tumor size and grade of patients were not significantly correlated with the expression level of CUL4B,whereas CUL4B expression was significantly related to lymph node metastasis of the patient.(3)We found that the expression of CUL4B in CCSCs was significantly higher than that in differentiated cells by Western blotting and RT-PCR.We confirmed that the co-expression of cancer stem cell markers ALDH1 and CUL4B in HCT116CSCs and HT29CSCs was significantly higher than the exclusiveness by immunofluorescence experiments.To analysis the effect of CUL4B on sphere formation of CCSCs and PDOs,we knocked down or overexpressed CUL4B in HCT116,HT29,and PDOs,and found that CUL4B knockdown decreased,while CUL4B overexpression increased spheriod formation of CCSCs and PDOs.(4)We analyze whether CUL4B plays an important role in CCSC self-renewal.We knocked down or overexpressed CUL4B in HCT116CSCs and HT29CSCs,and found that ablation of CUL4B significantly reduced the ability of spheroid formation in HCT116CSCs and HT29CSCs,and overexpression of CUL4B significantly increased the formation of HCT116CSCs.(5)Nude mice(BALB/c nude mice)tumorigenesis assays indicated that knockdown of CUL4B significantly reduced tumorigenicity in HCT116CSCs and HT29CSCs.(6)We injected CUL4B-overexpressed PDOs and their controls into severe immunodeficiency mice(NOD-Prkdcem26I12rgem26/Nju mice,NCG)through the spleen,and then observed the liver and lung metastases of the mice.The results showed that overexpression of CUL4B could significantly increase liver metastasis and lung metastasis of PDOs.Cells with CUL4B ablation and its controls of HCT116 and HT29 were injected from tail vein,and the lung metastatic ability of nude mice was observed.The results showed that knockdown of CUL4B could significantly reduce lung metastasis of HCT116 and HT29.(7)Sertraline and fluoxetine were used to evaluate the effect of CUL4B on drug resistance.The results showed that knockdown of CUL4B in#16 PDOs and#02 PDOs could significantly increase the sensitivity to sertraline and fluoxetine compared to their controls,and that overexpression of CUL4B in#16 PDOs could significantly decrease sensitivity to sertraline and fluoxetine compared to their controls.Part Ⅱ CRL4B coordinates with PRC2 to repress miR34a,and upregulates the expression of its downstream target genesIn the first part we showed that CUL4B regulates tumor malignant behavior by regulating CCSCs.To explore the molecular mechanism of CUL4B promoting colorectal cancer,we performed transcriptome sequencing(RNA-seq),miRNA sequencing(miRNA-seq)and chromatin immunoprecipitation(ChIP)experiments,got the following results:(1)We performed RNA-seq of CUL4B knockdown and its control PDOs from three individuals.Eight genes were identified to be significantly down-regulated by CUL4B knockdown in all three lines of PDOs,including CUL4B,NRCAM,EDAR,UPK3A,FAM3D,CYP2T3P,LCN2 and MYCN.The 8 genes and 13 genes whose expression was significantly down-regulated in at least two of three CUL4B knockdown PDOs were verified,and seven genes were identified to be significantly down-regulated by CUL4B knockdown in PDOs,HCT116CSCs and HT29CSCs,including ID1,UPK3A,CYP2T3P,FAM3D,NPTX2,AGR3,and MYCN.(2)The MYCN gene was verified in PDOs and CCSCs,and it was found that CUL4B positively regulated MYCN expression.(3)We performed miRNA-seq of CUL4B overexpressed and its control PDOs.Overlapping the differently expressed miRNAs by CUL4B with the miRNAs predicted by TargetScan database,miR34a and let7e were identified in both lists.RT-PCR assay verified that CUL4B negatively regulated miR34a,but not let7e.Double luciferase reporter assay and rescue experiments confirmed that CUL4B positively regulates MYCN expression by repressing miR34a expression,(4)CRL4B coordinates with PRC2 complex to repress miR34a expression:Our analysis showed that CUL4B regulated miR34a independent of p53 and the methylation status of CpG islands in the miR34a promoter region.ChIP assays confirmed the coocupancy of CUL4B,DDB1 and EZH2 at the miR34a promoter region.Knockdown of CUL4B led to reduced binding of CRL4B and PRC2 complexes,consequently decreased H2AK119ubl,H3K27me3,and increased H3K4me3 in the miR34a promoter region,supporting that CRL4B coordinates with PRC2 to repress miR34a expression.Rescue experiments indicated that inhibition of miR34a remarkably restored the sphere-forming capacity and migration in CUL4B knockdown colorectal cancer cells,indicating that CUL4B contributes to maintaining colorectal cancer cells sternness through repression of miR34a.(5)Other target genes of miR34a including CD44,NOTCH1 and NUMB were tested in CUL4B knockdown CSCs.Knockdown of CUL4B significantly reduced their levels.Double luciferase reporter assay confirmed that CUL4B promotes NOTCH1 expression by inhibiting miR34a.(6)We collected 38 tumor samples and paired adjacent normal tissues.The downregulation of miR34a was observed in 61%(23/38)of tumor tissues,while upregulation of CUL4B and MYCN was observed in 68%(26/38)and 71%(27/38)of tumor tissues,respectively.Importantly,negative correlation was observed between miR34a levels and CUL4B as well as MYCN protein,while the positive correlation was detected between CUL4B and CD44 as well as MYCN.