Lack Of CUL4B In Myeloid-derived Suppressive Cells Increases The Aggressiveness Of Cancer Cells

Cullin 4B(CUL4B)is a scaffold protein in the Cullin 4B-RING E3 ligase complex(CRL4B),which plays a biological role in ubiquitin-mediated protein degradation and epigenetic processes.It has been found that CUL4B acts as an important regulatory role in various life activities such as embryonic development,neural development,DNA damage repair,cell cycle,cell differentiation,and tumorigenesis.It is reported that CUL4B is highly expressed in tumor tissues such as esophageal cancer,breast cancer,lung cancer,liver cancer,colon cancer,gastric cancer and ovarian cancer,suggesting that CUL4B functiona as an oncogene in solid tumor.Myeloid-derived suppressor cells(MDSCs)are derived from hematopoietic stem cells of bone marrow.They are immature myeloid cells that accumulates and activates under various pathological conditions including inflammation,autoimmune disease and tumor,suppressing the immune ability of the body.In previous studies,we found that conditional ablation of CUL4B in the hematopoietic system resulted in significantly enhanced accumulation and activity of CD11b+Gr-1+ cells.Lack of CUL4B in the host hematopoietic system promoted the growth and metastasis of transplanted tumors,suggesting that CUL4B funcitons as tumor suppressor in tumor microenvironment.In order to explore whether the absence of CUL4B in myeloid cells also promotes the growth and metastasis of transplanted tumors,and the molecular mechanism of Cul4b knockout MDSCs in promoting tumor growth and metastasis,we examined the function of CUL4B in myeloid cells.Part I Lack of CUL4B in MDSCs increases tumor aggressivenessTo investigate the role of Cul4b in myeloid cells,we first generated conditional Cul4b knockout mice(LysM-Cre),in which Cul4b was specifically deleted in myeloid cells,and then carried on the following analysis:1.Generation of myeloid cell-specific Cul4b knockout miceWe crossed Cul4bflox/flox mice to LysM-Cre transgenic mice to obtain myeloid cell-specific Cul4b knockout mice(Cul4bflox/Y LysM-Cre+/-,referred to as MKO).CUL4B was effectively deleted in MDSCs of MKO mice as demonstrated by Western-blot and real-time qPCR.We detected the CD11b+ Gr1+ cells by flow cytomety,and found that the proportion was increased dramatically in blood from MKO mice compared to the WT littermates.The spleen of MKO mice was slightly larger than WT mice.2.Lack of CUL4B in the myeloid cells promotes tumor growth and MDSCs accumulationThrough transplantation of B16/F0 tumor cells,tumor growth was significantly increased in MKO mice when compared to WT mice,which is consistent with the results in TKO mice.Corresponding to accelerated tumor growth,MDSCs were expanded more dramatically in MKO mice.Allogeneic tumor-bearing experiment showed that the ability of rejection allogeneic 4T1 cells from BALB/c mice was significantly lower in MKO mice compared to WT mice.3.Lack of CUL4B in MDSCs increases tumor aggressivenessTo conform MKO mice increasing tumor aggressiveness is mediated by MDSCs,we co-injected tumor cells with MDSCs into C57BL/6 mice.MDSCs from MKO mice were significantly more potent in promoting tumor growth than those from WT mice.We detected the proportion of G-MDSCs and M-MDSCs by flow cytomety,and found there was no significant difference in proportion of the two subgroups between MKO-MDSCs amd WT-MDSCs.These results show that myeloid cell-specific Cul4b depletion promotes the growth of transplanted tumors,and this effect is mediated by increasing MDSC activity.Part ? CUL4B-depletion in MDSCs drive tumor growth independent of theirimmunosuppressive activitiesMDSCs are not only responsible for building an immune-suppressive environment,but also directly act on tumor cells.To determine whether CUL4B deletion in MDSCs could also drive tumor growth in T-cell independent manner,the following analyses were conducted.1.Nude mice model was used to examine the effect of CUL4B deletion in MDSCs on driving tumor growthWe co-injected B16/F0 and MDSCs(ratio of 1:3)sorted from the spleens of tumor-bearing MKO and WT mice,into BALB/c nude mice subcutaneously,and found MKO-MDSCs were more potent in promoting tumor growth greater in the absence of T cells.2.Co-culture system was used to examine the effect of CUL4B-deficent MDSCs on driving tumor growthIn order to further confirm that CUL4B-deficient MDSCs can drive tumor progression by directly acting on tumor cells,we co-cultured MDSCs with 4T1 cells,and found that CUL4B-deficient MDSCs significantly increased colony of 4T1 cells and reduced apoptosis of B16/F0 cells when compared to WT-MDSCs.Flow cytometry assay showed that MKO-MDSCs significantly increased the percentage of tumor cells sternness markers.Furthermore,CUL4B-deficient MDSCs significantly increased sphere formation and the expression of Sox2,Nanog and Oct4 genes.Importantly,when B16/F0 cells cocultured with MDSCs were subcutaneously inoculated into C57BL/6 mice,tumor masses formed from B16/F0 cells co-cultured with CUL4B-deficient MDSCs were significantly larger than those with WT-MDSCs.3.The effect of CUL4B deletion in MDSCs on regulating tumor aggressiveness was detected by conditional medium:In order to explore the mechanism by which CUL4B-deficient MDSCs affect tumor progression,we performed colony-formation assay using conditional medium prepared from MDSCs and mouse embryonic fibroblasts(MEFs).Conditional medium from MKO-MDSCs,but not from CUL4B-null MEFs,significantly increased colony of 4T1 cells compared to the medium from WT control.Flow cytometry assay showed that treatment with conditioned medium from MKO MDSCs significantly increased the percentage of the tumor stem cells.Increased sphere formation and the expression of Sox2,Nanog and Oct4 were detected in tumor cells cultured in MKO-MDSCs conditional medium.Moreover,when B16/F0 cells incubated with MDSCs conditional medium were injected into mice,increased tumor growth and more metastic tumor foci were detected with tumor cells exposed to MKO MDSD-derived conditioned medium.Collectively,these results suggest that the alterations in the secretory phenotype of CUL4B-deficient MDSCs may be responsible for increased potency in promoting tumor growth and metastasis.Part III CUL4B-deficient MDSCs promote tumor stemness through IL-6/STAT3 pathwayIn order to identify the MDSC-derived soluble factors,the following analyses were conducted.1.The CUL4B-deficient MDSCs secreted more IL-6:To identify the MDSC-derived soluble factors that enhance tumor progression,the MDSC-conditional medium was fractioned by<3kDa and>3kDa,and the activities of both fractions were tested.The result showed that>3kDa fraction from CUL4B-deficient MDSCs significantly increased colony formation,but this difference disappeared if the medium was pre-boiled.Cytokines in conditional medium and serum were detected by ELISA.IL-6 level was significantly higher in the medium from MKO-MDSCs,as well as plasma from tumor-bearing MKO mice when compared with the WT controls.To further explore whether IL-6 released by MKO-MDSCs is responsible for accelerated tumor progression,IL-6 was blocked by neutralizing antibody.The results showed that the addition of neutralizing antibodies against IL-6 significantly reduced both colony and sphere formation as well as the expression of stemness-associated genes.2.IL-6 activates the STAT3 signaling pathway in tumor cells:IL-6 functions through activating the downstream key signaling pathways in tumor cells by binding to cell membrane receptors.We detected the changes in JAK/STAT3 signaling pathway,which was a downstream of IL-6.The results showed that the level of phosphorylated STAT3(Y705)was increased in tumors formed from the B16/FO cells co-cultured with MKO-MDSCs.Increased levels of phosphor-STAT3(Y705)and phosphor-JAK2(S1007/1008)were also observed in tumor cells treated with MKO-MDSCs conditional medium.To confirm the ability of MKO-MDSCs to promoting tumor cells proliferation and sternness is through STAT3 signaling pathway,p-STAT3 inhibitor and knockdown STAT3 in 4T1 cells were used to rescue experiment.The result showed that p-STAT3 inhibitor and knockdown STAT3 attenuated the increase in colony formation,tumor sphere formation and the expression of stem cell genes caused by CUL4B depletion3.CRL4B/HDAC/PRC2 complex binds to and represses 1l-6 transcriptionTo detect the mechanism underlying the increased IL-6 secretion in conditional medium from CUL4B-deficient MDSCs,we examined the expression of IL-6 levels in WT and CUL4B-deficient MDSCs.Both mRNA and protein levels of Il-6 were significantly upregulated in MKO-MDSCs and CUL4B knockdown Raw264.7 cells,as detected by qPCR and Western-blot experiment.The expression levels of IL-6 were negatively correlated with CUL4B levels in MDSCs form tumor-bearing mice as well as from blood of cancer patients,indicating that CUL4B represses the transcription of Il-6.To research the molecular mechanisms responsible for the repression of IL-6 transcription by CUL4B,we used ChIP assay to examine the binding of these compexes to IL-6 promoter.We found that CUL4B,as well as H2AK119ub1,EZH2,DNMT3A,HDAC1 and HDAC3,could directly bind to the region at-1023 to-1140 upstream Il-6 transcription start site.ChIP-qPCR showed that CUL4B regulate the transcription of Il-6 transcription cooperating with PRC2 and HDAC complexes Rescue experiment showed that HDAC complex inhibitor(TSA)and PRC2 complex inhibitor(Dzep)could block the reduction in IL-6 transcription led by CUL4B overexpression.Taken together,our study have demonstrated the following conclusions:1.Lack of CUL4B in the myeloid cells accelerates accumulation and activity of MDSCs2.CUL4B-deficiency in MDSCs increases tumor aggressiveness in T cell independent manner3.Increased IL-6 production by CUL4B-deficient MDSCs renders cancer cells stem cell properties by activating STAT3 signaling in tumor cells4.CRL4B represses IL-6 transcription cooperating with PRC2 and HDAC complexes.