| Cancer initiation and progression requires a permissive microenvironment that shields cancer from the immunosurveilance of the host. In addition to many soluble factors and extracellular matrix secreted by tumor cells and host cells, the tumor microenvironment is composed of many kinds of non-neoplastic cells, such as endothelial cells, pericytes, mesenchymal cells and immune cells. Through the interactions between the molecules and cells, tumor microenvironment can provide not only the need for tumor growth, development and metastasis (cellular and molecular), but also can effectively protect tumor cells from the host immune surveillance and attack. Myeloid-derived suppressor cells (MDSCs) have emerged as one of the major players that enable cancer to escape immunosurveillance.MDSCs represent a heterogenic population of myeloid progenitor cells and immaturemyeloid cells (IMCs) that are characterized by their ability to suppress immune responses in many pathological conditions, including cancers.In healthy individuals, the population of immature myeloid cells that generated from bone marrow can quickly differentiate into three groups of terminally differentiated myeloid cells-macrophages, dendritic cells (DCs) and granulocytes, which are essential for the normal functions of the innate and adaptive immune systems. By contrast, in pathological conditions, suchas cancer, various infectious diseases, sepsis, trauma, bone marrow transplantation and some autoimmune diseases, a partial block in the differentiation of IMCs into mature myeloid cells results in the expansion and activation of this population. In mice, MDSCs are characterized by the co-expression of CD11b and Gr-1 markers. The immunosuppressive activity of MDSC is attributable to high activity of arginase 1 (ARG1) and/or inducible NO synthase (iNOS), production of NO and reactive oxygen species (ROS), as well as release of IL-10 and TGF-β. Based on the expression of Ly6C and Ly6G, MDSCs can be subdivided into monocytic (M-MDSC) and granulocytic (G-MDSC) subset, which inhibit immune responses through different mechanisms. Although many studies have focused on the phenotypic characterization of MDSCs and unraveling mechanisms by which these cells mediate tumor progression, little is known about the intracellular signaling pathways involved in regulating the activation and accumulation of MDSCs.Cullin 4B (CUL4B), as a scaffold protein in the Cullin 4B-RING E3 ligase complex (CRL4B), belongs to the CULLIN gene family. Cullin-Ring E3 ligase complexes (CRLs), which contain Cullin protein, RING protein, and substrate-recognition subunit as the core components, represent the largest known class of unbiquitin ligases and participate in a broad variety of biological processes, including cell cycle, signal transduction, transcriptional regulation, development, tumorigenesis and viral infection. Recently, CUL4B has been shown to be substantially up-regulated in various types of solid tumors and possess potent oncogenic properties by transcriptionally repressing a subset of tumor suppressors. As an important epigenetic regulator, CRL4B regulates histone methylation and DNA methylation by catalyzing H2AK119 mono-ubiquitination and coordinating with PRC2 and DNMT/HP1/SUV39H1 complexes. Mutations in human CUL4B cause X-linked mental retardation syndrome (XLMR). To date, more that 20 families have been reported to be attributable to the loss-of-function mutations in CUL4B. Constitutive knockout of Cul4b in mice led to embryonic lethality, while heterozygous mice exhibited developmental delay with impaired vascularization in placentas. These underscore the functional importance of CUL4B during the development. In this study, we examined the roles of CUL4B in angiogenesis and hematopoiesis.Part I Generation and characterization of the conditional Cul4b knockout miceOur previous results showed that impaired vascularization in placentas could contribute to the developmental delay of Cul4b heterozygous mice. We also noted that patients with CUL4B mutation exhibited an increased numbers of monocytes. These results led us to hypothesize that CUL4B might be involved in angiogenesis and hematopoiesis. To test this hypothesis, we did the following analyses.1. Generation of conditional Cul4b knockout mice (CKO; Cul4bflox/Y Tek-Cre+/-):we generated a conditional Cul4b knockout mice (CKO; Cul4bflox/Y Tek-Cre+/-), by crossing the Cul4b-floxed mice to Tek-Cre transgenic mice. The Cre recombinase in Tek-Cre transgenic mice was driven by the Tek kinase promoter and enhancer, and expressed in all endothelial cells and hematopoietic progenitors. CUL4B was effectively ablated in endothelial cells and hematopoietic tissues such as bone marrow, spleen and blood in Cul4b CKO mice, as shown by immunohistochemical staining, Western blotting and quantitative RT-PCR.2. CD 11 b+Gr-1+cells were overrepresented and activated in Cul4b CKO mice:While the CKO mice did not exhibit overt histological defects and were comparable to control littermates in peripheral white blood cell counts and in the percentages of granulocytes, lymphocytes and monocytes, flow cytometry analyses revealed a significant increase in the population of CD11b+Gr-1+ cells, referred to as MDSCs hereinafter, in peripheral blood, spleen and bone marrow of Cul4b CKO mice. What’s more, Cul4b CKO CD11b+Gr-1+ cells can inhibit the proliferation of T cells in vitro culture.Part Ⅱ Lack of CUL4B in hematopoietic cells promotes tumor growth accompanied by an enhanced MDSCs accumulation and activation MDSCs, identified as CD11b+Gr-1+ cells in mice, are well known to expand under pathological conditions such as cancers, and suppress antitumor immune responses. Overrepresented CD11b+Gr-1+ cells in Cul4b CKO mice prompted us to ask whether lack expression of CUL4B in hematopoietic progenitors favors tumor establishment and progression accompanied by an enhanced accumulation of MDSCs. By employing xenograft tumor model, we did the following studies.1. Lack of CUL4B in heamapoietic system promote the tumor growth:We subcutaneously injected B16/F0 melanoma cells and EL4 lymphoma cells, respectively, into the flanks of 6-8 week-old Cul4b CKO and WT littermates, and analyzed the tumor growth kinetics and immune responses. The tumor masses formed by injected tumor cells, B16/F0 melanoma cells or EL4 lymphoma cells, were significantly larger in Cul4b CKO mice than in WT mice. Consistently, tumors excised from CKO mice weighed significantly more than those from WT mice. 2. Lack of CUL4B in hematopoietic progenitors led to enhanced accumulation of both subsets of MDSCs:In accordance with accelerated tumor growth in Cul4b CKO mice, both subsets of MDSCs were expanded more drastically. Even with similar tumor masses, the increase in the population of MDSCs was more pronounced in CKO than in WT mice.3. Enhanced accumulation of MDSCs are responsible for accelerated tumor growth in CKO mice:To determine whether the increased number of MDSCs are responsible for enhanced tumor growth in Cul4b CKO mice, we mixed EL4 tumor cells with MDSCs isolated from tumor-bearing WT and CKO mice and implanted into syngeneic WT C57BL/6 recipients. MDSCs from Cul4b CKO mice were significantly more potent in promoting tumor growth than MDSCs from WT mice. Consistently, when adoptively transferred into tumor-bearing CD45.1 WT recipients, CD45.2 Cul4b-deficient MDSCs were more potent than their WT counterparts in promoting tumor growth. The fact that MDSCs from CKO mice possessed a stronger tumor-promoting property, whereas their retention in recipients was indistinct from that of their WT counterparts.4. Lack of CUL4B in hematopoietic tissues promotes immune tolerance:As MDSCs are recognized to promote immune tolerance, we also tested whether Cul4b CKO mice can tolerate allogeneic tumors by subcutaneously implanting BALB/c 4T1 mammary cancer cells into the C57BL/6 Cul4b CKO and WT mice, respectively. Strikingly, while tumor engraftments were all rejected in WT mice, all Cul4b CKO mice tolerated the allogeneic tumors.5. Lack of CUL4B in hematopoietic progenitors enhances the immunosuppressive activity of MDSCs:We next determined the immunosuppressive effect of MDSCs derived from Cul4b CKO mice on T cells in vivo and vitro. Remarkably, both subsets of MDSCs from Cul4b CKO mice were more suppressive on CD4+and CD8+T cell proliferation than those from WT mice. The frequencies of CD4+and CD8+T cells in tumors from Cul4b CKO mice were significantly lower than those in WT mice. Consistently, the frequencies of CD4+and CD8+T cells in peripheral blood and spleen were significantly decreased in tumor-bearing Cul4b CKO mice. Furthermore, Cul4b CKO MDSCs exhibited higher levels of ArgI, Nox2, Nos2, and Cox2, as well as IL-10. Consistant with this, MDSCs from Cul4b CKO mice produced ROS and NO at significantly higher levels when treated with or without PMA and LPS, respectively. Taken together, these results indicate that lack of CUL4B not only permit a greater accumulation of MDSCs but also enhance their immunosuppressive activity.6. Lack of CUL4B impaires the differentiation of MDSCs:To determine whether the increased accumulation of MDSCs in Cul4b CKO mice was due to increased proliferation or decreased apoptosis, we injected BrdU intraperitoneally into EL4 tumor-bearing mice and measured its incorporation into MDSCs in bone marrow cells 24 h later. There was no difference in BrdU incorporation into bone marrow MDSCs between EL4 tumor-bearing CKO and WT mice. Similarly, no significant difference was detected in the level of apoptosis between the two groups. These results indicate that the enhanced accumulation of MDSCs in Cul4b CKO mice is probably not caused by their increase proliferation or reduced apoptosis.We next examined whether MDSCs are more favorably generated from hematopoietic progenitors in the absence of CUL4B. Significantly increased generation of MDSCs was detected in cultures with the bone marrow progenitors from Cul4b CKO mice compared with those from WT mice, indicating that MDSC generation from bone marrow progenitors is restricted by CUL4B. Because the accumulation of MDSC induced by tumor was often attributed to its impaired mature differentiation, we tested whether the increased accumulation of MDSCs in Cul4b CKO mice could also be caused by impaired differentiation of MDSCs into dendritic cells or macrophages. Indeed, when treated with GM-CSF for 3 or 5 days, Cul4b-deficient MDSCs gave rise to lower percentages of CD11b+ CDllc+ and CD11b+F4/80+ cells than WT MDSCs. Taken together, these results indicate that increased production of MDSCs from hematopoietic progenitors and their impaired differentiation into mature myeloid cells could be responsible for enhanced accumulation of MDSCs in Cul4b CKO mice.Part Ⅲ CUL4B constrains MDSCs by activating AKT/β-catenin pathwayThe results above suggest that CUL4B plays important roles in regulating the accumulation and activation of MDSCs. To understand the underlying mechanisms, we did the following analyses:1. Aberrant accumulation of MDSCs was mediated by down-regulation of P-catenin: It was reported that β-catenin is involved in regulating MDSCs accumulation and function. We examined the effect of Cul4b ablation on the level of β-catenin in MDSCs. Immunoblotting showed that lack of CUL4B resulted in a significantly decreased level of β-catenin in naive MDSCs as well as in MDSCs from tumor-bearing mice. Furthermore, we found that the levels of GSK3β phosphorylated at Ser9, an inactive form of GSK3β, were significantly reduced in Cul4b-deficient MDSCs, indicating that increased kinase activity of GSK3β, which phosphorylates β-catenin for its degradation, could be responsible for the increased accumulation of MDSCs caused by lack of CUL4B. Indeed, two inhibitors of GSK3P, SB216763 and lithium chloride (LiCl), could efficiently block the reduction of β-catenin level as well as MDSCs accumulation caused by Cul4b ablation. Consistently, increased generation of MDSCs from Lin-’ progenitors and the impaired differentiation of MDSCs into CD11b+CDllc+ and CD11b+F4/80+ cells caused by lack of CUL4B were rescued by either of the two inhibitors as well as by ectopic expression of constitutively active β-catenin. The ability of LiCl to decrease the accumulation of MDSCs was also demonstrated in CKO mice in vivo. Strikingly, LiCl greatly impeded the growth of tumor xenografts in Cul4b CKO mice as well as tumor-induced expansion of MDSCs. Taken together, these results indicate that increased GSK3β activity, and consequently β-catenin down-regulation, is causally linked to the enhanced accumulation of MDSCs in Cul4b CKO mice.2. Up-regulation of AKT phosphatases PP2A and PHLPP1/2 in the absence of CUL4B:Since Ser9 of GSK3β is phosphorylated by AKT, we next determined the level of AKT phosphorylated at Thr308 and Ser473, which marks maximal AKT activity. We found that the levels of phosphorylated AKT were markedly decreased in Cul4b-deficient MDSCs, whereas the amount of total AKT was not altered. Interestingly, when candidate AKT phosphatases were examined, the expression levels of PP2A (AKT phosphatase for Thr308), PHLPP1 and PHLPP2 (AKT phosphatases for Ser473), were found to be significantly elevated in Cul4b-deficient MDSCs. Quantitative real-time PCR (qRT-PCR) revealed that the mRNA levels of genes encoding different isoforms of PP2A, Ppp2r1a, Ppp2r1b, Ppp2ca and Ppp2cb, as well as those of Phlppl and Phlpp2 were much higher in Cul4b-deficient MDSCs than in WT MDSCs. Moreover, chromatin immunoprecipitation (ChIP) assay showed that CUL4B could directly bind to the promoters of the phosphatase genes. Quantitative ChIP assay showed that CUL4B, HDAC, and EZH2 were all enriched on the promoters of these genes in control cells, knockdown of Cul4b led to their significantly reduced recruitment to the promoters. Consistently, the levels of H3K27me3 and H2AK119ubl were also markedly decreased, whereas those of acetylated H3 and H4 were increased at those promoters. Taken together, these results indicate that CRL4B/PRC2/HDAC complexes can repress AKT phosphatases PP2A and PHLPP1/2 by promoting H2AK119 monoubiquitination, H3K27 trimethylation, as well as H3 and H4 deacetylation, en route to activate AKT/GSK3β/β-catenin signaling pathway.3. CUL4B/AKT/β-catenin axis is down-regulated in MDSCs:The data shown above clearly demonstrated a critical role of CUL4B in activating the AKT/β-catenin cascade that limits the accumulation of MDSCs. To determine whether this finding bears general implications, we compared the levels of CUL4B, PHLPP1/2, PP2A, β-catenin, p-GSK3β, and p-AKT between naive IMCs isolated from tumor-free mice and MDSCs from tumor-bearing mice. This idea was supported by our observation of a significant reduction in the levels of CUL4B, PHLPP1/2, PP2A, β-catenin, p-GSK3β, and p-AKT in MDSCs isolated from tumor-bearing mice when compared with those from tumor-free mice. Consistently, the levels of AKT phosphatases PP2A and PHLPP1/2 were significantly increased. We next determined whether CUL4B/AKT/β-catenin signaling pathway is altered in MDSCs of patients with cancer. Taken together, these results indicate that CUL4B/AKT/β-catenin signaling pathway is generally down-regulated in MDSCs during tumor progression.In summary, we demonstrated that CUL4B functions to limit the expansion and activity of MDSCs that are an integral part of the immunosuppressive tumor microenvironment. The increased accumulation of MDSCs in mice that lack of CUL4B in hematopoietic cells greatly facilitated the growth of syngeneic tumor xenografts, and even tolerated allogeneic tumor growth. The aberrant accumulation of MDSCs is mediated by the down-regulation of AKT/β-catenin pathway. CUL4B functions to sustain the AKT/β-catenin pathway by repressing phosphatases PP2A and PHLPP1/2 that dephosphorylate and inactivate AKT. Importantly, the CUL4B/AKT/β-catenin axis is down-regulated in MDSCs of healthy individuals and is further suppressed in tumor-bearing mice and cancer patients. Thus, while CUL4B drives malignancy of cancer cells, it impedes the formation of a tumor-supportive microenvironment. Our results establish a functional dichotomy of CUL4B in cancer cells and in tumor microenvironment. |
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