The Cul4b/akt/β-catenin Axis Restricts the Accumulation of Myeloid-derived Suppressor Cells to Prohibit the Establishment of a Tumor Permissive Microenvironment

Running title: CUL4B constrains MDSCs through AKT/β-catenin pathway Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Conflict of interest: The authors disclose no potential conflicts of interest. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Cancer progression requires a permissive microenvironment that shields cancer from the host immunosurveillance. The presence of myeloid-derived suppressor cells (MDSCs) is a key feature of a tumor-permissive microenvironment. Cullin 4B (CUL4B), a scaffold protein in the Cullin 4B-RING E3 ligase complex (CRL4B), represses tumor suppressors through diverse epigenetic mechanisms and is overexpressed in many malignancies. We report here that CUL4B unexpectedly functions as a negative regulator of MDSC functions in multiple tumor settings. Conditional ablation of CUL4B in the hematopoietic system, driven by Tek-Cre, resulted in significantly enhanced accumulation and activity of MDSCs. Mechanistically, we demonstrate that the aberrant abundance of MDSCs in the absence of CUL4B was mediated by the downregulation of the AKT/β-catenin pathway. Moreover, CUL4B repressed the phosphatases PP2A and PHLPP1/2 that dephosphorylate and inactivate AKT to sustain pathway activation. Importantly, the CUL4B/AKT/β-catenin axis was downregulated in MDSCs of healthy individuals and was further suppressed in tumor-bearing mice and cancer patients. Thus, our findings point to a pro-and antitumorigenic role for CUL4B in malignancy, in which its ability to impede the formation of a tumor-supportive microenvironment may be context-specific. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 Surprising findings describe a previously uncharacterized antitumorigenic role for CUL4B in the hematopoietic system where it restricts the accumulation and activity of myeloid-derived suppressor cells to prevent the establishment of a tumor permissive microenvironment, underscoring mechanisms by which immunosurveillance may be compromised by certain therapeutic strategies. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

b-Catenin is critical for Wnt signaling, a pathway that governs many developmental processes, including specification of cell fate and the maintenance of stem cell pluripotency (10,11).Many studies indicated that a tight regulation of Wnt signaling is required for normal hematopoiesis and lymphopoiesis (12)(13)(14)(15).b-Catenin was recently shown to negatively regulate the functions of immunosuppressive cells, including regulatory T cell (Treg) and MDSCs (16)(17)(18).However, how Wnt signaling is regulated in those immunosuppressive cells remains to be elucidated.
Cullin 4B (CUL4B), as a scaffold protein in the Cullin 4B-RING E3 ligase complex (CRL4B), participates in the regulation of diverse physiologically and developmentally controlled processes (19)(20)(21)(22).Mutations in human CUL4B cause X-linked mental retardation (23,24).Recently, CUL4B has been shown to be substantially upregulated in various types of solid tumors and possess potent oncogenic properties.H2AK119 monoubiquitination catalyzed by CRL4B is critical for epigenetic inactivation of tumor suppressors by PRC2, SUV39H1/HP1/DNMT and SIN3A/histone deacetylase (HDAC) complexes (25)(26)(27).In this study, we demonstrated that CUL4B, AKT and b-catenin function in an axis that negatively regulates MDSCs.CUL4B sustains the activation of AKT/b-catenin pathway by repressing the antagonists of AKT.The CUL4B/AKT/b-catenin axis was downregulated in MDSCs and was further suppressed in tumorbearing hosts.Our results establish a functional dichotomy of CUL4B in cancer cells and in tumor microenvironment.

Mice
Cul4b flox/flox mice, as previously described (28), were crossed to Tek-Cre transgenic mice to produce Cul4b flox/Y Tek-Cre þ/À (CKO) mice and Cul4b flox/Y Tek-Cre À/À (WT) littermate controls.Mice were housed in a specific pathogen-free animal facility at Shandong University.All procedures involving mice were approved by the Animal Care and Use Committee of Shandong University School of Medicine.

Histology
Tissues from Cul4b flox/Y Tek-Cre þ/À R26R mice were fixed in paraformaldehyde.Seven micron thick sections were stained with X-gal and counterstained with eosin.Photomicrographs were taken using an Olympus IX71 with DP71 camera.

Isolation and differentiation assay of mouse MDSCs and Lin À cells
MDSCs were purified by immunomagnetic separation according to the manufacturer's protocol (Miltenyi Biotec).Cell purity (>95%) was determined by flow cytometric analysis using anti-CD11b and Gr-1 antibodies.MDSCs isolated from tumor-bearing mice were induced to differentiate in the presence or absence of 10 ng/mL GM-CSF (Sigma-Aldrich) for 3 and 5 days.Lin À cells were purified using a biotinylated lineage cell depletion cocktail (Miltenyi Biotec).The differentiation of Lin À cells was as described (18).

Isolation of human MDSCs
Human peripheral blood was collected before treatment from cancer patients aged between 45 and 65.Control samples were collected from age-and gender-matched healthy controls.MDSCs were isolated from freshly peripheral blood mononuclear cells by using HLA-DR-negative selection and CD33positive selection magnetic beads (Miltenyi Biotec).Purity was confirmed by flow cytometry using anti-HLA-DR and CD33 antibodies (BioLegend; >90%).Use of human specimens was approved by the Institutional Review Board at Shandong University School of Medicine.Informed consent was obtained from all blood donors.

Flow cytometric analysis
Single-cell suspensions were freshly prepared from mouse tissues.Cells were stained as described (17) with the antibodies listed in Supplementary Table S1.Data were analyzed with FlowJo 7.6.5 (Treestar) software.

NO and ROS measurements
The measurements of NO and ROS were as previously described (17).DCFDA (Invitrogen) was used to measure ROS production by purified G-MDSCs.Nitrite was quantified by a standard Greiss reaction (Invitrogen).

Lentiviral plasmid, viral production, and infection
Lentivirus shCul4b, PLOC-CUL4B, PLOC-CA-b-catenin, shPhlpp2, and shPpp2cb as well as their controls were prepared in HEK293T cells, as described previously (29).For the differentiation assay of mouse MDSCs and Lin À cells, 24 hours after infected with viral particles, the cells were cultured in fresh medium containing GM-CSF or GM-CSF and IL4 for 5 days.

Gene expression and chromatin immunoprecipitation analysis
Quantitative real-time PCR, Western blotting, and chromatin immunoprecipitation (ChIP) assays were performed as previously described (20,25).Primers and antibodies are listed in Supplementary Tables S1 and S2.

Statistical analysis
Statistical analysis was performed using unpaired Student t test to calculate a two-tailed P value between two groups.Differences were considered significant at P < 0.05.The data are recorded as the mean AE SEM of the indicated number of mice or biologic replicates and as the mean AE SD of the indicated number of technical replicates.

Results
Increased accumulation of CD11b þ Gr-1 þ cells in Cul4b CKO mice Cul4b heterozygous mice exhibit impaired vascularization in placentas (28).An elevation in monocytes was also detected in patients with CUL4B mutation (24).To test the function of CUL4B in angiogenesis and hematopoiesis, we generated Cul4b flox/Y Tek-Cre þ/À mice, referred to as Cul4b CKO hereinafter.CUL4B was effectively ablated in endothelial cells and hematopoietic tissues such as blood, spleen and bone marrow in Cul4b CKO mice (Fig. 1A and B).Cul4b CKO mice did not exhibit overt histologic defects (data not shown), and the percentages of lymphocytes, dendritic cells, macrophages and natural killer cells in spleen and the various hematopoietic progenitors in bone marrow were similar between two groups (Supplementary Tables S3 and S4).(30), were increased in the spleen of Cul4b CKO mice.Consistently, flow cytometry analyses revealed a significant increase in the population of CD11b þ Gr-1 þ cells in peripheral blood, spleen, and bone marrow of Cul4b CKO mice (Fig. 1C).
To determine whether CD11b þ Gr-1 þ cells in Cul4b CKO mice possess immunosuppressive activity, we examined their inhibitory effects on T-cell proliferation in vitro.As shown in Fig. 1D, Gr-1 þ cells from Cul4b CKO mice, but not those from Cul4b WT mice, significantly inhibited CD8 þ T-cell proliferation under both antigen-and mitogen-driven stimulatory conditions.These results suggest that the CD11b þ Gr-1 þ cells that are highly accumulated in Cul4b CKO mice probably represent MDSCs.

Cul4b CKO mice confer a tumor-supportive microenvironment that is dependent on
Because MDSCs are generally expanded in the presence of tumor and favor tumor establishment and progression, we next determined whether the increased abundance of CD11b þ Gr-1 þ cells in Cul4b CKO mice would promote tumor growth.The tumor masses formed by injected tumor cells, EL4 lymphoma cells, or B16/F0 melanoma cells, were significantly larger in Cul4b CKO mice than in WT mice (Fig. 2A and Supplementary Fig. S1A).In accordance with accelerated tumor growth in Cul4b CKO mice, MDSCs were expanded more drastically (Fig. 2B and Supplementary Fig. S1B).We next determined whether lack of CUL4B has distinct effects on the two MDSC subsets and found that both granulocytic (G-MDSCs) and monocytic (M-MDSCs) MDSCs were significantly elevated in Cul4b CKO mice compared with WT mice (Fig. 2C), suggesting that CUL4B has a negative effect on both subpopulations.
To determine whether the increased abundance of MDSCs is responsible for enhanced tumor growth in Cul4b CKO mice, we tested the effect of MDSCs admixed with EL4 cells.MDSCs from Cul4b CKO mice were significantly more potent in promoting tumor growth than those from WT mice (Supplementary Fig. S1C).Consistently, when adoptively transferred into tumor-bearing CD45.1 þ WT recipients, CD45.2 þ Cul4b-deficient MDSCs were more potent than their WT counterparts in promoting tumor growth (Fig. 2D).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 (Fig. 2E) suggests that Cul4b-deficient MDSCs individually have stronger tumor-promoting property.Thus, the MDSCs in CKO mice could have contributed to a tumor-permissive environment by their increased abundance and by the enhanced activity of the individual cells.The function of CD8 þ T cells and natural killer cells, on the other hand, appeared to be unimpaired by lack of CUL4B (Supplementary Fig. S2).Strikingly, even BALB/c 4T1 mammary cancer cells were able to form tumor grafts and grow in C57BL/6 Cul4b CKO (Fig. 2F), suggesting that Cul4b-deficient mice are greatly immunocompromised.We also determined the effect of CUL4B depletion on metastasis of intravenously injected B16/F0 melanoma cells to lung.As shown in Fig. 2G, both the number and the volume of micrometastatic nodules were markedly increased in the Cul4b CKO mice.
The immunosuppressive activities of CD11b þ Gr-1 þ cells are negatively regulated by CUL4B We next determined whether tumor-associated MDSCs are functionally more potent in Cul4b CKO mice than in WT.Remarkably, G-and M-MDSCs from Cul4b CKO tumor-bearing mice were more suppressive on CD8 þ T-cell proliferation than those from WT mice in both antigen-and mitogen-driven T-cell stimulatory conditions (Fig. 3A).The frequencies of CD4 þ and CD8 þ T cells in tumors from Cul4b CKO mice were significantly lower than those in WT mice (Fig. 3B).Consistently, the frequencies of CD4 þ and CD8 þ T cells in spleen were significantly decreased in tumor-bearing Cul4b CKO mice (Fig. 3B).We also examined the frequencies of Tregs in spleen, thymus and tumor.While the numbers of Tregs remained largely unchanged in thymus and spleen of Cul4b CKO mice, an increased abundance of Tregs was detected in tumor grafts of Cul4b CKO mice (Fig. 3C).To determine whether increased Tregs in tumor were secondary to the increased accumulation and activity of MDSCs, we examined the effect of MDSCs on Treg induction.As shown in Fig. 3D, MDSCs isolated from Cul4b CKO mice were more efficient in inducing Tregs than those from WT mice.(8).Compared with those isolated from WT mice, MDSCs from Cul4b CKO mice produced ROS and NO at significantly higher levels (Fig. 3E-G).Furthermore, Cul4b CKO MDSCs exhibited higher levels of Arg1, Nox2, Nos2, Cox2, and IL-10 transcripts (Fig. 3H).Taken together, these results indicate that lack of CUL4B does not only permit a greater accumulation of MDSCs but also enhances the immunosuppressive activity per cell, thus conferring a more permissive microenvironment for tumor progression.

The differentiation of MDSCs is impaired in Cul4b CKO mice
We evaluated the proliferation and apoptosis of MDSCs in bone marrow in vivo in EL4 tumor-bearing Cul4b CKO and  WT mice, but found no difference (Fig. 4A and B).Similarly, no significant differences in the proliferation or apoptosis of CD11b þ Gr-1 þ cells were observed in naive WT and CKO mice (Supplementary Fig. S3A and S3B).These results indicate that the enhanced accumulation of MDSCs in Cul4b CKO mice may not be caused by abnormal proliferation or apoptosis.
We next examined whether MDSCs are more favorably generated from hematopoietic progenitors in the absence of CUL4B.Lineage-negative (Lin À ) hematopoietic progenitor cells isolated from bone marrow were cultured for 5 days in the presence of IL4 and GM-CSF to induce myeloid differentiation.There was a significantly increased generation of MDSCs, marked by the expression CD11b and Gr-1, from Cul4b-deficient progenitors compared with WT progenitors (Fig. 4C).
In accordance with the impaired differentiation in Cul4b CKO mice, immunoblotting showed that the level of CUL4B was dramatically reduced when Lin À hematopoietic progenitor cells were induced to undergo myeloid differentiation.Conversely, CUL4B expression was increased when MDSCs were induced to undergo maturation (Fig. 4G).Taken together, these results indicate that the increased production of MDSCs from hematopoietic progenitors and their impaired differentiation into mature myeloid cells could be both responsible for the enhanced accumulation of MDSCs in Cul4b CKO mice.

Aberrant accumulation of MDSCs was mediated by downregulation of b-catenin
It was reported that loss of b-catenin in myeloid cells resulted in accelerated tumor growth and a greater accumulation of MDSCs, whereas expression of a constitutively active b-catenin mutant in myeloid cells inhibited tumor growth and reduced accumulation of MDSCs (17).Our recent study showed that CUL4B could activate Wnt/b-catenin signaling in human hepatocellular carcinoma (29).Thus, we next examined the effect of Cul4b ablation on the level of b-catenin in MDSCs.Immunoblotting showed that lack of CUL4B resulted in a significantly decreased level of b-catenin in naive Gr-1 þ cells as well as in two subsets of MDSCs from tumor-bearing mice (Fig. 5A).Because phosphorylation of b-catenin by GSK3b is crucial for its degradation and the phosphorylation of GSK3b itself reflects its inactivation, we next examined the phosphorylation status of GSK3b in MDSCs from Cul4b CKO and WT mice.As shown in Fig. 5A, the levels of GSK3b phosphorylated at Ser9, an inactive form of GSK3b, were significantly reduced in Cul4bdeficient MDSCs, indicating that increased kinase activity of GSK3b, which phosphorylates b-catenin for its degradation, could be responsible for the increased accumulation of MDSCs caused by lack of CUL4B.Indeed, when proteasome inhibitor MG132 was applied to Cul4b-deficient MDSCs, the reduction of b-catenin level was greatly attenuated (Supplementary Fig. S4A).Furthermore, two inhibitors of GSK3b, SB216763 and lithium chloride (LiCl), could efficiently block the reduction of b-catenin level caused by Cul4b ablation (Fig. 5B).Consistently, increased generation of MDSCs from Lin À progenitors and the impaired differentiation of MDSCs into CD11b þ CD11c þ and CD11b þ F4/80 þ cells caused by lack of CUL4B were rescued by either of the two inhibitors as well as by ectopic expression of constitutively active b-catenin (Supplementary Fig. S4B and Fig. 5C).The ability of LiCl to decrease the accumulation of MDSCs was also demonstrated in CKO mice in vivo (Fig. 5D).Strikingly, LiCl greatly impeded the tumor growth in Cul4b CKO mice (Fig. 5E) as well as tumor-induced expansion of MDSCs (Fig. 5F).Taken together, these results indicate that increased GSK3b activity, and consequently b-catenin downregulation, may mediate the enhanced accumulation of MDSCs in Cul4b CKO mice.

Upregulation of AKT phosphatases PP2A and PHLPP1/2 in the absence of CUL4B
As Ser9 of GSK3b is phosphorylated by AKT (33), we next determined the level of AKT phosphorylated at Thr308 and Ser473, which marks maximal AKT activity (34,35).We found that the levels of phosphorylated AKT were markedly decreased in naive Gr-1 þ cells and in both subsets of MDSCs in CKO mice, whereas the amount of total AKT was not altered (Fig. 6A), indicating that the decreased GSK3b phosphorylation at Ser9 is due to a decreased AKT kinase activity.Decreased level of AKT phosphorylation could be attributable to decreased kinase activities upstream of AKT or increased activities of phosphatases that dephosphorylate AKT.However, no significant difference in the levels of phosphorylated PDK1 (the kinase of AKT at the site of Thr308) and PTEN (a negative regulator of AKT) was found between Cul4b-deificient and control MDSCs (Supplementary Fig. S5).Interestingly, when AKT phosphatases were examined, the expression levels of PP2A (AKT phosphatase for Thr308), PHLPP1 and PHLPP2 (AKT phosphatases for Ser473; refs.[35][36][37], were found to be significantly elevated in  Cul4b-deficient MDSCs (Fig. 6B).Consistently, the phosphorylation level of PKCbII, another substrate of PHLPP1/2 (38), was significantly decreased in Cul4b-deficient MDSCs (Supplementary Fig. S6).
Because CUL4B is a transcriptional corepressor (25), we next examined the mRNA levels of the genes encoding these phosphatases.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 Phlpp1 and Phlpp2 were much higher in Cul4b-deficient MDSCs than in WT MDSCs (Fig. 6C).Moreover, ChIP assay showed that CUL4B could directly bind to the promoters of the phosphatase genes in MDSCs (Supplementary Fig. S7A).To determine whether the enhanced accumulation of MDSCs in Cul4b CKO mice is due to the upregulation of AKT phosphatases, we examined the generation of MDSCs from Phlpp2-or Ppp2cb-depleted Lin À cells.Importantly, depletion of either Phlpp2 or Ppp2cb significantly attenuated the generation of MDSCs from Cul4b-deficient Lin À cells (Supplementary Fig. S7B and Fig. 6D), suggesting that downregulation of AKT mediates the enhanced production of MDSCs.
The negative regulation of PP2A and PHLPP1/2 by CUL4B was also confirmed in Cul4b knockdown RAW264.7 cells as well as in cells overexpressing human CUL4B (Fig. 6E).Quantitative ChIP assay showed that depletion of Cul4b in RAW264.7 cells resulted in a significantly reduced recruitment of CUL4B, HDAC and EZH2 to the promoters (Fig. 6F and Supplementary Fig. S7C).Consistently, the levels of H3K27me3 and H2AK119ub1 were also markedly decreased, whereas those of acetylated H3 and H4 were increased, providing further evidence that CUL4B is critical for the transcriptional repression of these phosphatases.Furthermore, while overexpression of CUL4B could lead to a remarkable decrease in the expression of Phlpp1, Phlpp2, Ppp2ca, and Ppp2cb, treatment with HDACs inhibitor TSA, EZH2 inhibitor DNZep or both could efficiently attenuate the reduction in the expression of these genes (Fig. 6G).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/GSK3b/ b-catenin signaling pathway.

CUL4B/AKT/b-catenin axis is downregulated in MDSCs
The data shown above clearly demonstrated a critical role of CUL4B in activating the AKT/b-catenin cascade that limits the accumulation of MDSCs.To determine whether this finding bears general implications, we measured the levels of CUL4B in MDSCs and other hematopoietic cells in wild-type mice.As shown in Fig. 7A, Cul4b was remarkably downregulated in bone marrow and spleen MDSCs when compared with other hematopoietic cells.Similarly, CUL4B in MDSCs isolated from human peripheral blood, based on the absence of HLA-DR and expression of CD33, was also significantly downregulated when compared with other leukocytes (Fig. 7B).We speculated that the CUL4B/AKT/b-catenin pathway may generally restrain the population of MDSCs and that a downregulation of CUL4B is probably required for the generation of MDSCs from their progenitors and for the maintenance of their immunosuppressive ability.Indeed, there was a significant reduction in the levels of CUL4B, b-catenin, p-GSK3b, and p-AKT in MDSCs isolated from tumorbearing mice when compared with those from tumor-free mice (Fig. 7C).Consistently, the levels of AKT phosphatases PP2A and PHLPP1/2 were significantly increased (Fig. 7C).
We next determined whether CUL4B/AKT/b-catenin signaling pathway is altered in MDSCs of patients with cancer.Consistent with previous reports (2-4), the percentages of MDSCs were significantly higher in 22 patients with cancer than in agematched healthy donors (Fig. 7D).Furthermore, MDSCs from cancer patients were more suppressive on CD4 þ and CD8 þ T-cell proliferation than those from healthy controls (Fig. 7E).Importantly, the expression levels of CUL4B, measured by qRT-PCR, were negatively correlated with the percentages of MDSCs (Fig. 7F).Western blot analysis showed a significant reduction in the levels of CUL4B, b-catenin, p-GSK3b (Ser9) in MDSCs of cancer patients compared with those of healthy donors (Fig. 7G).Correspondingly, PP2A was significantly upregulated (Fig. 7G).Taken together, these results indicate that CUL4B/AKT/b-catenin signaling pathway is generally downregulated in MDSCs during tumor progression.

Discussion
CUL4B has recently been shown to be highly expressed in a variety of human malignancy and is positively correlated with tumor progression (25,26,29,39,40).In the current study, 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 CUL4B in hematopoietic cells greatly facilitated tumor growth and metastasis, and even tolerated allogeneic tumor growth.Thus, while CUL4B may drive the malignancy of cancer cells, it negatively regulates a cancer-supporting microenvironment in hematopoietic system.Thus, the function of CUL4B in cancer development is dichotomous.
Our finding that the negative regulation of MDSCs by CUL4B is mediated by b-catenin is consistent with two recent reports of b-catenin being a negative regulator of MDSCs (17,18).We showed that pharmacologic inhibition of GSK3b drastically reduced the growth of tumor grafts in Cul4b CKO mice.Importantly, our findings provided an insight into the mechanism by which b-catenin is regulated.We demonstrated that AKT, which phosphorylates and inactivates GSK3b (33), is positively regulated by CUL4B.CUL4B functions to sustain AKT activity by transcriptionally repressing AKT phosphatases, such as PP2A and PHLPP1/2, via H2AK119 monoubiquitination.The fact that the repression of those phosphatases by CUL4B can be compromised by HDAC and EZH2 inhibitors suggests that the functions of PRC2 and HDACs, which are physically associated with CRL4B, are also essential for the epigenetic silencing of those phosphatases.Interestingly, unlike in solid tumors in which EZH2 acts as an oncogene, inactivating somatic mutations in EZH2 cause myelodysplastic/myeloproliferative disorders, implicating EZH2 as a tumor suppressor in myeloid malignancy (41,42).Thus, EZH2 appears to behave similarly in this regard (41)(42)(43).Because EZH2 is essential for the function of PRC2, it is possible that an EZH2/AKT/b-catenin axis may operate in suppressing myeloid malignancy.The negative regulation of MDSCs by AKT described here bears some analogy to the reduction of AKT activity required for the development and function of Tregs, another kind of immunosuppressive cells contributing to tumor immune evasion (44,45), although dephosphorylation of AKT occurs only at Ser473, but not at Thr308, in activated Tregs.These findings suggest that the AKT signaling is regulated differently in Treg and MDSCs.Indeed, the numbers of Tregs remained largely unchanged in thymus and spleen of Cul4b CKO mice.Although there was an increased abundance of Tregs in tumor grafts in Cul4b CKO mice, it could be secondary to the increased accumulation and activity of MDSCs.PTEN, a primary negative regulator of AKT, was shown to be repressed by CUL4B in cancer cells (25).However, the level of PTEN was not found to be elevated in Cul4b-deficient hematopoietic cells, including MDSCs.Thus, the CUL4B/AKT/b-catenin axis described here could be cell type specific.
Our studies showed that whether CUL4B functions as a promoter or as a suppressor of tumorigenesis is context dependent.Because AKT/b-catenin signaling cascades in carcinomas are often sought as therapeutic targets, systemic downregulation of those pathways may sabotage the antitumor immune defense due to the expansion and activation of immunosuppressive cells such as MDSCs and Tregs.On the other hand, the aberrant accumulation of MDSCs, and its promotion of tumor growth, in Cul4b CKO mice could be managed by the use of GSK3b inhibitors.Lithium, commonly used for treatment of bipolar disorders, may help restore cancer-targeting immunosurveillance.

Figure 1 .
Figure 1.Lack of CUL4B in endothelial and hematopoitic cells leads to enhanced accumulation of CD11b þ Gr-1 þ cells.A, X-gal staining of spleen and heart from Cul4b loxP/Y

Figure 2 . 2 þ
Figure 2. Lack of CUL4B in hematopoietic system promotes tumor growth and metastasis.A, EL4 lymphoma cells were injected subcutaneously into 6-to 8-week-old WT or CKO mice, and tumor growth was monitored for 16 days.Then the tumors were resected and weighed.B, percentages of MDSCs in the blood, spleen, bone marrow, and tumor site from mice bearing EL4 tumors.C, flow cytometry analysis of CD11b þ Ly6G À Ly6C high and CD11b þ Ly6G þ Ly6C low cells from spleen and bone marrow from EL4-bearing WT or CKO mice.Numbers in the plots indicate the percentage of gated cells.D, adoptive transfer of CD45.2 þ Cul4b CKO or WT MDSCs into tumor-bearing CD45.1 WT recipients were performed on days 3 and 5 after tumor transplantation.Tumor growth was evaluated for 2 weeks.E, CD45.2 þ (donor) and CD45.1 þ (host) MDSCs in blood of tumor-bearing mice on days 7 after tumor transplantation.F, growth of 4T1 mammary tumors in WT or Cul4b CKO mice.G, B16/F0 metastasis to lung in WT and Cul4b CKO mice.Results represent mean AE SEM (n ¼ 6-10).Data are representative of more than three independent experiments.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 3 .Foxp3þFoxp3þ
Figure 3. CUL4B inhibits the immunosuppressive activity of MDSCs.A, flow cytometry analysis of CD4 þ and CD8 þ T cell proliferation (shown as CFSE dilution) in the presence of WT (blue line) and Cul4b CKO (red line) M-MDSCs or G-MDSC.Numbers indicate the percentages of gated proliferative cells (right).B, flow cytometry analysis of CD4 þ and CD8 þ T cells from tumors and spleens of mice bearing EL4.The summary is shown on the right.C, percentages of CD4 þ

Figure 4 .
Figure 4. Increased generation and impaired maturation of MDSCs in Cul4b CKO mice.A, flow cytometry analysis of BrdUrd incorporation in bone marrow CD11b þ Gr-1 þ cells (left).Each dot in the plots indicates one individual mouse (right).B, splenocytes were prepared 16 days after EL4 cell inoculation and then stained for CD11b, Gr-1, and Annexin V.Each dot in the plots indicates one individual mouse (right).C, enriched Lin À cells isolated from bone marrow of WT and CKO mice were cultured with GM-CSF and IL4 for 5 days in complete culture medium.The cells were then analyzed for CD11b and Gr-1 markers using flow cytometry.D, differentiation of bone marrow Gr-1 þ cells into CD11c þ CD11b þ and F4/80 þ CD11b þ cells in vitro.E, quantitative summary of MDSC differentiation shown in D. F, expression of S100A8 and S100A9 in MDSCs isolated from spleens of tumor-bearing mice.Results are presented as the fold change over WT controls.G, dynamic changes of CUL4B level in myeloid cells.Lane 1, Lin À cells; lane 2, Lin À cells treated with IL-4 and GM-CSF for 5 days; lane 3, Gr-1 þ cells; lane 4 and lane 5, Gr-1 þ cells treated with GM-CSF for 3 and 5 days, respectively.Results represent mean AE SEM (n ¼ 5-8).Data are representative of three independent experiments.ÃÃ , P < 0.01; ÃÃÃ , P < 0.001, NS, not significant.

Figure 6 .þ- 1 þ
Figure 6.Lack of CUL4B leads to upregulation of AKT phosphatases PP2A and PHLPP1/2.A and B, Western blot analysis of phosphorylated AKT and AKT phosphatases in MDSCs.C, mRNA levels of AKT phosphatases in MDSCs from Cul4b CKO mice.Data are shown as mean AE SEM (n ¼ 6).D, generation of CD11b þ Gr-1 þ

Figure 7 .
Figure 7. CUL4B/AKT/b-catenin axis is downregulated in mouse and human MDSCs.A, Cul4b expression in Gr-1 þ and Gr-1 À cells isolated from bone marrow (BM) and spleen (SP) of WT mice.B, CUL4B expression in MDSCs and non-MDSCs from healthy donors.C, Western blots of the indicated proteins in MDSCs isolated from spleen of na€ ve (N), B16, and EL4 tumor-bearing WT mice.D, the percentages of CD33 þ HLA-DR À cells in the peripheral blood of healthy donors and cancer patients determined by flow cytometry.n ¼ 25 for healthy donors; n ¼ 22 for cancer patients.E, HLA-DR À CD33 þ cells isolated from peripheral blood mononuclear cells of healthy donors and cancer patients were cocultured for 72 hours with CSFE-labeled T cells from healthy donors and stimulated with anti-CD3/anti-CD28 in mitogen-driven experiments.Data are shown as mean AE SEM (n ¼ 4).F, negative correlation of CUL4B expression, measured by qRT-PCR, with the percentages of MDSCs in cancer patients (n ¼ 22).G, Western blots of the indicated proteins in MDSCs isolated from healthy donors and cancer patients (left).Graph (right) shows protein levels based on band intensity after normalization to loading control, and healthy donors were assigned as 1.Data are shown as mean AE SEM (n ¼ 8).Data are representative of three independent experiments.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.