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Infiltration of immune cells in primary tumors and metastatic sites is known to influence tumor progression and metastasis. Macrophages represent the most abundant immune cells in the tumor microenvironment, and evidence has shown that macrophages promote seeding, extravasation, and persistent growth of tumor cells at metastatic sites. miR-155 plays an essential role in immune cell development/function, and its aberrant expression is associated with lymphomas and several solid tumor types. However, it is unknown how miR-155 expression in immune cells affects solid tumor growth and metastasis. To this end, bone marrow transplantation was performed using miR-155–deficient mice as bone marrow donors and wild-type (WT) mice as recipients, and the chimeric mice were inoculated with tumor cells. We demonstrate that bone marrow lacking miR-155 significantly enhanced lung metastasis without a substantial effect on primary tumor growth. Relative to mice with WT bone marrow, miR-155–deficient bone marrow accumulated more macrophages in the spleen and lungs. Further analysis revealed that miR-155–deficient macrophages in metastatic sites exhibited a tumor-promoting M2 phenotype. In vitro study suggested that miR-155–null macrophages were prone to M2 polarization upon incubation with tumor cell–conditioned medium, due to elevated expression of C/EBPβ, an identified miR-155 target. These data, for the first time, demonstrate that miR-155 in host immune cells plays a vital role in modulating solid tumor metastasis by affecting the recruitment and polarization of bone marrow–derived macrophages.Implications: Targeted inhibition of miR-155 delays tumor development but inhibition in host immune cells may encourage metastasis. Mol Cancer Res; 11(8); 923–36. ©2013 AACR.

PDF file - 252K, S1. miR-155 expression in spleen of WT and miR-155-/- chimeric mice. S2. Effects on bone marrow miR-155 deficiency on bone marrow cell mobilization in tumor-bearing mice. S3. The effects of bone marrow miR-155 deficiency on the leukocyte subpopulations in spleen of tumor-bearing mice. S4. The effects on bone marrow miR-155 deficiency on the expression of tumor-derived factors. S5. A, Expression level of miR-155 in LLC and B16-F10 cells. Quantitative real-time PCR was performed to measure the expression of miR-155 in the lung tissue of WT mice, and cultured LLC and B16-F10 cells. Data are presented as the mean plus-minus SEM of three replicates. **, p

Our previous studies demonstrated that the natural compound emodin blocks the tumor-promoting feedforward interactions between cancer cells and macrophages, and thus ameliorates the immunosuppressive state of the tumor microenvironment. Since tumor-associated macrophages (TAMs) also affect epithelial mesenchymal-transition (EMT) and cancer stem cell (CSC) formation, here we aimed to test if emodin as a neoadjuvant therapy halts breast cancer metastasis by attenuating TAM-induced EMT and CSC formation of breast cancer cells. Methods: Bioinformatical analysis was performed to examine the correlation between macrophage abundance and EMT/CSC markers in human breast tumors. Cell culture and co-culture studies were performed to test if emodin suppresses TGF-β1 or macrophage-induced EMT and CSC formation of breast cancer cells, and if it inhibits breast cancer cell migration and invasion. Using mouse models, we tested if short-term administration of emodin before surgical removal of breast tumors halts breast cancer post-surgery metastatic recurrence in the lungs. The effects of emodin on TGF-β1 signaling pathways in breast cancer cells were examined by western blots and immunofluorescent imaging. Results: Macrophage abundance positively correlates with EMT and CSC markers in human breast tumors. Emodin suppressed TGF-β1 production in breast cancer cells and macrophages and attenuated TGF-β1 or macrophage-induced EMT and CSC formation of breast cancer cells. Short-term administration of emodin before surgery halted breast cancer post-surgery metastatic recurrence in the lungs by reducing tumor-promoting macrophages and suppressing EMT and CSC formation in the primary tumors. Mechanistic studies revealed that emodin inhibited both canonical and noncanonical TGF-β1 signaling pathways in breast cancer cells and suppressed transcription factors key to EMT and CSC. Conclusion: Natural compound emodin suppresses EMT and CSC formation of breast cancer cells by blocking TGF-β1-mediated crosstalk between TAMs and breast cancer cells. Our study provides evidence suggesting that emodin harbors the potential for clinical development as a new effective and safe agent to halt metastatic recurrence of breast cancer.

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5+CD44+EpCAM+ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5+CD44+EpCAM+ colorectal CSCs.

MicroRNA 155 (miR-155) plays important roles in the regulation of the development and functions of a variety of immune cells. We previously revealed a vital role of miR-155 in regulating the function of dendritic cells (DCs) in breast cancer. miR-155 deficiency in DCs impaired their maturation, migration, cytokine production, and ability to activate T cells. In the current study, to exploit the therapeutic value of miR-155 for breast cancer, we examined the impact of overexpression of miR-155 on antitumor responses generated by DC vaccines. We boosted miR-155 expression in DCs by generating a miR-155 transgenic mouse strain (miR-155tg) or using lentivirus transduction. DCs overexpressing miR-155 exhibited enhanced functions in response to tumor antigens. Using miR-155 overexpressing DCs, we generated a DC vaccine and found that the vaccine resulted in enhanced antitumor immunity against established breast cancers in mice, demonstrated by increased effector T cells in the mice, suppressed tumor growth, and drastically reduced lung metastasis. Our current study suggests that in future DC vaccine development for breast cancer or other solid tumors, introducing forced miR155 overexpression in DCs via various approaches such as viral transduction or nanoparticle delivery, as well as including other adjuvant agents such as TLR ligands or immune stimulating cytokines, may unleash the full therapeutic potential of the DC vaccines.

Asthma is a chronic inflammatory disease of the airways and the mechanisms are not fully understood. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of monocytes, granulocyte and myeloid cells at early stage of differentiation. They possess phenotypic plasticity and regulate airway inflammation. We recently reported that Kruppel-like factor 4 (KLF4) regulates MDSC differentiation into fibrocytes, emerging effectors in chronic inflammation. However, the role of KLF4 in asthma is not known. Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine and a key initiator of allergic airway inflammation. Given the fact that TSLP promotes Th2 cytokine production that increases MDSC differentiation into fibrocytes, we postulate that KLF4 regulates asthma in a TSLP-dependent manner. In this study, we utilized a model of allergic asthma with ovalbumin challenge (OVA). We found that upon OVA treatment the wild type mice had increased MDSC infiltration into the lung, up-regulation of KLF4 and TSLP gene expression, and higher levels of Th2 cytokines including IL4 and IL13. Consistently, lack of KLF4 expression in monocytes and lung epithelial cells resulted in decreased TSLP expression and lower levels of Th2 cytokines in mice, and fibrocyte generation was compromised. KLF4 deficiency in these cells also led to decreased airway hyperresponsiveness (AHR), a cardinal feature of asthma, as assessed by whole body plethysmography. Moreover, lung fibrosis as measured by trichome staining was attenuated and the population of CD45 + COL1A1+ fibrocytes was diminished in this setting. Together, our results suggest that KLF4 regulates asthma development in a TSLP- and fibrocyte-dependent manner.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b +Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b + Ly6G + cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+IMCs in early cancer development. © 2011 Nature America, Inc. All rights reserved.

KLF4 is a transcription factor that is highly expressed in the gastrointestinal tract. Previously we have demonstrated that KLF4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1 binding GC box and downstream gastrin responsive elements. However, the mechanism by which KLF4 inhibits HDC promoter is not well defined. In the current study, by using yeast two-hybrid screening, Tip60 was identified as a KLF4 interacting protein. Further coimmunoprecipitation and functional reporter assays support the interaction between these two proteins. In addition, Tip60 and HDAC7, previously shown to interact with each other and repress transcription, inhibited HDC promoter activity in a dose-dependent fashion. Consistently, knock down of Tip60 or HDAC7 gene expression by specific shRNA increased endogenous HDC mRNA level. Co-immunoprecipitation assays showed that HDAC7 was pulled down by KLF4 and Tip60, suggesting that these three proteins form a repressive complex. Further chromatin immuno-precipitation indicated that all three proteins associated with HDC promoter. Two-hour gastrin treatment, known to activate HDC gene expression, significantly decreased the association of KLF4, Tip60 and HDAC7 with HDC promoter, suggesting that gastrin activates HDC gene expression at least partly by decreasing the formation of KLF4/Tip60/HDAC7 repressive complexes at the HDC promoter.

The Kruppel-like factor family is a group of zinc finger containing transcription factors, which are highly homologous with the Drosophila Kruppel protein. The feature that distin‐ guishes the KLF family from other zinc finger containing transcriptional factors is the pres‐ ence of three highly conserved C2H2 containing zinc finger motifs at the C-terminus [1-3]. These fingers enable KLFs to bind to the GC-box or CACCC-boxes on DNA with different affinities [4]. KLF4, as a member of KLF family, expresses in a wide range of tissues in mam‐ mals, and plays a critical role in regulating a diverse array of cellular processes including proliferation, differentiation, development, maintenance of normal tissue homeostasis and apoptosis. KLF4 can also acts either as a tumor suppressor or an oncogene depending on differing cellular context and cancer types.

Gamma-secretase inhibitors (GSIs), which block the activation of the NOTCH1 receptor, are currently being tested in the treatment of T-cell lymphoblastic leukemias (T-ALL) with activating mutations in the NOTCH1 gene. However, inhibition of NOTCH1 signaling induces only a delayed cytostatic antileukemic effect with little or no apoptosis and GSI treatment is associated with severe gastrointestinal toxicity, which limits the clinical application of these molecularly targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids reverses glucocorticoid resistance and is highly effective against T-ALL. Inhibition of NOTCH1 signaling with CompE, a highly active GSI, in combination with dexamethasone, induced apoptotic cell death in glucocorticoid-resistant T-ALL cell lines. The synergistic antileukemic effect of GSIs and glucocorticoids was effectively rescued by forced expression of ICN1, an intracellular form of NOTCH1 that does not require gamma-secretase processing; or by treatment with the glucocorticoid receptor antagonist RU486. Reversal of glucocorticoid resistance by CompE treatment was mediated by transcriptional upregulation of BIM, a BH3-only proapototic factor previously involved in glucocorticoid induced cell death. Thus, shRNA knock down of BIM effectively impaired the ability of CompE plus dexamethasone to induce programmed cell death in leukemic lymphoblasts. Using a mouse xenograft model of glucocorticoid-resistant T-ALL we demonstrated that the combination of DBZ, a GSI with well established activity in vivo, and dexamethasone is highly effective against glucocorticoid resistant T-ALL in vivo. All animals treated with DBZ plus dexamethasone showed complete clearance of leukemic lymphoblasts and improved survival, while mice treated with vehicle or dexamethasone alone died because of disease progression (P