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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.

Background/Aims: Although EpCAM+CD44+ cells exhibit more stem-like properties than did EpCAM-CD44- cells, the specificity of EpCAM combined with CD44 in defining CSCs needs further improvement. Lgr5 is used as a biomarker to isolate cancer stem cells (CSCs) in colorectal cancer. However, it remains unclear whether Lgr5, along with EpCAM and CD44, can further identify and define CSCs in colorectal cancer. Methods: Lgr5+CD44+EpCAM+, Lgr5+CD44+EpCAM-, Lgr5+CD44-EpCAM+, Lgr5-CD44+EpCAM+, and Lgr5-CD44-EpCAM-cells were separately isolated using fluorescence-activated cell sorting (FACS). Colony formation, self-renewal, differentiation, and tumorigenic properties of these cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression of stemness genes and CSC- and epithelial-mesenchymal transition (EMT)-related genes, such as KLF4, Oct4, Sox2, Nanog, CD133, CD44, CD166, ALDH1, Lgr5, E-cadherin, ZO-1, Vimentin, Snail, Slug, and Twist, was examined using real-time PCR. Results: Lgr5-positive subpopulations exhibited higher capacities for colony formation, self-renewal, differentiation, and tumorigenicity as well as higher expression of stemness genes and mesenchymal genes and lower expression of epithelial genes than did Lgr5-negative subpopulations. Conclusion: Our data revealed that tumorigenic cells were highly restricted to Lgr5-positive subpopulations. Most importantly, Lgr5+CD44+EpCAM+ cells exhibited more pronounced CSC-like traits than did any other subpopulation, indicating that Lgr5 combined with CD44 and EpCAM can further improve the stem-like traits of CSCs in colorectal cancer.

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.