下野 洋平

Adipocyte-cancer cell interactions promote tumor development and progression. Previously, we identified adipsin (CFD) and its downstream effector, hepatocyte growth factor (HGF), as adipokines that enhance adipocyte-breast cancer stem cell interactions. Here, we show that adipsin-dependent adipocyte maturation and the subsequent upregulation of HGF promote tumor invasion in breast cancers. Mature adipocytes, but not their precursors, significantly induced breast tumor cell migration and invasion in an adipsin expression-dependent manner. Promoters of tumor invasion, galectin 7 and matrix metalloproteinases, were significantly upregulated in cancer cells cocultured with mature adipocytes; meanwhile, their expression levels in cancer cells cocultured with adipocytes were reduced by adipsin knockout (Cfd KO) or a competitive inhibitor of CFD. Tumor growth and distant metastasis of mammary cancer cells were significantly suppressed when syngeneic mammary cancer cells were transplanted into Cfd KO mice. Histological analyses revealed reductions in capsular formation and tumor invasion at the cancer-adipocyte interface in the mammary tumors formed in Cfd KO mice. These findings indicate that adipsin-dependent adipocyte maturation may play an important role in adipocyte-cancer cell interaction and breast cancer progression.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. METHODS: "Bottom-of-the-crypt" (EPCAM+/CD44+/CD66alow) and "top-of-the-crypt" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX). RESULTS: Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. CONCLUSION: In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

Adipose tissue is a component of the tumor microenvironment and is involved in tumor progression. We have previously shown that adipokine adipsin (CFD) functions as an enhancer of tumor proliferation and cancer stem cell (CSC) properties in breast cancers. We established the Cfd-knockout (KO) mice and the mammary adipose tissue-derived stem cells (mADSCs) from them. Cfd-KO in mADSCs significantly reduced their ability to enhance tumorsphere formation of breast cancer patient-derived xenograft (PDX) cells, which was restored by the addition of Cfd in the culture medium. Hepatocyte growth factor (HGF) was expressed and secreted from mADSCs in a Cfd-dependent manner. HGF rescued the reduced ability of Cfd-KO mADSCs to promote tumorsphere formation in vitro and tumor formation in vivo by breast cancer PDX cells. These results suggest that HGF is a downstream effector of Cfd in mADSCs that enhances the CSC properties in breast cancers.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell-related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR-25, miR-93, and miR-106b) in the miR-106b-25 cluster were much lower in the CD44+ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR-93 suppressed invasive ability and 3D-organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott-Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR-93: overexpression of miR-93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR-93-mediated suppression of breast cancer cell invasion. These findings suggest that miR-93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.

Tumor microenvironment plays a key role for tumor development and progression. Although adipose tissue is a predominant component of stroma in mammary tissues and secretes various cytokines, chemokines and growth factors, roles of adipocytes in breast cancers remain to be elucidated. In this study, we found that adipsin, an adipokine secreted from mammary adipose tissues, enhanced proliferation and cancer stem cell (CSC)-like properties of human breast cancer patient-derived xenograft (PDX) cells. Adipsin was predominantly expressed in both adipose tissues of the surgical specimens of breast cancer patients and adipose-derived stem cells (ADSCs) isolated from them, and its expression level was significantly higher in obese patients. ADSCs significantly enhanced the sphere-forming ability of breast cancer PDX cells derived from both estrogen receptor-positive and -negative breast cancer PDX cells. Suppression of adipsin-mediated signaling by a specific inhibitor or adipsin knockdown in ADSCs significantly decreased the sphere-forming ability and the expression of CSC markers in co-cultured breast cancer PDX cells. Growth of breast cancer PDX tumors was significantly enhanced by co-transplantation with ADSCs in vivo, and it was weakened when co-transplanted with the adipsin knocked-down ADSCs. These results suggest that adipsin is an important adipokine secreted from mammary adipose tissue that functions as a component of tumor microenvironment and a CSC niche in breast cancers.

Organoid culture is a three-dimensional culture method that enables ex vivo analysis of stem cell behavior and differentiation. This method is also applicable to the studies on stem cell characters of human cancer stem cells. The components of organoid culture include Matrigel® and a culture medium containing growth factor cocktails that mimic the microenvironments of organ stem cell niches. Here, we describe the basic methods for the organoid culture of dissociated or FACS-sorted human cancer stem cells. Then, we introduce a method to dissociate the organoids for serial passage and propagation.

MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells. In addition, these miRNAs are similarly upregulated or downregulated in normal mammary stem/progenitor cells. In this review, we mainly describe the miRNAs that are dysregulated in human breast CSCs directly isolated from clinical specimens. The miRNAs and their clusters, such as the miR-200 clusters, miR-183 cluster, miR-221-222 cluster, let-7, miR-142 and miR-214, target the genes and pathways important for stem cell maintenance, such as the self-renewal gene BMI1, apoptosis, Wnt signaling, Notch signaling, and epithelial-to-mesenchymal transition. In addition, the current evidence shows that metastatic breast CSCs acquire a phenotype that is different from the CSCs in a primary site. Thus, clarifying the miRNA regulation of the metastatic breast CSCs will further advance our understanding of the roles of human breast CSCs in tumor progression.