Nobuyoshi Kosaka

Secretory mechanisms and intercellular transfer of microRNAs in living cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis.

In the past several years, the importance of microRNA (miRNA) in cancer cells has been recognized. Proper control of miRNA expression is essential for maintaining a steady state of the cellular machinery. Recently, it was discovered that extracellular miRNAs circulate in the blood of both healthy and diseased patients, although ribonuclease is present in both plasma and serum. Most of the circulating miRNAs are included in lipid or lipoprotein complexes, such as apoptotic bodies, microvesicles, or exosomes, and are, therefore, highly stable. The existence of circulating miRNAs in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function, as well as the meaning of the existence of extracellular miRNAs, remain largely unclear. In this review, we summarize the usefulness of circulating miRNA for cancer diagnosis, prognosis, and therapeutics. Furthermore, we propose a mechanism for the secretion and incorporation of miRNA into the cells. (Cancer Sci 2010)

Systemic Delivery of Synthetic MicroRNA-16 Inhibits the Growth of Metastatic Prostate Tumors via Downregulation of Multiple Cell-cycle Genes

Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.

microRNA as a new immune-regulatory agent in breast milk

BackgroundBreast milk is a complex liquid that provides nutrition to the infant and facilitates the maturation of the infants immune system. Recent studies indicated that microRNA (miRNA) exists in human body fluid. Because miRNAs are known to regulate various immune systems, we hypothesized that human breast milk contains miRNAs that may be important for the development of the infants immune system.FindingsWe profiled miRNA expression in human breast milk and detected high expression levels of immune-related miRNAs in the first 6 months of lactation. Furthermore, these miRNA molecules are stable even in very acidic conditions, indicating that breast milk allows dietary intake of miRNAs by infants.ConclusionsOur findings provide new insight into how breast milk can modulate the development of the infants immune system. This study suggests the transfer of genetic material as miRNA from human to human occurs by means other than through sexual reproduction.

Neutral Sphingomyelinase 2 (nSMase2)-dependent Exosomal Transfer of Angiogenic MicroRNAs Regulate Cancer Cell Metastasis

Background: Contribution of exosomal microRNAs to cancer metastasis remains unknown. Results: Exosomal angiogenic microRNAs secreted by metastatic cancer cells promote the metastasis through the activation of endothelial cells. Conclusion: Horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell. Significance: This is the first to connect cancer metastasis to the exosomal microRNA in vivo. The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis; however, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Here, we demonstrate that the neutral sphyngomyelinase 2 (nSMase2) regulates exosomal microRNA (miRNA) secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis. We demonstrate a requirement for nSMase2-mediated cancer cell exosomal miRNAs in the regulation of metastasis through the induction of angiogenesis in inoculated tumors. In addition, miR-210, released by metastatic cancer cells, was shown to transport to endothelial cells and suppress the expression of specific target genes, which resulted in enhanced angiogenesis. These findings suggest that the horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell, like “on demand system” for cancer cells.

Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells

The bone marrow delivers dormancy signals in exosomes to metastatic cancer cells. Promoting Cancer Cell Dormancy with Exosomes Metastatic disease often develops long after treatment of the primary tumor because of cells that spread into metastatic niches, particularly the bone marrow. Because these “dormant” cells have a slow rate of cell cycling, they are not killed by traditional chemotherapies that target rapidly dividing cells. Ono et al. examined how cells present in the bone marrow niche trigger dormancy of metastatic breast cancer cells. They found that exosomes (vesicles shed by cells) from bone marrow–derived mesenchymal stem cells were taken up by cancer cells, in which they inhibited proliferation and promoted other characteristics associated with dormancy, in part through the delivery of a microRNA that suppressed the expression of a gene encoding a protein that promotes cell proliferation and motility. Thus, the bone marrow niche releases exosomes that deliver signals promoting metastatic cancer cell dormancy. Breast cancer patients often develop metastatic disease years after resection of the primary tumor. The patients are asymptomatic because the disseminated cells appear to become dormant and are undetectable. Because the proliferation of these cells is slowed, dormant cells are often unresponsive to traditional chemotherapies that exploit the rapid cell cycling of most cancer cells. We generated a bone marrow–metastatic human breast cancer cell line (BM2) by tracking and isolating fluorescent-labeled MDA-MB-231 cells that disseminated to the bone marrow in mice. Coculturing BM2 cells with bone marrow mesenchymal stem cells (BM-MSCs) isolated from human donors revealed that BM-MSCs suppressed the proliferation of BM2 cells, decreased the abundance of stem cell–like surface markers, inhibited their invasion through Matrigel Transwells, and decreased their sensitivity to docetaxel, a common chemotherapy agent. Acquisition of these dormant phenotypes in BM2 cells was also observed by culturing the cells in BM-MSC–conditioned medium or with exosomes isolated from BM-MSC cultures, which were taken up by BM2 cells. Among various microRNAs (miRNAs) increased in BM-MSC–derived exosomes compared with those from adult fibroblasts, overexpression of miR-23b in BM2 cells induced dormant phenotypes through the suppression of a target gene, MARCKS, which encodes a protein that promotes cell cycling and motility. Metastatic breast cancer cells in patient bone marrow had increased miR-23b and decreased MARCKS expression. Together, these findings suggest that exosomal transfer of miRNAs from the bone marrow may promote breast cancer cell dormancy in a metastatic niche.

The progression of liver fibrosis is related with overexpression of the miR-199 and 200 families

Background Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples. Methodology In a CCL4-induced mouse liver fibrosis model, we compared the miRNA expression profile from CCL4 and olive oil administrated liver specimens on 4, 6, and 8 weeks. We also measured expression profiles of human miRNAs in the liver biopsy specimens from 105 CH type C patients without a history of anti-viral therapy. Principle Findings Eleven mouse miRNAs were significantly elevated in progressed liver fibrosis relative to control. By using a large amount of human material in CH analysis, we determined the miRNA expression pattern according to the grade of liver fibrosis. We detected several human miRNAs whose expression levels were correlated with the degree of progression of liver fibrosis. In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were positively and significantly correlated to the progressed liver fibrosis. The expression level of fibrosis related genes in hepatic stellate cells (HSC), were significantly increased by overexpression of these miRNAs. Conclusion Four miRNAs are tightly related to the grade of liver fibrosis in both human and mouse was shown. This information may uncover the critical mechanism of progression of liver fibrosis. miRNA expression profiling has potential for diagnostic and therapeutic applications.

Competitive Interactions of Cancer Cells and Normal Cells via Secretory MicroRNAs

Background: Homeostatic cell competitive system between cancerous cells and non-cancerous cells is considered as the reason for tumor initiation. Results: Exosomal tumor-suppressive microRNAs secreted by non-cancerous cells inhibit the proliferation of cancerous cells. Conclusion: Exosomal tumor-suppressive microRNAs act as an inhibitory signal for cancer cells in a cell-competitive process. Significance: This provides a novel insight into a tumor initiation mechanism. Normal epithelial cells regulate the secretion of autocrine and paracrine factors that prevent aberrant growth of neighboring cells, leading to healthy development and normal metabolism. One reason for tumor initiation is considered to be a failure of this homeostatic cell competitive system. Here we identify tumor-suppressive microRNAs (miRNAs) secreted by normal cells as anti-proliferative signal entities. Culture supernatant of normal epithelial prostate PNT-2 cells attenuated proliferation of PC-3M-luc cells, prostate cancer cells. Global analysis of miRNA expression signature revealed that a variety of tumor-suppressive miRNAs are released from PNT-2 cells. Of these miRNAs, secretory miR-143 could induce growth inhibition exclusively in cancer cells in vitro and in vivo. These results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process. This study provides a novel insight into a tumor initiation mechanism.

MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrix metalloprotease-13 expression

Pulmonary metastases are the main cause of death in patients with osteosarcoma, however, the molecular mechanisms of metastasis are not well understood. To detect lung metastasis-related microRNA (miRNA) in human osteosarcoma, we compared parental (HOS) and its subclone (143B) human osteosarcoma cell lines showing lung metastasis in a mouse model. miR-143 was the most downregulated miRNA (P < 0.01), and transfection of miR-143 into 143B significantly decreased its invasiveness, but not cell proliferation. Noninvasive optical imaging technologies revealed that intravenous injection of miR-143, but not negative control miRNA, significantly suppressed lung metastasis of 143B (P < 0.01). To search for miR-143 target mRNA in 143B, microarray analyses were performed using an independent RNA pool extracted by two different comprehensive miR-143-target mRNA collecting systems. Western blot analyses revealed that MMP-13 was mostly protein downregulated by miR-143. Immunohistochemistry using clinical samples clearly revealed MMP-13-positive cells in lung metastasis-positive cases, but not in at least three cases showing higher miR-143 expression in the no metastasis group. Taken together, these data indicated that the downregulation of miR-143 correlates with the lung metastasis of human osteosarcoma cells by promoting cellular invasion, probably via MMP-13 upregulation, suggesting that miRNA could be used to develop new molecular targets for osteosarcoma metastasis.

MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC.