Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.

Project description:Abstract: Background & Aims: Unusual hypervascularity is a hallmark of human hepatocellular carcinoma (HCC). Although microRNA-214 (miR-214) is upregulated in other human cancers, it is downregulated in HCC. We elucidated the biological and clinical significance of miR-214 downregulation in HCC. Methods: MicroRNAs deregulated in HCC were identified using array-based MicroRNA profiling. A luciferase reporter assay confirmed target association between miR-214 and hepatoma-derived growth factor (HDGF). Tube formation and in vivo angiogenesis assays validated the roles of miR-214/HDGF in angiogenesis. Results: MiR-214 downregulation was associated with higher tumor recurrence and worse clinical outcomes. Ectopic expression of miR-214 suppressed xenograft tumor growth and microvascularity of the tumor and its surrounding tissues. The genes downregulated by ectopic expression of miR-214 were involved in the regulation of apoptosis, cell cycle, and angiogenesis. Integrated analysis disclosed HDGF as a downstream target of miR-214. Conditioned medium of HCC cells contained bioactivity to stimulate tube formation of human umbilical vein endothelial cells, which was abolished by pretreatment of the conditioned media with HDGF antibodies, silencing of HDGF expression or ectopic expression of miR-214 in the donor HCC cells. The angiogenic activity of the conditioned media lost by ectopic expression of miR-214 in the donor cells was restored by supplementation with recombinant HDGF. In vivo tumor angiogenesis assays showed significant suppression of tumor vascularity by ectopic expression of miR-214. Conclusions: A novel role of microRNA in tumrigenesis is identified. Downregulation of miR-214 contributes to unusual hypervascularity of HCC via activation of the HDGF paracrine pathway for tumor angiogenesis. To identify miRNAs that are deregulated in human HCC, 68 HCC and 21 non-tumor liver tissues were subjected to profiling of miRNA expression using miRNA arrays containing 739 human miRNA probes. Differentially expressed microRNAs were identified.

Project description:Exososmes, potent intercellular communicators, are supposed to contribute to metastasis formation, which we confirmed for exosomes of the metastatic rat pancreatic adenocarcinoma line BSp73ASML that promote metastatic settlement in lymph nodes and lung of poorly metastatic BSp73ASML cells with a selective CD44v4-v7 (BSp73ASML-CD44vkd) knockdown. To define the molecular pathway(s), whereby exosomes contribute to premetastatic niche preparation, we profiled mRNA miRNA of BSp73ASMLwt and BSp73ASML-CD44vkd- exosomes and evaluated the impact on potential target cells. BSp73ASML exosomes are recovered in the draining lymph node after subcutaneous injection. In vitro, they preferentially bind and are taken-up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. BSp73ASMLwt and BSp73ASML-CD44kd exosomes contain a restricted repertoire of mRNA and miRNA, hwere the lattter differe significantly between the two lines and even more pronounced, exosomes derived thereof with a not yet explored dominance of tumor-suppressor miRNA in ASML-CD44kd cells and exosomes. Both, exosomal mRNA and miRNA are recovered in target cells and exosome-uptake is accompanied by significant changes in gene expression. We didn't observe a correlation between exosomal mRNA and changes in target cell mRNA or proteins. Instead transferred miRNA significantly affected target cell mRNA translation as demonstrated for selected, most abundant ASML exosomal miRNA besides others, miR-494 known target MAL (myelin and lymphocytes protein)/cadherin17, and miR-542-3p which targets TRAF/cadherin17. Furthermore, MMP transcription suggested to accompany cadherin17 dwon-regulation was upregulated in miR-494 or miR542-3p transfected or exosome co-cultured LnStr. Taken together, tumor exosomes target in vivo non-transformed cells in premetastatic organs. Exosome uptake induced altered target celll gene expression is strongly promoted by exosomal miRNA where we demonstrate for the first time that exosomes/exosomal miRNA from a metastasizing tumor line can modulate stroma cells from premetastatic organs. Endothelial cells lines were treated with pancreatic adenocarcinoma (AS) derived exosomes or pancreatic adenocarcinoma derived exosomes expressing tetraspanin 8. Total RNA was isolated and used to perform the Agilent gene expression microarrays. In this assay a replicate of endothelial cell lines treated with ASTspan8 were also included. Moreover, total RNA from both base line expression of endothelial cells and rat endothelial fibroblasts were also used to perfrom gene expression microarrays. RNA isolated from Rat endothelial fibroblasts treated with the exosomes derived from rat pancreatic adenocarcinoma and exosomes derived from rat pancreatic adenocarcinoma expressing tetraspanin8 were individually used to perfrom gene expression microarrays. RNA isolated from exosomes derived from rat pancreatic adenocarcinoma cell lines expressing tetraspanin were used to peform gene expresiion to see the base line expression. Another replicate were also used. RNA isolated from base line or control of rat pancreatic adenocarcinoma wild type cells and also base line RNA isolated from rat pancreatic adenocarcinoma cells lines where CD44 was knock-down.

Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy.

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Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells

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