BackgroundmicroRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring.MethodsExosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis.ResultsSmall RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels.ConclusionsOur results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

Clioquinol, a metal chelator, has been used for many years as an antimicrobial agent and more recently as a potential treatment for Alzheimer's disease. Because it binds copper and zinc, metals essential for the activity of the enzyme superoxide dismutase-1 (SOD1), a potential target for anticancer drug development, we investigated its effects on human cancer cells. Treatment with clioquinol reduced the viability of eight different human cancer cell lines in a concentration-dependent manner, with IC 50 values in the low micromolar range. Biochemical analysis revealed that clioquinol induced cancer cell death through apoptotic pathways that require caspase activity. Although clioquinol induced modest inhibition of SOD1 activity in treated cells, comparable inhibition by a known SOD1 inhibitor, diethyldithiocarbamate, did not result in cytotoxicity. The addition of copper, iron, or zinc did not rescue cells from cliquinol-induced cytotoxicity but enhanced its killing, arguing against metal chelation as its major mechanism of action. To test if clioquinol might act as an ionophore, a fluorescent probe was used to monitor intracellular zinc concentrations. The addition of clioquinol resulted in elevated levels of intracellular zinc, indicating that clioquinol acts as a zinc ionophore. In an in vivo xenografts mouse model, clioquinol inhibited tumor growth of xenografts over a 6-week period, without inducing visible toxicity. Our results show that clioquinol has anticancer effects both in vitro and in vivo. Transition metal ionophores may be a subclass of metal chelators with anticancer activity deserving of further development. (Cancer Res 2005; 65(8): 3389-95)

Background Altered expression of microRNAs (miRNAs) is known to contribute to cancer progression. miR-23b and miR-27b, encoded within the same miRNA cluster, are reported to have both tumor suppressive and oncogenic activity across human cancers, including breast cancer. Methods To clarify this dichotomous role in breast cancer, miR-23b and miR-27b were knocked out using CRISPR/Cas9 gene knockout technology, and the role of endogenous miR-23b and miR-27b was examined in a breast cancer model system in vitro and in vivo. Results Characterization of the knockout cells in vitro demonstrated that miR-23b and miR-27b are indeed oncogenic miRNAs in MCF7 breast cancer cells. miR-23b and miR-27b knockout reduced tumor growth in xenograft nude mice fed a standard diet, supporting their oncogenic role in vivo. However, when xenograft mice were provided a fish-oil diet, miR-27b depletion, but not miR-23b depletion, compromised fish-oil-induced suppression of xenograft growth, indicating a context-dependent nature of miR-27b oncogenic activity. Conclusions Our results demonstrate that miR-23b and miR-27b are primarily oncogenic in MCF7 breast cancer cells and that miR-27b may have tumor suppressive activity under certain circumstances.

miR-1246 is considered an oncomiR in various cancer types. However, the origin and biogenesis of miR-1246 remain controversial which often leads to misinterpretation of its detection and biological function, and inevitably masking its mechanisms of action. Using next generation small RNA sequencing, CRISPR-Cas9 knockout, siRNA knockdown and the poly-A tailing SYBR qRT-PCR, we examined the biogenesis of exosomal miR-1246 in human cancer cell model systems. We found that miR-1246 is highly enriched in exosomes derived from human cancer cells and that it originates from RNU2-1, a small nuclear RNA and essential component of the U2 complex of the spliceosome. Knockdown of Drosha and Dicer did not reduce exosomal miR-1246 levels, indicating that exosomal miR-1246 is generated in a Drosha-and Dicer-independent manner. Direct digestion of cellular lysate by RNase A and knockdown of the RNU2-1 binding protein SmB/B' demonstrated that exosomal miR-1246 is a RNU2-1 degradation product. Furthermore, the GCAG motif present in the RUN2-1 transcript was shown to mediate miR-1246 enrichment in cancer exosomes. We conclude that exosome miR-1246 is derived from RNU2-1 degradation through a non-canonical microRNA biogenesis process. These findings reveal the origin of an oncomiR in human cancer cells, providing guidance in understanding miR-1246 detection and biological function.

Extracellular vesicles (EVs) are small lipid bilayer-delimited nanoparticles released from all types of cells examined thus far. Several groups of EVs, including exosomes, microvesicles, and apoptotic bodies, have been identified according to their size and biogenesis. With extensive investigations on EVs over the last decade, it is now recognized that EVs play a pleiotropic role in various physiological processes as well as pathological conditions through mediating intercellular communication. Most notably, EVs have been shown to be involved in cancer initiation and progression and EV signaling in cancer are viewed as potential therapeutic targets. Furthermore, as membrane nanoparticles, EVs are natural products with some of them, such as tumor exosomes, possessing tumor homing propensity, thus leading to strategies utilizing EVs as drug carriers to effectively deliver cancer therapeutics. In this review, we summarize recent reports on exploring EVs signaling as potential therapeutic targets in cancer as well as on developing EVs as therapeutic delivery carriers for cancer therapy. Findings from preclinical studies are primarily discussed, with early phase clinical trials reviewed. We hope to provide readers updated information on the development of EVs as cancer therapeutic targets or therapeutic carriers.