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.

Background Exosomes are small membrane-bound vesicles that contribute to tumor progression and metastasis by mediating cell-to-cell communication and modifying the tumor microenvironment at both local and distant sites. However, little is known about the predominant factors in exosomes that contribute to breast cancer (BC) progression. MTA1 is a transcriptional co-regulator that can act as both a co-activator and co-repressor to regulate pathways that contribute to cancer development. MTA1 is also one of the most up-regulated proteins in cancer, whose expression correlates with cancer progression, poor prognosis and increased metastatic potential. Methods We identified MTA1 in BC exosomes by antibody array and confirmed expression of exosome-MTA1 across five breast cancer cells lines. Ectopic expression of tdTomato-tagged MTA1 and exosome transfer were examined by fluorescent microscopy. CRISPR/Cas9 genetic engineering was implemented to knockout MTA1 in MCF7 and MDA-MB-231 breast cancer cells. Reporter assays were used to monitor hypoxia and estrogen receptor signaling regulation by exosome-MTA1 transfer. Results Ectopic overexpression of tdTomato-MTA1 in BC cell lines demonstrated exosome transfer of MTA1 to BC and vascular endothelial cells. MTA1 knockout in BC cells reduced cell proliferation and attenuated the hypoxic response in these cells, presumably through its co-repressor function, which could be rescued by the addition of exosomes containing MTA1. On the other hand, consistent with its co-activator function, estrogen receptor signaling was enhanced in MTA1 knockout cells and could be reversed by addition of MTA1-exosomes. Importantly, MTA1 knockout sensitized hormone receptor negative cells to 4-hydroxy tamoxifen treatment, which could be reversed by the addition of MTA1-exosomes. Conclusions This is the first report showing that BC exosomes contain MTA1 and can transfer it to other cells resulting in changes to hypoxia and estrogen receptor signaling in the tumor microenvironment. These results, collectively, provide evidence suggesting that exosome-mediated transfer of MTA1 contributes to BC progression by modifying cellular responses to important signaling pathways and that exosome-MTA1 may be developed as a biomarker and therapeutic target for BC. Electronic supplementary material The online version of this article (10.1186/s12964-019-0325-7) contains supplementary material, which is available to authorized users.

Background Exosomes are extracellular vesicles containing a variety of biological molecules including microRNAs (miRNAs). We have recently demonstrated that certain miRNA species are selectively and highly enriched in pancreatic cancer exosomes with miR-1246 being the most abundant. Exosome miRNAs have been shown to mediate intercellular communication in the tumor microenvironment and promote cancer progression. Therefore, understanding how exosomes selectively enrich specific miRNAs to initiate exosome miRNA signaling in cancer cells is critical to advancing cancer exosome biology. Results The aim of this study was to identify RNA binding proteins responsible for selective enrichment of exosome miRNAs in cancer cells. A biotin-labeled miR-1246 probe was used to capture RNA binding proteins (RBPs) from PANC-1 cells. Among the RBPs identified through proteomic analysis, SRSF1, EIF3B and TIA1 were highly associated with the miR-1246 probe. RNA immunoprecipitation (RIP) and electrophoretic mobility shift assay (EMSA) confirmed the binding of SRSF1 to miR-1246. Lentivirus shRNA knockdown of SRSF1 in pancreatic cancer cells selectively reduced exosome miRNA enrichment whereas GFP-SRSF1 overexpression enhanced the enrichment as analyzed by next generation small RNA sequencing and qRT-PCR. miRNA sequence motif analysis identified a common motif shared by 36/45 of SRSF1-associated exosome miRNAs. EMSA confirmed that shared motif decoys inhibit the binding of SRSF1 to the miR-1246 sequence. Conclusions We conclude that SRSF1 mediates selective exosome miRNA enrichment in pancreatic cancer cells by binding to a commonly shared miRNA sequence motif. Graphical abstract

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.

Ductal carcinoma in situ (DCIS) is a benign breast condition that increases the risk of invasive breast cancer (IBC). Clinically, not all DCIS will progress to IBC and the major drivers of progression are not well understood. The role of small extracellular vesicles, also known as exosomes, is well described in advanced cancers, however, little is known about their role in the invasive progression of in situ breast disease. In this study, we sought to evaluate the role of exosomes and their RNA contents in the progression of DCIS, using the MCF10 isogenic series, in vitro assays and the mouse intraductal model of breast cancer progression. We observed a significant increase of exosome loading of microRNAs (miRNAs) between normal and benign states that increased with each stage. We demonstrate that pro-invasive programs were attenuated when overall exosome and miRNA secretion was inhibited by Rab27A and Dicer knockdown, respectively. Comparisons of exosome miRNA expression changes revealed the greatest differential expression between in situ and invasive cells, with miR-205 being the most significantly different, suggesting a mechanistic role for exosome miRNAs in invasive progression. Modulation of miR-205 levels affected epithelial to mesenchymal transition and the invasive programs in vitro. Progression of DCIS in vivo resulted in increased levels of specific circulating exosome miRNAs, while inhibition of exosome secretion (Rab27A knockdown) attenuated invasive progression and reduced circulating levels of exosome miRNAs. These observations suggest that exosome miRNAs are differentially loaded and expressed, and promote the invasive progression of DCIS and that circulating exosome miRNAs are a potential source of biomarkers of the DCIS to invasive transition. Citation Format: Bethany Hannafon, Samrita Dogra, Sugantha Priya Elayapillai, James Lausen, Matthew Bruns, Fariba Behbod, Amy Gin, Chao Xu, Roy Zhang, Wei-Qun Ding. Exosome microRNAs are progressively altered and contribute to invasive progression of ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B012.