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KeywordsRecipient Cell Extracellular Vesicle Exosome Secretion Metastatic Breast Cancer Cell Line Inhibit Breast Cancer Cell Growth
Exosomes, shuttling from donor cells to recipient cells, are cell-derived extracellular vesicles (EV) with 30–100 nm in diameter and dish- or classic cup-shaped morphology. Exosomes transport bioactive cargo carrying selective proteins, lipids, DNA, messenger RNAs (mRNAs), and small and large noncoding RNAs such as microRNAs (miRNAs). miRNA embedded in exosomes is termed as exosomal miRNA. Exosomal miRNAs regulate diverse biological processes in recipient cells.
Biogenesis of Exosomes and Exosomal miRNA Packaging
As for today, there are several models to clarify the mechanisms of the package of exosomal miRNA. Firstly, the specific short sequence located on miRNAs can guide their loading into exosomes. For example, in B lymphocytes, 3′ ends of uridylated endogenous miRNAs are secreted into B lymphocyte-derived exosomes, while 3′ ends of adenylated endogenous miRNAs remain in B lymphocytes. Secondly, the package of miRNAs into exosomes requires the assistance of some intracellular functional proteins. For instance, the overexpression of neural sphingomyelinase 2 (nSMase2), the first reported protein functioning in the miRNA selection into exosomes, can increase the exosomal miRNA number. In addition, argonaute 2 (AGO2) prefers to bind uracil or adenine on the 5′ end of miRNAs. AGO2 deletion can decrease the expression of exosomal miRNAs including miR-150 and miR-451. Moreover, some modified RNA-binding protein contributes to the recognition of specific sequence on miRNAs. Sumoylated protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) binds to the specific 4 nt motifs (GGAG) in the 3′ region of miRNA to mediate miRNA transporting to MVBs and then packaging into exosomes. KRAS is also engaged in the process of miRNA package into exosomes. KRAS mutant colorectal cancer (CRC) cells have more miR-100 sorted into exosomes, while wild type cells have more exosomal miR-10. Thus, specific motif located on miRNAs and intracellular functional molecules are both critical to the package of exosomal miRNAs. Apart from those two kinds of elements, the affinity between MVB membrane and cellular miRNAs as well as cell-activation-dependent miRNA-targeted transcript level changes in donor cells also contribute to sorting of miRNAs into exosomes (Squadrito et al. 2014).
Exosomal miRNA is secreted into extracellular environment by employing exosome as vesicle. To date, the investigation of the underlying secretion mechanism is still going on. The secretion of exosomes in parent cells is dependent on important regulatory molecules such as small GTPase Rab family including Rab27, Rab28, Rab31, and Rab11 and Rab effector molecules (SYTL4 and SLAC2B). The tumor suppressor protein p53 modulates transcription of the downstream genes such as tsap6 and chmp4c that regulate the endosomal compartment and lead to elevated exosome secretion. Additionally, nSMase2 and calcium ionophore also regulate exosome secretion.
Bio-functions of Exosomal miRNA in Cancer
Exosomes utilize surface receptor such as MHC interaction or plasma membrane fusion to transfer contents in recipient cells, where exosomal miRNAs can bind to the 3′ untranslated region (3′ UTR) of target mRNAs. By repressing the expression of direct targets in recipient cells at posttranscription level, exosomal miRNAs carry out their functions in acceptor cells, most of which are closely linked to human cancers. Exosomal miRNAs can function as oncogenic exosomal miRNAs in tumor invasion, metastases, tumor angiogenesis, and immune suppression in various cancers. In breast cancer, exosomal miRNAs can promote breast cancer cell metastasis by direct targeting downstream genes in recipient cells. For example, exosomal miR-105, characteristically overexpressed and produced by metastatic breast cancer cell line MDA-MB-231, can directly repress downstream target tight junction-related gene (ZO-1) in endothelial cells, damaging the natural barrier integrity against breast cancer metastasis. IL4-activated macrophages-derived exosomal miR-223 can promote invasion of human breast cancer cell line SKBR3. In addition, exosomal miRNAs can also modulate tumor microenvironment. Stromal cell, an important component in tumor microenvironment, can produce exosomal miRNAs to assist breast cancer cells to be resistant to cancer therapy through activating STAT1 signaling pathway and NOTCH3 on breast cancer cells (Miller and Grunewald 2015). Also, exosomal miRNAs can promote angiogenesis. Blood vessel formation is essential for tumor cell growth. Melanoma cell-derived miR-9 induces the migration of endothelial cells and promotes cancer angiogenesis by regulating JAK-STAT pathway. Exosomal miR-135b derived from hypoxic multiple myeloma cells can block its target factor-inhibiting hypoxia-inducible factor 1 (HIF-FIH-1) when delivered to human umbilical vein endothelial cells (HUVECs). Hypoxic exosomal miRNAs enhance angiogenesis via the HIF-FIH signaling pathway under the condition of hypoxia. Besides, human endothelial cell line HMEC-1 s-derived exosomal miR-214 stimulates migration program and angiogenesis in recipient HMEC-1 cells. Exosomal miRNAs might also assist cancer cells to escape from immune cell detection. For instance, TW03-derived exosomes can block T-cell proliferation and T helper cell differentiation, leading to inhibit the function of T cells in human nasopharyngeal carcinoma (NPC) (Ye et al. 2014). Furthermore, five exosomal miRNAs have been identified to be abundant in the patient sera, and they modulate the MARK1 signaling pathway to affect cell proliferation. By contrast, some exosomal miRNAs are under-expressed in multiple cancers. These are known as tumor-suppressive exosomal miRNAs. Tumor-suppressive exosomal miRNAs have characteristics similar with tumor-suppressive genes that can inhibit cancer cell proliferation, metastasis, and induce apoptosis. For instance, it is well known that Let-7 miRNAs usually play a tumor-suppressive role by repressing oncogenes such as RAS and HMGA2, a metastatic gastric cancer cell line AZ-P7a could keep their oncogenic capacity through releasing tumor-suppressive Let-7 miRNAs via exosomes into the extracellular environment. In addition, tumor-suppressive exosomal miR-143 that derived from noncancerous cells can suppress the growth of prostate cancer cells by inhibiting its target genes including KRAS and ERK5 both in vitro and in vivo. Thus, exosomal miRNAs participate in tumorigenesis. Oncogenic exosomal miRNAs and tumor-suppressive exosomal miRNAs can promote and inhibit process of tumorigenesis, respectively (Fig. 1).
Exosomal miRNAs in Cancer Diagnostic and Clinical Use
Exosomal miRNAs play fundamental characters during cancer progression. So far, more and more evidence shows exosomal miRNAs could be used as diagnostic biomarkers for various cancers. For colorectal cancer, exosomal levels of seven miRNAs including miR-21 and miR-223 are significantly hyperactivated in primary cancer patients compared with healthy controls, with significantly downregulation after surgical resection of colorectal tumors. Thus, exosomal miRNA signatures could be utilized to mirror pathological process of colorectal cancer. For lung cancer, the expression of some exosomal miRNAs in patients’ sera is much higher than non-lung cancer tissues, indicating circulating exosomal miRNAs might be useful as biomarker for lung adenocarcinoma as well. For breast cancer, exosomal miRNAs can also be used as a diagnostic biomarker. Certain serum miRNAs are also highly correlated with breast cancer tissues. For instance, oncogenic miR-21 and miR-155 are significantly abundant in breast cancer specimens, whereas miR-126 is dramatically under-expressed. In ovarian cancer, tumor-derived exosomal miRNA signatures exhibit dramatically different profiles compared with that from benign tissues. So, exosomal miRNAs can be applied as diagnostic markers of ovarian cancer and references for tumor stages. Additionally, expression of exosomal miR-21 is correlated with tumor progression stages in esophageal squamous carcinoma, indicating that it may be a useful target for cancer therapy. Exosomal miR-1290 and miR-375 can be used as prognostic marker in castration-resistant prostate cancer as well.
Exosomes have already been employed by human virus to transfer miRNAs to noninfected cells, thereby assisting virus spread, which tells us exosomes may be applied as therapeutic vesicles and function as a good delivery system for tumor-suppressive exosomal miRNAs in cancer therapy. For example, delivering tumor-suppressive exosomal miR-143 leads to the shrink of development of prostate cancer cells in nude mice (Kosaka et al. 2013). Tumor-suppressive miRNA Let-7a can inhibit breast cancer cell growth when introduced into EGFR-expressing cells. Moreover, exosomes can be able to cross the blood–brain barrier, which could enhance the efficiency of delivery of medications to cancer cells, whereas there are still some questions need to be addressed when employing tumor-suppressive exosomal miRNAs in cancer therapy. For instance, how to facilitate more specific tumor-suppressive exosomal miRNA loading and packaging into exosomes? How to enhance the uptake efficiency of exosome by recipient cells? Secondly, apart from tumor-suppressive exosomal miRNAs, oncogenic exosomal miRNAs can also be applied in tumor therapy. Due to their functions in tumor angiogenesis and tumor invasion, oncogenic exosomal miRNAs might be utilized as cancer vaccines, whereas their cancer-promoting effects should be monitored during cancer research trials. Lastly, the small size of exosomal miRNAs also benefits them as an ideal target for drug designing. Collectively, exosomal miRNAs could potentially be served as clinical tools for cancer diagnostic and clinical use for various cancers. However, any side effects when using bioengineered or naturally occurring exosomal miRNAs need to be considered. In a word, using exosomal miRNAs might be an exciting but challenging application in cancer therapy in the near future.
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