miRNA Trafficking Research Articles

Abstract PO4-28-03: Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion

Abstract Ductal carcinoma in situ (DCIS) is the most common form of early-stage breast cancer. Anywhere from 10%–30% of DCIS cases progress to invasive ductal carcinoma (IDC), resulting in poor prognosis and clinical challenges. Still, the cellular basis of this DCIS transition remains poorly understood. To escape the primary site during local invasion, DCIS cells navigate through the extracellular matrix and a diverse collection of stromal cells in the surrounding microenvironment. Cancer associated fibroblasts (CAFs) are a stromal cell population that can facilitate tumor invasion in part through extracellular matrix remodeling. CAFs at the primary tumor are largely derived from fibroblasts that have been reprogrammed to an activated state. In addition, several physical, cellular and metabolic changes within the tumor microenvironment (TME) are known to alter the breast cancer invasive capacity. Here we report that forced inhibition of cell invasion, by blocking the activity of known regulators of cell invasion in the ductal epithelial cell line, MCF10ADCIS.com, (herein referred to as DCIS.com), is accompanied by a marked reduction in extracellular vesicle (EV) secretion. Moreover, we show that DCIS.com cells conditioned with secreted EVs exhibit significantly enhanced invasive capacity relative to naïve DCIS.com cells. It is now well documented that breast and other tumor cells release heterogenous populations of EVs that can function as mediators of intercellular communication in the tumor microenvironment. EVs contain a host of bioactive cargo, such as membrane and cytosolic proteins, various RNA species including microRNAs, as well as dsDNA. These shed vesicles may deposit paracrine information and can also be taken up by stromal cells such as fibroblasts causing the recipient cells to undergo phenotypic changes that profoundly impact various facets of tumor progression. Conversely, EVs shed from stromal cells also affect tumor cell behavior. This unique form of intercellular crosstalk helps condition the TME and promotes both invasive and metastatic activity. In this regard, we found that incubation of human fibroblast cells with EVs shed from tumor cells leads to a loss of SMAD-7 expression (previously implicated in CAF reprogramming), and this loss is abrogated when shedding cells are depleted of miR-21 or regulators of miRNA trafficking to surface-derived EVs. Further, using spheroid invasion assays we demonstrate that fibroblast-derived EVs facilitate directed and leader-follower modes of migration, suggesting the possibility that CAFs likely deposit chemotactic cues to promote directional movement in DCIS. While the regulation of this behavior is still being explored, our observations suggest that EVs, in addition to facilitating distal proteolysis, may also support both autocrine and paracrine signaling during cell invasion. Finally, we are investigating how these pathways may also contribute to the ‘field effect’ of breast cancerization, a poorly understood process that introduces genetic and phenotypic changes in normal epithelia, potentially contributing to disease onset and/or recurrence. Citation Format: Madison Schmidtmann, Grace Richmond, Shireen Jayman, James Clancy, Crislyn D'Souza-Schorey. Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-03.

Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from endoplasmic reticulum to endosomes is a rate determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein HuR (Human antigen R). This activity ensures export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.

Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth.

ABSTRACTIn response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host.

RILP gets cleaved and exosomes leave.

Wozniak and colleagues reveal that cleavage of the lysosomal protein RILP following activation of the inflammasome enhances exosome release and regulates the trafficking of miRNAs into exosomes by FMR1.

Distinct communication patterns of trophoblastic miRNA among the maternal-placental-fetal compartments

IntroductionThe placenta produces microRNAs (miRNA) that may traffic to the maternal or fetal compartments and influence the physiology of pregnancy. The trafficking patterns of miRNA expressed from the large human chromosome 19 and chromosome 14 clusters (C19MC and C14MC), remains unclear. We interrogated the cross-sectional landscape of miRNA expression within the human placenta, fetal and maternal plasma to elucidate miRNA trafficking. We hypothesized that C19MC and C14MC miRNAs have similar expression patterns across the maternal-fetal compartments. MethodsPlacental biopsies, maternal and fetal venous plasma were collected from 25 pregnancies, and RNA was quantified using next generation sequencing. We identified expression and correlations differences among the compartments, and uncovered distinct miRNA expression patterns using consensus clustering. ResultsWe found that the placenta exhibits the highest total abundance, average miRNA expression and lowest variance of both C19MC and C14MC miRNAs. The C19MC miRNAs had a comparable expression and variance in fetal and maternal plasma and higher expression in the placenta. In contrast, the C14MC miRNAs had comparable expression between the placenta and fetal plasma, which was higher than the maternal plasma. We also identified 5 distinct groups of trophoblastic miRNAs with different expression patterns in each compartment. DiscussionThis is the first comprehensive analysis of C19MC and C14MC miRNA expression patterns in the human placental, maternal and fetal compartments. Our findings suggest that C14MC miRNAs are produced by both the fetus and placenta, but C19MC miRNAs are produced primarily in the placenta and are trafficked to the fetal and maternal compartments.

Stroma-derived extracellular vesicles deliver tumor-suppressive miRNAs to pancreatic cancer cells.

The biology of tumor-associated stroma (TAS) in pancreatic ductal adenocarcinoma (PDAC) is not well understood. The paradoxical observation that stroma-depletion strategies lead to progression of PDAC reinforced the need to critically evaluate the functional contribution of TAS in the initiation and progression of PDAC. PDAC and TAS cells are unique in their expression of specific miRNAs, and this specific miRNA expression pattern alters host to tumor microenvironment interactions. Using primary human pancreatic TAS cells and primary xenograft PDAC cells co-culture, we provide evidence of miRNA trafficking and exchanging between TAS and PDAC cells, in a two-way, cell-contact independent fashion, via extracellular vesicles (EVs) transportation. Selective packaging of miRNAs into EVs led to enrichment of stromal specific miR-145 in EVs secreted by TAS cells. Exosomes, but not microvesicles, derived from human TAS cells demonstrated a tumor suppressive role by inducing PDAC cell apoptosis. This effect was mitigated by anti-miR-145 sequences. Our data suggest that TAS-derived miRNAs are delivered to adjacent PDAC cells via exosomes and suppress tumor cell growth. These data highlight that TAS cells secrete exosomes carrying tumor suppressive genetic materials, a possible anti-tumor capacity. Future work of the development of patient-derived exosomes could have therapeutic implications for unresectable PDAC.

Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution

Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery.

IntroductionExosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current “gold standard” surrogate biomarker of myocardial damage.Methods and ResultsThe concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed (miR-1, miR-24, miR-133a/b, miR-208a/b, miR-210), non-cardiovascular (miR-122) and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs.ConclusionsThe plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.

Current State of Circulating MicroRNAs as Cancer Biomarkers.

Numerous studies have demonstrated the existence of stable regulatory RNAs, microRNAs (miRNAs), in the circulation and have shown that the spectrum of these extracellular miRNAs is affected by various pathologic conditions including cancers. Circulating miRNAs have been the focus of numerous cancer biomarker discovery efforts over the past few years; however, a considerable number of these studies have yielded inconsistent and irreproducible findings. Here, we have summarized and compared the results of studies covering 8 different cancer types to address key questions, including the possibility of using circulating miRNA to detect cancers and what factors may affect miRNA signatures. Although identifying circulating miRNA signatures to detect specific types of early stage cancers can be challenging, study results suggest that it may be possible to use miRNAs to detect cancers in general. Circulating miRNA is a rich source for potential disease biomarkers; however, factors, both intrinsic and extrinsic, that may affect measurement of circulating miRNA have not been fully characterized. Better understanding of intra- and intercellular miRNA trafficking and the fundamental biology of cancer cell-derived lipid vesicles may facilitate the development of circulating miRNA-based biomarkers for cancer detection and classification.

MiRNA trafficking across the maternal-placental-fetal compartments

MiRNA trafficking across the maternal-placental-fetal compartments

MiRNA nano-packages for maternal-placental-fetal communication

Positioned at the maternal-fetal interface, the placenta generates regulatory signals and serves as a communication junction that governs pregnancy health. In addition to hormones, growth factors and diverse proteins, we and others have recently shown that small non-coding RNA molecules, including microRNAs (miRNAs), traffic among the maternalplacental-fetal compartments, and may have a central role in maternalfetal adaptation to homeostatic perturbations. Although common and unique types of miRNAs are expressed by the placenta during pregnancy, the function of placental miRNA species remains largely unknown. We recently showed that primary human trophoblasts are resistant to infection by a heterogeneous panel of viruses, and that this resistance can be partly conferred to non-trophoblast cells by exogenous expression of trophoblast-specific miRNAs, expressed from the chromosome 19 miRNA cluster (C19MC). This effect was not observed during cell infection by nonviral placental pathogens, including Listeria monocytogenes, and Toxoplasma gondii. Trophoblastic miRNAs can be released to the extracellular space within several types of vesicles, including cell fragments, apoptotic bodies, microvesicles, and nanovesicles (exosomes), or can be packaged with non-vesicular protein complexes, such as argonaute-2. To examine the trafficking of trophoblastic miRNAs to maternal-fetal tissues we engineered a transgenic mouse that expresses a 160 kb segment of human genomic DNA harboring the human C19MC cluster. We coupled this technology with RNAseq analysis of miRNA expression in triads of placenta, maternal plasma and fetal plasma from pregnant women at term. Together, these analyses shed new light on transplacental bidirectional miRNA trafficking patterns (Supported by NIH HD06589, HD075665, HD071707, AI081759, and the Pennsylvania Department of Health Research Formula Funds)

Wolbachia interferes with the intracellular distribution of Argonaute 1 in the dengue vector Aedes aegypti by manipulating the host microRNAs

Argonaute proteins (AGOs) are vital components of the RNA-induced silencing complex in gene silencing. AGOs are indispensable for microRNA (miRNA) biogenesis as well as function, and are intracellularly localized to both the cytoplasm and the nucleus. Cytoplasmic AGO-miRNA complexes are mainly involved in cleavage or translational repression of target mRNAs while the nuclear ones are engaged in transcriptional gene silencing, methylation, chromatin remodeling, and splicing. In insects, AGO1 and AGO2 are involved in RNA interference and miRNA pathways but the components involved in their trafficking between the nucleus and the cytoplasm are not known. In this study, we found that importin β-4 facilitates AGO1 distribution to the nucleus, which is regulated by aae-miR-981 miRNA. The results also revealed association of prohibitin with AGO1 that may play an important role in its stability. Importantly, we found that AGO1 distribution to the nucleus is blocked by Wolbachia, an endosymbiotic bacterium introduced into the Dengue vector, Aedes aegypti. Our results provide basic mechanisms on intracellular trafficking of AGO1 in insects and how this may be altered by Wolbachia, which may affect trafficking of miRNAs to the nucleus leading to alteration in epigenetic effects.

MicroRNAs as regulators of tumor-associated stromal cells

The interplay between tumor and stromal cells represents an essential component of tumor progression and metastasis, and has become a focal point of current research efforts. Tumor-stromal interactions support early events in tumorigenesis, mediate the formation of pre-metastatic niches, and regulate the initiation and progression of distant metastases. Developing a better understanding of stromal regulation will be necessary for future therapeutic strategies, although progress has traditionally been hindered due to the complexity of the stromal microenvironment. Despite this, multiple studies have now examined the regulation of stromal microRNAs (miRNAs) during tumor progression, revealing the potential for miRNA-based therapeutic strategies to combat metastasis. We have recently described broad miRNA expression changes in osteoclasts, essential bone-resorbing stromal cells, during bone metastasis (Ell et al, Cancer Cell, 2013, 24;542-556). Osteoclastic activity drives lytic breast cancer bone metastasis, degrading bone and releasing bone-derived growth factors that enhance tumor growth. Thus, inhibiting osteoclast differentiation, through the blockage of osteoclast differentiation factors such as RANKL, or via direct inhibition with bisphosphonates, has proven to be a reliable method of limiting bone metastasis progression. Paramount within our study was the discovery that tumor-secreted factors could induce broad miRNA changes within osteoclasts, mimicking miRNA changes seen in physiological osteoclast differentiation. Mechanistically, osteoclast miRNA changes were induced through downstream signaling from soluble ICAM1 and other tumor-secreted factors. Importantly, specific ectopic expression of miR-141 or miR-219 was capable of inhibiting osteoclast differentiation and limiting bone metastasis in a mouse model of breast cancer metastasis. A similar study (Squadrito et al, Cell Reports, 2012, 1;141-154) discovered activity of miR-511-3p in tumor-associated macrophages (TAMs), well known mediators of tumor progression. The authors discovered that ectopic expression of miR-511-3p reduced the pro-tumoral activity of TAMs and inhibited the growth of Lewis lung carcinoma in mice. Interestingly, a comparable study has revealed a miRNA signature capable of recapitulating a cancer-associated fibroblast (CAF) phenotype in normal omental fibroblasts (Mitra et al, Cancer Discovery, 2012, 2;1100). Normal fibroblasts transfected with anti-miR-31, anti-miR-214, and miR-155 displayed enhanced migration, as well as elevated invasion and colony formation of co-cultured ovarian cancer cells, and increased tumor growth in vivo. Importantly, in an opposite set of experiments, elevating expression of miR-31 and miR-214 while inhibiting miR-155, abrogated the CAF phenotype. Together, these studies reveal the potential for utilizing miRNAs to target the stromal compartment and thereby limit tumor progression. An additional discovery within our study was the capability to utilize serum levels of miRNAs as biomarkers for bone metastasis progression in patients. It is currently difficult to predict which patients will develop bone metastasis, and standard biomarkers, such as N-terminal telopeptide, show only modest specificity as diagnostic markers. Interestingly, we found that two miRNAs, miR-16 and miR-378, were elevated in bone metastasis patients relative to healthy donors. It is not currently known exactly how miRNAs are secreted into the serum, although it has been hypothesized that miRNA trafficking could occur through the secretion of exosomes. While the miRNAs examined in our study did not show an overt effect on osteoclast differentiation or tumor cell proliferation, it is possible that secreted miRNAs can be laterally transferred between stromal and tumor cells, leading to enhanced metastatic potential. Indeed, TAM secreted miR-223 can enhance breast cancer invasiveness in vitro, and might indicate a novel mechanism of communication between macrophages and tumor cells (Yang et al, Molecular Cancer 2011, 10;117). These studies raise a number of interesting questions in regard to the therapeutic potential for miRNAs. Can miRNAs be useful as therapeutics, not only as direct inhibitors of metastatic cells, but also to disrupt tumor-stromal interactions? These preliminary studies show promising results, although rigorous follow-up studies will be necessary before advancing to clinical trials. Additionally, our preliminary results suggest a potential additive effect when miRNAs were combined with the bisphosphonate Zometa. It will be interesting to examine combinatorial treatments with miRNAs and current antiresorptive therapies, as well as the usefulness of miRNAs as inhibitors of other diseases associated with pathological osteoclast activity, including Paget's disease and osteoporosis. Beyond osteoclasts, can miRNAs be useful as inhibitors of other stromal cells, including TAMs, fibroblasts, and endothelial cells, and will these treatments be effective in limiting metastasis within their respective tumor models? Finally, a major concern within miRNA treatments involves off-target effects, and it will be essential to confirm specificity and low risk of side effects as development of these therapeutics progresses. In summary, these studies reveal the potential for utilizing miRNAs as therapeutic targets to limit the activity of tumor-associated stromal cells. In addition, secreted stromal miRNAs might prove useful as functional mediators of metastatic progression or clinical biomarkers. While a number of questions remain as to the utility, efficacy, and safety of miRNA-based therapeutic strategies, we hope that our efforts will facilitate the development of novel treatments for metastasis.

Alzheimer's disease and the microbiome

OPINION article Front. Cell. Neurosci., 17 September 2013Sec. Cellular Neuropathology Volume 7 - 2013 | https://doi.org/10.3389/fncel.2013.00153

Export of microRNAs and microRNA-protective protein by mammalian cells

The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questions about how this regulation functions and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell–cell communication. We report here that after serum deprivation several human cell lines tested promptly export a substantial amount of miRNAs into the culture medium and the export process is largely energy dependent. The exported miRNAs are found both within and outside of the 16.5 and 120 K centrifugation pellets which contain most of the known cell-derived vesicles, the microvesicles and exosomes. We have identified some candidate proteins involved in this system, and one of these proteins may also play a role in protecting extracellular miRNAs from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that is consistent with the cell–cell communication hypothesis.

Mammalian cells in culture actively export specific microRNAs

AbstractThe discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in blood plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations; and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication^[1-5]^. Such a system would be expected to export specific miRNAs from cells in response to specific biological stimuli. We report here that after serum deprivation several human cell lines tested do export a spectrum of miRNAs into the culture medium. The export response is substantial and prompt. The exported miRNAs are found both within and outside of microvesicles and exosomes. We have identified some candidate protein components of this system outside the cell, and found one exported protein that plays a role in protecting miRNA from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that may involve cell-cell communication.