Extracellular RNA in oncogenesis, metastasis and drug resistance.

Extracellular vesicles and nanoparticles (EVPs) are now recognized as a novel form of cell-cell communication. All cells release a wide array of heterogeneous EVPs with distinct protein, lipid, and RNA content, dependent on the pathophysiological state of the donor cell. The overall cargo content in EVPs is not equivalent to cellular levels, implying a regulated pathway for selection and export. In cancer, release and uptake of EVPs within the tumour microenvironment can influence growth, proliferation, invasiveness, and immune evasion. Secreted EVPs can also have distant, systemic effects that can promote metastasis. Here, we review current knowledge of EVP biogenesis and cargo selection with a focus on the role that extracellular RNA plays in oncogenesis and metastasis. Almost all subtypes of RNA have been identified in EVPs, with miRNAs being the best characterized. We review the roles of specific miRNAs that have been detected in EVPs and that play a role in oncogenesis and metastasis.

miR-100 and miR-125b Contribute to Enhanced 3D Growth and Invasiveness and can be Functionally Transferred to Silence Target Genes in Recipient Cells.

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles.

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

MOTIVATION: Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. METHODS: We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. RESULTS: We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. CONCLUSION: This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites.

RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.

Interaction of lncRNA MIR100HG with hnRNPA2B1 facilitates m6A-dependent stabilization of TCF7L2 mRNA and colorectal cancer progression.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown. METHODS: The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining. RESULTS: The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/ß-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (m6A) site of TCF7L2 mRNA in the presence of MIR100HG. TCF7L2, in turn, activated MIR100HG transcription, forming a feed forward regulatory loop. The MIR100HG/hnRNPA2B1/TCF7L2 axis was augmented in specimens from CRC patients who either developed local or distant metastasis or had disease progression that was associated with cetuximab resistance. CONCLUSIONS: MIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Depletion of METTL3 alters cellular and extracellular levels of miRNAs containing m6A consensus sequences.

Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs.

Induction of a proliferative response in the zebrafish retina by injection of extracellular vesicles.

Ongoing research using cell transplantation and viral-mediated gene therapy has been making progress to restore vision by retinal repair, but targeted delivery and complete cellular integration remain challenging. An alternative approach is to induce endogenous Müller glia (MG) to regenerate lost neurons and photoreceptors, as occurs spontaneously in teleost fish and amphibians. Extracellular vesicles (EVs) can transfer protein and RNA cargo between cells serving as a novel means of cell-cell communication. We conducted an in vivo screen in zebrafish to identify sources of EVs that could induce MG to dedifferentiate and generate proliferating progenitor cells after intravitreal injection into otherwise undamaged zebrafish eyes. Small EVs (sEVs) from C6 glioma cells were the most consistent at inducing MG-derived proliferating cells. Ascl1a expression increased after intravitreal injection of C6 sEVs and knockdown of ascl1a inhibited the induction of proliferation. Proteomic and RNAseq analyses of EV cargo content were performed to begin to identify key factors that might target EVs to MG and initiate retina regeneration.

Rab13 regulates sEV secretion in mutant KRAS colorectal cancer cells.

Small extracellular vesicles (sEVs), 50-150 nm in diameter, have been proposed to mediate cell-cell communication with important implications in tumor microenvironment interactions, tumor growth, and metastasis. We previously showed that mutant KRAS colorectal cancer (CRC) cells release sEVs containing Rab13 protein and mRNA. Previous work had shown that disruption of intracellular Rab13 trafficking inhibits epithelial cell proliferation and invasiveness. Here, we show that Rab13 additionally regulates the secretion of sEVs corresponding to both traditional exosomes and a novel subset of vesicles containing both ß1-integrin and Rab13. We find that exposure of recipient cells to sEVs from KRAS mutant donor cells increases proliferation and tumorigenesis and that knockdown of Rab13 blocks these effects. Thus, Rab13 serves as both a cargo protein and as a regulator of sEV secretion. Our data support a model whereby Rab13 can mediate its effects on cell proliferation and invasiveness via autocrine and paracrine signaling.

Argonautes in Extracellular Vesicles: Artifact or Selected Cargo?

Argonaute-2 (Ago2) is a key component of the RNA-induced silencing complex that mediates downregulation of mRNA by miRNAs. Its presence in extracellular vesicles (EV) has been postulated to be important for the activity of EV-carried miRNA in modulating gene expression in recipient cells. However, whether it is in fact contained within EVs or is instead an extravesicular contaminant is controversial. In this opinion piece, we argue that the ability to detect Ago2 in EVs is a result of multiple factors, including cell source, cell signaling control of Ago2 trafficking to EVs, experimental conditions, and detection methods.

Reassessment of Exosome Composition.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.

Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.

Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells.

The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.

lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/ß-catenin signaling.

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/ß-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.

Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.

Identification of a Paralog-Specific Notch1 Intracellular Domain Degron.

Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.

KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.

Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.

KRAS-dependent sorting of miRNA to exosomes.

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.

Technical brief: Constant intense light exposure to lesion and initiate regeneration in normally pigmented zebrafish.

Zebrafish are capable of robust and spontaneous regeneration of injured retina. Constant intense light exposure to adult albino zebrafish specifically causes apoptosis of rod and cone photoreceptor cells and is an excellent model to study the molecular mechanisms underlying photoreceptor regeneration. However, this paradigm has only been applied to lesion zebrafish of the nonpigmented albino genetic background, which precludes the use of numerous transgenic reporter lines that are widely used to study regeneration. Here, we explored the effectiveness of constant intense light exposure in causing photoreceptor apoptosis and stimulating regeneration in normally pigmented zebrafish retinas. We show that constant intense light exposure causes widespread photoreceptor damage in the dorsal-central retinas of pigmented zebrafish. Photoreceptor loss triggers dedifferentiation and proliferation of Müller glia as well as progenitor cell proliferation. We also demonstrate that the timeline of regeneration response is comparable between the albino and the pigmented retinas.

Strain-specific suppression of microRNA-320 by carcinogenic Helicobacter pylori promotes expression of the antiapoptotic protein Mcl-1.

Helicobacter pylori is the strongest risk factor for gastric cancer, and strains harboring the cag pathogenicity island, which translocates the oncoprotein CagA into host cells, further augment cancer risk. We previously reported that in vivo adaptation of a noncarcinogenic H. pylori strain (B128) generated a derivative strain (7.13) with the ability to induce adenocarcinoma, providing a unique opportunity to define mechanisms that mediate gastric carcinogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that regulate expression of oncogenes or tumor suppressors and are frequently dysregulated in carcinogenesis. To identify miRNAs and their targets involved in H. pylori-mediated carcinogenesis, miRNA microarrays were performed on RNA isolated from gastric epithelial cells cocultured with H. pylori strains B128, 7.13, or a 7.13 cagA(-) isogenic mutant. Among 61 miRNAs differentially expressed in a cagA-dependent manner, the tumor suppressor miR-320 was significantly downregulated by strain 7.13. Since miR-320 negatively regulates the antiapoptotic protein Mcl-1, we demonstrated that H. pylori significantly induced Mcl-1 expression in a cagA-dependent manner and that suppression of Mcl-1 results in increased apoptosis. To extend these results, mice were challenged with H. pylori strain 7.13 or its cagA(-) mutant; consistent with cell culture data, H. pylori induced Mcl-1 expression in a cagA-dependent manner. In human subjects, cag(+) strains induced significantly higher levels of Mcl-1 than cag(-) strains, and Mcl-1 expression levels paralleled the severity of neoplastic lesions. Collectively, these results indicate that H. pylori suppresses miR-320, upregulates Mcl-1, and decreases apoptosis in a cagA-dependent manner, which likely confers an increased risk for gastric carcinogenesis.