RNA regulatory mechanisms controlling TGF-ß signaling and EMT in cancer.

Epithelial-mesenchymal transition (EMT) is a major contributor to metastatic progression and is prominently regulated by TGF-ß signalling. Both EMT and TGF-ß pathway components are tightly controlled by non-coding RNAs - including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) - that collectively have major impacts on gene expression and resulting cellular states. While miRNAs are the best characterised regulators of EMT and TGF-ß signaling and the miR-200-ZEB1/2 feedback loop plays a central role, important functions for lncRNAs and circRNAs are also now emerging. This review will summarise our current understanding of the roles of non-coding RNAs in EMT and TGF-ß signaling with a focus on their functions in cancer progression.

Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis.

Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. "Basal-like" breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10-15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

Quaking isoforms cooperate to promote the mesenchymal phenotype.

The RNA-binding protein Quaking (QKI) has widespread effects on mRNA regulation including alternative splicing, stability, translation, and localization of target mRNAs. Recently, QKI was found to be induced during epithelial-mesenchymal transition (EMT), where it promotes a mesenchymal alternative splicing signature that contributes to the mesenchymal phenotype. QKI is itself alternatively spliced to produce three major isoforms, QKI-5, QKI-6, and QKI-7. While QKI-5 is primarily localized to the nucleus where it controls mesenchymal splicing during EMT, the functions of the two predominantly cytoplasmic isoforms, QKI-6 and QKI-7, in this context remain uncharacterized. Here we used CRISPR-mediated depletion of QKI in a human mammary epithelial cell model of EMT and studied the effects of expressing the QKI isoforms in isolation and in combination. QKI-5 was required to induce mesenchymal morphology, while combined expression of QKI-5 with either QKI-6 or QKI-7 further enhanced mesenchymal morphology and cell migration. In addition, we found that QKI-6 and QKI-7 can partially localize to the nucleus and contribute to alternative splicing of QKI target genes. These findings indicate that the QKI isoforms function in a dynamic and cooperative manner to promote the mesenchymal phenotype.

The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-ß-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

Basonuclin-2 regulates extracellular matrix production and degradation.

Epithelial-mesenchymal transition is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the ECM-a complex environment of proteoglycans and fibrous proteins essential for tissue homeostasis, signaling in response to chemical and biomechanical stimuli, and is often dysregulated under conditions such as cancer, fibrosis, and chronic wounds. Here, we demonstrate that basonuclin-2 (BNC2), a mesenchymal-expressed gene, that is, strongly associated with cancer and developmental defects across genome-wide association studies, is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases, and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the production and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers, which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.

On the rules of engagement for microRNAs targeting protein coding regions.

MiRNAs post-transcriptionally repress gene expression by binding to mRNA 3'UTRs, but the extent to which they act through protein coding regions (CDS regions) is less well established. MiRNA interaction studies show a substantial proportion of binding occurs in CDS regions, however sequencing studies show much weaker effects on mRNA levels than from 3'UTR interactions, presumably due to competition from the translating ribosome. Consequently, most target prediction algorithms consider only 3'UTR interactions. However, the consequences of CDS interactions may have been underestimated, with the reporting of a novel mode of miRNA-CDS interaction requiring base pairing of the miRNA 3' end, but not the canonical seed site, leading to repression of translation with little effect on mRNA turnover. Using extensive reporter, western blotting and bioinformatic analyses, we confirm that miRNAs can indeed suppress genes through CDS-interaction in special circumstances. However, in contrast to that previously reported, we find repression requires extensive base-pairing, including of the canonical seed, but does not strictly require base pairing of the 3' miRNA terminus and is mediated through reducing mRNA levels. We conclude that suppression of endogenous genes can occur through miRNAs binding to CDS, but the requirement for extensive base-pairing likely limits the regulatory impacts to modest effects on a small subset of targets.

MicroRNA-21 is immunosuppressive and pro-metastatic via separate mechanisms.

MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.

The Quaking RNA-binding proteins as regulators of cell differentiation.

The RNA-binding protein Quaking (QKI) has emerged as a potent regulator of cellular differentiation in developmental and pathological processes. The QKI gene is itself alternatively spliced to produce three major isoforms, QKI-5, QKI-6, and QKI-7, that possess very distinct functions. Here, we highlight roles of the different QKI isoforms in neuronal, vascular, muscle, and monocyte cell differentiation, and during epithelial-mesenchymal transition in cancer progression. QKI isoforms control cell differentiation through regulating alternative splicing, mRNA stability and translation, with activities in gene transcription now also becoming evident. These diverse functions of the QKI isoforms contribute to their broad influences on RNA metabolism and cellular differentiation. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Development.

Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation.

BACKGROUND: Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC. METHODS: The clinical prognostic value of NRP1 was determined by Kaplan-Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting. RESULTS: High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors. CONCLUSIONS: These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

Selective Microfluidic Capture and Detection of Prostate Cancer Cells from Urine without Digital Rectal Examination.

Urine-based biomarkers have shown suitable diagnostic potential for prostate cancer (PCa) detection. Yet, until now, prostatic massage remains required prior to urine sampling. Here, we test a potential diagnostic approach using voided urine collected without prior digital rectal examination (DRE). In this study, we evaluated the diagnostic performance of a microfluidic-based platform that combines the principle of photodynamic diagnostic with immunocapture for the detection of PCa cells. The functionality and sensitivity of this platform were validated using both cultured cells and PCa patient urine samples. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) demonstrated this platform had a detection limit of fewer than 10 cells per 60 µL and successfully validated the presence of a PCa biomarker in the urine of cancer patients without prior DRE. This biosensing platform exhibits a sensitivity of 72.4% and a specificity of 71.4%, in suitable agreement with qRT-PCR data. The results of this study constitute a stepping stone in the future development of noninvasive prostate cancer diagnostic technologies that do not require DRE.

Post-transcriptional Gene Regulation by MicroRNA-194 Promotes Neuroendocrine Transdifferentiation in Prostate Cancer.

Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer samples, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signaling. miR-194 facilitated the emergence of neuroendocrine features in prostate cancer cells, a process mediated by its ability to directly target a suite of genes involved in cell plasticity. One such target was FOXA1, which encodes a transcription factor with a vital role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor blocked epithelial-neuroendocrine transdifferentiation and inhibited the growth of cell lines and patient-derived organoids possessing neuroendocrine features. Overall, our study reveals a post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in NEPC.

Insufficiently complex unique-molecular identifiers (UMIs) distort small RNA sequencing.

The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.

MicroRNA-143-3p targets pyruvate carboxylase expression and controls proliferation and migration of MDA-MB-231 cells.

Pyruvate carboxylase (PC) is a biotin-containing enzyme that converts pyruvate to oxaloacetate. We have previously shown that PC is overexpressed in highly invasive cancer cell lines where it supports biosynthesis during rapid cell growth. Here, we show that miR-143-3p suppresses the expression of PC in MDA-MB-231 cells by targeting its conserved binding site in the 3'-untranslated region (UTR) of human PC mRNA. Incorporation of the PC 3'UTR into a luciferase reporter gene inhibited expression of luciferase by 50% while mutation of the miR-143-3p binding site abrogated this inhibitory effect in MDA-MB-231 cells but not in low aggressive MCF-7 cell line. Transfection of miR-143-3p mimic or overexpression of miR-143-3p using tetracycline-inducible system in MDA-MB-231 cells down-regulated expression of both endogenous PC mRNA and protein by 40% and 50% respectively, confirming the regulatory role of miR-143-3p in PC expression. Induction of miR-143-3p expression at low and high levels lowered proliferation, metabolic activity and migration of MDA-MB-231 cells, in a dose-dependent manner. Re-expression of PC in MDA-MB-231 cells which were induced to express miR-143-3p partially restored migration but not proliferation, indicating that miR-143-3p regulates proliferation and migration through multiple pathways.

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-ß or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.

Combinatorial Targeting by MicroRNAs Co-ordinates Post-transcriptional Control of EMT.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, functioning in part by facilitating the degradation of target mRNAs. They have an established role in controlling epithelial-mesenchymal transition (EMT), a reversible phenotypic program underlying normal and pathological processes. Many studies demonstrate the role of individual miRNAs using overexpression at levels greatly exceeding physiological abundance. This can influence transcripts with relatively poor targeting and may in part explain why over 130 different miRNAs are directly implicated as EMT regulators. Analyzing a human mammary cell model of EMT we found evidence that a set of miRNAs, including the miR-200 and miR-182/183 family members, co-operate in post-transcriptional regulation, both reinforcing and buffering transcriptional output. Investigating this, we demonstrate that combinatorial treatment altered cellular phenotype with miRNA concentrations much closer to endogenous levels and with less off-target effects. This suggests that co-operative targeting by miRNAs is important for their physiological function and future work classifying miRNAs should consider such combinatorial effects.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Regulation of splicing and circularisation of RNA in epithelial mesenchymal plasticity.

Interconversions between epithelial and mesenchymal states, often referred to as epithelial mesenchymal transition (EMT) and its reverse MET, play important roles in embryonic development and are recapitulated in various adult pathologies including cancer progression. These conversions are regulated by complex transcriptional and post-transcriptional mechanisms including programs of alternative splicing which are orchestrated by specific splicing factors. This review will focus on the latest developments in our understanding of the splicing factors regulating epithelial mesenchymal plasticity associated with cancer progression and the induction of pluripotency, including potential roles for circular RNAs (circRNAs) which have been recently implicated in these processes.