Here, we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such ...RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein-coding mRNAs.
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► A microRNA target prediction algorithm predicts candidate PTEN ceRNAs ► Expression of PTEN ceRNAs significantly correlates with PTEN expression in vivo ► PTEN ceRNAs modulate PTEN and P-Akt levels in a microRNA-dependent manner ► PTEN ceRNAs display tumor-suppressive properties and are lost in human cancer
A large network of messenger RNAs regulates tumor-suppressive properties of PTEN by controlling the availability of microRNAs targeting PTEN.
We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ...ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAFV600E to promote melanomagenesis.
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► A Sleeping Beauty screen followed by MuTaME analysis discovered putative PTEN ceRNAs ► The PTEN ceRNA ZEB2 regulates PTEN in a miRNA-dependent manner ► ZEB2 loss activates PI3K/AKT signaling and promotes cell transformation ► Attenuated ZEB2 expression is found in melanoma and other human cancers
A transposon-based mutagenesis screen uncovers a striking enrichment of putative PTEN ceRNAs among mutated genes that accelerate tumorigenesis in a mouse model of melanoma. One candidate gene, ZEB2, regulates PI3K/AKT signaling and tumor-suppressive properties of PTEN.
Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines ...transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screen’s 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.
•Basal-like TNBC cell lines are addicted to the proteasome and MCL-1•Proteasome inhibition blocks T-IC functions in basal-like TNBCs•Proteasome addiction in these cells is mediated by NOXA and linked to MCL-1•Proteasome inhibitors inhibit basal-like tumor growth and metastasis in mice
Tumor suppressor genes (TSGs) are often concomitantly lost or mutated in human cancers and have been shown to act synergistically to promote tumorigenesis. In addition to genomic ...alterations, posttranscriptional regulation by microRNAs (miRNAs) represents another mechanism by which TSG expression is dysregulated in cancers. Although miRNAs that target critical TSGs such as PTEN or p53 have been identified, little is known about miRNAs that concomitantly regulate both these key TSGs. In this study, we characterize microRNA 518c∗ (miR-518c∗) and miR-638 as dual PTEN- and p53-targeting miRNAs that are upregulated in multiple human cancers. We focus on miR-638 and show that it associates independently with these two tumor suppressor transcripts as well as BRCA1, a known miR-638 target. We find that miR-638 overexpression promotes tumorigenesis and demonstrate cooperativity between miR-638 and its host gene Dnm2, suggesting that the Dnm2 locus encodes two distinct oncogenic components that play important roles in tumorigenesis.
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•MicroRNAs miR-518c∗ and miR-638 target multiple tumor suppressor genes•These microRNAs are upregulated in multiple human cancers•Their overexpression increases cell migration, invasion, and proliferation•miR-638 cooperates with its host gene Dnm2 to promote tumorigenesis
Tumor suppressor genes (TSGs) are often lost or mutated in human cancers and often act synergistically to promote tumorigenesis. In addition to genomic alterations, posttranscriptional regulation by microRNAs represents another mechanism by which TSG expression is dysregulated in cancers. In this study, Tay et al. characterize microRNAs that concomitantly regulate the key TSGs PTEN and p53. They focus on microRNA 638 (miR-638) and demonstrate that the Dnm2/miR-638 locus encodes two distinct components that play important roles in tumorigenesis.
Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical “seed” base-pairing rules, making ...identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.
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•Pull-down of biotin-miRNA-bound RNAs without crosslinking identifies miRNA targets•Sequencing of RNase-treated pulled-down miRNA targets identifies MREs•miR-522 MREs do not obey the canonical 3′ UTR seed-pairing rules•The oncomir miR-522 promotes detachment, invasion, and mesenchymal gene expression
Identifying miRNA-regulated genes is key to understanding miRNA function. Here, Tan et al. developed an unbiased method to identify with high specificity the target genes, miRNA recognition elements (MREs), and functions of primate-specific miR-522. Transcripts pulled down (without crosslinking) with a transfected biotinylated miRNA were sequenced to identify miRNA-regulated genes. Addition of RNase treatment identified MREs. Bioinformatics analysis of the transcription factors that regulate the genes, together with gene ontology analysis, provided a straightforward path toward uncovering miRNA function.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate the stability and expression of target RNAs in a sequence-dependent manner. Identifying miRNA-regulated genes is key to understanding miRNA ...function. Here, we describe an unbiased biochemical pulldown method to identify with high-specificity miRNA targets. Regulated transcripts are enriched in streptavidin-captured mRNAs that bind to a transfected biotinylated miRNA mimic. The method is relatively simple, does not involve cross-linking and can be performed with only a million cells. Addition of an on-bead RNase digestion step also identifies miRNA recognition elements (MRE).
As the projected incidence and mortality of cancer in Sub-Saharan Africa (SSA) rises to epidemic proportions, it is imperative that more is done to identify the genomic differences and commonalities ...between patients of African and European ancestry to fulfil the promise of precision oncology. Here, we summarize the utility of precision oncology approaches, with a focus on comprehensive genomic profiling (CGP) and consolidate examples of national and international consortia that are driving the field forward. We describe the importance of genomic diversity and its relevance in cancer, and propose recommendations, success factors and desired outcomes for precision oncology consortia to adopt in SSA. Through this, we hope to catalyze the initiation of such projects and to contribute to improving cancer patient outcomes in the region.
Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, ...metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.
Lin28 is an evolutionary conserved RNA-binding protein that regulates miRNAs and mRNAs; Lin28 overexpression is linked to poor prognosis in cancer.
Lin28 binds to guanine-rich miRNAs and mRNAs that contain G-quartet structural features and remodels these structures.
Lin28 recognition of G-quartets is a unifying feature of Lin28-regulated RNAs.
Small molecules that bind to G-quartets might be useful inhibitors of Lin28 function.
LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of ...translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.
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