Search

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 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.

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).

SUMMARY 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 -a ndp53-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.

SummaryIdentifying 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.

SummaryWe 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.

Human embryonic stem cells, or hESCs, are pluripotent cells isolated from blastocryststage preimplantation embryos that have great potential in basic research, drug development and cell replacement therapy. However, advances in many facets of hESC research have to be attained before these goals can be reached. We describe here the development of two technologies that will aid in this regard: genetic manipulation and stem cell selection. DOCTOR OF PHILOSOPHY (SBS)

The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage lambda integrase, phage P1 Cre recombinase, and mutant gammadelta resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent lambda integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.

There is no targeted therapy for triple negative breast cancers (TNBC), which have the worst prognosis of human breast cancers. TNBCs are prone to relapse and metastasize after cytotoxic drug treatment. This group of cancers, defined by low or absent expression of estrogen, progesterone and Her2 receptors, are heterogeneous in genetic, epigenetic and phenotypic features, making it difficult to identify specific drug targets suitable for this group of cancers as a whole. About half or more of TNBCs have an epithelial phenotype (classified based on gene expression profiling as basal-like or basal-A) and a large minority of the remaining tumors are mesenchymal (classified as basal-B). To identify selective genetic dependencies of the basal-like subtype of TNBC that are not required for survival of normal breast epithelial cells, which might be good targets for targeted drug therapy, we first performed a genome-wide siRNA lethality screen that compared two human breast epithelial TNBC cell lines transformed with the same genes in different media - basal-like BPLER and myoepithelial HMLER. BPLER are highly malignant and enriched for tumor-initiating cells, forming tumors in nude mice after injection of just 50 cells, while the more differentiated HMLER cell line only forms tumors when more than 50,000 cells are injected. The screen identified 154 genes on which BPLER, but not HMLER, depended. Expression of the 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Of note, no oncogenic kinases, which are often chosen as cancer drug targets, were identified in the screen. Proteasome genes were overrepresented hits. Basal-like TNBC lines as a group were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal breast cancer and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiation and metastasis. Tumor-initiating cells (T-ICs) within basal-like cancers did not survive proteasome inhibition. Developing ways to target T-ICs is a priority of cancer drug development. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence, which was also a common dependency gene of basal-like TNBCs. Because of the heterogeneity of TNBCs, we next assessed how many of the BPLER dependencies were shared with other breast cancers. A targeted screen was performed comparing the viability of 10 breast cancer cell lines (3 luminal A, 5 basal-A and 2 basal-B TNBCs) and telomerase-immortalized normal breast epithelial cells after knockdown of the 154 BPLER dependency genes. This screen confirmed the selective dependence of basal-A TNBCs on the proteasome and MCL-1. This screen identified 17 additional hits that were shared dependencies in at least 4 of 5 basal-like TNBC cell lines. A novel hit, required for survival of 4 of 5 basal-A TNBC cell lines, but not normal breast epithelial cells, was the spindle checkpoint regulator NDC80. This work was supported by Department of Defense grants to FP and JL and by a grant from the Harvard Stem Cell Institute. The authors have no financial conflicts of interest to disclose. Citation Format: Fabio Petrocca, Gabriel Altschuler, Shen Mynn Tan, Marc L. Mendillo, Haoheng Yan, D. Joseph Jerry, Andrew L. Kung, Winston Hide, Tan A. Ince, Judy Lieberman. Screening for triple negative breast cancer vulnerabilities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY43-03. doi:10.1158/1538-7445.AM2014-SY43-03

Undifferentiated transcription factor 1 (UTF1) was identified first in mouse embryonic stem cells and is also expressed in human embryonic and adult stem cells. UTF1 transcription ceases at the onset of differentiation, which clearly distinguishes it from less sensitive pluripotency markers, such as Oct4 or Nanog. We present here two transgenic hESC lines, named ZUN. Each line harbors one copy of the UTF1 promoter/enhancer driving a resistance gene and yielded highly homogeneous cultures under selection pressure, with a larger proportion of Oct4 and Sox2 positive cells. While ZUN cultures, like parental HUES8 cultures, retained the capacity to differentiate into tissues of all three germ layers using a SICD mouse teratoma model, they surprisingly exhibited an increased refractoriness to various differentiation cues in vitro. Together with its small size of only 2.4 kb for the entire cassette, these features render our selection system a powerful novel tool for many stem cell applications and human somatic cell reprogramming strategies.

SummaryBasal-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.

SUMMARY 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.