Your browser doesn't support javascript.
loading
: 20 | 50 | 100
1 - 20 de 32
1.
Nat Commun ; 15(1): 2287, 2024 Mar 13.
Article En | MEDLINE | ID: mdl-38480701

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.


Breast Neoplasms , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , RNA Polymerase II , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Drug Resistance, Neoplasm/genetics , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism
2.
Cell Rep ; 42(10): 113280, 2023 10 31.
Article En | MEDLINE | ID: mdl-37851577

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.


DNA-Binding Proteins , Protein Biosynthesis , Ribosomal Proteins , Ribosome Subunits, Small, Eukaryotic , Transcription Factors , Cell Nucleolus/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosomes/metabolism , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Humans , Animals , Rats , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Res Sq ; 2023 Jul 10.
Article En | MEDLINE | ID: mdl-37502925

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer acquired resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Using a genome-wide CRISPR screen, we identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout (RBKO) breast cancer cells. PRMT5 inhibition blocked cell cycle G1-to-S transition independent of RB, thus arresting growth of RBKO cells. Proteomics analysis uncovered fused in sarcoma (FUS) as a downstream effector of PRMT5. Pharmacological inhibition of PRMT5 resulted in dissociation of FUS from RNA polymerase II (Pol II), Ser2 Pol II hyperphosphorylation, and intron retention in genes that promote DNA synthesis. Treatment with the PRMT5i inhibitor pemrametostat and fulvestrant synergistically inhibited growth of ER+/RB-deficient patient-derived xenografts, suggesting dual ER and PRMT5 blockade as a novel therapeutic strategy to treat ER+/RB-deficient breast cancer.

4.
Cell Rep ; 42(2): 112076, 2023 02 28.
Article En | MEDLINE | ID: mdl-36753415

During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.


COVID-19 , Frameshifting, Ribosomal , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , COVID-19/metabolism , Codon, Terminator/genetics , Codon, Terminator/metabolism , Pandemics , Virus Replication/genetics , Ribosomes/metabolism , RNA, Viral/metabolism
5.
Genes Dev ; 36(9-10): 550-565, 2022 05 01.
Article En | MEDLINE | ID: mdl-35589130

Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX-NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.


Nucleocytoplasmic Transport Proteins , RNA, Viral , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA Transport , RNA, Viral/metabolism
7.
Cell Rep ; 29(10): 3134-3146.e6, 2019 12 03.
Article En | MEDLINE | ID: mdl-31801078

Sequences within 5' UTRs dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhances upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript, where this results in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced use of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells but also to maintain the fidelity of translation initiation.


Codon, Initiator/genetics , Peptide Initiation Factors/genetics , RNA-Binding Proteins/genetics , Ribosomes/metabolism , 5' Untranslated Regions/genetics , Cell Line , Cell Line, Tumor , HCT116 Cells , HEK293 Cells , Humans , Peptide Chain Elongation, Translational/genetics , Protein Biosynthesis/genetics , Proto-Oncogene Proteins c-myc/genetics , Eukaryotic Translation Initiation Factor 5A
8.
Elife ; 82019 07 25.
Article En | MEDLINE | ID: mdl-31343408

NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice (Kopp et al., 2019). Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance.


Genomic Instability , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Cell Line , Humans , Protein Binding , Protein Interaction Maps
9.
Nature ; 570(7761): E51, 2019 Jun.
Article En | MEDLINE | ID: mdl-31127195

Change history: In this Letter, the citation to 'Fig. 4e, f' in the main text should be 'Fig. 3e, f'. This has not been corrected online.

10.
Dev Cell ; 49(4): 618-631.e5, 2019 05 20.
Article En | MEDLINE | ID: mdl-30982661

MicroRNAs (miRNAs) are processed from primary miRNA transcripts (pri-miRNAs), many of which are annotated as long noncoding RNAs (lncRNAs). We assessed whether MIR205HG, the host gene for miR-205, has independent functions as an lncRNA. Comparing mice with targeted deletions of MIR205HG and miR-205 revealed a functional role for the lncRNA in the anterior pituitary. Mice lacking MIR205HG had a temporal reduction in Pit1, growth hormone, and prolactin. This was mediated, in part, through the ability of this lncRNA to bind and regulate the transcriptional activity of Pit1 in conjunction with Zbtb20. Knockdown of MIR205HG in lactotropes decreased the expression of Pit1, Zbtb20, prolactin, and growth hormone, while its overexpression enhanced the levels of these transcripts. The effects of MIR205HG on the pituitary were independent of miR-205. The data support a role for MIR205HG as an lncRNA that regulates growth hormone and prolactin production in the anterior pituitary.


Growth Hormone/biosynthesis , MicroRNAs/metabolism , Pituitary Gland, Anterior/metabolism , Prolactin/biosynthesis , RNA, Long Noncoding/metabolism , Animals , Growth Hormone/genetics , Growth Hormone/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Prolactin/genetics , Prolactin/metabolism , RNA, Long Noncoding/genetics , Rats , Transcription Factor Pit-1/genetics , Transcription Factor Pit-1/metabolism , Transcriptome
11.
Methods Mol Biol ; 1823: 1-9, 2018.
Article En | MEDLINE | ID: mdl-29959669

Proper control of microRNA (miRNA) expression is critical for normal development and physiology, while abnormal miRNA expression is a common feature of many diseases. Dissecting mechanisms of miRNA regulation, however, is complicated by the generally poor annotation of miRNA primary transcripts (pri-miRNAs). Although some miRNAs are processed from well-defined protein coding genes, the majority of pri-miRNAs are poorly characterized noncoding RNAs, with incomplete annotation of promoters, splice sites, and polyadenylation signals. Due to the efficiency of DROSHA processing, the abundance of pri-miRNAs is very low at steady state, thereby complicating the elucidation of pri-miRNA structures. Here we describe a strategy to enrich intact pri-miRNAs and improve their coverage in RNA sequencing (RNA-seq) experiments. In addition, we outline a computational approach for reconstruction of pri-miRNA structures. This pipeline begins with raw RNA-seq reads and concludes with publication-ready visualization of pri-miRNA annotations. Together, these approaches allow the user to define and explore miRNA gene structures in a cell-type or organism of interest.


Gene Expression Regulation , MicroRNAs , Sequence Analysis, RNA/methods , HEK293 Cells , Humans , MicroRNAs/biosynthesis , MicroRNAs/genetics , Molecular Sequence Annotation/methods
12.
Nature ; 542(7640): 197-202, 2017 02 09.
Article En | MEDLINE | ID: mdl-28114302

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.


Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Gene Silencing , MicroRNAs/genetics , Amino Acid Sequence , Argonaute Proteins/chemistry , CRISPR-Cas Systems/genetics , Casein Kinase II/metabolism , HCT116 Cells , Humans , MicroRNAs/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphorylation/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Substrate Specificity
14.
Cell ; 164(1-2): 69-80, 2016 Jan 14.
Article En | MEDLINE | ID: mdl-26724866

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.


Genomic Instability , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , Animals , Base Sequence , Chromosomal Instability , HCT116 Cells , Humans , Mice , Ploidies , RNA, Long Noncoding/chemistry , RNA, Long Noncoding/genetics
15.
Genome Res ; 25(9): 1401-9, 2015 Sep.
Article En | MEDLINE | ID: mdl-26290535

Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.


Gene Expression Regulation , Genome-Wide Association Study , MicroRNAs/chemistry , MicroRNAs/genetics , Molecular Sequence Annotation , Nucleic Acid Conformation , Alternative Splicing , Evolution, Molecular , Genome-Wide Association Study/methods , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/metabolism , Multigene Family , Open Reading Frames , Promoter Regions, Genetic , RNA, Untranslated , Ribonuclease III/metabolism , Sequence Analysis, RNA , Transcription, Genetic
16.
Nat Biotechnol ; 33(3): 290-5, 2015 Mar.
Article En | MEDLINE | ID: mdl-25690850

Methods used to sequence the transcriptome often produce more than 200 million short sequences. We introduce StringTie, a computational method that applies a network flow algorithm originally developed in optimization theory, together with optional de novo assembly, to assemble these complex data sets into transcripts. When used to analyze both simulated and real data sets, StringTie produces more complete and accurate reconstructions of genes and better estimates of expression levels, compared with other leading transcript assembly programs including Cufflinks, IsoLasso, Scripture and Traph. For example, on 90 million reads from human blood, StringTie correctly assembled 10,990 transcripts, whereas the next best assembly was of 7,187 transcripts by Cufflinks, which is a 53% increase in transcripts assembled. On a simulated data set, StringTie correctly assembled 7,559 transcripts, which is 20% more than the 6,310 assembled by Cufflinks. As well as producing a more complete transcriptome assembly, StringTie runs faster on all data sets tested to date compared with other assembly software, including Cufflinks.


Sequence Analysis, RNA/methods , Software , Transcriptome/genetics , Algorithms , HEK293 Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism
17.
Nat Commun ; 2: 4802, 2014 Sep 05.
Article En | MEDLINE | ID: mdl-25190313

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.


DEAD-box RNA Helicases/genetics , Kidney Neoplasms/genetics , MicroRNAs/metabolism , Ribonuclease III/genetics , Wilms Tumor/genetics , Child , Child, Preschool , Cohort Studies , Female , HEK293 Cells , Humans , Infant , Male , Mutation, Missense , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA
18.
Nature ; 512(7515): 431-5, 2014 Aug 28.
Article En | MEDLINE | ID: mdl-25043055

Bone-resorbing osteoclasts significantly contribute to osteoporosis and bone metastases of cancer. MicroRNAs play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how microRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is downregulated during osteoclast differentiation. Osteoclastic miR-34a-overexpressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify transforming growth factor-ß-induced factor 2 (Tgif2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.


Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Cell Differentiation/genetics , MicroRNAs/genetics , Osteoclasts/pathology , Osteoporosis/prevention & control , Repressor Proteins/deficiency , Animals , Base Sequence , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Resorption/drug therapy , Bone Resorption/genetics , Cell Differentiation/drug effects , Cell Line, Tumor , Disease Models, Animal , Female , Gene Deletion , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Male , Mammary Neoplasms, Animal/pathology , Mice , Mice, Transgenic , MicroRNAs/pharmacology , MicroRNAs/therapeutic use , Neoplasm Transplantation , Organ Size/drug effects , Osteoclasts/drug effects , Osteoporosis/genetics , Osteoporosis/pathology , Ovariectomy , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Repressor Proteins/metabolism , Skin Neoplasms/pathology , Transgenes , Xenograft Model Antitumor Assays
19.
Cell ; 137(6): 1005-17, 2009 Jun 12.
Article En | MEDLINE | ID: mdl-19524505

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.


Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Liver Neoplasms/genetics , Liver Neoplasms/therapy , MicroRNAs/therapeutic use , Animals , Cyclin D2 , Cyclins/metabolism , Dependovirus/genetics , Disease Models, Animal , Genetic Vectors , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Proto-Oncogene Proteins c-myc/genetics
20.
Proc Natl Acad Sci U S A ; 106(9): 3384-9, 2009 Mar 03.
Article En | MEDLINE | ID: mdl-19211792

Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.


Down-Regulation/genetics , MicroRNAs/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcriptional Activation/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-myc/genetics
...