Publications by authors named "Remco Nagel"

20 Publications

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A comprehensive enhancer screen identifies TRAM2 as a key and novel mediator of YAP oncogenesis.

Genome Biol 2021 Jan 29;22(1):54. Epub 2021 Jan 29.

Division of Oncogenomics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.

Background: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions.

Results: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on Enhancer, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness.

Conclusions: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.
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http://dx.doi.org/10.1186/s13059-021-02272-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845134PMC
January 2021

Anti-tumour immunity induces aberrant peptide presentation in melanoma.

Nature 2021 02 16;590(7845):332-337. Epub 2020 Dec 16.

Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.
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http://dx.doi.org/10.1038/s41586-020-03054-1DOI Listing
February 2021

Proteomic patterns associated with response to breast cancer neoadjuvant treatment.

Mol Syst Biol 2020 09;16(9):e9443

Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.
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http://dx.doi.org/10.15252/msb.20209443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507992PMC
September 2020

SLC1A3 contributes to L-asparaginase resistance in solid tumors.

EMBO J 2019 10 16;38(21):e102147. Epub 2019 Sep 16.

Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.
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http://dx.doi.org/10.15252/embj.2019102147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826201PMC
October 2019

Inhibition of the Replication Stress Response Is a Synthetic Vulnerability in SCLC That Acts Synergistically in Combination with Cisplatin.

Mol Cancer Ther 2019 04 14;18(4):762-770. Epub 2019 Mar 14.

Oncode Institute, Amsterdam, the Netherlands.

Small cell lung cancer (SCLC) is generally regarded as very difficult to treat, mostly due to the development of metastases early in the disease and a quick relapse with resistant disease. SCLC patients initially show a good response to treatment with the DNA damaging agents cisplatin and etoposide. This is, however, quickly followed by the development of resistant disease, which urges the development of novel therapies for this type of cancer. In this study, we set out to compile a comprehensive overview of the vulnerabilities of SCLC. A functional genome-wide screen where all individual genes were knocked out was performed to identify novel vulnerabilities of SCLC. By analysis of the knockouts that were lethal to these cancer cells, we identified several processes to be synthetic vulnerabilities in SCLC. We were able to validate the vulnerability to inhibition of the replication stress response machinery by use of Chk1 and ATR inhibitors. Strikingly, SCLC cells were more sensitive to these inhibitors than nontransformed cells. In addition, these inhibitors work synergistically with either etoposide and cisplatin, where the interaction is largest with the latter. ATR inhibition by VE-822 treatment in combination with cisplatin also outperforms the combination of cisplatin with etoposide Altogether, our study uncovered a critical dependence of SCLC on the replication stress response and urges the validation of ATR inhibitors in combination with cisplatin in a clinical setting.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451635PMC
April 2019

Proteome analysis of non-small cell lung cancer cell line secretomes and patient sputum reveals biofluid biomarker candidates for cisplatin response prediction.

J Proteomics 2019 03 30;196:106-119. Epub 2019 Jan 30.

Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Medical Oncology, Cancer Center Amsterdam, 1081 HV Amsterdam, the Netherlands. Electronic address:

Molecular markers are urgently needed to select non-small cell lung cancer (NSCLC) patients most likely to benefit from platinum-based chemotherapies. Of particular interest are proteins that can be found in biofluids like sputum for non-invasive detection. Therefore, we profiled the secretomes of 6 NSCLC cell lines with varying IC50-values for cisplatin, using label-free GeLC-MS/MS-based proteomics. Out of a total dataset of 2610 proteins, 304 proteins showed significant differences in expression levels between cisplatin sensitive and insensitive cell lines. Functional data mining revealed that the secretion of typically extracellular factors was associated with a higher sensitivity towards cisplatin, while cisplatin insensitivity correlated with increased secretion of theoretically intra-cellular proteins. Stringent statistical analysis and quantitative filtering yielded 58 biomarker candidates, 34 of which could be detected in clinical biofluids of lung cancer patients such as sputum using label-free LC-MS/MS-based proteomics. To assess performance of these biofluid biomarker candidates, we correlated protein expression with patient survival using a publically available clinical gene expression data set (GSE14814). We thus identified 3 top candidates with potential predictive value in determining cisplatin response (UGGT1, COL6A1 and MAP4) for future development as non-invasive biomarkers to guide treatment decisions. SIGNIFICANCE: Platinum-based chemotherapies are still the standard of care for NSCLC and other lung cancer types in the clinic today. However, due to chemoresistance, many patients suffer from the toxic side effects of these treatments without gaining any benefit in terms of survival. To date, no molecular biomarkers are available to predict clinical outcome of platinum-based chemotherapy. Because proteins present the functional read-out of genetic, epigenetic and translational events in the cell, a protein test is likely to be particularly suitable for response prediction. Of high relevance are proteins that are shed or secreted from cells, for example at primary tumor sites, and can be found in easily accessible biofluids like sputum for non-invasive detection. Here, we report the proteome profiling of the conditioned media (secretomes) of a panel of NSCLC cell lines in relation to cisplatin IC50 values, as a pre-clinical model, and of patient sputum as a clinical, lung cancer relevant biofluid. Using this approach in conjunction with exploration of the predictive potential in a transcriptome lung cancer patient dataset, we reveal biofluid biomarker candidates that, with further validation, may be used for non-invasive cisplatin response prediction in the future.
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http://dx.doi.org/10.1016/j.jprot.2019.01.018DOI Listing
March 2019

CUEDC1 is a primary target of ERα essential for the growth of breast cancer cells.

Cancer Lett 2018 11 23;436:87-95. Epub 2018 Aug 23.

Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; Department of Molecular Genetics, Erasmus University Medical Center, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands. Electronic address:

Breast cancer is the most prevalent type of malignancy in women with ∼1.7 million new cases diagnosed annually, of which the majority express ERα (ESR1), a ligand-dependent transcription factor. Genome-wide chromatin binding maps suggest that ERα may control the expression of thousands of genes, posing a great challenge in identifying functional targets. Recently, we developed a CRISPR-Cas9 functional genetic screening approach to identify enhancers required for ERα-positive breast cancer cell proliferation. We validated several candidates, including CUTE, a putative ERα-responsive enhancer located in the first intron of CUEDC1 (CUE-domain containing protein). Here, we show that CUTE controls CUEDC1 expression, and that this interaction is essential for ERα-mediated cell proliferation. Moreover, ectopic expression of CUEDC1, but not a CUE-domain mutant, rescues the defects in CUTE activity. Finally, CUEDC1 expression correlates positively with ERα in breast cancer. Thus, CUEDC1 is a functional target gene of ERα and is required for breast cancer cell proliferation.
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http://dx.doi.org/10.1016/j.canlet.2018.08.018DOI Listing
November 2018

Drugging the addict: non-oncogene addiction as a target for cancer therapy.

EMBO Rep 2016 11 4;17(11):1516-1531. Epub 2016 Oct 4.

Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Historically, cancers have been treated with chemotherapeutics aimed to have profound effects on tumor cells with only limited effects on normal tissue. This approach was followed by the development of small-molecule inhibitors that can target oncogenic pathways critical for the survival of tumor cells. The clinical targeting of these so-called oncogene addictions, however, is in many instances hampered by the outgrowth of resistant clones. More recently, the proper functioning of non-mutated genes has been shown to enhance the survival of many cancers, a phenomenon called non-oncogene addiction. In the current review, we will focus on the distinct non-oncogenic addictions found in cancer cells, including synthetic lethal interactions, the underlying stress phenotypes, and arising therapeutic opportunities.
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http://dx.doi.org/10.15252/embr.201643030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090709PMC
November 2016

Origins, genetic landscape, and emerging therapies of small cell lung cancer.

Genes Dev 2015 Jul;29(14):1447-62

Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

Lung cancer is the leading cause of cancer deaths, with small cell lung cancer (SCLC) representing the most aggressive subtype. Standard treatments have not changed in decades, and the 5-year survival rate has remained <7%. Genomic analyses have identified key driver mutations of SCLC that were subsequently validated in animal models of SCLC. To provide better treatment options, a deeper understanding of the cellular and molecular mechanisms underlying SCLC initiation, progression, metastasis, and acquisition of resistance is required. In this review, we describe the genetic landscape of SCLC, features of the cell of origin, and targeted therapeutic approaches.
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http://dx.doi.org/10.1101/gad.263145.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526731PMC
July 2015

Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC.

Mol Cancer Ther 2015 Jun 25;14(6):1434-44. Epub 2015 Mar 25.

Department of Otolaryngology-Head and Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands.

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non-small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle-related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2-M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0846DOI Listing
June 2015

Treatment response of HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines.

Oral Oncol 2013 Jun 8;49(6):560-6. Epub 2013 Apr 8.

Department of Otolaryngology/Head-Neck Surgery, VU Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands.

Objectives: Infection with the human papillomavirus (HPV) is an important risk factor for development of head and neck squamous cell carcinoma (HNSCC). Strikingly, HPV-positive HNSCCs have a more favorable prognosis than their HPV-negative counterparts. The current study was designed to explain this favorable prognosis of HPV-positive HNSCC.

Materials And Methods: This was performed by investigating the response of four HPV-positive and fourteen HPV-negative HNSCC cell lines to cisplatin, cetuximab and radiation.

Results: Analysis of the responses of this cell line panel indicated that HPV-positive cells are more resistant to cisplatin treatment than the HPV-negative HNSCCs, whereas the response to radiation and cetuximab did not differ.

Conclusions: The current study suggests that the favorable prognosis for patients with HPV-positive HNSCC does not seem to be related to an intrinsic sensitivity of these tumor cells to chemotherapy or radiation in vitro.
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http://dx.doi.org/10.1016/j.oraloncology.2013.03.446DOI Listing
June 2013

Functional genetic screens identify genes essential for tumor cell survival in head and neck and lung cancer.

Clin Cancer Res 2013 Apr 26;19(8):1994-2003. Epub 2013 Feb 26.

AuDepartment of Otolaryngology/Head-Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands.

Purpose: Despite continuous improvement of treatment regimes, the mortality rates for non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) remain disappointingly high and novel anticancer agents are urgently awaited.

Experimental Design: We combined the data from genome-wide siRNA screens on tumor cell lethality in a lung and a head and neck cancer cell line.

Results: We identified 71 target genes that seem essential for the survival of both cancer types. We identified a cluster of 20 genes that play an important role during G2-M phase transition, underlining the importance of this cell-cycle checkpoint for tumor cell survival. Five genes from this cluster (CKAP5, KPNB1, RAN, TPX2, and KIF11) were evaluated in more detail and have been shown to be essential for tumor cell survival in both tumor types, but most particularly in HNSCC. Phenotypes that were observed following siRNA-mediated knockdown of KIF11 (kinesin family member 11) were reproduced by inhibition of KIF11 using the small-molecule inhibitor ispinesib (SB-715992). We showed that ispinesib induces a G2 arrest, causes aberrant chromosome segregation, and induces cell death in HNSCC in vitro, whereas primary keratinocytes are less sensitive. Furthermore, growth of HNSCC cells engrafted in immunodeficient mice was significantly inhibited after ispinesib treatment.

Conclusion: This study identified a wide array of druggable genes for both lung and head and neck cancer. In particular, multiple genes involved in the G2-M checkpoint were shown to be essential for tumor cell survival, indicating their potential as anticancer targets.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-2539DOI Listing
April 2013

The miRNA-192/194 cluster regulates the Period gene family and the circadian clock.

FEBS J 2009 Oct 13;276(19):5447-55. Epub 2009 Aug 13.

Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Several biological functions in mammals are regulated in a circadian fashion. The molecular mechanisms orchestrating these circadian rhythms have been unravelled. The biological clock, with its core transcriptional unit Bmal1/CLOCK, is composed of several self-sustaining feedback loops. In this study, we describe another mechanism impinging on the core components of the circadian clock. Using a forward genetic screen, we identified the miR-192/194 cluster as a potent inhibitor of the entire Period gene family. In accordance, the exogenous expression of miR-192/194 leads to an altered circadian rhythm. Thus, our results have uncovered a new mechanism for the control of the circadian clock at the post-transcriptional level.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07229.xDOI Listing
October 2009

Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer.

Cancer Res 2008 Jul;68(14):5795-802

The Netherlands Cancer Institute, Division of Tumor Biology, Amsterdam, the Netherlands.

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost beta-catenin binding sites. APC-free beta-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3' untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-0951DOI Listing
July 2008

The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis.

Nat Cell Biol 2008 Feb 13;10(2):202-10. Epub 2008 Jan 13.

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently and still remains largely unexplored. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.
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http://dx.doi.org/10.1038/ncb1681DOI Listing
February 2008

RNA-binding protein Dnd1 inhibits microRNA access to target mRNA.

Cell 2007 Dec;131(7):1273-86

The Netherlands Cancer Institute, Division of Tumor Biology, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.

MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.
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http://dx.doi.org/10.1016/j.cell.2007.11.034DOI Listing
December 2007

Diverse ways to control p27Kip1 function: miRNAs come into play.

Cell Cycle 2007 Nov 16;6(22):2742-9. Epub 2007 Aug 16.

Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

MicroRNAs (miRNAs) are small RNA molecules controlling messenger RNA (mRNA) and protein abundance. Since their discovery, research has been aimed at identifying the functions and target genes of miRNAs. A number of computer algorithms have been developed capable of predicting putative targets for any given miRNA. However, they might predict many false-positive targets and on top of that some true targets could be missed. This reflects the incomplete knowledge we still have concerning the rules governing true and effective miRNA-mRNA interactions. To experimentally identify miRNA-target genes, we have recently developed a genetic approach and employed it on p27(Kip1), a hapo-insufficient tumor suppressor and cell cycle inhibitor. Here we review the difficulties interpreting the data from available computer algorithms, and critically address the pros and cons of our genetic screening method. Additionally, we focus on the different ways in which p27 is managed, argue how miRNAs could be involved in the regulation of p27 in both normal and malignant conditions, and discuss possible use of this knowledge for cancer therapy.
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http://dx.doi.org/10.4161/cc.6.22.4900DOI Listing
November 2007

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Adv Exp Med Biol 2007 ;604:17-46

Division of Tumour Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.
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http://dx.doi.org/10.1007/978-0-387-69116-9_2DOI Listing
September 2007

Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation.

EMBO J 2007 Aug 12;26(15):3699-708. Epub 2007 Jul 12.

Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27(Kip1), a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27(Kip1) levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27(Kip1) protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27(Kip1).
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http://dx.doi.org/10.1038/sj.emboj.7601790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949005PMC
August 2007

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Cell 2006 Mar;124(6):1169-81

Division of Tumour Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.
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http://dx.doi.org/10.1016/j.cell.2006.02.037DOI Listing
March 2006