The E2F1-miRNA cancer progression network Knoll, Susanne; Emmrich, Stephan; Pützer, Brigitte M
Advances in experimental medicine and biology,
01/2013, Letnik:
774
Journal Article
Recenzirano
The transcription factor E2F1 exhibits dual properties, acting as a tumor suppressor and oncogene. Cellular stress such as DNA damage or mitogenic signaling leads to the activation of E2F1 as a ...mediator of apoptosis in the context of a conserved cellular anti-tumorigenic safeguard mechanism. However in highly aggressive chemoresistant tumors like malignant melanoma and prostate/bladder cancer it switches off this role and acts as promoter of cancer progression. Possible reasons for E2F1 mediated aggressiveness are defects in cell death pathways caused by epigenetic inactivation of important tumor suppressor genes, which often occur in late stage cancer and contribute to chemoresistance. Nevertheless exact mechanisms underlying E2Fs role in invasiveness and metastasis are largely unknown. Different reports hint towards the existence of feedback loops between E2F1 and microRNAs (miRNAs or miRs). MiRs are activated by E2F1 and either the transcription factor itself or cellular genes necessary for the growth regulating function of E2F1 are inhibited by different miRNAs. This mutual regulation possibly influences the balance between E2F1s proapoptotic versus prosurvival function. In the following we will summarize some miRNA-E2F1-interactions contributing to a complex regulatory network.
E2F1 in Melanoma Progression and Metastasis Alla, Vijay; Engelmann, David; Niemetz, Annett ...
JNCI : Journal of the National Cancer Institute,
01/2010, Letnik:
102, Številka:
2
Journal Article
Recenzirano
Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors ...that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n = 3 mice per group; 5% vs 46%, difference = 41%, 95% confidence interval = 15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1–epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers.
Transient leukemia (TL) is evident in 5-10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate ...abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34(+)-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1(Δe2) knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
Long non-coding RNAs (lncRNAs) and miRNAs have emerged as crucial regulators of gene expression, epigenetics and cell fate decisions. Here we present an integrated quantitative and functional ...analysis of the miRNA-, lncRNA- and mRNA-transcriptome of purified human hematopoietic stem cells (HSCs) and their differentiated progenies, including granulocytes, monocytes, T-cells, NK-cells, B-cells, megakaryocytes and erythroid precursors, which we correlated with the ncRNA expression profile of 46 pediatric AML samples to establish a core lncRNA stem cell signature in AML.
For each blood cell population, RNA from 5 healthy donors was hybridized onto three microarray platforms (Arraystar lncRNA V2.0, NCode™-miRNA/-ncRNA), yielding a coverage of more than 40,000 lncRNAs, 25,000 mRNAs and 900 miRNAs on 146 arrays. Compared to mRNAs, the mean expression level of lncRNAs was nearly 2-fold lower (p<2.2x10-16), highlighting the challenge for RNA-Seq to provide adequate coverage of these rare transcripts. Hierarchical clustering, PCA (principle components analysis) and t-SNE (t-distributed stochastic neighbor embedding) on lncRNA and miRNA genes robustly structured the dataset into groups of samples that matched the input populations, demonstrating their unique ncRNA expression profiles. Using LIMMA (linear models for microarrays) and PAM (prediction analysis of microarrays) we extracted a robust lncRNA/miRNA core signature for each population. Self-organizing maps (SOM) structured the gene space into spot clusters of lncRNAs and mRNAs that were coordinately expressed during maturation from HSCs into the different lineages. Using a guilt-by-association approach, we assigned potential functionality to lncRNAs through gene set enrichment analysis of co-expressed mRNAs (validated for a set of lncRNAs by qRT-PCR). To demonstrate their functionality, we knocked down two lncRNAs (LINC00173 and DY635655) from the granulocytic core signature using two independent shRNA constructs, which resulted in diminished granulocytic in vitro differentiation (2-fold reduction in percentage of CD66b+/CD13+ granulocytes, p≤0.05), myeloid colony-formation (1.5-2-fold, p≤0.05) and nuclear lobulation (MGG-staining). Accordingly, CRISPR-mediated transcriptional repression of nuclear localized LINC00173 and cytoplasmic localized DY635655 (RNA-FISH validated by qRT-RNA of fractionated RNA) using dCas9-KRAB and three sgRNAs per locus reduced proliferation of myeloid NB4 cells (2-3-fold, p≤0.01).
By plotting ncRNA expression over the genomic coordinates of all chromosomes, we uncovered a strong and highly coordinated upregulation of miRNAs, small nucleolar RNAs (snoRNAs) and lncRNAs (MEG3, MEG8 and MEG9) within the DLK1-DIO3 locus on chromosome 14 (hsa14) specifically in megakaryocytes (average log2-FC >5, p<10-8; confirmed by qRT-PCR). shRNA-mediated knock-down of MEG3 reduced erythroid colony-formation and megakaryocytic cell proliferation in vitro (p≤0.05), implicating the functional importance of this ncRNA locus.
Having established a global human hematopoietic lncRNA expression resource, we extended our findings to malignant hematopoiesis. Linear (PCA) and nonlinear (t-SNE) dimensionality reduction of 46 pediatric AML samples including Down syndrome AMKL, core-binding factor AMLs (inv16 or t8;21) and MLL-rearranged leukemias mapped most samples to a space between HSCs and differentiated cells together with the myeloid progenitors. A subset of AML-samples mapped closely to healthy HSCs, including most of the DS-AMKLs and MLL-AMLs. We identified a stem-cell associated lncRNA signature that was absent in healthy differentiated progenies, but upregulated in AML samples. Interestingly, AML samples that did not show upregulation of this set of lncRNAs suppressed the upregulation of differentiation-associated lncRNAs, thereby keeping the global identity of myeloid progenitors as shown by t-SNE.
Thus, our study provides a comprehensive resource for the exploration of the mRNA-, lncRNA- and miRNA-transcriptome across the human hematopoietic hierarchy, including malignant hematopoiesis. The definition of a core lncRNA stem cell signature in normal HSCs and AML blasts will guide our way towards an improved understanding of self-renewal and the underlying transcriptional program, which is hijacked during malignant transformation.
No relevant conflicts of interest to declare.
iPhones, Emotions, Mediations Manja Stephan-Emmrich
Mobilities, Boundaries, and Travelling Ideas,
04/2018
Book Chapter
Odprti dostop
Mona, a thirty-year-old Tajik woman whom I met during my field trip to the United Arab Emirates in November and December 2013, lives with her family in a comfortable apartment complex in the emirate ...of Sharjah. Ma’ruf, her husband, is a graduate of Sana’a University in Yemen, where he studied Koran exegesis (tafsir) and Islamic law (fiq) from 1999 until 2006. In 2001, he married Mona and encouraged her to join him in his religious studies. Five years later and after the birth of their first son, they returned to Tajikistan and Ma’ruf started a journalism course at the National
HOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive ...research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA– and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)–mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.
•The lncRNA HOXA10-AS is highly expressed in KMT2A-rearranged AMLs and exerts its oncogenic effects by inducing NF-κB target genes.•Knockdown of HOXA10-AS reduces the leukemic growth of KMT2A-rearranged AML blasts in vivo.
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The process of megakaryopoiesis culminates in the release of platelets, the pivotal cellular component for hemostasis and wound healing. The regulatory architecture including the modulatory role of ...microRNAs, which underlies megakaryocytic maturation and platelet formation, is incompletely understood, precluding the ex vivo generation of sufficient platelet numbers for transfusion medicine. We derived a highly efficient differentiation protocol to produce mature polyploid megakaryocytes and functional platelets from CD34
+
-hematopoietic stem and progenitor cells by comparing previously published approaches. Our megakaryocytic culture conditions using the cytokines SCF, TPO, IL-9, and IL-6 include nicotinamide and Rho-associated kinase (ROCK) inhibitor Y27632 as contextual additives. The potency of our novel megakaryocytic differentiation protocol was validated using cord blood and peripheral blood human hematopoietic stem and progenitor cells. Using this novel megakaryocytic differentiation protocol, we characterized the modulatory capacity of several miRNAs highly expressed in normal megakaryocytic cells or malignant blasts from patients with megakaryoblastic leukemia. Overexpression of candidate microRNAs was achieved by lentiviral transduction of CD34
+
-hematopoietic stem and progenitor cells prior to differentiation. We revealed
miR-125b
and
miR-660
as enhancers of polyploidization, as well as platelet output of megakaryocytes. The oncogene
miR-125b
markedly expanded the number of megakaryocytes during in vitro culture. Conversely, the
miR-23a/27a/24-2
cluster, which is highly expressed in normal megakaryocytes, blocked maturation and platelet formation. Our study on the utilization of microRNAs in conjunction with a highly efficient differentiation protocol constitutes another step towards ex vivo platelet manufacturing on a clinically relevant scale.
The microRNA (miRNA) family miR-193 consists of two members, miR-193a and miR-193b, which share identical seed regions and are therefore thought to be functionally redundant. However, the target ...spectrum of a miRNA is defined by the mRNA spectrum of the tissue, where it is expressed as well as dynamic strand preferences (miRNA-5p vs miRNA-3p) (Kuchenbauer et al., Blood, 2011). We and others previously showed that miR-193b is a tumor suppressor in acute myeloid leukemia (AML) (Bhayadia et al., JCO, 2018) and a potent regulator of hematopoietic stem cell expansion (Haetscher et al., Nat Com, 2015). However, the role of miR-193a in normal and malignant hematopoiesis is still unclear.
First, we profiled the expression of miR-193a-5p and miR-193a-3p in human hematopoietic subpopulations from healthy donors via quantitative real-time PCR (qRT-PCR). Unlike miR-193b, which is enriched in hematopoietic stem cells (HSC) (Haetscher et al., Nat Com, 2015), miR-193a expression was restricted to differentiated cells of the myeloid lineage (granulocytes, monocytes, and erythrocytes). To investigate the function of miR-193a during normal hematopoiesis, we analyzed its effect on a murine myeloid progenitor cell line (32D cells) upon engineered overexpression of miR-193a in combination with granulocyte-stimulating growth factor (G-CSF) treatment (n=3-7). MiR-193a strongly promoted granulocytic differentiation of 32D cells already after two days compared to the control arm (p=0.006) as assessed by flow cytometry and morphological analysis. To analyze the effect of miR-193a on HSC function, we ectopically expressed miR-193a in highly purified E-SLAM (CD45+EPCR+CD48-CD150+) cells and transplanted them into lethally irradiated recipient mice (n=5 mice/arm). HSCs overexpressing miR-193a failed to reconstitute hematopoiesis (p=0.038). The negative effect on stem cell properties could be translated to human CD34+ cord blood (CB) cells, where miR-193a overexpression significantly reduced colony counts. Taken together, these results suggest, that miR-193a has pro-differentiation and anti-stemness functions.
To assess the role of miR-193a in AML, we profiled miR-193a-5p and miR-193a-3p in two cohorts of de novo pediatric (n=187) and de novo adult AML patients (n=40) by qRT-PCR. We found that both miRNA strands (miR-193a-5p and miR-193a-3p) are present at similar levels, which is in contrast to the almost undetectable levels of miR-193b-5p strand, hinting at additional functional roles for miR-193a through its 5p arm. MiR-193a was significantly downregulated in adult and pediatric AML compared to healthy donor samples, suggesting a possible tumor suppressor function. To investigate the role of miR-193a in AML, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9/Meis1 (aggressive, short latency in vivo) with low endogenous miR-193a levels. Restoration of miR-193a by lentiviral overexpression delayed Hoxa9/Meis1 mediated leukemogenesis in vivo (p=0.01, n=8-10). Furthermore, miR-193a overexpression reduced leukemic growth of human AML cell lines (n=6) and decreased colony-forming capacity of primary AML patient samples (n=4; p=0.029) in vitro. To identify novel targets of miR-193a, we performed a proteomics screen in human NOMO1 AML cells overexpressing miR-193a compared to an empty control vector (n=5). We identified and verified stathmin (STMN1), a tubulin-associated, intracellular phosphoprotein previously linked to proliferation of AML cells, as a novel putative miR-193a target, further explaining its tumor suppressor effect.
Taken together, we characterized miR-193a as a positive regulator of myeloid differentiation and negative modulator of HSCs. Based on the balanced presence of both miRNA arms, we hypothesize that each arm is functionally active and has different functions such as pro-differentiation and anti-stemness. Furthermore, this work is the first characterization of a miRNA family that exerts cooperative effects at both early and late hematopoietic differentiation stages, highlighting a novel mechanism of balancing anti-stemness and pro-differentiation.
Döhner:Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau.
Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from ...differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells CB-HSPCs) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs.
The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis.
Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs.
Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA).
For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays.
Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes.
No relevant conflicts of interest to declare.
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