DICER1 Function Research Articles

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

The molecular landscape of ETMR at diagnosis and relapse.

ETMRs are aggressive pediatric embryonal brain tumors with universally dismal outcome1. We collected 193 primary ETMRs and 23 matched relapses to investigate the genomic landscape of this distinct entity. We found that patients having tumors without C19MC amplification, the proposed driver3–5, frequently harbor DICER1 germline mutations or other miRNA-related aberrations including somatic miR-17–92 amplifications. Whole-genome sequencing revealed an overall low recurrence of SNVs, but prevalent R-loop-associated chromosomal instability, of which we show that this can be induced by loss of DICER1 function. Comparing primary tumors and matched relapses revealed a strong conservation of SVs but low conservation of SNVs. Moreover, many newly acquired SNVs are associated to a new cisplatin treatment related mutational signature. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

Abstract IA10: Notch signaling and cancer

Abstract Cancer can be seen as disease of perturbed self-renewal. In the last decades it became clear that many of the signaling pathways known to be important during embryonic development also play important roles in regulating self-renewing tissues. Deregulation of the self-renewal process results in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis all of which are hallmarks of cancer. The major question is how do these deregulated signaling cascades mechanistically contribute to cancer and are they suitable for targeting therapy? The Notch pathway is one such cascade required for normal stem cell maintenance and development of different organs. Over activation of this pathway due to mutations in the Notch receptor are found in more than 50% of human T-cell leukemia and deregulated Notch signaling has been shown to promote tumor progression of various organs. The oncogenic mediators of aberrant Notch function in T-ALL and other Notch-driven cancers appear to be manifold and complex. The modulatory function of individual miRNAs in Notch driven T-ALLs has recently been established. However, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. We used conditional and inducible genetic loss of function approaches to show that development and maintenance of Notch-driven T-ALL is dependent on Dicer1 function. Lineage tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4. Moreover, we will report on the identification of a novel small molecule inhibitor (CB103) that blocks Notch signaling by interfering with the transcriptional activation complex. The compound CB103 has shown remarkable ability to block Notch signaling in human cancer cell lines as well as primary human T-ALL cells, thus abrogating their proliferative properties. In addition, compound CB103 exhibit in vivo activity as demonstrated by its ability to impinge upon Notch dependent developmental processes and by impeding tumor growth in xenograft models of human leukemia and breast cancer. Citation Format: Fabian Junker, Rajwinder Lehal, Viktoras Frimantas, Beat C. Bornhauser, Jean-Pierre Bourquin, Ute Koch, Freddy Radtke. Notch signaling and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA10.

Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21–mediated tumor suppressor Pdcd4 repression

AbstractThe modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.

Cell- and developmental stage-specific Dicer1 ablation in the lung epithelium models cystic pleuropulmonary blastoma.

Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss-of-function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause-effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Here we report the generation of a murine model for PPB and demonstrate that precise temporal and cell type-specific Dicer1 ablation is necessary and sufficient for the development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB, whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilatation accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type-specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for the development of cystic PPB. These results also provide evidence that PPB arise through a novel mechanism of non-cell-autonomous tumour initiation, in which the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign-appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation.

Abstract 2312: A genomics approach to determining the role of DICER1 in tumorigenesis: Interferon responses in the cancer cell

Abstract DICER1 is required for generation of mature microRNAs (miRNAs) and other short noncoding RNAs. Reduced DICER1 levels are associated with adverse outcomes in a variety of cancers and multiple lines of evidence suggest DICER1 is a tumor suppressor. The effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. To determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels had no effect on cell proliferation but were associated with enhanced cell migration and increased growth in soft agar. MiRNA profiling for the KLE cancer cell line DICER1 knockdowns (two different DICER1 shRNA constructs) revealed that miRNA levels were decreased overall. Not surprisingly, many mRNA transcripts were upregulated in the DICER1 knockdowns. A striking number of interferon stimulated genes (ISGs) showed upregulation. Upregulation of six ISG transcripts was validated by qRT-PCR in three independent knockdowns of DICER1 in KLE cells. IFNα, a key upstream regulator of the interferon response, was significantly increased in DICER1 knockdowns (KLE and additional endometrial cancer cell lines). IFNα protein secreted in media of DICER1 knockdown cells activated an interferon response in HT29 colon carcinoma cells. The majority of ISGs with increased transcript levels lacked miRNA target sequences and as such it seemed unlikely that the increase in expression levels was secondary to reduced miRNA levels. We tested the hypothesis that defects in miRNA processing could trigger an interferon response. We showed that DICER1 knockdowns in which miRNA processing is reduced have increased levels of pre-microRNAs in the cytoplasm. Pre-microRNAs are double-stranded molecules and long enough (60-100 nt) to activate the dsRNA sensors in the cytoplasm, such as Mda5/IFIH1, that typically sense viral dsRNA (> 30 nt) and stimulate the interferon response. Knockdown of DROSHA, necessary for processing of pri-miRNAs to pre-miRNAs, did not result in accumulation of pre-miRNAs and did not cause an interferon response. Our findings suggest elevated pre-microRNA levels trigger the interferon response to double stranded RNA (incompletely processed miRNA and other species). Rescue experiments (restoring DICER1 function) are currently underway to shed additional light on how reduced double-stranded RNA processing might elicit an interferon response. Work is ongoing to determine the relationship between the observed interferon response and the migration and soft agar phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2312. doi:1538-7445.AM2012-2312

Reduced DICER1 Elicits an Interferon Response in Endometrial Cancer Cells

DICER1 is essential for the generation of mature miRNAs and other short noncoding RNAs. Several lines of investigation implicate DICER1 as a tumor suppressor. Reduced DICER1 levels and changes in miRNA abundance have been associated with aggressive tumor phenotypes. The global effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. We used short hairpin RNA to stably knock down DICER1 in endometrial cancer cell lines to begin to determine how reduced DICER1 activity contributes to tumor phenotypes. DICER1 knockdown did not affect cell proliferation but caused enhanced cell migration and growth in soft agar. miRNA and mRNA profiling in KLE cells revealed overall decreases in miRNA levels and changes in the relative abundance of many mRNAs. One of the most striking changes in mRNA levels was the upregulation of IFN-stimulated genes (ISG), the majority of which lack known miRNA target sequences. IFNβ, a key upstream regulator of the IFN response, was significantly increased in DICER1 knockdowns in the AN3CA, Ishikawa, and KLE endometrial cancer cell lines and in the normal endometrial cell line EM-E6/E7/TERT. IFNβ secreted in media from KLE and EM-E6/E7/TERT shDcr cells was sufficient to activate an IFN response in HT29 cells. The reduced miRNA processing in DICER1 knockdowns was associated with increases in pre-miRNAs in the cytoplasm. Our findings suggest that elevated pre-miRNA levels trigger the IFN response to double-stranded RNA. We thus report a novel effect of reduced DICER1 function in cancer cells.

Recurrent Somatic DICER1 Mutations in Nonepithelial Ovarian Cancers

BackgroundGermline truncating mutations in DICER1, an endoribonuclease in the RNase III family that is essential for processing microRNAs, have been observed in families with the pleuropulmonary blastoma–family tumor and dysplasia syndrome. Mutation carriers are at risk for nonepithelial ovarian tumors, notably sex cord–stromal tumors.MethodsWe sequenced the whole transcriptomes or exomes of 14 nonepithelial ovarian tumors and noted closely clustered mutations in the region of DICER1 encoding the RNase IIIb domain of DICER1 in four samples. We then sequenced this region of DICER1 in additional ovarian tumors and in certain other tumors and queried the effect of the mutations on the enzymatic activity of DICER1 using in vitro RNA cleavage assays.ResultsDICER1 mutations in the RNase IIIb domain were found in 30 of 102 nonepithelial ovarian tumors (29%), predominantly in Sertoli–Leydig cell tumors (26 of 43, or 60%), including 4 tumors with additional germline DICER1 mutations. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for microRNA interaction and cleavage, and were somatic in all 16 samples in which germline DNA was available for testing. We also detected mutations in 1 of 14 nonseminomatous testicular germ-cell tumors, in 2 of 5 embryonal rhabdomyosarcomas, and in 1 of 266 epithelial ovarian and endometrial carcinomas. The mutant DICER1 proteins had reduced RNase IIIb activity but retained RNase IIIa activity.ConclusionsSomatic missense mutations affecting the RNase IIIb domain of DICER1 are common in nonepithelial ovarian tumors. These mutations do not obliterate DICER1 function but alter it in specific cell types, a novel mechanism through which perturbation of microRNA processing may be oncogenic. (Funded by the Terry Fox Research Institute and others.)

Monoallelic but not biallelic loss of Dicer1 promotes tumorigenesis in vivo

Human tumors are characterized by widespread reduction in microRNA (miRNA) expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells. A defect in miRNA processing is one possible mechanism for global downregulation. To explore this possibility in more detail in vivo, we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only a partial decrease in miRNA processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer etiology and cancer therapy.

A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNA (mRNA) transcripts. Recently, a miRNA expression profile of human tumors has been characterized by an overall miRNA downregulation. Explanations for this observation include a failure of miRNA post-transcriptional regulation, transcriptional silencing associated with hypermethylation of CpG island promoters and miRNA transcriptional repression by oncogenic factors. Another possibility is that the enzymes and cofactors involved in miRNA processing pathways may themselves be targets of genetic disruption, further enhancing cellular transformation. However, no loss-of-function genetic alterations in the genes encoding these proteins have been reported. Here we have identified truncating mutations in TARBP2 (TAR RNA-binding protein 2), encoding an integral component of a DICER1-containing complex, in sporadic and hereditary carcinomas with microsatellite instability. The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs. The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth. Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein. These results provide, for a subset of human tumors, an explanation for the observed defects in the expression of mature miRNAs.

MicroRNA Processing Pathway Regulates Olfactory Neuron Morphogenesis

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

Hepatic function is preserved in the absence of mature microRNAs #

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression through partial or complete complementarity with target messenger RNAs. The function of miRNAs in normal liver physiology is largely unknown. We address the role of Dicer1 in the differentiated liver. We derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA via quantitative polymerase chain reaction. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed. Mice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2 and 4 months of age exhibited progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large-scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes. Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in the mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes.

Impaired microRNA processing causes corpus luteum insufficiency and infertility in mice

The microRNA (miRNA) processing enzyme Dicer1 is required for zygotic and embryonic development, but the early embryonic lethality of Dicer1 null alleles in mice has limited our ability to address the role of Dicer1 in normal mouse growth and development. To address this question, we used a mouse mutant with a hypomorphic Dicer1 allele (Dicer(d/d)) and found that Dicer1 deficiency resulted in female infertility. This defect in female Dicer(d/d) mice was caused by corpus luteum (CL) insufficiency and resulted, at least in part, from the impaired growth of new capillary vessels in the ovary. We found that the impaired CL angiogenesis in Dicer(d/d) mice was associated with a lack of miR17-5p and let7b, 2 miRNAs that participate in angiogenesis by regulating the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1. Furthermore, injection of miR17-5p and let7b into the ovaries of Dicer(d/d) mice partially normalized tissue inhibitor of metalloproteinase 1 expression and CL angiogenesis. Our data indicate that the development and function of the ovarian CL is a physiological process that appears to be regulated by miRNAs and requires Dicer1 function.

Stage-Specific Differences in the Requirements for Germline Stem Cell Maintenance in the Drosophila Ovary

In this study, we uncover a role for microRNAs in Drosophila germline stem cell (GSC) maintenance. Disruption of Dicer-1 function in GSCs during adult life results in GSC loss. Surprisingly, however, loss of Dicer-1 during development does not result in a GSC maintenance defect, although a defect is seen if both Dicer-1 and Dicer-2 function are disrupted. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam plays a key role in GSC self-renewal. Mad, a component of the TGF-beta pathway, behaves similarly to Dicer-1: adult GSC maintenance requires Mad if it is lost during adult life, but not if it is lost during pupal development. Overall, these results show stage-specific differential sensitivity of GSC maintenance to certain perturbations, and suggest that there may be Dcr-2 dependent redundancy of GSC maintenance mechanisms during development that is lost in later life.