Knockdown Mouse Model Research Articles

Abstract 200: Targeting the Highly Abundant Circular RNA, circHeart, in Cardiomyocytes Attenuates Pressure Overload Induced Hypertrophy

Objectives: Circular RNAs (circRNAs) are single-stranded non-coding RNAs that form a covalent closed looped structure and are generated during gene transcription. In our genome-wide analysis of cardiac circRNA expression in both human and mouse hearts we identified a highly abundant circRNA, named here as circHeart and hypothesize an important role for circHeart in cardiomyocyte physiology and homeostasis. Materials and method: We undertook FISH to visualize the localization of circHeart . Online tools have predicted multiple potential miRNA binding sites on circHeart , including the cardiac-enriched microRNA-133a (miR-133a). We assessed the interactions of circHeart with these miRNAs by luciferase and pull-down assays and performed AAV9-mediated RNAi knockdown and overexpression of circHeart in mice in vivo . In vivo phenotype was tracked by echocardiography, electrocardiogram and histological studies. Transverse aortic constriction (TAC) was also performed. Results: FISH imaging showed circHeart localized largely to the cardiomyocyte cytosol. Luciferase and reciprocal pull-down assays suggest circHeart can interact with miR-133a. MiR-133a is a well-known regulator of cardiac hypertrophy and antagomir-133a treatment has been shown to induce cardiac hypertrophy in vivo . In our studies, while AAV9 RNAi-mediated knockdown of circHeart has no apparent baseline effect in the heart it suppresses pathological cardiac hypertrophy post-TAC. More strikingly, AAV9-mediated overexpression of circHeart in vivo resulted in mouse mortality between 3-4 days post-transduction and surviving mice showed evidence of dilated cardiomyopathy. Known targets of miR-133a were concordantly modulated in the knockdown and overexpression mouse model. Conclusion: CircRNAs have huge potential role as therapeutic targets and/or disease biomarkers and represent a new avenue for cardiovascular research. Taken together, our data are consistent with a mechanistic model of circHeart sequestering miR-133a. Inhibiting circHeart abundance presents a potential target for treating cardiac hypertrophy.

Abstract 579: Involvement of the Alpha 1A-Adrenergic Receptor in the Cardiac Adaptation to Physiological Stress

The ‘fight or flight’ response, involving sympathetic nervous system activation and catecholamine release, is central to survival. Resulting activation of adrenergic receptors (ARs) drives physiological responses, such as positive inotropy, which to date has been attributed solely to β 1 -AR/Gs/adenylyl cyclase/protein kinase A pathway-activation. We previously demonstrated that overexpression of the α 1A -AR is cardioprotective in rodents in the setting of pathological cardiac stress; a finding in agreement with studies of α 1A -AR inactivation. However, it remains unknown if α 1A -ARs also protect the heart from physiological stress. Using a tamoxifen-inducible, cardiomyocyte-specific α 1A -AR knockdown mouse model, we evaluated cardiac responses to swim exercise. Treatment of adult (8-12 week) mice with 4-OH tamoxifen (Tam) resulted in a 7.4 fold decrease (p<0.001) in cardiac α 1A -AR mRNA levels, with no compensatory changes in expression of β 1 -, β 2 - or α 2 -AR subtypes. A 90 minute, twice daily, four-week swim challenge induced eccentric cardiac hypertrophy, with Tam- and vehicle-treated mice displaying similar increases in heart weight/tibia length (mean increase: 1.4 and 1.6mg/mm, respectively; p<0.001 vs sedentary mice) and left ventricular chamber radius (mean increase: 0.16 and 0.17mm, respectively; p<0.001 vs sedentary mice) with no change in wall thickness. Consistent with physiological hypertrophy, Igf-1 mRNA increased 1.7 and 0.7 fold (p=0.004 vs sedentary mice), respectively, with no increase in Nppb , Myh7 or Acta1 mRNA. Cardiac function, assessed by micromanometry and echocardiography, showed increases in dP/dt min/EDV (mean: -117 vs -150 mmHg/s/μL; p=0.011), end-diastolic volume (mean: 76.9 vs 68.0μL; p=0.038) and end-systolic volume (mean: 29.4 vs 21.9μL; p=0.018), as well as a decrease in ejection fraction (mean: 63 vs 68%; p=0.046) in Tam- compared with vehicle-treated mice, respectively. Thus, despite a similar hypertrophic response, mice with cardiomyocyte-specific α 1A -AR inactivation showed an impaired contractile response to a sustained swim challenge. These findings are consistent with cardiomyocyte α 1A -ARs playing an important role in maintaining cardiac contractility in response to physiological stress.

TARGETING P53 AND NUCLEOLAR STRESS IN DIAMOND-BLACKFAN ANAEMIA

Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.

First behavioural assessment of a novel Immp2l knockdown mouse model with relevance for Gilles de la Tourette syndrome and Autism spectrum disorder

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder, which shares some clinical features with Autism spectrum disorder (ASD). The genetic factors relevant to the development of both disorders are yet to be fully understood, however, some genetic association studies have identified inner mitochondrial membrane peptidase subunit 2 (IMMP2L) as a potential risk gene for both GTS and ASD. The impact of Immp2l deficiency on behavioural domains is currently unknown.A new genetic mouse model for Immp2l was developed. Adult heterozygous (HET) and homozygous (HOMO) Immp2l knockdown (Immp2l KD) mice of both sexes were compared to wild type-like (WT) littermates in the open field (OF), social interaction, novel object recognition, marble burying, and prepulse inhibition (PPI). The effect of acute dexamphetamine (2 mg/kg) on OF behaviour was also determined.OF locomotion was significantly higher in HET compared to HOMO male littermates. Male and female HOMO mice were much more sensitive to the locomotor-stimulating effects of dexamphetamine (DEX), whereas only HOMO males exhibited significant increased DEX-induced OF exploration compared to control groups. HOMO females failed to habituate to an acoustic startle stimulus. Furthermore, compared to HOMO females, HET females showed reduced social interaction, and a similar trend was seen in HET males.The Immp2l KD mouse model possesses moderate face validity for preclinical research into GTS and ASD, in particular as dysfunctional dopaminergic neurotransmission appears to be one mechanism leading to disease presentation. The sex-dependent differences observed in most findings reinforce the strong influence of sex in the pathophysiology of GTS and ASD.

Convergent neural substrates of inattention in bipolar disorder patients and dopamine transporter-deficient mice using the 5-choice CPT.

Bipolar disorder (BD) is a debilitating psychiatric illness affecting 2%-5% of the population. Although mania is the cardinal feature of BD, inattention and related cognitive dysfunction are observed across all stages. Since cognitive dysfunction confers poor functional outcome in patients, understanding the relevant neural mechanisms remains key to developing novel-targeted therapeutics. The 5-choice continuous performance test (5C-CPT) is a mouse and fMRI-compatible human attentional task, requiring responding to target stimuli while inhibiting responding to nontarget stimuli, as in clinical CPTs. This task was used to delineate systems-level neural deficits in BD contributing to inattentive performance in human subjects with BD as well as mouse models with either parietal cortex (PC) lesions or reduced dopamine transporter (DAT) expression. Mania BD participants exhibited severe 5C-CPT impairment. Euthymic BD patients exhibited modestly impaired 5C-CPT. High impulsivity BD subjects exhibited reduced PC activation during target and nontarget responding compared with healthy participants. In mice, bilateral PC lesions impaired both target and nontarget responding. In the DAT knockdown mouse model of BD mania, knockdown mice exhibited severely impaired 5C-CPT performance versus wildtype littermates. These data support the role of the PC in inattention in BD-specifically regarding identifying the appropriate response to target vs nontarget stimuli. Moreover, the findings indicate that severely reduced DAT function/hyperdopaminergia recreates the attentional deficits observed in BD mania patients. Determining the contribution of DAT in the PC to attention may provide a future target for treatment development.

Measurement of lactate levels in postmortem brain, iPSCs, and animal models of schizophrenia

Converging evidence suggests bioenergetic defects contribute to the pathophysiology of schizophrenia and may underlie cognitive dysfunction. The transport and metabolism of lactate energetically couples astrocytes and neurons and supports brain bioenergetics. We examined the concentration of lactate in postmortem brain (dorsolateral prefrontal cortex) in subjects with schizophrenia, in two animal models of schizophrenia, the GluN1 knockdown mouse model and mutant disrupted in schizophrenia 1 (DISC1) mouse model, as well as inducible pluripotent stem cells (iPSCs) from a schizophrenia subject with the DISC1 mutation. We found increased lactate in the dorsolateral prefrontal cortex (p = 0.043, n = 16/group) in schizophrenia, as well as in frontal cortical neurons differentiated from a subject with schizophrenia with the DISC1 mutation (p = 0.032). We also found a decrease in lactate in mice with induced expression of mutant human DISC1 specifically in astrocytes (p = 0.049). These results build upon the body of evidence supporting bioenergetic dysfunction in schizophrenia, and suggests changes in lactate are a key feature of this often devastating severe mental illness.

Vascular peroxidase 1 is a novel regulator of cardiac fibrosis after myocardial infarction

Cardiac fibrosis is the most important mechanism contributing to cardiac remodeling after myocardial infarction (MI). VPO1 is a heme enzyme that uses hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl). Our previous study has demonstrated that VPO1 regulates myocardial ischemic reperfusion and renal fibrosis. We investigated the role of VPO1 in cardiac fibrosis after MI. The results showed that VPO1 expression was robustly upregulated in the failing human heart with ischemic cardiomyopathy and in a murine model of MI accompanied by severe cardiac fibrosis. Most importantly, knockdown of VPO1 by tail vein injection of VPO1 siRNA significantly reduced cardiac fibrosis and improved cardiac function and survival rate. In VPO1 knockdown mouse model and cardiac fibroblasts cultured with TGF-β1, VPO1 contributes to cardiac fibroblasts differentiation, migration, collagen I synthesis and proliferation. Mechanistically, the fibrotic effects following MI of VPO1 manifested partially through HOCl formation to activate Smad2/3 and ERK1/2. Thus, we conclude that VPO1 is a crucial regulator of cardiac fibrosis after MI by mediating HOCl/Smad2/3 and ERK1/2 signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

The microRNA-23a cluster regulates the developmental HoxA cluster function during osteoblast differentiation

MicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3'-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a ClWT/ZIP) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stage-specific HoxA factor expression during osteogenesis.

HGF alleviates renal interstitial fibrosis via inhibiting the TGF-β1/SMAD pathway.

To study the role of HGF (stem cell growth factor) in renal interstitial fibrosis and to explore its underlying mechanism. A unilateral ureteral obstruction (UUO) mouse model was first constructed, and kidney samples of mice were then collected. Fibrosis-related indicators in UUO mice kidney were detected by Western blot. The mRNA and protein levels of HGF in UUO mice were detected by quantitative Real-time-polymerase chain reaction (qRT-PCR) and Western blot, respectively. The HGF overexpression mouse model was established by using UUO mice. For in vitro experiments, fibrosis-related indicators and the expression of HGF were detected in transforming growth factor-β1 (TGF-β1)-induced NRK-52E cells. Finally, a p-SMAD3 knockdown mouse model was established to confirm whether p-SMAD3 was involved in HGF-regulated renal interstitial fibrosis. The expression levels of HGF and α-SMA (α-smooth muscle actin) were both significantly increased in UUO mice, while E-cadherin expression was significantly decreased, which were consistent with results of in vitro experiments. Overexpression of HGF remarkably decreased the protein and mRNA levels of α-SMA in fibrotic NRK-52E cells. After overexpression of HGF in UUO mice, α-SMA was remarkably downregulated, whereas E-cadherin was significantly upregulated. Further, results also demonstrated that HGF was upregulated and α-SMA was downregulated after p-SMAD3 knockdown in UUO mice. HGF is highly expressed during renal interstitial fibrosis, which may suppress renal interstitial fibrosis by inhibiting the TGF-β1/SMAD signaling pathway.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Development of a novel conditional knockdown mouse based on YB-1 protein degradation.

To clarify the pathogenic mechanism of disease and establish effective therapies, animal disease models that can be dynamically analyzed are urgently required. Knockout mouse models and conditional genetically engineered mouse models were developed to analyze genes and proteins involved in disease. However, these methods have drawbacks, including embryonic lethality, side effects and low efficiency. To address this issue, we created a novel transgenic mouse model in which the YB1 gene was fused with a destabilizing domain (DD), named the YB1-DD mouse. YB-1 is widely expressed throughout development and has been implicated as a cell survival factor. Newly synthesized DD proteins are degraded through the proteasome pathway, but their degradation can be blocked with trimethoprim (TMP). In this study, we established a novel conditional knockdown mouse model that enables targeting of protein degradation directly; this model resulted in dose-dependent regulation of the target protein YB-1 by the ligand TMP in YB1 heterozygous mice. Since this conditional knockdown mouse model appears to be functional, it has potential as a useful disease model based on direct protein degradation control.

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.98AAV-mediated gene transfer of FKRP for therapy of LGMD2I

Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan α, a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins whose functions are not fully elucidated. In particular, mutations in the FKRP gene encoding Fukutin related protein lead to hypoglycosylation of α, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We and others have published the proof of concept of FKRP gene transfer using an AAV vector for treating FKRP deficiencies. As the knock-in mouse models used in these studies are not severe enough to evaluate properly the dose-effect of AAV-FKRP administration, we developed a muscle specific FKRP knock-down mouse model. This new mouse model, named HSA-FKRPdel, presents a much more severe phenotype than observed in knock-in mouse. Defects of glycosylation of αDG and of its binding to laminin were observed, as well as histological dystrophic signs as centronucleation and inflammation. Functional evaluation also showed a reduced force of HSA-FKRPdel mice. AAV-FKRP was systemically administrated to this new mouse model at different doses. Depending of the dose, positive effects were observed at the molecular, histological and functional levels. Full animal characterization and therapeutic effects will be presented. Development of AAV production at the GMP level is now on going. These data will be included in an IND for AAV-mediated transfer of FKRP in patients.

Epigenetic remodeling and modification to preserve skeletogenesis in vivo

ABSTRACTCurrent studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a ClZIP) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo.

Developmental abnormalities in supporting cell phalangeal processes and cytoskeleton in the Gjb2 knockdown mouse model

ABSTRACTMutations in the GJB2 gene [which encodes connexin 26 (Cx26)] are the most common causes of hereditary hearing loss in humans, and previous studies showed postnatal development arrest of the organ of Corti in different Cx26-null mouse models. To explore the pathological changes and the mechanism behind the cochlear abnormalities in these mice further, we established transgenic mouse models by conditional knockdown of cochlear Cx26 at postnatal day (P) 0 and P8. Auditory brainstem responses were recorded and the morphological features in the organ of Corti were analyzed 18 days after Cx26 knockdown. Mice in the P0 knockdown group displayed severe hearing loss at all frequencies, whereas mice in the P8 knockdown group showed nearly normal hearing. In the P8 knockdown group, the organ of Corti displayed normal architecture, and no ultrastructural changes were observed. In the P0 knockdown group, the phalangeal processes of Deiter's cells did not develop into finger-like structures, and the formation of microtubules in the pillar cells was significantly reduced; moreover, the amount of acetylated α-tubulin was reduced in pillar cells. Our results indicate that Gjb2 participates in postnatal development of the cytoskeleton in pillar cells during structural maturation of the organ of Corti. In P0 knockdown mice, the reduction in microtubules in pillar cells might be responsible for the failure of the tunnel of Corti to open, and the malformed phalangeal processes might negatively affect the supporting framework of the organ of Corti, which would be a new mechanism of Gjb2-related hearing loss.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Deficiency of Cux1, Encoded on Human Chromosome 7q, Causes Aberrant Hematopoietic Stem Cell Function and Spontaneous Myeloproliferative Disease in Mice

Loss of all or part of chromosome 7 [-7/del(7q)] is a common, high-risk cytogenetic abnormality identified in a variety myeloid malignancies, including myelodysplastic syndrome (MDS), acute myeloid leukemia, therapy-related myeloid neoplasms, juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). CUX1 is a non-clustered, homeodomain-containing transcription factor encoded on the commonly deleted region of 7q22. CUX1 inactivating mutations occur across solid tumors and myeloid neoplasms, and carry a poor prognosis in myeloid malignancies independently of 7q status. Previously, we reported that CUX1 is a conserved, haploinsufficient, myeloid tumor suppressor. To determine the mechanism by which CUX1 regulates leukemogenesis, we generated an inducible, shRNA-transgenic, Cux1 -knockdown mouse model. Insufficient Cux1 levels caused early mortality in mice, due to a spontaneous myeloproliferative disorder with features of CMML/JMML and MDS. Cux1 deficient mice had a significant increase in myelomonocytic cell numbers in the peripheral blood, bone marrow, and spleen. Cux1 deficiency led to extramedullary hematopoiesis, splenomegaly, and an increase in megakaryocyte numbers and megakaryocyte dysplasia. In addition, Cux1 is required for normal red cell development, as Cux1 knockdown led to a normocytic anemia due to an erythroblast maturation arrest. To identify CUX1 transcriptional targets, we performed gene expression profiling after CUX1 knockdown in CD34+ human hematopoietic stem and progenitor cells (HSPCs). Differentially expressed genes were enriched for genes deregulated in JMML and -7/del(7q) MDS, in addition to altered expression of quiescent, proliferative, and myeloid differentiation pathways. Consistent with these gene signatures, Cux1 maintains hematopoietic stem cell quiescence and represses HSPC proliferation, self-renewal, and granulocyte/monocyte differentiation. Thus Cux1 has pleotropic roles in multiple myeloid lineages. Deletion of CUX1, in the context of -7/del(7q), may facilitate myeloid transformation through aberrant transcriptional regulation of HSPC differentiation and proliferation. DisclosuresNo relevant conflicts of interest to declare.

Roles of BCCIP deficiency in mammary tumorigenesis

BackgroundDysregulated DNA repair and cell proliferation controls are essential driving forces in mammary tumorigenesis. BCCIP was originally identified as a BRCA2 and CDKN1A interacting protein that has been implicated in maintenance of genomic stability, cell cycle regulation, and microtubule dynamics. The aims of this study were to determine whether BCCIP deficiency contributes to mammary tumorigenesis, especially for a subset of breast cancers with 53BP1 abnormality, and to reveal the mechanistic implications of BCCIP in breast cancer interventions.MethodsWe analyzed the BCCIP protein level in 470 cases of human breast cancer to determine the associations between BCCIP and 53BP1, p53, and subtypes of breast cancer. We further constructed a unique BCCIP knockdown mouse model to determine whether a partial BCCIP deficiency leads to spontaneous breast cancer formation.ResultsWe found that the BCCIP protein level is downregulated in 49% of triple-negative breast cancer and 25% of nontriple-negative breast cancer. The downregulation of BCCIP is mutually exclusive with p53 mutations but concurrent with 53BP1 loss in triple-negative breast cancer. In a K14-Cre-mediated conditional BCCIP knockdown mouse model, we found that BCCIP downregulation causes a formation of benign modules in the mammary glands, resembling the epidermal inclusion cyst of the breast. However, the majority of these benign lesions remain indolent, and only ~ 10% of them evolve into malignant tumors after a long latency. This tumor progression is associated with a loss of 53BP1 and p16 expression. BCCIP knockdown did not alter the latency of mammary tumor formation induced by conditional Trp53 deletion.ConclusionsOur data suggest a confounding role of BCCIP deficiency in modulating breast cancer development by enhancing tumor initiation but hindering progression. Furthermore, secondary genetic alternations may overcome the progression suppression imposed by BCCIP deficiency through a synthetic viability mechanism.

Thrombospondin-1 contributes to slower aortic aneurysm growth by inhibiting maladaptive remodeling of extracellular matrix.

In this issue of Clinical Science, Krishna and colleagues describe recent work on thrombospondin-1 (TSP-1) maturation and its association with slower growth of aortic aneurysm in TSP-1 knockdown mouse models. The authors conclude that TSP-1 deficiency promotes maladaptive remodeling of the extracellular matrix (ECM) leading to accelerated aortic aneurysm progression. We comment on a causal relation between TSP-1 and the progression of aortic aneurysm.

Inhibition of microRNA‐17 Promotes the Progression of Thyroid Cancer in a Xenograft Murine Cancer Model

MicroRNAs (miRs) are small non‐coding RNAs that bind to target mRNAs and negatively regulate their expression. The expression of miRs are altered in many cancers and cancer cell types. We previously generated a miR‐17‐92 knockdown mouse model using the Plasmid based MicroRNA Inhibitor System (PMIS). With this new PMIS system we found that thyroid development was abnormal or absence in these mice. Thus, thyroid development requires miR‐17 for normal development. We investigated the role of miR‐17 in SW579 thyroid cancer cells, which are derived from human thyroid tissue. PMIS‐miR‐17, specifically targeting miR‐17‐5p completely inhibited the activity of miR‐17‐5p in SW579 cells. However, inhibiting miR‐17‐5p activity in SW579 thyroid cancer cells also promotes tumor growth in a nude mouse model for thyroid cancer. The tumor formation was due to increase cell proliferation and aberrant gene expression. RNA‐seq data shows knocking down miR‐17 in thyroid cancer cells induces large‐scale changes in miR‐17‐5p target gene expression, including PDGFR and MMP2, which may play oncogenic roles in tumorigenesis. Bioinformatics analyses has identified several new gene expression networks regulated by miR‐17 in thyroid cancer. We have identified several new gene expression mechanisms for miR‐17 and more importantly regulation of miR‐17‐92 expression and processing of the miR‐17‐92 cluster during thyroid development and thyroid cancer.Support or Funding InformationIowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA