Conditional Transgenic Mouse Model Research Articles

PR-Set7 is Degraded in a Conditional Cul4A Transgenic Mouse Model of Lung Cancer.

Background and objectiveMaintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and afer DNA damage. Tis might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity afer DNA damage.MethodsWe developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the frst time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identifed higher protein level changes of γ-tubulin and pericentrin by IHC.ResultsTe level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identifed higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre.ConclusionPR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.

MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism

The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization-mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.

Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL.Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system.Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias.Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency.Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7.In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies. DisclosuresNo relevant conflicts of interest to declare.

Assessing recovery from neurodegeneration in spinocerebellar ataxia 1: Comparison of in vivo magnetic resonance spectroscopy with motor testing, gene expression and histology

Suppression of transgene expression in a conditional transgenic mouse model of spinocerebellar ataxia 1 (SCA1) reverses the Purkinje cell pathology and motor dysfunction that are hallmarks of SCA1. We previously showed that cerebellar neurochemical levels measured by magnetic resonance spectroscopy (MRS) correlate with progression of pathology and clinical status of patients and that abnormal neurochemical levels normalize upon suppression of transgene expression, indicating their potential as robust surrogate markers of treatment effects. Here we investigated the relative sensitivities of MRS, histology, transgene expression and motor behavioral testing to disease reversal in conditional SCA1 mice. Transgene expression was suppressed by doxycycline administration and treated and untreated mice were assessed by MRS at 9.4tesla before and after treatment and with an accelerating Rotarod, histology and quantitative polymerase chain reaction (qPCR) for ataxin-1 transgene expression following doxycycline treatment. The MRS-measured N-acetylaspartate-to-myo-inositol ratio (NAA/Ins) correlated significantly with the molecular layer (ML) thickness and transgene expression. NAA/Ins, ML thickness and transgene expression were highly significantly different between the treated vs. untreated groups (p<0.0001), while the Rotarod assessment showed a trend for treatment effect. MRS, qPCR and histology had high sensitivity/specificity to distinguish treated from untreated mice, all with areas under the curve (AUC)=0.97–0.98 in receiver operating characteristic (ROC) analyses, while Rotarod had significantly lower sensitivity and specificity (AUC=0.72). Therefore, MRS accurately reflects the extent of recovery from neurodegeneration with sensitivity similar to invasive measures, further validating its potential as a surrogate marker in pre-clinical and clinical treatment trials.

Mechanisms of Aberrant Organization of Growth Plates in Conditional Transgenic Mouse Model of Spondyloepiphyseal Dysplasia Associated with the R992C Substitution in Collagen II

Mutations in collagen II, a main structural protein of cartilage, are associated with various forms of spondyloepiphyseal dysplasia (SED), whose main features include aberrations of linear growth. Here, we analyzed the pathomechanisms responsible for growth alterations in transgenic mice with conditional expression of the R992C collagen II mutation. Specifically, we studied the alterations of the growth plates of mutant mice in which chondrocytes lacked their typical columnar arrangement. Our studies demonstrated that chondrocytes expressing the thermolabile R992C mutant collagen II molecules endured endoplasmic reticulum stress, had atypical polarization, and had reduced proliferation. Moreover, we demonstrated aberrant organization and morphology of primary cilia. Analyses of the extracellular collagenous deposits in mice expressing the R992C mutant collagen II molecules indicated their poor formation and distribution. By contrast, transgenic mice expressing wild-type collagen II and mice in which the expression of the transgene encoding the R992C collagen II was switched off were characterized by normal growth, and the morphology of their growth plates was correct. Our study with the use of a conditional mouse SED model not only indicates a direct relation between the observed aberration of skeletal tissues and the presence of mutant collagen II, but also identifies cellular and matrix elements of the pathomechanism of SED.

Abstract 5495: BET bromodomain inhibitors abrogate cell cycle progression and induces apoptosis in Myc-induced mouse lymphoma cells without affecting MYC transcription

Abstract Overexpression of one of the three MYC genes is a hallmark of many human cancers. They encode transcription factors that regulate expression of a number of genes critical for tumor development. Conditional transgenic mouse models have shown that Myc inhibition causes tumor regression; Myc is therefore an attractive target for therapy, and effective pharmacological Myc inhibition has been a long-standing goal in cancer research. Recent publication have shown that selective inhibition of BET bromodomain and extra-terminal (BET) domain family of proteins, that bind to acetylated lysines on histones, show promise as potent anti-cancer drugs via down-regulation of the MYC oncogene. Here we confirm that two structurally different BET protein inhibitors (BETi), JQ1 (proto-type) and RVX2135, inhibit the proliferation and induce apoptosis of lymphoma cells arising in Myc-transgenic mice. BETi have been reported to directly inhibit MYC transcription. Surprisingly, in our system BET inhibition had no effect on MYC transcription, despite exhibiting broad transcriptional effects evident from expression profiling. Our data challenge the prevailing view that BETi operate primarily via suppression of MYC transcription in hematological malignancies. Instead we suggest that BET proteins have Myc-independent pleiotropic effects that should be exploited to treat a wide range of cancers. Myc still remains a challenging target for therapy to date. Citation Format: Joydeep Bhadury, Lisa M. Nilsson, Lydia Green, Zhoulei Li, Emily M. Gesner, Somsundar Veppil Muralidharan, Henrik C. Hansen, Ulrich B. Keller, Kevin G. Mclure, Jonas A. Nilsson. BET bromodomain inhibitors abrogate cell cycle progression and induces apoptosis in Myc-induced mouse lymphoma cells without affecting MYC transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2014-5495

Abstract 1822: HER2/PIK3CAH1047R transgenic tumors develop acquired resistance to triple therapy with trastuzumab, pertuzumab, and PI3K inhibitors via multiple mechanisms

Abstract The HER2 (ERBB2) oncogene is amplified in 20-25% of breast cancers and is associated with poor patient outcome. Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), occur in ∼30% of breast cancers. HER2 amplification and PIK3CA mutations often co-occur in breast cancer. Aberrant activation of the PI3K pathway correlates with a diminished response to HER2-directed therapies. We previously generated a conditional transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. We showed that PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers and generates resistance to FDA-approved combinations of anti-HER2 therapies (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. We utilized tumor transplants derived from two HER2+/PIK3CA transgenic mice, #564 and #635. Tumor transplants from model 564 were initially growth inhibited by TPB, but did not regress. A subset of 564 transplants (3/11) resumed growth in the presence of continuous TPB therapy. Resistance was maintained following passaging in mice and tumors were cross-resistant to trastuzumab/pertuzumab/BYL719, a p110α-specific inhibitor. P-AKT remained suppressed in resistant tumors, whereas P-ERK was elevated. All transplants (n=9) from model 635 regressed to a volume of &amp;lt;100 mm3 within 6 weeks of treatment. All tumors recurred within 2 months; 2 tumors continued growth when re-treated with TPB. Unlike the 564 resistant tumors, P-AKT was restored in the 635 resistant tumors, while short-term TPB treatment strongly inhibited P-AKT in the 635 tumors. TPB-resistant tumor 635-2 expressed p95 HER2, which was not detected in untreated tumors. In contrast, HER2 expression was significantly reduced in TPB-resistant tumor 635-3. We are currently performing whole-exome sequencing on TPB-resistant vs. untreated tumors in order to determine the mechanisms of resistance. Both 564 and 635 TPB-resistant tumor transplants displayed resistance to the antibody-drug conjugate trastuzumab-DM1, despite maintenance of HER2 overexpression. These early data suggest that multiple mechanisms may contribute to resistance to dual HER2 blockade in combination with PI3K inhibitors. In parallel, we are currently establishing human HER2+, PIK3CA-mutant cell lines resistant to TPB. We speculate that a similar heterogeneity of mechanisms of acquired resistance may occur in different HER2+/PIK3CA-mutant metastases in patients. Citation Format: Ariella B. Hanker, Christian D. Young, Thomas P. Stricker, Rebecca S. Cook, Carlos L. Arteaga. HER2/PIK3CAH1047R transgenic tumors develop acquired resistance to triple therapy with trastuzumab, pertuzumab, and PI3K inhibitors via multiple mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1822. doi:10.1158/1538-7445.AM2014-1822

Constitutively active Notch1 signaling promotes endothelial‑mesenchymal transition in a conditional transgenic mouse model.

Endothelial-mesenchymal transition (EndoMT) is a process in which endothelial cells lose their cell-type-specific characteristics and gain a mesenchymal cell phenotype. The Notch signaling pathway is crucial in the regulation of EndoMT; however, its roles have not been fully studied in vivo. In a previous study, we reported the generation of transgenic mice with a floxed β-geo/stop signal between a CMV promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1) linked with a human placental alkaline phosphatase (hPLAP) reporter (ZAP-IC-Notch1). In this study, we examined the results of activating IC-Notch1 in endothelial cells. ZAP-IC-Notch1 mice were crossed with Tie2-Cre mice to activate IC-Notch1 expression specifically in endothelial cells. The ZAP-IC-Notch1/Tie2-Cre double transgenic embryos died at E9.5–10.5 with disruption of vasculature and enlargement of myocardium. VE-cadherin expression was decreased and EphrinB2 expression was increased in the heart of these embryos. Mesenchymal cell marker α-smooth muscle actin (SMA) was expressed in IC-Notch1-expressing endothelial cells. In addition, upregulation of Snail, the key effector in mediating EndoMT, was identified in the cardiac cushion of the double transgenic murine embryo heart. The results of the present study demonstrate that constitutively active Notch signaling promotes EndoMT and differentially regulates endothelial/mesenchymal cell markers during cardiac development.

C/EBP homologous protein inhibits tissue repair in response to gut injury and is inversely regulated with chronic inflammation.

Loss of intestinal epithelial cell (IEC) homeostasis and apoptosis negatively affect intestinal barrier function. Uncontrolled activation of the unfolded protein response (UPR) in IEC contributes to an impaired barrier and is implicated in the pathogenesis of inflammatory bowel diseases. However, the contribution of the UPR target gene C/EBP homologous protein (CHOP), an apoptosis-associated transcription factor, to inflammation-related disease susceptibility remains unclear. Consistent with observations in patients with ulcerative colitis, we show that despite UPR activation in the epithelium, CHOP expression was reduced in mouse models of T-cell-mediated and bacteria-driven colitis. To elucidate the molecular mechanisms of IEC-specific CHOP expression, we generated a conditional transgenic mouse model (Chop(IEC Tg/Tg)). Chop overexpression increased the susceptibility toward dextran sodium sulfate (DSS)-induced intestinal inflammation and mucosal tissue injury. Furthermore, a delayed recovery from DSS-induced colitis and impaired closure of mechanically induced mucosal wounds was observed. Interestingly, these findings seemed to be independent of CHOP-mediated apoptosis. In vitro and in vivo cell cycle analyses rather indicated a role for CHOP in epithelial cell proliferation. In conclusion, these data show that IEC-specific overexpression impairs epithelial cell proliferation and mucosal tissue regeneration, suggesting an important role for CHOP beyond mediating apoptosis.

Modeling effects of mutant huntingtin (HTT)–associated pathology with a conditional transgenic mouse model

Modeling effects of mutant huntingtin (HTT)–associated pathology with a conditional transgenic mouse model

Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington's disease.

Huntington’s disease (HD) is a fatal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion leading to an elongated polyglutamine stretch in Huntingtin1. Mutant Huntingtin (mHTT) is ubiquitously expressed but elicits selective cortical and striatal neurodegeneration in HD2. The mechanistic basis for such selective neuronal vulnerability remains unclear. A necessary step towards resolving this enigma is to define the cell types in which mHTT expression is causally linked to the disease pathogenesis. Using a conditional human genomic transgenic mouse model of HD expressing full-length mHTT (BACHD)3, we genetically reduced mHTT expression in striatal, cortical, or both neuronal populations. We show that cortical mHTT reduction in BACHD partially improves motor and psychiatric-like behavioral deficits, but does not improve neurodegeneration, while mHTT reduction in both neuronal populations consistently ameliorates all behavioral deficits and selective brain atrophy in this HD model. Furthermore, mHTT reduction in cortical or striatal neurons partially ameliorates cortico-striatal synaptic deficits, while further restoration of striatal synaptic function is achieved by mHTT reduction in both neuronal cell types. Our study demonstrates distinct, but interacting roles of cortical and striatal mHTT in disease pathogenesis and suggests that optimal HD therapeutics may require targeting mHTT in both cortical and striatal neurons.

Pim1 Kinase Cooperates with Hormone Treatment to Promote Bladder and Ureteral Urothelial Hyperplasia

The Pim serine/threonine kinases have been shown to be overexpressed in cancer. Elevated levels of Pim1 kinase were demonstrated in human leukemia and lymphomas, as well as in solid tumors such as pancreatic, prostate and bladder cancers, and have been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic mouse models, they have only weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in bladder and ureteral urothelial cancer, we generated a conditional Pim1 transgenic mouse model and found that prostate specific antigen-(PSA)-driven Cre expression lead to transgene expression in the bladder upon (testosterone/estrogen) hormone treatment. We then explored the effect of Pim1 overexpression on hormone treatment, either alone or in combination with Pten haploinsufficiency. We found that Pim1 overexpression increased the severity of bladder and ureteral urothelial hyperplasias in both backgrounds, leading to pyelonephritis in transgenic animals. Our data suggest that Pim1 might contribute to progression, rather than initiation, and that the hyperplasias also contribute to the development of pyelonephritis.

Abstract A007: Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies

Abstract The HER2 (ERBB2) oncogene is amplified in 20-25% of breast cancers and is associated with poor patient outcome. Mutational activation of PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), occurs in ~30% of breast cancers. HER2 amplification and PIK3CA mutations often co-occur in breast cancer. Aberrant activation of the PI3K pathway has been shown to correlate with a diminished response to HER2-directed therapies. We sought to directly determine whether mutant PI3K enhances HER2-induced tumor progression and promotes resistance to HER2 inhibition in vivo. For this purpose, we generated a conditional transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared to mice expressing PIK3CA alone (p=0.0012) or HER2 alone (p&amp;lt;0.0001). HER2 and mutant PIK3CA also strongly cooperated to promote lung metastases following tail vein injection of cells derived from transgenic tumors (p&amp;lt;0.01). PIK3CA and HER2+/PIK3CA tumors were histologically diverse (i.e., adenocarcinoma, adenomyoepithelioma, adenocarcinoma with squamous metaplasia, and metaplastic carcinoma), expressed luminal and basal cytokeratins by IHC, and were associated with the claudin-low subtype of human breast cancers by microarray analysis. Accordingly, HER2+/PIK3CA tumors expressed elevated transcripts encoding EMT and stem cell markers, such as Snail, Slug, Twist, Bmi1, ITGB1 (CD29), and Procr. Further, single cells from HER2+/PIK3CA tumors formed more and larger mammospheres compared to cells from HER2-driven tumors. HER2+/PIK3CA mammospheres and tumors were completely resistant to the HER2 monocolonal antibody trastuzumab, alone and in combination with the HER2 tyrosine kinase inhibitor lapatinib or with pertuzumab, an antibody that inhibits ligand-induced HER2 dimerization. Both drug resistance and enhanced mammosphere formation were reversed by treatment with the pan-PI3K inhibitor BKM120. BKM120 treatment strongly inhibited T308 and S473 pAKT, whereas pAKT levels were unaffected by trastuzumab in combination with lapatinib or with pertuzumab. We further generated a model of resistance to the combination of trastuzumab, pertuzumab, and BKM120 by long-term treatment of HER2+/PIK3CA tumor transplants. Tumors initially responded, but a subset (3/11) resumed growth in the presence of drugs within 2 weeks of treatment initiation. Resistance was maintained following passaging in mice. We are currently performing whole-exome sequencing on resistant vs. untreated tumors in order to determine the mechanism(s) of resistance. In sum, PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers and generates resistance to FDA-approved combinations of anti-HER2 therapies. We propose the mouse model presented herein will be a valuable tool to investigate the underlying biology of HER2+/PI3K-mutant tumors and for the preclinical testing of therapeutic strategies against this subtype of breast cancer. Citation Format: Ariella B. Hanker, Adam Pfefferle, Justin M. Balko, Maria Gabriela Kuba, Christian D. Young, Violeta Sanchez, Cammie R. Sutton, Hailing Cheng, Charles M. Perou, Jean J. Zhao, Rebecca S. Cook, Carlos L. Arteaga. Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A007.

97. Impact of psychological stress on the adaptive immune system and disease progression in a triple negative breast and a transgenic ovarian cancer mouse model

Immune suppression plays a critical role in tumor progression, particularly in triple negative breast cancer (TNBC) and ovarian cancer. However the role of stress in adaptive immunity is still unclear. We hypothesize that psychological stress can regulate T cell mediated immunity, thus contributing to disease outcome. To test this hypothesis, we used female BALB/c mice injected with TNBC 4T1 mammary adenocarcinoma cells into the mammary fat pad and a LSLS-KrasG12D/+PtenloxP/loxp conditional transgenic mouse model for endometrioid ovarian cancer. For the TNBC model, mice were randomized into 4 groups (n = 10); (1) social isolation; (2) restraint stress for 1 h,3x/week; (3) restraint stress for 2 h, daily and (4) no stress. For the ovarian model, mice were restrained for 1 h,3x week. In the TNBC model, the stress groups demonstrated significantly reduced survival compared to non-stressed controls (p = 0.013) with none of the social isolation mice surviving. There was a significant difference across the groups for CD3 (p = 0.042), CD3CD69 (p = 0.047) and CD4FOXP3 (Tregs; p = 0.0014) in mouse spleens. In the ovarian mouse model, we found that stressed mice demonstrated early onset of tumors and a significant decrease in CD3CD69 (p = 0.006). These findings indicate that stress regulates T cell immunity in mice with TNBC and ovarian cancer and demonstrate the complexity of the stress response in tumor-bearing, immune competent mice.

MicroRNA-Antagonism Regulates Breast Cancer Stemness and Metastasis via TET-Family-Dependent Chromatin Remodeling

Tumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis inmouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoterof EMT and breast cancer stemness toward metastasis.

262 VENTRICULAR DYSFUNCTION PRECEDES CARDIAC HYPERTROPHY IN RESPONSE TO ENHANCED ANGIOTENSIN II TYPE 1 RECEPTOR ACTIVITY IN THE DIFFERENTIATED CARDIOMYOCYTE

Left ventricular hypertrophy (LVH) is frequently associated with elevated blood pressure and ventricular dysfunction in response to modulation of the renin-angiotensin system (RAS). Ultimately this leads to pathological remodelling with...

Abstract 267: MYC maintains the self-renewal of cancer stem cells through the regulation of the glutathione redox system.

Abstract Cancer stem cells (CSCs) are a rare population of self-renewing cells that can persist even after extensive therapeutic intervention. The molecular mechanism of CSC self-renewal is not yet clear. Here we show that MYC is causally involved in CSC self-renewal through the regulation of glutathione (GSH) redox system. In a MYC-driven conditional transgenic mouse model of T-cell acute lymphoblastic leukemia (T-ALL), we identified Sca-1+ CSCs. We found that these CSCs express the glutathione (GSH) redox system related genes including glutamate cysteine ligase (GCL) and Pyruvate Dehydrogenase Kinase 1 (PDK1) but low levels of expression of the mitochondrial redox enzyme, Superoxide Dismutase 2 (SOD2). Even a brief period of MYC inactivation in the Sca1+ CSCs led to their apoptosis associated with the downregulation of GCL and PDK1, but the upregulation of SOD2, in turn resulting in ROS-generation and mitochondrial damage. Moreover, suppression of SOD2 by siRNA silencing or the use of the peroxide scavenger, ascorbic acid, prevented MYC inactivation from inducing apoptosis in the Sca-1+ CSCs. Finally, in human CSCs, including primary human Chronic Myeloid Leukemia (CML), and human colon cancers, we confirmed that MYC expression was required for high GCL, PDK1 and low SOD2. Even the brief inactivation of MYC in human CSCs resulted in the loss of their self-renewal, as well as the global chance in redox state by decrease in GCL, PDK1 and increase in SOD2. We conclude that high levels of continued MYC expression are essential for CSC self-renewal through regulation of redox state. Citation Format: Bikul Das, Rika Tsuchida, Reza Bayat-Mokhtari, Jason R. Gotlib, Renee Reijo-Pera, Herman Yeger, Felsher W. Dean. MYC maintains the self-renewal of cancer stem cells through the regulation of the glutathione redox system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 267. doi:10.1158/1538-7445.AM2013-267 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar toESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon invitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

Functional Effects of GRM1 Suppression in Human Melanoma Cells

Ectopic expression of a neuronal receptor, metabotropic glutamate receptor 1 (Grm1), in melanocytes has been implicated in melanoma development in mouse models. The human relevance of this receptor's involvement in melanoma pathogenesis was shown by detecting GRM1 expression in subsets of human melanomas, an observation lacking in benign nevi or normal melanocytes. Grm1-transformed mouse melanocytes and a conditional Grm1 transgenic mouse model confirmed a requirement for sustained expression of Grm1 for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. Here, we investigate if continued GRM1 expression is also required in human melanoma cell lines by using two inducible, silencing RNA systems: the ecdysone/Ponasterone A and tetracycline on/off approaches to regulate GRM1 expression in the presence of each inducer. Various in vitro assays were conducted to assess the consequences of a reduction in GRM1 expression on cell proliferation, apoptosis, downstream targeted signaling pathways, and in vivo tumorigenesis. We showed that suppression of GRM1 expression in several human melanoma cell lines resulted in a reduction in the number of viable cells and a decrease in stimulated mitogen-activated protein kinase (MAPK) and PI3K/AKT and suppressed tumor progression in vivo. These results reinforce earlier observations where a reduction in cell growth in vitro and tumorigenesis in vivo were correlated with decreased GRM1 activities by pharmacologic inhibitors of the receptor, supporting the notion that GRM1 plays a role in the maintenance of transformed phenotypes in human melanoma cells in vitro and in vivo and could be a potential therapeutic target for the treatment of melanoma.

A New Mouse Model of Dry Eye Disease

Oxidative damage and inflammation are proposed to be involved in the age-related functional decline of lacrimal glands. The molecular mechanism(s) of how oxidative stress affects the secretory function of lacrimal glands was investigated because this is currently unclear. We used a novel mev-1 conditional transgenic mouse model (Tet-mev-1) with a modified tetracycline system. The mev-1 gene encodes the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria. Expression of the mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in aqueous secretory function. Tear volume in Tet-mev-1 mice was lower than in wild-type mice, and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. This new model provides evidence that mitochondria-induced oxidative damage in the lacrimal gland induces lacrimal dysfunction, resulting in dry eye disease. Our findings strongly suggest that oxidative stress can be a causative factor in the development of dry eye disease.