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Nature communications  2015;6:5987.
Triple-negative breast cancer (TNBC) has poor prognostic outcome compared to other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and in normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC targeted therapies.
PMCID: PMC4338552  PMID: 25574598
2.  The Tetraspanin Protein Peripherin-2 Forms a Complex with Melanoregulin, a Putative Membrane Fusion Regulator† 
Biochemistry  2007;46(5):1256-1272.
Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/− retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2–ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST–PerCter protein selectively pulled down His–MREG and His–MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341–Gly346), and kinetic data predicted a KD of 80 nM for PerCter–MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk–plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.
PMCID: PMC4472003  PMID: 17260955
3.  BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells 
Nature Communications  2015;6:5987.
Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies.
Triple-negative breast cancers (TNBCs) tend to have poor prognosis; however, the mechanisms underlying TNBC pathology are not well understood. Here the authors utilize epidemiologic data and animal models to demonstrate an important role for BCL11A in the genesis and propagation of TNBCs.
PMCID: PMC4338552  PMID: 25574598
4.  Mutation of Murine Sox4 Untranslated Regions Results in Partially Penetrant Perinatal Lethality 
In vivo (Athens, Greece)  2014;28(5):709-718.
Sox4 is an essential gene, and genetic deletion results in embryonic lethality. In an effort to develop mice with tissue-specific deletion, we bred conditional knockout mice bearing LoxP recombination sites flanking the Sox4 gene, with the LoxP sites located in the Sox4 5’UTR and 3’UTR.
The number of mice homozygous for this LoxP-flanked conditional knockout allele was far below the expected number, suggesting embryonic lethality with reduced penetrance. From over 200 animals bred, only 11% were homozygous Sox4flox/flox mice compared to the expected Mendelian ratio of 25% (p < 0.001). Moreover, there was a significant reduction in the number of female Sox4flox/flox mice (26%) relative to male Sox4flox/flox mice (p = 0.0371). Reduced Sox4 expression in homozygous embryos was confirmed by in-situ hybridization and Quantitative real-time polymerase chain reaction (QPCR).
LoxP sites in the 5’ and 3’ UTR of both alleles of Sox4 resulted in reduced, but variable expression of Sox4 message.
PMCID: PMC4237010  PMID: 25189881
Mouse; Sox4; perinatal lethality; transcription
5.  Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse 
Developmental biology  2013;384(1):10.1016/j.ydbio.2013.09.027.
The integrity and function of epithelial tissues depends on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.
PMCID: PMC3853123  PMID: 24095903
Scrib; PDZ proteins; Lens development; Epithelial to mesenchymal transition; Cell adhesion; Cell polarity
6.  Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model 
Nature genetics  2013;46(1):24-32.
The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.
PMCID: PMC4131144  PMID: 24316982
7.  Response and Resistance to MEK Inhibition in Leukaemias Initiated by Hyperactive Ras 
Nature  2009;461(7262):411-414.
The Raf/MEK/ERK cascade is a therapeutic target in human cancers with deregulated Ras signaling, which includes tumours that have inactivated the Nf1 tumour suppressor1. Nf1 encodes neurofibromin, a GTPase activating protein that terminates Ras signalling by stimulating hydrolysis of Ras•GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed due to outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4, and Mapk14, which encodes p38α. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.
PMCID: PMC4119783  PMID: 19727076
8.  Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development 
Development (Cambridge, England)  2012;139(10):1831-1841.
Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C2H2 zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.
PMCID: PMC4067532  PMID: 22491945
Spinal cord; Transcription factor; Neuronal differentiation; Bcl11a (CTIP1); Mouse
9.  Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects 
PLoS ONE  2014;9(2):e89397.
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1δex3/δex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1δex3/δex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1δex3/δex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
PMCID: PMC3937339  PMID: 24586749
10.  Musashi1-CreERT2: A New Cre Line for Conditional Mutagenesis in Neural Stem Cells 
Genesis (New York, N.Y. : 2000)  2012;51(2):128-134.
The RNA-binding protein Musashi1 (Msi1) is one of two mammalian homologues of Drosophila Musashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS) Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long-term neurogenesis in the olfactory bulb (OB) but it remains unclear whether Msi1-expressing cells have self-renewing potential and can contribute to neurogenesis in the adult. Here we describe the generation of Msi1-CreERT2 knock-in mice and show by cell lineage tracing that Msi1-CreERT2-expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1-CreERT2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development.
PMCID: PMC3577968  PMID: 23132814
Msi1; NSCs; SVZ; Olfactory epithelium; Knock-in mice
11.  LIM Domain Only-2 (LMO2) Induces T-Cell Leukemia by Two Distinct Pathways 
PLoS ONE  2014;9(1):e85883.
The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.
PMCID: PMC3897537  PMID: 24465765
12.  Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages 
Absence of the transcriptional regulator Zfp521 results in decreased bone formation by osteoblasts and increased osteoclast differentiation, largely via Zfp521’s regulation of the transcription factor Ebf1.
Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521+/− mice was rescued in Zfp521+/−:Ebf1+/− mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis.
PMCID: PMC3646489  PMID: 23569325
13.  A modified Sleeping Beauty transposon system that can be used to model a wide variety of human cancers in mice 
Cancer research  2009;69(20):8150-8156.
Recent advances in cancer therapeutics stress the need for a better understanding of the molecular mechanisms driving tumor formation. This can be accomplished by obtaining a more complete description of the genes that contribute to cancer. We previously described an approach using the Sleeping Beauty transposon system to model hematopoietic malignancies in mice. Here we describe modifications of the SB system that provide additional flexibility in generating mouse models of cancer. First, we describe a Cre-inducible SBase allele, RosaSBaseLsL, that allows restriction of transposon mutagenesis to a specific tissue of interest. This allele was used to generate a model of germinal center B-cell lymphoma by activating SBase expression with a Aid-Cre allele. In a second approach, a novel transposon was generated, T2/Onc3, in which the CMV enhancer/β-actin (CAG) promoter drives oncogene expression. When combined with ubiquitous SBase expression, the T2/Onc3 transposon produced nearly 200 independent tumors of over 20 different types in a cohort of 62 mice. Analysis of transposon insertion sites identified novel candidate genes, including Zmiz1 and Rian, involved in squamous cell carcinoma and hepatocellular carcinoma, respectively. These novel alleles provide additional tools for the SB system and provide some insight into how this mutagenesis system can be manipulated to model cancer in mice.
PMCID: PMC3700628  PMID: 19808965
14.  Bcl11a is essential for lymphoid development and negatively regulates p53 
The Journal of Experimental Medicine  2012;209(13):2467-2483.
Bcl11a regulates development of lymphoid cells in adult mice in part by inhibiting expression of p53.
Transcription factors play important roles in lymphopoiesis. We have previously demonstrated that Bcl11a is essential for normal lymphocyte development in the mouse embryo. We report here that, in the adult mouse, Bcl11a is expressed in most hematopoietic cells and is highly enriched in B cells, early T cell progenitors, common lymphoid progenitors (CLPs), and hematopoietic stem cells (HSCs). In the adult mouse, Bcl11a deletion causes apoptosis in early B cells and CLPs and completely abolishes the lymphoid development potential of HSCs to B, T, and NK cells. Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a. Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities. Overexpression of Bcl2 and Mdm2, or p53 deficiency, rescues both lethality and proliferative defects in Bcl11a-deficient early B cells and enables the mutant CLPs to differentiate to lymphocytes. Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities. Deletion of Bcl11a may represent a new approach for generating a mouse model that completely lacks an adaptive immune system.
PMCID: PMC3526365  PMID: 23230003
15.  Reprogramming of T Cells to Natural Killer–Like Cells upon Bcl11b Deletion 
Science (New York, N.Y.)  2010;329(5987):85-89.
T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction and defense against microbes and viruses. Here we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell–associated gene expression. These induced T-to–natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.
PMCID: PMC3628452  PMID: 20538915
16.  Identification of Rtl1, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis 
PLoS Genetics  2013;9(4):e1003441.
We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB–induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.
Author Summary
HCC is the third deadliest cancer worldwide, largely due to a lack of effective treatment options. Therapeutic approaches targeted at the molecular mechanisms underlying tumor formation and progression have shown great efficacy for treating other tumor types. Unfortunately, however, much remains to be learned about the molecular pathogenesis of HCC. There is an urgent need to identify and characterize genetic alterations that drive HCC in order to facilitate the development of more effective targeted therapeutics for patients. Here, we present data showing that recurrent mutations identified in a mouse model of HCC result in overexpression of the Rtl1 gene. We have validated Rtl1 as a driver of HCC by demonstrating that its overexpression in mouse liver causes tumor formation. We also detected overexpression of this gene in a significant proportion of human HCC samples, suggesting that it may be a relevant therapeutic target for patients with this disease.
PMCID: PMC3616914  PMID: 23593033
17.  Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes 
Biankin, Andrew V. | Waddell, Nicola | Kassahn, Karin S. | Gingras, Marie-Claude | Muthuswamy, Lakshmi B. | Johns, Amber L. | Miller, David K. | Wilson, Peter J. | Patch, Ann-Marie | Wu, Jianmin | Chang, David K. | Cowley, Mark J. | Gardiner, Brooke B. | Song, Sarah | Harliwong, Ivon | Idrisoglu, Senel | Nourse, Craig | Nourbakhsh, Ehsan | Manning, Suzanne | Wani, Shivangi | Gongora, Milena | Pajic, Marina | Scarlett, Christopher J. | Gill, Anthony J. | Pinho, Andreia V. | Rooman, Ilse | Anderson, Matthew | Holmes, Oliver | Leonard, Conrad | Taylor, Darrin | Wood, Scott | Xu, Qinying | Nones, Katia | Fink, J. Lynn | Christ, Angelika | Bruxner, Tim | Cloonan, Nicole | Kolle, Gabriel | Newell, Felicity | Pinese, Mark | Mead, R. Scott | Humphris, Jeremy L. | Kaplan, Warren | Jones, Marc D. | Colvin, Emily K. | Nagrial, Adnan M. | Humphrey, Emily S. | Chou, Angela | Chin, Venessa T. | Chantrill, Lorraine A. | Mawson, Amanda | Samra, Jaswinder S. | Kench, James G. | Lovell, Jessica A. | Daly, Roger J. | Merrett, Neil D. | Toon, Christopher | Epari, Krishna | Nguyen, Nam Q. | Barbour, Andrew | Zeps, Nikolajs | Kakkar, Nipun | Zhao, Fengmei | Wu, Yuan Qing | Wang, Min | Muzny, Donna M. | Fisher, William E. | Brunicardi, F. Charles | Hodges, Sally E. | Reid, Jeffrey G. | Drummond, Jennifer | Chang, Kyle | Han, Yi | Lewis, Lora R. | Dinh, Huyen | Buhay, Christian J. | Beck, Timothy | Timms, Lee | Sam, Michelle | Begley, Kimberly | Brown, Andrew | Pai, Deepa | Panchal, Ami | Buchner, Nicholas | De Borja, Richard | Denroche, Robert E. | Yung, Christina K. | Serra, Stefano | Onetto, Nicole | Mukhopadhyay, Debabrata | Tsao, Ming-Sound | Shaw, Patricia A. | Petersen, Gloria M. | Gallinger, Steven | Hruban, Ralph H. | Maitra, Anirban | Iacobuzio-Donahue, Christine A. | Schulick, Richard D. | Wolfgang, Christopher L. | Morgan, Richard A. | Lawlor, Rita T. | Capelli, Paola | Corbo, Vincenzo | Scardoni, Maria | Tortora, Giampaolo | Tempero, Margaret A. | Mann, Karen M. | Jenkins, Nancy A. | Perez-Mancera, Pedro A. | Adams, David J. | Largaespada, David A. | Wessels, Lodewyk F. A. | Rust, Alistair G. | Stein, Lincoln D. | Tuveson, David A. | Copeland, Neal G. | Musgrove, Elizabeth A. | Scarpa, Aldo | Eshleman, James R. | Hudson, Thomas J. | Sutherland, Robert L. | Wheeler, David A. | Pearson, John V. | McPherson, John D. | Gibbs, Richard A. | Grimmond, Sean M.
Nature  2012;491(7424):399-405.
Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
PMCID: PMC3530898  PMID: 23103869
18.  Whole-body Sleeping Beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality 
Cancer research  2009;69(21):8429-8437.
The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system can be used to identify new genes involved in lymphomagenesis/leukemiogenesis.
PMCID: PMC2771123  PMID: 19843846
Transposon; leukemia; glioma
19.  Toll-Like Receptor 3 Expressing Tumor Parenchyma and Infiltrating Natural Killer Cells in Hepatocellular Carcinoma Patients 
Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.
HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3–9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan–Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.
TLR3 activation increased cell death in the TLR3+ SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).
TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.
PMCID: PMC3814220  PMID: 23197495
20.  Aberrant Epithelial-Mesenchymal Hedgehog Signaling Characterizes Barrett's Metaplasia 
Gastroenterology  2010;138(5):1810-1822.
Background & Aims
The molecular mechanism underlying epithelial metaplasia in Barrett's esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barrett's esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium.
Immunohistochemistry, immunofluorescence, and quantitative real-time PCR were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice.
Marked upregulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barrett's epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barrett's epithelium. We further show that expression of DMBT1, the human homologue of the columnar cell factor Hensin, occurs in Barrett's epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9 and columnar cytokeratins.
Epithelial Hedgehog ligand expression may contribute to the initiation of Barrett's esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype.
PMCID: PMC3422577  PMID: 20138038
Hedgehog signaling; Barrett's esophagus; BMP4; SOX9
21.  A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis 
The EMBO Journal  2012;31(13):2922-2936.
The transcription factor Bcl11b/Ctip2 promotes hippocampal progenitor proliferation and neural differentiation in a non-cell autonomous manner by regulating the expression of the cell adhesion molecule Desmoplakin. Forebrain-specific ablation causes defective spatial learning and memory.
The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.
PMCID: PMC3395096  PMID: 22588081
Bcl11b/Ctip2; dentate gyrus; neurogenesis; transcription factor
22.  Critical role of Bcl11b in suppressor function of T regulatory cells and prevention of inflammatory bowel disease 
The Journal of Experimental Medicine  2011;208(10):2069-2081.
Bcl11b is required for optimal FoxP3 expression and suppressor function by regulatory T cells and for the generation of inducible regulatory T cells.
Dysregulated CD4+ T cell responses and alterations in T regulatory cells (Treg cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in Treg cells causes IBD because of proinflammatory cytokine-producing CD4+ T cells infiltrating the colon. Provision of WT Treg cells prevented IBD, demonstrating that alterations in Treg cells are responsible for the disease. Furthermore, Bcl11b-deficient Treg cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced Treg cells (iTreg cells) after Tgf-β treatment of conventional CD4+ T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for Treg suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4+ T cells in response to Tgf-β and generation of iTreg cells.
PMCID: PMC3182057  PMID: 21875956
23.  Targeted Inactivation of Kinesin-1 in Pancreatic β-Cells In Vivo Leads to Insulin Secretory Deficiency 
Diabetes  2010;60(1):320-330.
Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in β-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.
A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic β-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls.
Mice with β-cell specific deletion of Kif5b (Kif5bfl/−:RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5bfl/−:RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of β-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size. However, compared with controls, pancreas of Kif5bfl/−:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.
In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.
PMCID: PMC3012189  PMID: 20870970
24.  Zfp521 Is a Target Gene and Key Effector of Parathyroid Hormone-Related Peptide Signaling in Growth Plate Chondrocytes 
Developmental cell  2010;19(4):533-546.
In the growth plate, the interplay between Parathyroid Hormone-Related Peptide (PTHrP) and Indian Hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional co-regulator, in pre-hypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP-/- and chondrocyte-specific PTHR1-/- mice, with decreased chondrocyte proliferation, early hypertrophic transition and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased cyclin D1 and Bcl-2 expression while increasing caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to up-regulate cyclin D1 and to antagonize Runx2, Ihh and Collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.
PMCID: PMC2958174  PMID: 20951345
25.  Synphilin-1 attenuates neuronal degeneration in the A53T α-synuclein transgenic mouse model 
Human Molecular Genetics  2010;19(11):2087-2098.
Genetic alterations in α-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an α-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in α-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T α-synuclein double-transgenic mice survived longer than A53T α-synuclein single-transgenic mice. There were attenuated A53T α-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of α-synucleinopathy and play a neuroprotective role against A53T α-synuclein toxicity in vivo.
PMCID: PMC2865369  PMID: 20185556

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