Andrew Wakeham

University Health Network, Toronto, Ontario, Canada

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Publications (109)1902.19 Total impact

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    ABSTRACT: Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size.
    Molecular cell 03/2014; 53(6):904-15. · 14.61 Impact Factor
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    ABSTRACT: Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase-AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.
    Proceedings of the National Academy of Sciences 02/2014; · 9.81 Impact Factor
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    ABSTRACT: The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility.
    Proceedings of the National Academy of Sciences 01/2014; · 9.81 Impact Factor
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    ABSTRACT: Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.
    Journal of Experimental Medicine 07/2013; · 13.21 Impact Factor
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    ABSTRACT: Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.Oncogene advance online publication, 27 May 2013; doi:10.1038/onc.2013.183.
    Oncogene 05/2013; · 8.56 Impact Factor
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    ABSTRACT: Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.
    Genes & development 05/2013; 27(10):1101-1114. · 12.08 Impact Factor
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    ABSTRACT: It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.24.
    Oncogene 02/2013; · 8.56 Impact Factor
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    ABSTRACT: Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.
    Proceedings of the National Academy of Sciences 01/2013; · 9.81 Impact Factor
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    ABSTRACT: The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBβ in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.
    Nature Immunology 11/2012; · 26.20 Impact Factor
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    ABSTRACT: Flotillin microdomains, specialized lipid raft domains in cell membranes, serve as physical platforms for many different molecules important in crucial intracellular signaling pathways. Flotillin-2 (Flot2), together with flotillin-1, is a marker for lipid raft microdomains distinct from caveolar lipid rafts, and has been implicated in the progression of cancer and metastasis formation. Based largely on studies in xenograft models, flotillin-2 has been implicated in the progression of multiple types of human tumors, including breast cancer. In our studies, we identified flotillin-2 as highly amplified in a high-throughput comparative genomic hybridization screen of human breast cancer cell lines and breast tumor samples. Short hairpin RNA-mediated reduction of flotillin-2 protein levels significantly reduced the tumorigenicity and metastatic capability of a human breast cancer cell line in vivo. We generated mice deficient for flotillin-2 and also found a reduction of flotillin-1 protein levels and complete absence of flotillin-specific membrane microdomains in these mice. To examine the role of Flot2 in mammary tumorigenesis and lung metastasis, we used an in vivo molecular genetics approach, crossing a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with gene-targeted Flot2(-/-) mice. Flotillin-2 deficiency lead to a striking reduction in the number of lung metastasis observed, but had no influence on primary tumor formation in this model. Our results indicate, using a novel in vivo animal model approach, that Flot2 is an important regulator of mammary tumor-derived lung metastasis.Oncogene advance online publication, 12 November 2012; doi:10.1038/onc.2012.499.
    Oncogene 11/2012; · 8.56 Impact Factor
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    ABSTRACT: Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.
    Genes & development 08/2012; 26(18):2038-49. · 12.08 Impact Factor
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    ABSTRACT: T cell immunoglobulin and mucin domain-containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (T(H)1) cells from galectin-9-mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen-specific CD4(+) T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3(hi), interferon-γ (IFN-γ)(lo)CD4(+) cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3(+) T cells from mouse tumors and HIV-1-infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3-dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.
    Nature medicine 08/2012; · 27.14 Impact Factor
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    ABSTRACT: Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.
    Nature 07/2012; 488(7413):656-9. · 38.60 Impact Factor
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    ABSTRACT: Innate immune responses are vital for pathogen defense but can result in septic shock when excessive. A key mediator of septic shock is tumor necrosis factor-α (TNFα), which is shed from the plasma membrane after cleavage by the TNFα convertase (TACE). We report that the rhomboid family member iRhom2 interacted with TACE and regulated TNFα shedding. iRhom2 was critical for TACE maturation and trafficking to the cell surface in hematopoietic cells. Gene-targeted iRhom2-deficient mice showed reduced serum TNFα in response to lipopolysaccharide (LPS) and could survive a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to control the replication of Listeria monocytogenes. Our study has identified iRhom2 as a regulator of innate immunity that may be an important target for modulating sepsis and pathogen defense.
    Science 01/2012; 335(6065):229-32. · 31.20 Impact Factor
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    ABSTRACT: The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.
    Proceedings of the National Academy of Sciences 06/2011; 108(25):10243-8. · 9.81 Impact Factor
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    ABSTRACT: Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.
    Genes & development 05/2011; 25(10):1041-51. · 12.08 Impact Factor
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    ABSTRACT: 14-3-3σ regulates cytokinesis and cell cycle arrest induced by DNA damage but its role in the immune system is unknown. Using gene-targeted 14-3-3σ-deficient (i.e., KO) mice, we studied the role of 14-3-3σ in B-cell functions. Total numbers of B cells were reduced by spontaneous apoptosis of peripheral B cells. Upon B-cell antigen receptor engagement in vitro, KO B cells did not proliferate properly or up-regulate CD86. In response to T cell-independent antigens, KO B cells showed poor secretion of antigen-specific IgM. This deficit led to increased lethality of KO mice after vesicular stomatitis virus infection. KO B cells showed elevated total FOXO transcriptional activity but also increased FOXO1 degradation. Coimmunoprecipitation revealed that endogenous 14-3-3σ protein formed a complex with FOXO1 protein. Our results suggest that 14-3-3σ maintains FOXO1 at a consistent level critical for normal B-cell antigen receptor signaling and B-cell survival.
    Proceedings of the National Academy of Sciences 01/2011; 108(4):1555-60. · 9.81 Impact Factor
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    ABSTRACT: Estrogen is a key regulator of normal function of female reproductive system and plays a pivotal role in the development and progression of breast cancer. Here, we demonstrate that JMJD2B (also known as KDM4B) constitutes a key component of the estrogen signaling pathway. JMJD2B is expressed in a high proportion of human breast tumors, and that expression levels significantly correlate with estrogen receptor (ER) positivity. In addition, 17-beta-estradiol (E2) induces JMJD2B expression in an ERα dependent manner. JMJD2B interacts with ERα and components of the SWI/SNF-B chromatin remodeling complex. JMJD2B is recruited to ERα target sites, demethylates H3K9me3 and facilitates transcription of ER responsive genes including MYB, MYC and CCND1. As a consequence, knockdown of JMJD2B severely impairs estrogen-induced cell proliferation and the tumor formation capacity of breast cancer cells. Furthermore, Jmjd2b-deletion in mammary epithelial cells exhibits delayed mammary gland development in female mice. Taken together, these findings suggest an essential role for JMJD2B in the estrogen signaling, and identify JMJD2B as a potential therapeutic target in breast cancer.
    PLoS ONE 01/2011; 6(3):e17830. · 3.73 Impact Factor
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    ABSTRACT: Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.
    Blood 11/2010; 116(19):3792-801. · 9.78 Impact Factor
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    ABSTRACT: Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.
    Proceedings of the National Academy of Sciences 07/2010; 107(27):12186-91. · 9.81 Impact Factor

Publication Stats

21k Citations
1,902.19 Total Impact Points

Institutions

  • 2004–2014
    • University Health Network
      • Campbell Family Institute for Breast Cancer Research
      Toronto, Ontario, Canada
  • 2013
    • Università degli Studi di Torino
      Torino, Piedmont, Italy
  • 1993–2013
    • University of Toronto
      • Department of Medical Biophysics
      Toronto, Ontario, Canada
  • 2004–2012
    • Ontario Institute for Cancer Research
      Toronto, Ontario, Canada
  • 2010–2011
    • National Cancer Centre Singapore
      • Division of Cellular and Molecular Research
      Singapore
  • 2008–2010
    • The Princess Margaret Hospital
      Toronto, Ontario, Canada
  • 2007
    • Princeton University
      Princeton, New Jersey, United States
  • 2003
    • Kyushu University
      • Department of Pediatrics
      Hukuoka, Fukuoka, Japan
  • 2001
    • Osaka University
      • Division of Molecular and Cellular Pharmacology
      Ōsaka-shi, Osaka-fu, Japan
  • 1996–2001
    • Amgen Canada
      Mississauga, Ontario, Canada
  • 2000
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada
    • Samuel Lunenfeld Research Institute
      Toronto, Ontario, Canada