Suzuki T, Shen H, Akagi K, Morse HC, Malley JD, Naiman DQ.. New genes involved in cancer identified by retroviral tagging. Nat Genet 32: 166-174

Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland 21702, USA.
Nature Genetics (Impact Factor: 29.35). 10/2002; 32(1):166-74. DOI: 10.1038/ng949
Source: PubMed


Retroviral insertional mutagenesis in BXH2 and AKXD mice induces a high incidence of myeloid leukemia and B- and T-cell lymphoma, respectively. The retroviral integration sites (RISs) in these tumors thus provide powerful genetic tags for the discovery of genes involved in cancer. Here we report the first large-scale use of retroviral tagging for cancer gene discovery in the post-genome era. Using high throughput inverse PCR, we cloned and analyzed the sequences of 884 RISs from a tumor panel composed primarily of B-cell lymphomas. We then compared these sequences, and another 415 RIS sequences previously cloned from BXH2 myeloid leukemias and from a few AKXD lymphomas, against the recently assembled mouse genome sequence. These studies identified 152 loci that are targets of retroviral integration in more than one tumor (common retroviral integration sites, CISs) and therefore likely to encode a cancer gene. Thirty-six CISs encode genes that are known or predicted to be genes involved in human cancer or their homologs, whereas others encode candidate genes that have not yet been examined for a role in human cancer. Our studies demonstrate the power of retroviral tagging for cancer gene discovery in the post-genome era and indicate a largely unrecognized complexity in mouse and presumably human cancer.

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Available from: Herbert C Morse, Oct 06, 2015
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    • "Efforts to identify the nature and role of the Ras-related intracellular signalization pathways have recently suggested that not only the downstream mediators but also the upstream regulators of Ras might play a role on these mechanisms. In perfect agreement with this proposal, members of RasGRP family are suggested to function as oncogenes in multiple cancers [10]. Indeed, RasGRP3 is highly expressed in human Burkitt’s lymphoma, human pre-B-cell leukemia, and natural killer-like T-cell leukemia. "
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    ABSTRACT: Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe TM DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells. Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.
    Molecular Cancer 04/2014; 13(1):96. DOI:10.1186/1476-4598-13-96 · 4.26 Impact Factor
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    • "Common insertion sites (CIS) are integration hotspots that may represent an early readout of insertional mutagenesis or an intrinsic bias of the vector of interest. By means of a specific analysis (42), we identified a single CIS in a 56-Kb region encompassing the galc gene that was targeted by 17 integrations, exclusively in exons and only in bdLV.GALC-injected mice. In agreement with a previous report on LV-injected adult brain tissues (36), we found that ∼60% of LV integrations mapped within genes (Fig. 8B). "
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    ABSTRACT: Globoid Cell Leukodystrophy (GLD) is an inherited lysosomal storage disease caused by β-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in CNS tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30-50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective, and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders.
    Human Molecular Genetics 01/2014; 23(12). DOI:10.1093/hmg/ddu034 · 6.39 Impact Factor
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    • "Dysregulated expression of Notch can affect HSC cell differentiation and lead to skewed differentiation of hematopoietic lineages [58]. A study of murine tumor retroviral insertion sites by Suzuki and colleagues identified Notch as a CIS [59]. Based on the role of Notch family genes in hematopoietic differentiation, dysregulation by proviral insertional mutagenesis has the potential to enhance hematopoietic repopulation. "
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    ABSTRACT: Hematopoietic stem cell (HSC) therapy using replication-incompetent retroviral vectors is a promising approach to provide life-long correction for genetic defects. HSC gene therapy clinical studies have resulted in functional cures for several diseases, but in some studies clonal expansion or leukemia has occurred. This is due to the dyregulation of endogenous host gene expression from vector provirus insertional mutagenesis. Insertional mutagenesis screens using replicating retroviruses have been used extensively to identify genes that influence oncogenesis. However, retroviral mutagenesis screens can also be used to determine the role of genes in biological processes such as stem cell engraftment. The aim of this review is to describe the potential for vector insertion site data from gene therapy studies to provide novel insights into mechanisms of HSC engraftment. In HSC gene therapy studies dysregulation of host genes by replication-incompetent vector proviruses may lead to enrichment of repopulating clones with vector integrants near genes that influence engraftment. Thus, data from HSC gene therapy studies can be used to identify novel candidate engraftment genes. As HSC gene therapy use continues to expand, the vector insertion site data collected will be of great interest to help identify novel engraftment genes and may ultimately lead to new therapies to improve engraftment.
    09/2013; 2013(Suppl 3). DOI:10.4172/2157-7633.S3-004
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