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Sleeping beauty: a novel cancer gene discovery tool
Abstract
The National Cancer Institute and the National Human Genome Research Institute recently announced a 3-year 100-million-dollar pilot study to use large-scale resequencing of genes in human tumors to identify new cancer genes. The hope is that some of these genes can be used as drug targets for developing better therapeutics for treating cancer. Although this effort will identify new cancer genes, it could be made more efficient by preferentially resequencing genes identified as novel candidate cancer genes in animal models of cancer. Although retroviral insertional mutagenesis has proven to be an effective tool for identifying novel cancer genes in the mouse, these studies are limited by the fact that retroviral mutagenesis primarily induces hematopoietic and mammary cancer, but little else, while the majority of cancers affecting humans are solid tumors. Recently, two groups have shown that sleeping beauty (SB) transposon-based insertional mutagenesis can also identify novel candidate cancer genes in the mouse. Unlike retroviral infection, SB transposition can be controlled to mutagenize any target tissue and thus potentially induce many different kinds of cancer, including solid tumors. SB transposition in animal models of cancer could therefore greatly facilitate the identification of novel human cancer genes and the development of better cancer therapies.
... Due to these intrinsic properties, the mobile nature of DNA transposons has been successfully exploited to provide efficient insertional mutagens or sensors in functional genomics (de la Rosa et al., 2017;Dupuy et al., 2006;Ruf et al., 2011) as well as artificial gene carriers in transgenesis (Miskey et al., 2005;Skipper et al., 2013) and even in gene therapy applications (Kebriaei et al., 2016). One recent and exciting application is the high resolution mapping of nucleosome positions using transposon integrations into accessible chromatin followed by sequencing [called ATAC-seq method; (Buenrostro et al., 2013;Chen et al., 2016)]. ...
... Among Tc1/mariners, the Sleeping Beauty (SB) transposon (Ivics et al., 1997) is exceptionally efficient in inserting into vertebrate genomes (Dupuy et al., 2006), making it a prime genetic tool with applications spanning from transgenesis (Ammar et al., 2012), forward mutagenesis screening (de la Rosa et al., 2017;Dupuy et al., 2006;Ruf et al., 2011) to even cancer immunotherapy trials in humans (Kebriaei et al., 2016;Singh et al., 2015). However, the mechanistic principles underlying SB's transposition and distinctive performance have remained elusive, greatly hampering the rational design and further improvement of this transposon as genetic tool in research and medicine. ...
... Among Tc1/mariners, the Sleeping Beauty (SB) transposon (Ivics et al., 1997) is exceptionally efficient in inserting into vertebrate genomes (Dupuy et al., 2006), making it a prime genetic tool with applications spanning from transgenesis (Ammar et al., 2012), forward mutagenesis screening (de la Rosa et al., 2017;Dupuy et al., 2006;Ruf et al., 2011) to even cancer immunotherapy trials in humans (Kebriaei et al., 2016;Singh et al., 2015). However, the mechanistic principles underlying SB's transposition and distinctive performance have remained elusive, greatly hampering the rational design and further improvement of this transposon as genetic tool in research and medicine. ...
Transposons are DNA segments that autonomously move within and between genomes across the tree of life. Tc1/mariners in particular have frequently crossed species boundaries in nature and provide powerful broad-host-range genetic vectors. Among them, the Sleeping Beauty (SB) transposon inserts DNA in vertebrate genomes with extraordinarily high efficiency, making it a prime genetic tool with applications expanding to gene therapy clinical trials. Nevertheless, the molecular principles of SB’s distinctive activity remain elusive, greatly hampering its further development. In the first part of this thesis, I investigated the molecular mechanisms of the SB transposon in comparison to Human mariner 1 (Hsmar1), a representative transposon of the same superfamily. Using biochemical and biophysical techniques together with fluorescence-based assays, I have characterized the initial steps of SB and Hsmar1 transposition and shown that the two transposons assemble their molecular machineries (or transpososomes) differently. By combining crystallographic data and SAXS-based modelling, I visualized the structural basis of these differences and explained how transpososome assembly is coupled to catalysis in the Hsmar1 transposon. Moreover, the data demonstrated that the unique assembly pathway of SB largely contributes to its exceptional efficiency and that it can be chemically modulated to control insertion rates in living cells. I have further reconstituted in vitro the ordered series of events comprising SB transposition, including transposon end binding, cleavage, and integration, and dissected previously unrevealed molecular features of the process. In the second part of my work, building on these mechanistic insights, I developed a novel SB transposase variant (hsSB) by employing a structure-based protein design approach. Using hsSB allowed for establishing a new genome engineering method based on the direct delivery of recombinant SB protein to cells. We showed that this new method, named SBprotAct, provides safer and more controlled genome modification of several cell types (including stem cells and human T cells), as compared to the state-of-art technology. This work sheds first light on the molecular determinants of SB transposition and its hyper-activity, providing a unique resource for the rational design of improved genome engineering platforms for research and medicine.
... Such experiments provide an association between the selected phenotype and the locus/gene at which the insertion occurred. Insertional mutagenesis is increasingly popular in the cancer research community, in which researchers are using them for forward genetic screens to identify cancer-causing genes (Dupuy, Jenkins, & Copeland, 2006). ...
... Beauty can be activated in a tissue specific manner in the somatic cells of the host organism (Dupuy et al., 2006). Tumors are harvested, from which genomic DNA is extracted and sequenced. ...
... Although the system generated tumors, they were mostly hematopoietic. Most of the tumors prevalent in humans are solid tumors (Dupuy et al., 2006). (Dupuy et al., 2006). ...
... A widely used transposon system is SB, which is a synthetic element reconstructed from inactive copies in fish genomes 16 . Owing to its high insertion efficiency in vertebrates, SB has been applied for functional genomics, cancer gene discovery, transgenesis and gene therapy applications (reviewed elsewhere [17][18][19][20]. In the medical setting, SB is being used in several clinical phase 1 or 2 trials for ex vivo engineering of therapeutic cells, including production of CAR T cells for cancer immunotherapy 18,19,21,22 . ...
... This direct correlation was not observed when the protein was provided from an expression plasmid (Fig. 2e). Moreover, hsSB protein generated fewer insertions (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) per cell than the plasmidbased strategy (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) at similar transgenesis rates (Fig. 2b,e and Supplementary Fig. 2b). Because uncontrolled transgene integration can lead to disruption or misregulation of nearby genes, lowering the copy number is important to reduce the risk of genomic damage in target cells. ...
The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.
... Este ha sido el caso de la integrasa ΦC31 [30], que integra fragmentos acompañados de una secuencia de 285 pb llamada attB en sitios específicos del genoma (pseudo-attP) [31][32][33]. También, se ha propuesto el uso de transposones [34,35], los AAV recombinantes [36] El adenovirus fue ampliamente utilizado en las fuerzas armadas americanas como vacuna, donde se confirmó que producía gran número de síndromes respiratorios febriles [40,41]. Estudios epidemiológicos confirmaron que los adenovirus provocan una pequeña proporción de las enfermedades respiratorias en la población general y un 7-10% en niños [40,42]. ...
... Aunque simple, este método presenta gran dificultad ya que son escasas las dianas de restricción únicas localizadas en el genoma viral. Además, la clonación de fragmentos pequeños de ADN (0.5-3 Kpb) en plásmidos grandes (35)(36) es muy poco eficiente. El método de ligación directa requiere, frecuentemente, el uso del ensayo en placa para el aislamiento de clones positivos, debido a que muchos de los genomas amplificados suelen resultar adenovirus salvajes o por otro lado, nulos en cuanto a la expresión del transgén clonado. ...
... A significant number of mice that inherit both alleles die during embryogenesis, and the remaining mice develop aggressive tumors ( for review, see refs. 7,8). Mutations in SB-induced tumors are caused and tagged by transposon insertions, facilitating the identification of cancer genes. ...
... We developed an automated process to trim, map, and annotate each sequence called the Integration Analysis System. 7 We were able to identify an average of 166 unique integration sites in each SB-induced skin (Supplementary Table S4) and liver Table S5). Most of the mapped integration sites (50-80%) were represented by a single sequence. ...
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 (SB) 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, RosaSBase(LsL), that allows the 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 an Aid-Cre allele. In a second approach, a novel transposon was generated, T2/Onc3, in which the CMV enhancer/chicken beta-actin promoter drives oncogene expression. When combined with ubiquitous SBase expression, the T2/Onc3 transposon produced nearly 200 independent tumors of more than 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.
... Mice bred to carry both the Sleeping Beauty transposase and DNA transposon developed tumours after a short latency. When tumour DNA was analysed for the sites of integration, both known and novel oncogenes were identified Dupuy et al., 2005;Dupuy et al., 2006;Ivics et al., 1997). Use of the Sleeping Beauty transposon system in Blm knockout mice, which have an increased rate of mitotic recombination, has allowed the method to be extended to the identification of tumour suppressor genes (Dupuy et al., 2006). ...
... When tumour DNA was analysed for the sites of integration, both known and novel oncogenes were identified Dupuy et al., 2005;Dupuy et al., 2006;Ivics et al., 1997). Use of the Sleeping Beauty transposon system in Blm knockout mice, which have an increased rate of mitotic recombination, has allowed the method to be extended to the identification of tumour suppressor genes (Dupuy et al., 2006). ...
Sporadic diseases, which occur as single, scattered cases, are among the commonest causes of human morbidity and death. They result in a variety of diseases, including many cancers, premature aging, neurodegeneration and skeletal defects. They are often pathogenetically complex, involving a mosaic distribution of affected cells, and are difficult to model in the mouse. Faithful models of sporadic diseases require innovative forms of genetic manipulation to accurately recreate their initiation and pathogenesis. Such modelling is crucial to understanding these diseases and, by extension, to the development of therapeutic approaches to treat them. This article focuses on sporadic diseases with a genetic aetiology, the challenges they pose to biomedical researchers, and the different current and developing approaches used to model such disorders in the mouse.
... For example, modifying the cargo to enable only truncating mutations could increase the likelihood of identifying tumour suppressor genes [248]. Tissue-specific promoters can be integrated as cargo, making transposons an attractive mutagen for cancer gene discovery in a range of cancer types [256]. Spatial and temporal transposition could also be achieved by introducing a lox– stop–lox cassette between the SB transposase promoter and cDNA, such that transposition is induced upon the addition of Cre [256]. ...
... Tissue-specific promoters can be integrated as cargo, making transposons an attractive mutagen for cancer gene discovery in a range of cancer types [256]. Spatial and temporal transposition could also be achieved by introducing a lox– stop–lox cassette between the SB transposase promoter and cDNA, such that transposition is induced upon the addition of Cre [256]. Identification of cancer genes in SB mutagenesis follows much the same procedure as for retroviruses. ...
The elucidation of the human and mouse genome sequence and developments in high-throughput genome analysis, and in computational tools, have made it possible to profile entire cancer genomes. In parallel with these advances mouse models of cancer have evolved into a powerful tool for cancer gene discovery. Here we discuss the approaches that may be used for cancer gene identification in both human and mouse and discuss how a cross-species 'oncogenomics' approach to cancer gene discovery represents a powerful strategy for finding genes that drive tumourigenesis.
... screening is a powerful genetic tool for genome-wide identification of cancer driver genes and for understanding the evolutionary forces that promote cancer progression [11][12][13][14] . SB transposons continuously introduce insertional mutations so that cells with genetic mutations that are best adapted to the cancer microenvironment continue to be selected. ...
Chronic inflammation promotes development and progression of colorectal cancer (CRC). To comprehensively understand the molecular mechanisms underlying the development and progression of inflamed CRC, we perform in vivo screening and identify 142 genes that are frequently mutated in inflammation-associated colon tumors. These genes include senescence and TGFβ-activin signaling genes. We find that TNFα can induce stemness and activate senescence signaling by enhancing cell plasticity in colonic epithelial cells, which could act as a selective pressure to mutate senescence-related genes in inflammation-associated colonic tumors. Furthermore, we show the efficacy of the Cdk4/6 inhibitor in vivo for inflammation-associated colonic tumors. Finally, we functionally validate that Arhgap5 and Mecom are tumor suppressor genes, providing possible therapeutic targets for CRC. Thus, we demonstrate the importance of the inactivation of senescence pathways in CRC development and progression in an inflammatory microenvironment, which can help progress toward precision medicine.
... With regard to modeling human WM in transgenic mice, the findings suggested that inducible insertional mutagens that can be unleashed in B cells may be useful engineering tools. The Sleeping Beauty transposon may be particularly effective to that end [60] . ...
Waldenström macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma of mature IgM+ B-lymphocytes that warrants additional research to increase therapeutic options, enhance quality of life, and improve survival of patients with WM. Here we concluded a miniseries of short reviews on the diagnosis and treatment, natural history and putative cell-of-origin of WM with a brief survey of preclinical experimental model systems available for fundamental and translational research studies on this enigmatic neoplasm. The model systems comprise of: ① continuous tumor cell lines, three of which are well authenticated and demonstrated to be derived from the patient's index tumor; ② human-in-mouse xenografts that rely on immunodeficient laboratory mice, adapted to carry small fragments of implanted human bone, to provide a suitable microenvironment for incoming lymphoma cells; and ③ genetically engineered mouse models (GEMMs) of neoplastic B-cell development, in which WM-like tumors arise spontaneously in the presence of fully functional innate and adaptive immune systems. Because none of the models developed thus far are perfect, additional efforts are required to achieve a better preclinical representation of disease characteristics of WM. To achieve that goal, the active involvement of basic and clinical research experts from China is called for, so novel drugs and immunotherapies for WM will reach clinics sooner, thereby ensuring the future of patients with WM will be brighter.
... 'Sleeping Beauty' was a transposon found in fish which had been inactivated at least 10 million years ago, but was re-activated by genetic engineering (Wadman et al. 2005). It has been used for insertional and conditional gene trap mutagenesis in mice (Geurts et al. 2006) and also for the insertional mutagenesis of cancer genes where it can be made specific to particular tissues (Dupuy et al. 2006). It appears to be a promising new way of causing mutations in the mouse and possibly other species. ...
The remarkable advances in the science of genetics over the past few decades are having a strong impact on laboratory animal science. These advances are due to the development of a range of molecular techniques making it possible to map, clone, and sequence many genes, but progress has been facilitated by the classical period of mouse genetics which started soon after the rediscovery of Mendel’s work in 1900 and continued until about 1980. This laid a firm foundation on which the new molecular methods could be based. A large proportion of “Laboratory Animal Genetics” is in fact “Mouse Genetics.”1 This should not imply that the genetics of other species are unimportant, but simply that for many technical reasons, including small body weight, high reproductive performance, small space requirements, and the availability of a wide range of strains and mutants, the mouse has been used more extensively than any other mammalian species.
... However, such large-scale genomic approaches also identify a significant number of "passenger" mutations of uncertain functional relevance and disregard potential oncogenes whose expression is dysregulated in the absence of an identifiable genomic lesion. A powerful complementary approach to the discovery of novel oncogenes and tumor suppressors has been the use of retroviral and transposase-based insertional mutagenesis screens (Nusse and Varmus, 1982;Callahan, 1996;Collier et al., 2005;Dupuy et al., 2005Dupuy et al., , 2006Copeland and Jenkins, 2010;McIntyre et al., 2012). For instance, int-2/fibroblast growth factor-3 (FGF3), an oncogene that is recurrently amplified in human breast tumors, first came to prominence because of its dysregulation resulting from a shared integration site of mouse mammary tumor virus in murine models of breast cancer (Nusse and Varmus, 1982;Casey et al., 1986;Callahan, 1996). ...
A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2 -overexpressing cells.
... Complementary to large-scale genome sequencing studies, forward genetic mutagenesis screens in mice provide an unbiased approach to study the significance of gene mutations in tumorigenesis [7][8][9][10][11][12]. Previously, we utilized the Sleeping Beauty (SB) DNA transposon system to identify mutations that cooperate with MYC to accelerate liver tumorigenesis in mice. ...
Hepatocellular carcinoma (HCC) is the fifth most common solid tumor in the world and the third leading cause of cancer-associated deaths. A Sleeping Beauty-mediated transposon mutagenesis screen previously identified mutations that cooperate with MYC to accelerate liver tumorigenesis. This revealed a tumor suppressor role for Steroid Receptor Coactivator 2/Nuclear Receptor Coactivator 2 (Src-2/Ncoa2) in liver cancer. In contrast, SRC-2 promotes survival and metastasis in prostate cancer cells, suggesting a tissue-specific and context-dependent role for SRC-2 in tumorigenesis. To determine if genetic loss of SRC-2 is sufficient to accelerate MYC-mediated liver tumorigenesis, we bred Src-2-/- mice with a MYC-induced liver tumor model and observed a significant increase in liver tumor burden. RNA sequencing of liver tumors and in vivo chromatin immunoprecipitation assays revealed a set of direct target genes that are bound by SRC-2 and exhibit downregulated expression in Src-2-/- liver tumors. We demonstrate that activation of SHP (Small Heterodimer Partner), DKK4 (Dickkopf-4), and CADM4 (Cell Adhesion Molecule 4) by SRC-2 suppresses tumorigenesis in vitro and in vivo. These studies suggest that SRC-2 may exhibit oncogenic or tumor suppressor activity depending on the target genes and nuclear receptors that are expressed in distinct tissues and illuminate the mechanisms of tumor suppression by SRC-2 in liver.
... SB is a member of the widespread Tc1/mariner superfamily of DNA transposons that has been developed as a genome engineering tool in a broad range of organisms 1,2 . It offers up to 95% gene transfer efficiency in diverse vertebrate cell types and is widely used in forward genetic screens 3,4 as well as in ex vivo human gene therapy trials 5,6 . The transposon (tnp) DNA includes terminal inverted repeats (TIRs) at its ends and encodes a transposase protein, the workhorse of transposition, that catalyses all DNA cleavage and joining reactions required for transposition. ...
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB’s applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.
... Transgenic mice expressing the transposase under tissue-specific promoters have proven to be an advance over the traditional method of cancer gene discovery using retroviruses. 47 Although in these instances SB has been optimized to find the cancer genes, this application highlights the risk that even the more benign version of SB used in gene therapy could also cause problems by integrating near cancer genes. ...
Plasmid DNA vectors provide a way to sidestep many of the limitations of viral vectors, such as capsid immunogenicity. However, gene expression from unintegrated plasmid DNA is typically transient. DNA vectors that integrate into the chromosomes via either an integrase or transposase provide permanent gene addition. Two such systems are under intensive development. The Sleeping Beauty transposase integrates into TA din-ucleotides in an essentially random fashion, while φC31 integrase is a sequence-specific recombinase that integrates at a more limited number of endogenous sites in mammalian genomes. Both systems have mediated successful gene therapy in numerous animal models. The features of these DNA integrating systems are described, including safety issues and efforts to increase target site specificity.
... SB is a member of the widespread Tc1/mariner superfamily of DNA transposons that has been developed as a genome engineering tool in a broad range of organisms 1,2 . It offers up to 95% gene transfer efficiency in diverse vertebrate cell types and is widely used in forward genetic screens 3,4 as well as in ex vivo human gene therapy trials 5,6 . The transposon (tnp) DNA includes terminal inverted repeats (TIRs) at its ends and encodes a transposase protein, the workhorse of transposition, that catalyses all DNA cleavage and joining reactions required for transposition. ...
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.
... Once the tagged gene(s) have been identified, more traditional marker-assisted selection methods could be applied on fish wild type populations. For SB/ITR to do massive insertional mutagenesis in mice however, either an enormous number of initial transgenics needed to be made (Keng et al. 2005) or specific SB/ITR vectors need to be used (Dupuy et al. 2006). Thus, for massive insertional mutagenesis in mice, two limitations of the usual SB/ITR vectors had to be overcome: (1) only one or two transpositions were induced per mouse born (Fischer et al. 2001)and ...
... The advantages of SB transposon system in transgenic studies are a wide range of utilization in vertebrates and the short IRs (230bp) for recognition and binding of SB. It has been used in many different organisms (Ivics et al., 2007;Dupuy et al., 2006). Recently, Partow Kebriaei and Laurence ...
... Among the ten genes, seven (CNTN4, CTNNA3, EPHA6, FOXO1, MAGI1, PTPRD and SMAD4) were The Protein Domain Landscape of Cancer-Type-Specific Somatic Mutations predicted to be tumor suppressors. Using transposon insertion positions, the Sleeping Beauty study [60] had annotated three of these seven genes as loss of function (while not suggesting an annotation for the remaining four). We also reported two potential oncogenes, USP25 and GNA13 (Table 4). ...
Author Summary
Extensive tumor genome sequencing has provided raw material to understand mutational processes and identify cancer-associated somatic variants. However, fundamental problems remain to: i) separate ‘driver’ from ‘passenger’ mutations, ii) further understand the functional mechanisms and consequences of driver mutations, and iii) identify the cancer types in which each driver mutation is relevant. Here we analyze whole-genome and exome tumor sequencing data from the perspective of protein domains—the basic structural and functional units of proteins. Exploring the cancer-type-specific landscape of domain mutations across 21 cancer types, we identify both cancer-type-specific mutated domains and mutational hotspots. Frequently-mutated domains were identified for oncoproteins for which the ‘mutational hotspot’ phenomenon owing to the relative rarity of gain-of-function mutations is well known, and also for tumor suppressor proteins, for which more uniformly distributed loss-of-function driver mutations are expected. A given gene product may be perturbed differently in different cancers. Indeed, we observed systematic shifts between cancer types of the positions at which mutations occur within a given protein. Both known and novel candidate driver mutations were retrieved. Novel cancer gene candidates significantly overlapped with orthogonal systematic cancer screen hits, supporting the power of this approach to identify cancer genes.
... The Sleeping Beauty (SB) is the first DNA transposon system that has been described to work in mammalian cells and is also the most widely used transposon system for mammalian transgenesis and mutagenesis (Takeda et al., 2007;Wang et al., 2008). For example, SB has been used to identify new cancer genes (Collier et al., 2005;Dupuy et al., 2006) and generate transgene animals (Kitada et al., 2009). However, SB transposition application is limited by its relatively low transposition efficiency and its nonspecific integration (Wu et al., 2006;Yant et al., 2005). ...
The PiggyBac (PB) transposon system is a non-viral DNA-transfer system in which a transposase directs integration of a PB transposon into a TTAA site in the genome. Transgenic expression of porcine CD163 is necessary and sufficient to confer non-permissive cells susceptible to infection with porcine reproductive and respiratory syndrome virus (PRRSV). Such permissive cells can be used as a tool for PRRSV cellular receptor and other studies. One of the problems in studying PRRSV is the lack of porcine cell lines. In this study, efficient transfection and expression of porcine CD163 in PK-15 cells by PB transposition was demonstrated. The stable PK-15(CD163) cell line was used in PRRSV infection assays. The data indicated that the average PB transgene copy number per genome was approximately ten. In line with previous literature the integration of PB into the genome had a bias toward the TTAA chromosomal site. The PK-15(CD163) cell line was susceptible to infection by different PRRSV strains and the virus grew to similar titers compared to the Marc-145 cell line. This simplification of PK-15(CD163) cell line production will provide a valuable tool to facilitate PRRSV cellular receptor studies and to accelerate existing vectors for PK-15 cell-based gene transfer and expression.
... 'Sleeping Beauty' was a transposon found in fish which had been inactivated at least 10 million years ago, but was re-activated by genetic engineering (Wadman et al. 2005). It has been used for insertional and conditional gene trap mutagenesis in mice (Geurts et al. 2006) and also for the insertional mutagenesis of cancer genes where it can be made specific to particular tissues (Dupuy et al. 2006). It appears to be a promising new way of causing mutations in the mouse and possibly other species. ...
... For genetic screening in mammalian cell cultures and mice, systems of insertional mutagenesis based on transposons piggiBac ( Trichoplusia ni ) [17], Minos [18], and Sleeping Beauty (Salmonids) [19][20][21] have been developed. ...
In spite of a vast number of drug preparations used in medicine, advances in treating most socially important human diseases
remain modest. Historically, many drugs were developed without clear understanding of the mechanisms of their action and were
intended only for correcting symptoms of the disease. Identification of molecular targets in pharmacological screening new
pharmaceuticals plays a key role not only in defining the strategy of the treatment, but also in understanding the general
development of the disease. Sequencing of the genomes of various organisms, human in particular, and the development of new
modern techniques of research have created the prerequisites for targeted screening for genes that are potentially interesting
for development of new drugs.
... This proved to be a viable strategy for transient transgenic RNAi knockdown in mouse embryos using Prdm16-specific shRNAs, although the specialized training and facilities necessary for working with these pathogens reduces its attractiveness as a universal tool for these studies. The SB system is also a tractable means to perform in vitro and in vivo transgenic studies of many kinds, including cancer modeling, gene trapping, generation of transgenic mouse lines and insertional mutagenesis[33,34,35,36]. Several factors have been shown to affect the transposition efficiency of SB transposons in vitro. ...
RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for "transient transgenic" knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.
... Additionally, SB has also been used in functional genetic screens in mammals for the identification of genes implicated in diseases such as cancer. SB is used to induce insertional mutagenesis, and candidate genes identified through the analysis of insertion sites in tumors vs control tissues (in gain of function studies[115,116], reviewed in[117]). Although Sleeping Beauty is currently the most promising gene transfer system for vertebrate cells within the Tc1/mariner superfamily, other transposons from this family have been used as genomic tools as well. ...
Repeated DNA makes up a large fraction of a typical mammalian genome, and some repetitive elements are able to move within the genome (transposons and retrotransposons). DNA transposons move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant role in the evolution of many genomes. As genetic tools, DNA transposons can be used to introduce a piece of foreign DNA into a genome. Indeed, they have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Thus, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy. In this review, we will describe the nature of these elements and discuss recent advances in this field of research, as well as our evolving knowledge of the DNA transposons most widely used in these studies.
... The transposability of a few transposons has been demonstrated in mammalian cells. After molecular reconstruction of Sleeping Beauty (SB) transposon,4 SB has been widely used for mammalian genetic5,6 and preclinical studies7 because of its high transposability in mammalian cells. Recently, piggyBac (PB), a transposon derived from cabbage looper moth Trichoplusia ni,8 was shown to transpose more efficiently than other transposons including Tol2 (refs. ...
Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.
... Furthermore, the time-consuming preparation also disqualifies embryo cryopreservation for crisis intervention or rescuing a "last-of-line" when a large-scale disaster occurs in the animal colony. In addition, this technology is also unsuitable for archiving mutant mouse libraries obtained by randomized methods for genetic modification, e.g. by chemical induction or the application of transposon technology (Dupuy et al. 2006). ...
... The resurrection of an ancient vertebrate transposon (Tn) system, Sleeping Beauty (SB) has provided a nonviral vector system allowing efficient insertion of transgenes into the host genome. It has been used successfully for both gene replacement (15) and gene discovery in mice (16); and integrates transgenes at a frequency comparable with that of viral vectors in both quiescent and replicating cells (17,18). The Tn inserts into a genomic TA dinucleotide using a cut-and-paste mechanism catalyzed by the obligate SB transposase, which can be supplied either as a 2-plasmid (trans) system or as a single plasmid in cis carrying the expression cassette for the transposase external to the inverted repeat/direct repeat-flanked (IR/DR-flanked) transgene (19,20). ...
Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.
... Transposases mediate DNA movement in lower organisms by excising defined segments of DNA and then reinserting them at other locations in the genome, a process that can be repeated multiple times for a given segment (1,2). While transposase activity probably accounts for half of the present organization of the human genome, almost all of these sequences are pseudogenes, as unregulated DNA mobility would be deleterious to human cells, causing genome instability (1–4). ...
Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIalpha decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIalpha inhibitors.
... In their current formulations, however, the integration events remain uncontrolled and thus may suffer the same problems of integration-based viral delivery systems. In fact, transposon-based insertions are being developed as a tool for cancer gene discovery [31]. An active but still preliminary area of research is to try to create hybrid transposases to target transposon integration to defined regions of the genome. ...
The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise.
... The development of genome-scale libraries of RNAi reagents has facilitated loss-of-function approaches in mammalian cells that have identified candidate tumor-suppressor genes (Kolfschoten et al., 2005; Westbrook et al., 2005). In parallel to these loss-of-function approaches, candidate oncogenes have been identified using screening tools such as cDNA libraries derived from cancer cells or the use of viral integration to activate endogenous genes (Dupuy et al., 2006; Peeper et al., 2002). Here we have combined both gain-of-function and lossof-function approaches with whole-genome characterization of genetic alterations in cancer cell lines and tumors. ...
The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.
... Arpc3 SB , , [35] ...
The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has been transpositionally inactive for over 10 million years. The SB transposon system was awakened from inactive Tc1-like transposable elements by using molecular phylogenetic data in 1997. Recent studies on its transposition efficiency and mechanism have shown its broad applications in vertebrate animals for gene-screening, gene transfer, and human gene therapy. In this review, we summarize our current knowledge of Sleeping Beauty, such as structure, transposition mechanism and potential applications, and bring forward some means aiming at the limitations of transposon technology.
Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer. Traditionally, retroviruses have been used for this purpose, allowing the identification of genes that can cause various forms of leukemia or lymphoma with murine leukemia viruses or mammary cancer with mouse mammary tumor viruses. Recently, the Sleeping Beauty transposon system has emerged as a tool for cancer gene discovery in mouse models of human cancer. Transposons mobilized in the mouse soma can insertionally mutate cancer genes, and the transposon itself serves as a molecular "tag," which facilitates candidate cancer gene identification. We provide an overview of some general issues related to use of Sleeping Beauty for cancer genetic studies and present here the polymerase chain reaction-based method for cloning transposon-tagged sequences from tumors.
Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF-β) pathway. Importantly, the effects of TGF-β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-β signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-β signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-β signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-β receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/β-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC. This article is protected by copyright. All rights reserved.
Family history, reproductive factors, hormonal exposures, and subjective immunihistochemical evaluations of in situ lesions, and to a lesser extent age, remain the best clinical predictors of an individual's risk of developing breast cancer. Identification of early markers predictive of impending invasive breast cancer from in situ carcinoma is a long-term goal. The latent mammary cancer transgenic mouse model of human breast cancer, C57BL/6JTg(WapTag)1Knw (Waptag1), develops characteristic stages of tumorigenesis in a highly predictable manner: atypical hyperplasia advances to ductal carcinoma in situ (DCIS), which progresses to papillary adenocarcinomas and/or solid, invasive tumors. Microarray analyses of whole mammary glands and tumors across these stages, to detect transcriptional changes throughout tumorigenesis, revealed marked, phased stage-specific changes. In constrast, results from the laser capture microdissected tumor cells depict a moderately constant characteristic tumorigenic profile, irrespective of stage. Evaluation of differences in whole glands with those of microdissected samples suggests that paracrine signaling between tumor and stromal cells substantially alters the tumor microenvironment, early in progression. Strikingly, comparison of statistically significant microarray results between Waptag1 DCIS and human DCIS revealed 2,097 overlapping early transcriptional changes. When compared with species-specific controls, common abundant early gene alterations were associated with cell cycle, cell division, and DNA replication Gene Ontology categories, with a notable decrease in genes involved in aerobic energy metabolism, and significant increased transcription of retrotransposons and chromosome modification genes. Based on these initial experimental results, retrotransposons were identified as a potential marker for testing in several mouse models and in biopsies derived from breast cancer patients. Analysis of data from five independent mouse models of mammary cancer and five human breast cancer datasets revealed over expression of retrotransposons, mainly Class I and Class II LTR elements, as well as LINEs and SINEs, when compared with normal samples. Cross-species comparison of gene expression profiles suggests epigenetic alterations and chromatin remodeling changes coincide with retrotransposon over expression. Through validation of such mutual human and mouse changes in gene expression, these novel putative markers may allow earlier detection and therapeutic intervention, possibly reducing the incidence of invasion and metastases in patients with breast cancer.
Transposable elements (TEs) have been considered traditionally as junk DNA, i.e., DNA sequences that despite representing a high proportion of genomes had no evident cellular functions. However, over the last decades, it has become undeniable that not only TE-derived DNA sequences have (and had) a fundamental role during genome evolution, but also TEs have important implications in the origin and evolution of many genomic disorders. This concise review provides a brief overview of the different types of TEs that can be found in genomes, as well as a list of techniques and methods used to study their impact and mobilization. Some of these techniques will be covered in detail in this Method Book.
Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from
place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two
hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level
of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved
between genomes by horizontal transfer. We have generated an intact version of this element, SPINON, which is highly active in human cells. In vitro analysis of TcBuster and SPINON shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability
of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human
genome of TcBuster and SPINON and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPINON suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.
The concept of transposon tagging or insertional mutagenesis as a strategy for fishing out genes connected with the phenotype
of interest was emerging since the early 1980s. Study of genetic basis of tumorigenesis is one of the fields where insertional
mutagenesis proved to be exceptionally powerful. Crucial elements of this experimental approach have been retroviruses whose
unique properties have revolutionized the work in the field of oncogenesis. Retroviruses contributed to our knowledge of tumor
formation in two ways. First, some of them transduce oncogenes—mutants of normal cellular genes with an oncogenic potential.
And it was the comparison of viral and cellular alleles of these genes that allowed comprehending the principles of oncogenic
activation of genes. Second, retroviruses not carrying oncogenes can induce tumors by affecting host genes. Through integration
of their proviral DNA into chromosomes they can activate tumorigenic potential of oncogenes or inactivate tumor suppressor
genes. The mechanism is referred to as oncogenesis by insertional mutagenesis. The insertional mutagenesis by retroviruses
is very efficient. Perhaps each locus of a host genome can be hit by the provirus insertion in many cells of an infected tissue.
If any of these insertions or their combinations incites malignant transformation, the touched cell outgrows and can give
rise to a tumor. Affected host gene loci can be easily identified since they are tagged by integrated proviral sequences.
Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional
mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer.
Traditionally, retroviruses have been used for this purpose, allowing the identification of genes that can cause various forms
of leukemia or lymphoma with murine leukemia viruses or mammary cancer with mouse mammary tumor viruses. Recently, the Sleeping Beauty transposon system has emerged as a tool for cancer gene discovery in mouse models of human cancer. Transposons mobilized
in the mouse soma can insertionally mutate cancer genes, and the transposon itself serves as a molecular “tag,” which facilitates
candidate cancer gene identification. We provide an overview of some general issues related to use of Sleeping Beauty for cancer genetic studies and present here the polymerase chain reaction-based method for cloning transposon-tagged sequences
from tumors.
Key WordsCancer genetics–linker-mediated PCR–mouse transgenesis–Sleeping Beauty–somatic mutagenesis–transposon
In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated.
Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs
with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction
protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from
the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a
small recombination plasmid, which bears positive and negative selection markers and a cloned homologous “probe” region. This
probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover
recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore,
if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal
of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful
gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation
and mutagenesis have all been demonstrated with this method.
Key WordsBacteriophage-λ–embryonic stem cell–γ–gene targeting vector–library screening–phage-plasmid recombination–supF–suppressor tRNA–transplacement mutagenesis
The mobility of class II transposable elements (DNA transposons) can be experimentally controlled by separating the two functional components of the transposon: the terminal inverted repeat sequences that flank a gene of interest to be mobilized and the transposase protein that can be conditionally supplied to drive the transposition reaction. Thus, a DNA molecule of interest (e.g., a fluorescent marker, an shRNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be stably integrated into the genome in a regulated and highly efficient manner. Sleeping Beauty (SB) was the first transposon ever shown capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering in vertebrate species, including transgenesis, insertional mutagenesis, and therapeutic somatic gene, transfer both ex vivo and in vivo. This methodological paradigm opened up a number of avenues for genome manipulations for basic and applied research. This review highlights the state-of-the-art in SB transposon technology in diverse genetic applications with special emphasis on the transposon as well as transposase vectors currently available in the SB transposon toolbox.
Identifying the normal cell from which a tumor originates is crucial to understanding the etiology of that cancer. However, retrospective identification of the cell of origin in cancer is challenging because of the accumulation of genetic and epigenetic changes in tumor cells. The biologic state of the cell of origin likely influences the genetic events that drive transformation. We directly tested this hypothesis by performing a Sleeping Beauty transposon mutagenesis screen in which common insertion sites were identified in tumors that were produced by mutagenesis of cells at varying time points throughout the T lineage. Mutation and gene expression data derived from these tumors were then compared with data obtained from a panel of 84 human T-cell acute lymphoblastic leukemia samples, including copy number alterations and gene expression profiles. This revealed that altering the cell of origin produces tumors that model distinct subtypes of human T-cell acute lymphoblastic leukemia, suggesting that even subtle changes in the cell of origin dramatically affect genetic selection in tumors. These findings have broad implications for the genetic analysis of human cancers as well as the production of mouse models of cancer.
Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB transposon can be developed into powerful tools for chromosomal manipulations. Mutagenesis screens based on SB have numerous advantages including high throughput and easy identification of mutated alleles. Forward genetic approaches based on insertional mutagenesis by engineered SB transposons have the advantage of providing insight into genetic networks and pathways based on phenotype. Indeed, the SB transposon has become a highly instrumental tool to induce tumors in experimental animals in a tissue-specific -manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with SB transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models.
Insertional mutagenesis in mice is a potent instrument for cancer gene discovery. Until recently, retroviruses were the main experimental tools in this field and application of insertional mutagenesis was limited to tissues for which these agents have tropism, namely hemopoietic cells and mammary epithelium. However, the field has been revolutionized and greatly expanded with the recent reanimation of the transposons, a highly flexible group of insertional mutagens first discovered in maize, which have now been adapted for use in mammalian cells. Transposons do not only extend the application of insertional mutagenesis to any tissue of choice, but also allow a more extensive and unbiased coverage of the genome, can be designed to selectively activate or inactivate genes, and are highly amenable to temporal and spatial control. This chapter gives an overview of the design and application of transposons to cancer gene discovery in mice.
This review highlights the current techniques used to generate transgenic mouse models of cancer, with an emphasis on recent advances in the use of ubiquitous promoters, models that use Cre-loxP and Flip-FRT recombinase technology, inducible systems, RNAi to target genes, and transposon mutagenesis. A concluding section discusses new imaging systems that visualize tumor progression and the microenvironment in vivo. In this review, these techniques and strategies used in mouse models of cancer are highlighted, as they are pertinent and relevant to the development of zebrafish models of cancer.
The mouse is central to the goal of establishing a comprehensive functional annotation of the mammalian genome that will help elucidate various human disease genes and pathways. The mouse offers a unique combination of attributes, including an extensive genetic toolkit that underpins the creation and analysis of models of human disease. An international effort to generate mutations for every gene in the mouse genome is a first and essential step in this endeavor. However, the greater challenge will be the determination of the phenotype of every mutant. Large-scale phenotyping for genome-wide functional annotation presents numerous scientific, infrastructural, logistical, and informatics challenges. These include the use of standardized approaches to phenotyping procedures for the population of unified databases with comparable data sets. The ultimate goal is a comprehensive database of molecular interventions that allows us to create a framework for biological systems analysis in the mouse on which human biology and disease networks can be revealed.
Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the transposon systems.
The primary goal of mouse mutagenesis programmes is to develop a fundamental research infrastructure for mammalian functional genomics and to produce human disease models. Many large-scale programmes have been ongoing since 1997; these culminated in the International Knockout Mouse Consortium (IKMC) in 2007 with the aim to establish knockout and conditional mouse strains for all mouse genes. This article traces the origins and rationale of these large-scale mouse mutagenesis programmes.
Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.
Ever since their molecular isolation in eukaryotic organisms, transposons have been precious implements of the geneticist's toolkit. The properties that make transposons so useful are their ability to move from one chromosomal position to another and the relatively minimal requirements for transposition to occur in the test tube and in living cells (1). In particular, transposons can be harnessed to stably integrate sizeable pieces of DNA into a host's chromosome (2, 3). This quality provides a tremendous potential for transgenesis and large-scale insertional mutagenesis. Transposon-mediated DNA delivery, although first limited to a small number of invertebrate species, has become progressively more applicable to vertebrates, including mammalian cells (3–5). This progress has opened the door to the development of a new generation of vectors for human gene therapy and mammalian forward genetics that are potentially more easily controlled, more versatile, and safer than viral vectors (5). However, applications of transposon vectors for clinical trials in gene therapy, and the predictable manipulation of mammalian genomes, have been hindered by their low integration efficiency relative to viral vectors. In this issue of PNAS, Wu et al. (6) move one step further in the quest for a superior gene delivery tool in mammals. They identified piggyBac (PB) as the most active and flexible transposon system yet tested for transformation of mammalian cells.
Mobile group II introns are ribozymes and use a novel mechanism--target DNA-primed reverse transcription--to proliferate in DNA. Group II introns are a unique mobile element for their high sequence-specific, yet readily flexible target site recognition. Both the intron RNA and the intron-encoded protein (IEP) are involved in target site recognition, and the specificity is determined primarily by base pairing between the intron RNA and DNA target. Therefore, the intron RNA can be modified according to the desired target sequence for specific gene disruption. Group II intron knockout technology is mature in bacteria and is currently being developed in eukaryotes. This technology has great potential to revolutionize fields such as functional genomics, gene therapy, and cell line engineering.
Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use
in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene
transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes
on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size
was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large
genes.
Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.
Transposons have been successfully exploited as insertional mutagens for the efficient identification and isolation of genes (transposon tagging) in many organisms. Plants are no exception. The maize Activator and Suppressor-mutator transposons function when transferred into heterologous plant species, and many different gene tagging systems have been developed. These systems have recently been used to clone novel and important genes, including disease resistance loci from Nicotiana tabacum, tomato and flax.
Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context. Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism. On the other hand, it has long been speculated that members of the Tc1/mariner-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms. However, this prediction has not been realized in mice. We report here the chromosomal transposition of the Sleeping Beauty (SB) element in mouse embryonic stem cells, providing evidence that it can be used as an in vivo mutagen in mice.
Mouse strains expressing the site-specific recombinase Cre (or Flp) facilitate conditional ablation of gene function when one or several exons of the gene of interest are flanked by loxP (or FRT) sites1. Cre expression achieved by classic transgenesis or targeting to an appropriate locus might be tissue specific, temporally restricted or inducible2, 3. In such experimental outlines, it is necessary to monitor Cre activity at desired time points as well as to verify that Cre was not active previously during development. Other investigators have generated transgenic4, 5 or knock-in6 lines in which lacZ expression is conditional on the removal of an intervening segment. However, such lines are most useful if lacZ can be expressed in all cell types and hence is driven off a constitutively active promoter in the mouse.
The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.
Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.
The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis in vertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes. A mutagenesis approach was undertaken to improve various aspects of the transposon, including safety and overall efficiency of gene transfer in human cells. Deletional analysis of transposon sequences within first-generation SB vectors showed that the inverted repeats of the element are necessary and sufficient to mediate high-efficiency transposition. We constructed a "sandwich" transposon, in which the DNA to be mobilized is flanked by two complete SB elements arranged in an inverted orientation. The sandwich element has superior ability to transpose >10-kb transgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactive versions of the SB transposase by single-amino-acid substitutions. These mutations act synergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressed HMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition by almost an order of magnitude compared to the first-generation transposon system. The improved vector system should prove useful for efficient gene transfer in vertebrates.
Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.
We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation-site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)-trap transposon vectors, were outcrossed to wild-type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics. genesis 30:82–88, 2001. © 2001 Wiley-Liss, Inc.
INFECTION of fibroblasts by avian sarcoma virus (ASV) leads to
neoplastic transformation of the host cell. Genetic analyses have
implicated specific viral genes in the transforming
process1-4, and recent results suggest that a single viral
gene is responsible4. Normal chicken cells contain DNA
homologous to part of the ASV genome5-8 moreover, embryonic
fibroblasts from certain strains of chickens can produce low titres of
infectious type C viruses either spontaneously9 or in
response to various inducing agents10. None of the viruses
obtained from normal chicken cells, however, can transform fibroblasts,
and results with molecular hybridisation indicate that the nucleotide
sequences responsible for transformation by ASV are not part of the
genetic complement of the normal cell11. We demonstrate here
that the DNA of normal chicken cells contains nucleotide sequences
closely related to at least a portion of the transforming gene(s) of
ASV; in addition, we have found that similar sequences are widely
distributed among DNA of avian species and that they have diverged
roughly according to phylogenetic distances among the species. Our data
are relevant to current hypotheses of the origin of the genomes of RNA
tumour viruses12 and the potential role of these genomes in
oncogenesis13.
In mouse mammary tumor virus (MMTV) infected mice, three identifiable stages of mammary tumorigenesis can be biologically defined: preneoplastic hyperplastic nodules, malignant tumor, and distant metastatic lesions (primarily in the lung). MMTV is a biological carcinogen which induces somatic mutations as consequence of its integration into the host cellular genome. Each stage of mammary tumorigenesis appears to result from the clonal outgrowth of cells containing additional integrated proviral MMTV genomes. This phenomenon has provided the basis for an approach to identify genes which, when affected, may contribute to progression through the different stages of mammary tumorigenesis. Eight different genes (Wnt1, Wnt3, Wnt10b, Fgf3, Fgf4, Fgf8, Int3, and Int6) have been shown to be genetically altered in multiple mammary tumors as a consequence of MMTV integration. Although the significance of the human homologs of these genes as targets for somatic mutation during human breast carcinogenesis is only now being explored, it is clear that this work has led to a new appreciation of the complexity of the genetic circuitry that is involved in the control of normal mammary gland growth and development. It seems likely that some of the mutations induced by MMTV, and the signaling pathways in which these target genes take part, will be relevant to the progression from preneoplastic lesions to distant metastasis in human breast cancer.
Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.
The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF+/+ or ARF+/- MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.
During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.
We wish to thank Terry Schoop of Biomed Arts Associates, San Francisco, for preparation of the figures, Cori Bargmann and Zena Werb for insightful comments on the manuscript, and Normita Santore for editorial assistance. In addition, we are indebted to Joe Harford and Richard Klausner, who allowed us to adapt and expand their depiction of the cell signaling network, and we appreciate suggestions on signaling pathways from Randy Watnick, Brian Elenbas, Bill Lundberg, Dave Morgan, and Henry Bourne. R. A. W. is a Ludwig Foundation and American Cancer Society Professor of Biology. His work has been supported by the Department of the Army and the National Institutes of Health. D. H. acknowledges the support and encouragement of the National Cancer Institute. Editorial policy has rendered the citations illustrative but not comprehensive.
Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.
As a model system for the understanding of human cancer, the mouse has proved immensely valuable. Indeed, studies of mouse models have helped to define the nature of cancer as a genetic disease and demonstrated the causal role of genetic events found in tumors. As the scientific and medical community's understanding of human cancer becomes more sophisticated, however, limitations and potential weaknesses of existing models are revealed. How valid are these murine models for the understanding and treatment of human cancer? The answer, it appears, depends on the nature of the research requirement. Certain models are better suited for particular applications. Using novel molecular tools and genetic strategies, improved models have recently been described that accurately mimic many aspects of human cancer.
Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.
Slow transforming retroviruses, such as the Moloney murine leukemia virus (M-MuLV), induce tumors upon infection of a host after a relatively long latency period. The underlying mechanism leading to cell transformation is the activation of proto-oncogenes or inactivation of tumor suppressor genes as a consequence of proviral insertions into the host genome. Cells carrying proviral insertions that confer a selective advantage will preferentially grow out. This means that proviral insertions mark genes contributing to tumorigenesis, as was demonstrated by the identification of numerous proto-oncogenes in retrovirally induced tumors in the past. Since cancer is a complex multistep process, the proviral insertions in one clone of tumor cells also represent oncogenic events that cooperate in tumorigenesis. Novel advances, such as the launch of the complete mouse genome, high-throughput isolation of proviral flanking sequences, and genetically modified animals have revolutionized proviral tagging into an elegant and efficient approach to identify signaling pathways that collaborate in cancer.
Cancer drugs have traditionally been identified in screens designed to produce broad biological end points such as cell death. A serious undesired outcome of drugs discovered in these screens is that the mechanism of drug action is unknown and such drugs often have adverse side effects. Designing cancer drugs that act on specific targets offer the advantage that the mechanism of drug action can be understood and accurately monitored in clinical trials leading to development of better drugs. The pharmacological industry has recently shifted to a target directed drug discovery model. However, until recently potential cancer drug targets comprised of only a small fraction of the human genome. The human genome project and high-throughput structural and functional genomics have dramatically increased the number of cancer drug targets. Deciphering cancer drug targets requires the understanding of biochemical pathways that are affected in the cancer genome. It has been suggested that utilization of Single-nucleotide polymorphisms (SNPs) will aid in identifying individuals at high risk of developing certain cancers, and will also help in development of tailored medication or identify genetic profiles of specific drug action and toxicity. Achieving successful new cancer drug development schemes will require a merger of research disciplines that include pharmacology, genomics, comparative genomics, functional genomics, proteomics and bioinformatics. In this review the significance and challenges of these rapidly evolving technologies in cancer drug target discovery are discussed.
Twenty years after the epidermal growth factor receptor (EGFR) was identified as a potential anticancer target, the EGFR inhibitor gefitinib (Iressa; AstraZeneca) has been approved for the treatment of patients with advanced non-small-cell lung cancer in many countries. Studies have indicated its potential for treating patients with other types of solid tumours. Investigation of gefitinib has not only increased our knowledge about the biology of EGFR signalling, but is contributing to our evolving understanding of which tumours are EGFR dependent.
Imatinib has revolutionized drug therapy of chronic myeloid leukemia (CML). Preclinical studies were promising but the results of clinical trials by far exceeded expectations. Responses in chronic phase are unprecedented, with rates of complete cytogenetic response (CCR) of more than 40% in patients after failure of interferon-alpha (IFN) and more than 80% in newly diagnosed patients, a level of efficacy that led to regulatory approval in record time. While most of these responses are stable, resistance to treatment after an initial response is common in more advanced phases of the disease. Mutations in the kinase domain (KD) of BCR-ABL that impair imatinib binding have been identified as the leading cause of resistance. Patients with CCR who achieve a profound reduction of BCR-ABL mRNA have a very low risk of disease progression. However, residual disease usually remains detectable with reverse transcription-polymerase chain reaction (RT-PCR), indicating that disease eradication may pose a significant challenge. The mechanisms underlying the persistence of minimal residual disease are unknown. In this manuscript, we review the preclinical and clinical development of imatinib for the therapy of CML, resistance and strategies that may help to eliminate resistant or residual leukemia.
Mouse models of human cancer are vital to our understanding of the neoplastic process, and to advances in both basic and clinical research. Indeed, models of many of the major human tumours are now available and are subject to constant revision to more faithfully recapitulate human disease. Despite these advances, it is important to recognize that limitations do exist to the current range of models. The principal approach to modelling has relied upon the use of constitutive gene knockouts, which can often result in embryonic lethality, can potentially be affected by developmental compensation, and which do not mimic the sporadic development of a tumour expanding from a single cell in an otherwise normal environment. Furthermore, simple knockouts are usually designed to lead to loss of protein function, whereas a subset of cancer-causing mutations clearly results in gain of function. These drawbacks are well recognized and this review describes some of the approaches used to address these issues. Key amongst these is the development of conditional alleles that precisely mimic the mutations found in vivo, and which can be spatially and tissue-specifically controlled using 'smart' systems such as the tetracycline system and Cre-Lox technology. Examples of genes being manipulated in this way include Ki-Ras, Myc, and p53. These new developments in modelling mean that any mutant allele can potentially be turned on or off, or over- or under-expressed, in any tissue at any stage of the life-cycle of the mouse. This will no doubt lead to ever more accurate and powerful mouse models to dissect the genetic pathways that lead to cancer.
Epigenetic alterations, such as abnormal DNA-methylation patterns, are associated with many human tumour types. New techniques have been developed to perform genome-wide screening for alterations in DNA-methylation patterns, not only to identify tumour-suppressor genes, but also to find patterns that can be used in diagnosis and prognosis. However, interpretation of differential methylation has proven difficult because the significance of methylation alterations depends on the genomic region, and functions of CpG islands at specific sites have not been fully clarified. What techniques can be used to identify new tumour suppressors and diagnostic markers?
Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in specimens from patients with non-small-cell lung cancer who have a response to anilinoquinazoline EGFR inhibitors. Despite the dramatic responses to such inhibitors, most patients ultimately have a relapse. The mechanism of the drug resistance is unknown. Here we report the case of a patient with EGFR-mutant, gefitinib-responsive, advanced non-small-cell lung cancer who had a relapse after two years of complete remission during treatment with gefitinib. The DNA sequence of the EGFR gene in his tumor biopsy specimen at relapse revealed the presence of a second point mutation, resulting in threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.
Imatinib has tremendously changed the treatment of gastrointestinal stromal tumor (GIST). Research is currently focusing on its optimal use and the mechanisms of resistance that may emerge. A multidisciplinary approach including medical oncologists, surgeons, radiologists, and pathologists is crucial for the optimal management of these patients. Moreover, imatinib treatment in GIST represents an extraordinary model to expand our knowledge on the molecular mechanisms that are basic to the effects of molecularly targeted therapies. This review summarizes the existing knowledge of the imatinib treatment in GIST and describes directions for further development.
Cancer is a disease characterized by genomic instability. Comparative genomic hybridization (CGH) is a technique designed for detecting segmental genomic alterations. Recent advances in array-based CGH technology have enabled examination of chromosomal regions in unprecedented detail, revolutionizing our understanding of tumour genomes. A number of array-based technologies have been developed, aiming to improve the resolution of CGH, enabling researchers to refine and define regions in the genome that may be causal to cancer, and facilitating gene discovery at a rapid rate. This article reviews the various array CGH platforms and their use in the study of cancer genomes. In addition, the need for high-resolution analysis is discussed as well as the importance of studying early-stage disease to discover genetic alterations that may be causal to cancer progression and aetiology.
Breast cancers are a biologically heterogeneous group of mammary tumors with distinct natural histories and varied responses to established therapies. They have long been divided into those that are hormone sensitive [as defined by expression of the estrogen receptor alpha (ERalpha) and/or the progesterone receptor (PR)] and those that are not. Notably, only those breast cancers that express ERalpha and/or PR typically respond to hormonal therapy with tamoxifen, aromatase inhibitors, or the newer agent fulvestrant. More recently, the transmembrane tyrosine kinase receptor HER-2/neu was identified as an oncogene overexpressed by about 30% of breast cancers. These HER-2/neu-overexpressing breast cancers define a subset of breast tumors that are characteristically more aggressive, and women who develop them have a shorter survival. Trastuzumab (Herceptin), a humanized monoclonal antibody specific for HER-2/neu, has revolutionized the management of metastatic HER-2/neu-overexpressing breast cancers. As a single agent, it produces response rates similar to those of many single-agent chemotherapeutic agents active in metastatic breast cancer and has limited toxicity. Combining trastuzumab with chemotherapy can result in synergistic antitumor activity. The clear efficacy of trastuzumab against HER-2/neu-overexpressing metastatic breast cancer has led to a keen interest in testing its role in the management of early breast cancer, and multiple large clinical trials are currently in progress. This review summarizes the available clinical data on the use of trastuzumab in HER-neu-overexpressing breast cancer and briefly highlights emerging opportunities for innovative, trastuzumab-based breast cancer therapies.
Conventional chemotherapeutic drugs are ineffective in treatment of gastrointestinal stromal tumors (GISTs). Imatinib (STI571, Gleevec, Glivec; Novartis Pharmaceuticals, East Hanover, NJ), a selective inhibitor of KIT, ABL, BCR-ABL, PDGFRA, and PDGFRB, represents a new paradigm of targeted cancer therapy and has revolutionized the treatment of patients with chronic myelogenous leukemia and GISTs. Unfortunately, imatinib resistance has emerged. The reported mechanism of imatinib resistance in GISTs involves missense mutation in the kinase domain of KIT, including Thr670Ile, Tyr823Asp, and Val654Ala. The established mechanisms and potential mechanisms of imatinib resistance in GISTs, the imaging studies indicative of early development of imatinib resistance, and the management of imatinib-resistant GISTs are discussed.
The advancement of microarray technologies for characterizing tumors at the gene expression level has made a significant impact on the field of oncology. Profiling gene expression of various human tumors has led to the identification of gene expression patterns or signatures related to tumor classification, disease outcome and response to therapy. This technology can also be used to study the mechanism of action of specific therapeutics. Routine application of microarrays in clinical practice will require significant efforts to standardize the array manufacturing techniques, assay protocols and analytical methods used to interpret the data. Extensive, independent validation using large, statistically sound datasets will also be necessary. Studies on gene expression profiling of clinically relevant tissue samples with the aim of finding gene markers to support disease prognosis and therapy decisions are reviewed.
Retroviruses, acting as somatic cell insertional mutagens, have been widely used to identify cancer genes in the haematopoietic system and mammary gland. An insertional mutagen for use in other mouse somatic cells would facilitate the identification of genes involved in tumour formation in a wider variety of tissues. Here we report the ability of the Sleeping Beauty transposon to act as a somatic insertional mutagen to identify genes involved in solid tumour formation. A Sleeping Beauty transposon, engineered to elicit loss-of-function or gain-of-function mutations, transposed in all somatic tissues tested and accelerated tumour formation in mice predisposed to cancer. Cloning transposon insertion sites from these tumours revealed the presence of common integration sites, at known and candidate cancer genes, similar to those observed in retroviral mutagenesis screens. Sleeping Beauty is a new tool for unbiased, forward genetic screens for cancer genes in vivo.
Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.
Imatinib, a specific small molecule inhibitor of the Abl kinase, has become the standard drug therapy for chronic myelogenous leukemia in all phases. More than 80% of newly diagnosed patients with chronic phase attain a complete cytogenetic response (CCR). Although remissions in patients with early disease are generally durable, acquired resistance after an initial response is common in advanced disease. Reactivation of Bcr-Abl signaling is almost invariably present at the time of relapse, consistent with re-establishment of the initial pathogenetic mechanism. Mutations in the kinase domain (KD) of Bcr-Abl that impair drug binding and increased expression of Bcr-Abl have been identified as major mechanism of acquired drug resistance. The fact that Bcr-Abl remains central to disease pathogenesis at the time of relapse implies that it also remains the optimal drug target. Alternative Abl kinase inhibitors with increased potency and activity against most Bcr-Abl KD mutants are currently undergoing phase I/II clinical testing, with encouraging early results. Despite the high rates of CCR, persistence of residual leukemia as assessed by reverse transcription polymerase chain reaction is the rule even in patients with chronic phase, suggesting that even these patients may remain at risk of relapse. Understanding the mechanisms underlying disease persistence will be crucial for developing strategies to eradicate residual leukemia.
Trastuzumab (Herceptin) is an excellent model of rationally designed targeted cancer treatment. However, less than 35% of patients with ErbB2-positive breast tumors respond to trastuzumab as a single agent, and 2-5% of trastuzumab-treated patients suffer from severe side effects, including cardiac dysfunction. Recent progress in understanding the mechanisms of trastuzumab antitumor function and cellular defects leading to trastuzumab resistance is summarized. Also explored is the potential of combination therapies for reversing trastuzumab resistance.