Mouse Model Overview

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Mouse Models of Cancer

Dong-Joo Cheon and Sandra Orsulic
Womens Cancer Research Institute, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048; email: orsulics@cshs.org
Annu. Rev. Pathol. Mech. Dis. 2011.6:95-119. Downloaded from www.annualreviews.org by University of Texas - Houston Academy of Medicine on 04/05/13. For personal use only.
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Annu. Rev. Pathol. Mech. Dis. 2011. 6:95119 First published online as a Review in Advance on October 5, 2010 The Annual Review of Pathology: Mechanisms of Disease is online at pathol.annualreviews.org This articles doi: 10.1146/annurev.pathol.3.121806.154244 Copyright c 2011 by Annual Reviews. All rights reserved 1553-4006/11/0228-0095$20.00
Keywords
genetically engineered mice, gene targeting, oncogene, tumor-suppressor gene, cancer-gene discovery, oncogenomics
Abstract
Genetically engineered mouse models have signicantly contributed to our understanding of cancer biology. They have proven to be useful in validating gene functions, identifying novel cancer genes and tumor biomarkers, gaining insight into the molecular and cellular mechanisms underlying tumor initiation and multistage processes of tumorigenesis, and providing better clinical models in which to test novel therapeutic strategies. However, mice still have signicant limitations in modeling human cancer, including species-specic differences and inaccurate recapitulation of de novo human tumor development. Future challenges in mouse modeling include the generation of clinically relevant mouse models that recapitulate the molecular, cellular, and genomic events of human cancers and clinical response as well as the development of technologies that allow for efcient in vivo imaging and high-throughput screening in mice.
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INTRODUCTION
Cancers are thought to result from the accumulation of multiple genetic aberrations that transform cells, which allows for their abnormal growth, proliferation, and metastasis. Discovery of these aberrations and understanding how they contribute to the pathophysiology of cancer are necessary for improvements in diagnosis and therapy. More than any other model system, mice have revolutionized our ability to study gene function in vivo and understand the molecular mechanisms of cancer pathogenesis. As a model system, mice have several important advantages over other mammalian models: (a) They are small in size; (b) they are inexpensive to maintain; (c) they reproduce rapidly and have large litters; and (d ) they can be genetically manipulated. Advances in the genetic manipulation of mice have facilitated the introduction of dened genetic alterations that can be
controlled temporally and spatially in order to faithfully mimic the pathophysiology of human cancers. Currently, numerous techniques for genetic manipulation are available to mouse modelers. Selecting the correct set of techniques is an important rst step in generating mouse models of cancers. In this review, we describe the currently available techniques, their advantages and disadvantages, and the unique features that must be considered in the planning phase of mouse modeling (Figure 1). It is also important to understand the intended purpose of a specic mouse model. The use of a specic approach (forward or reverse genetics) and the level of tissue involvement (a single cell, a small number of cells, a whole tissue, or a whole organism) vary depending on the intended use of the mouse model, such as validation of a gene function, identication of novel cancer genes
Gene of interest
Loss of function
Gain of function
Knockdown
Knockout
Dominant-negative
Transgenic
Knockin
Virus-mediated delivery
RNAi
Need spatial and temporal control? No Yes
Transgenic Knockin
Need spatial and temporal control? No Need tissue-specific expression? No Transgenic with a ubiquitous promoter Yes Transgenic with a tissue-specific promoter Yes
Need spatial and temporal control? Yes No Conventional knockin to endogenous locus (e.g., point mutations)
Conventional knockout
Need reversible change? No Yes Inducible system
Conditional (loxP-STOP-loxP) Inducible
Need sporadic change? No Conditional knockout Yes
Virus-mediated Cre delivery
Figure 1 A strategy for the generation of genetically engineered mouse (GEM) models of cancer. The ow chart shows the decision-making process of selecting the correct approach to generating GEM models of cancers.
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Tissue scale
Single cell
A small number of cells
A specific group of tissue cells
Whole tissues and germ line
Methodology
Mosaic analysis with double markers system (MADM)
Hit and run Head-to-head loxP Virus-mediated delivery
Conditional and inducible knockout Transgenic with a tissue-specific promoter
Knockout Transgenic with a ubiquitous promoter
Figure 2 Different levels of genetic manipulation that can be used to generate genetically engineered mouse models. Genetic engineering technologies allow for genetic manipulation at different levels of tissue involvement, from a single cell to an entire organism.
and tumor biomarkers, or anticancer drug testing (Figure 2). We introduce examples of mouse model use in cancer research and describe the important lessons learned from mouse models. We also discuss the important remaining challenges in generating ideal mouse models of human cancers.
generating genetically altered mice are discussed in more detail in several reviews (68), we briey highlight the main features of these approaches and their applications in generating GEM models of cancer.
Transgenic Approaches
Transgenic mice are created by microinjection of foreign DNA into the pronuclei of fertilized zygotes. Once introduced, the transgene sequences are integrated into random sites of the mouse genome with variable frequency. The rst GEM models of human cancers were generated by overexpressing viral and cellular oncogenes in specic tissues (1, 2). The expression of the transgene in certain types of tissues at specic developmental stages depends on the nature of the promoter or regulatory element used. Certain promoters, such as the mouse mammary tumor virus (MMTV) and whey acidic protein (WAP) promoters, are well characterized and are still actively used to generate mouse models of breast cancer (9, 10). Tumor development in such transgenic mice indicates in vivo oncogenic potential of a gene of interest. In addition, transgenic mice can be intercrossed with other mouse lines or exposed to chemicals
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GENETICALLY ENGINEERED MOUSE MODELS

The development of transgenic and genetargeting technologies in mouse embryonic stem (ES) cells in the 1980s (15) facilitated the generation of genetically engineered mouse (GEM) models to study tumor biology through the manipulation of the mouse germ line. The most common ways to generate mouse models of cancers are to activate oncogenes or inactivate tumor-suppressor genes (or both) in vivo through the use of transgenic and gene-targeting approaches, such as knockouts and knockins. Loss-of-function studies typically employ knockout or conditional knockout alleles, whereas gain-of-function studies use transgenic, conditional transgenic, and knockin approaches. Although the technical aspects of
GEM: genetically engineered mouse MMTV: mouse mammary tumor virus
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BAC: bacterial articial chromosome
or retroviruses in order to identify cooperating genes during tumorigenesis (11, 12). A modied version of this approach is mouse ES cellbased transgenesis in which mouse ES cells are either infected with lentiviruses containing transgenes (13) or electroporated with transgene DNA (14). The advantages of the transgenic approach are the straightforward methodology to assess in vivo tumorigenic functions of a gene and the short time it takes to generate the mice in comparison to the gene-targeting strategy. However, this approach also has several disadvantages. The main disadvantage is the inability to control the level and pattern of transgene expression. Even with well-characterized promoters, the level and pattern of transgene expression vary among the transgenic founders because the copy number and the integration sites of the transgene are random. Random integration of the transgene is of particular concern because it can result in a lack of transgene expression due to positional effects or an unexpected phenotype resulting from secondary effects of transgene integration into sensitive genomic sites. Another caveat to this approach is the limited availability of tissue-specic promoters. In terms of tumor biology, transgenic tumor models deviate from human tumors in two ways: (a) The transgene is integrated into the mouse genome, where two copies of the wild-type alleles are present, whereas in human tumors, it is typical to have one wild-type and one mutant allele; and (b) because all tumor and stromal cells in the transgenic mice express the transgene, the mouse model cannot recapitulate the clonal and sporadic development of human tumors. Several strategies have been developed to address these limitations. The level and pattern of transgene expression can be partially controlled through ES cellbased transgenesis and the screening of ES cells for appropriate expression patterns and levels (14). The positional effect can be prevented by the use of insulators, which are DNA-sequence elements at gene boundaries that prevent the neighboring chromatin environment from disrupting the programmed pattern of expression of the enclosed
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gene (15). In addition, the development of new transgenic technology, such as site-specic integration in ES cells (16) and single-copy transgenesis through long interspersed element type 1 (17), allows for control of the transgene copy number. One method to introduce large genomic fragments into the mouse genome is bacterial articial chromosome (BAC) transgenesis. BAC transgenesis is typically used to rescue the mutant phenotype and to express a transgene under the control of its endogenous promoter and cis elements. This approach has been used to generate so-called humanized mice by replacing a whole mouse genomic locus with a syntenic human genomic locus (18). The caveat to this approach is the possibility that noncoding regions and regulatory elements could contribute to the phenotype. This is an important issue, especially when human BAC is introduced into the mouse genome.
Gene-Targeting Approaches
A specic endogenous locus of a gene can be disrupted or mutated or its expression pattern visualized by gene targeting. The gene-targeting approach is based on homologous recombination in ES cells to replace endogenous ES cell chromatin with a targeting vector that disrupts the allele (8). This approach involves multiple steps that result in either deletions of the coding sequence of a gene (knockout) or the introduction of exogenous sequences into the specic locus (knockin) (7). In the knockout approach, a coding region of the gene of interest (typically a few critical exons that are necessary for the function of the gene) is deleted or replaced with a neomycin-positive selection marker or a reporter gene cassette (e.g., lacZ or GFP), thereby creating a null allele. Several crucial tumor-suppressor genes, such as Rb (19), p53 (20), and Brca1 (21, 22), have been disrupted in mice through the use of this technique. Such knockout mice have signicantly contributed to our understanding of the functions of tumorsuppressor genes during embryonic development and tumorigenesis. This technique is
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particularly useful for modeling heritable tumor syndromes. For example, p53 knockout mice have provided models in which to study Li-Fraumeni syndrome, which is caused by germ-line mutations of p53 (20). The major disadvantage of this approach is the lack of spatial and temporal control over the gene of interest. This is of particular concern because disruption of oncogenes or tumor-suppressor genes in the mouse germ line often causes embryonic lethality (21, 22). Another disadvantage is the occasional occurrence of incomplete gene disruption due to alternative splicing or the presence of a cryptic promoter, which may result in a truncated protein that retains some biological activity, possibly causing wild-type, hypomorph, or dominant-negative phenotypes. Even when the gene of interest is completely disrupted, the model fails to mimic sporadic multistep tumorigenesis because the initiating mutation is present throughout the body and germ line from the beginning of development. Additionally, the tumor spectrum in knockout mice may vary depending on the mouse strain. Furthermore, studies of specic tumor types may be prevented due to the faster development of tumors in other tissues, which may result in precocious mouse morbidity (e.g., lymphoma in p53 knockout mice) (20). The knockin approach is used to introduce exogenous sequences into the specic locus of a gene, including point mutations, loxP sites, reporter gene cassettes, and transgenes. This approach is used to test the oncogenic potential of a mutated gene, visualize a gene of interest under the control of its endogenous promoter and regulatory elements, and generate a modied version of transgenic mice by targeting a specic oncogene into a ubiquitous locus (e.g., the ROSA26 locus) (23) or a specic locus driving tissue-specic gene expression. For example, p53R271H , which is a mutation commonly found in human cancer patients, was successfully introduced into an endogenous p53 locus, resulting in p53 knockin mice with a different pattern of tumor development and metastasis from p53 knockout mice (24). This approach offers better models of human cancers
than does transgenesis because the introduced gene or mutation is regulated under the endogenous promoter and regulatory elements. Additionally, subtle genetic changessuch as mutations, small insertions, or deletions that lead to the activation of proto-oncogenes, or the generation of dominant-negative forms of genesmimic genetic alterations in human cancers. The knockin approach can be further modied to include (a) conditional and inducible systems; (b) chromosomal engineering through two gene-targeting events in ES cells; or (c) the so-called hit-and-run strategy, which takes advantage of spontaneous recombination between duplicated genomic sequences at a low frequency during cell division (25). This strategy is of particular interest because it can recapitulate important aspects of human cancers, such as sporadic gene mutation and hosttumor cell interaction. For example, when two nonfunctional K-ras gene copies were introduced in tandem, the expression of activated K-ras occurred at a low frequency as a result of spontaneous intrachromosomal recombination between homologous sequences. Mice generated by use of this approach were predisposed to a variety of tumors, predominantly lung adenocarcinomas (25).
Dominant-negative: refers to a mutation whose gene product adversely affects the normal, wild-type gene within the same cell
Conditional and Inducible Systems

Conditional and inducible systems allow for the induction of somatic mutations in a tissuespecic and time-controlled manner. The most widely used are (a) the Cre-loxP system; (b) the ippase (FLP)ippase recognition target (FRT ) system; (c) the inducible Cre (e.g., CreERT ) system; and (d ) the tetracycline (tet)-inducible system. Recent mouse models use a combination of these technologies to more accurately mimic tumor onset and progression. The most common conditional geneexpression strategy in the mouse is the CreloxP system. Cre recombinase mediates sitedirected DNA recombination between two 34 base pair loxP sequences. The relative orientation of the two loxP sites determines whether
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recombination results in excision, inversion, or translocation of host DNA (26). Two sameoriented loxP sites are introduced into the specic gene locus through the knockin approach to generate either conditional knockout mice or conditional transgenic mice. Cre recombinase can be introduced by crossing these mice to transgenic mouse lines that express Cre in a specic tissue or by administering a lentivirus or adenovirus that expresses Cre. Conditional knockout mice are useful in uncovering the roles of genes that cause embryonic lethality in conventional knockouts. For example, Brca1 knockout mice die during embryonic development, but Brca1 conditional micewhen bred with WAP-Cre mice, which express Cre in the mammary gland epitheliadevelop mammary tumors (27). The Cre-loxP system can also be used to conditionally activate an oncogene. For example, conditional activation of Kras in LSL-K-rasG12D mice (28) was achieved by targeting loxP-STOP (three tandem polyA sequences)-loxP-K-rasG12D into the endogenous K-ras locus through the knockin approach. Oncogenic K-ras can only be expressed after Cre-mediated removal of the STOP sequences. The advantage of the Cre-loxP conditional system is that gene function can be studied in a specic tissue or cell type at a specic time point, thereby allowing more accurate modeling of sporadic mutational events in a subset of cells. In this regard, virus-mediated Cre delivery is complementary to Cre transgenic mice because it makes it possible to control the amount, time, and frequency of virus injection for the optimal Cre-mediated recombination. The major disadvantage of the conditional system is its irreversibility; once a deletion has occurred in a cell, all of its descendants will carry the recombined allele, even if the promoter that drives Cre is no longer active in the descendants. This can be useful in cell-fate mapping but does not accurately mimic the roles of oncogenes in different stages of tumor progression. Cre expression outside of the tissue of interest or at an early developmental stage can be problematic because it might cause unexpected phenotypes or embryonic lethality. Reporter mouse strains,
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such as the ROSA26-lacZ reporter (R26R) strain (29), allow for a thorough analysis of Cre expression throughout development. In addition, this approach could possibly yield a hypomorphic phenotype due to an incomplete deletion by Cre or an incorrect choice of critical exons. The limited availability of Cre mouse lines is another caveat to this approach. Although this problem may be largely overcome by the usage of lentivirus or adenovirus Cre, this alternative strategy also poses several problems. Depending on the tissue type, Cre delivery may be restricted or may require survival surgery. Leakage problems are a signicant issue because the Cre virus can also infect neighboring tissues, making tumor analysis more complicated. Additionally, a virus could cause a host immune response, depending on the dosage and frequency of delivery. Finally, prolonged Cre expression may have adverse effects, possibly due to recombination at pseudo-loxP sites in the mouse genome (30). Such cytotoxic activity can be overcome by a self-deleting lentiviral Cre or a cell-permeable Cre (30, 31). Inducible systems allow for temporal control over genetic changes. Currently, there are two widely used types of inducible systems: (a) the tamoxifen-inducible system and (b) the tet-inducible system, which is also known as the Tet-On/Tet-Off system. In the tamoxifeninducible system, the ligand-binding domain of a mutated estrogen receptor is fused to either Cre recombinase (Cre-ERT or Cre-ERT2 ) or oncogene complementary DNA (32). The fused protein can then be activated by administration of 4-hydroxytamoxifen (4-OHT) or inactivated by removal of 4-OHT. For tissue-specic genetic changes, a tissue-specic promoter and local injection of 4-OHT are commonly used. Systems that are not only inducible but also reversible are especially useful for studying the role of a gene of interest in tumor maintenance. For example, Pelengaris et al. (33) generated tumor mice by administering 4-OHT topically to mice that expressed an inducible form of c-myc (c-mycERTM ) under the control of the human involucrin promoter. Sustained c-myc activation in adult suprabasal
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epidermis was sufcient to induce papillomatosis; however, this premalignant lesion regressed upon deactivation of c-myc. The major advantage of this approach is the ability to control gene expression in a time-dependent manner. The major disadvantage of this system is the toxicity of tamoxifen and the fusion protein. Excess amounts of tamoxifen can be lethal or can cause damage to certain tissues, such as the uterus (34). In addition, the Cre-ER fusion protein is toxic in the hematopoietic system (35). Also, in the case of Cre-ERT , Cre expression without 4-OHT (leakiness) or weak Cre expression in a few cells after 4-OHT treatment (poor inducibility) can be problematic. However, this characteristic can be useful in mimicking sporadic genetic alterations in human tumors, which are thought to occur in a few cells rather than in entire groups of cells and tissues. The tetracyclin (tet)-inducible system is most commonly used to switch gene expression on and off. It is a binary system composed of the tet transactivator (tTA)/reverse tTA (rtTA) and the operator sequences of the tet operon (tetO). In the Tet-Off system, tTA cannot bind to the tetO sequences located upstream of the target transgene in the presence of tet, thereby turning off transgene expression. The Tet-On system works in the opposite way: rtTA binds the tetO sequences and turns on transgene expression only in the presence of tet (32). Tet or doxycycline (a less toxic version of tet) can be injected into mice or simply added to their drinking water. Tissue specicity is achieved by tissue-specic promoters that drive tTA or rtTA expression. This approach is very useful in studying the roles of sustained expression of oncogenes in tumor maintenance. Inactivation of mutant Ras by the withdrawal of doxycycline results in clinical and histological regression of melanoma and lung cancers, which demonstrates the role of Ras in tumor maintenance (36, 37). The main advantage of this inducible system is the ability to repeatedly turn transgene expression on and off. The major disadvantage is the need to generate two transgenic mouse
lines: one expressing tTA/rtTA and the other expressing the tetO oncogene.
Chromosome Engineering
Many human cancers have various chromosomal abnormalities, such as large deletions, inversions, and translocations. As such, an important aspect of tumor modeling is the recapitulation of genomic aberrations. Chromosome engineering allows for controlled generation of chromosomal abnormalities through the use of ES cellmediated gene targeting and the Cre-loxP system (38). In this approach, two loxP sites are sequentially introduced into two loci in the ES cell genome. The doubly targeted ES cells are exposed to Cre to induce recombination between the loxP sites and to generate the rearranged chromosome. ES cell clones with the desired rearranged chromosome are then microinjected into blastocysts to generate the mice. This approach allows nonhomologous chromosome segments to be recombined, thereby creating various chromosomal rearrangements, such as deletion, duplication, inversion, or translocation, depending on the chromosomal location (cis or trans) and relative orientation of the two loxP sites (38). This strategy was successfully used to replicate the t(8;21) translocation found in human acute myeloid leukemia (AML) (39). More recently, Bagchi et al. (40) used chromosome engineering to identify a novel tumor-suppressor gene, Chd5, by generating mouse strains with a deletion (df ) or duplication (dp) of the syntenic region to human 1p36, which is frequently deleted in various human cancers. The result of the df heterozygosity was increased proliferation and spontaneous tumor development, whereas the dp heterozygosity caused the opposite effect. A short hairpin RNA (shRNA)-mediated proliferation-suppression screening among the candidate genes in that genomic region identied Chd5 as a novel tumor-suppressor gene. Disadvantages of this approach include the need to generate double-targeted ES cells with two independent targeting events and the
AML: acute myeloid leukemia
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extremely low recombination frequency between distant chromosomal sites (38).

Cell-autonomous: describes a genetic trait in which only mutant cells exhibit the mutant phenotype Non-cellautonomous: describes a genetic trait in which mutant cells cause other cells to exhibit a mutant phenotype
RNA Interference
In vivo RNA interference (RNAi) is an alternative to gene targeting in ES cells and is used to rapidly assess the consequences of suppressing gene activity. For a stable knockdown in mammalian cells, plasmid-based RNAi is commonly used to target and silence specic genes by shRNA. To generate RNAi transgenic mice, RNAi vectors can be introduced via microinjection, electroporation, or viral infection into ES cells or zygotes (4145). The RNAi-transduced ES cells are subsequently injected into blastocysts or aggregated with tetraploid embryos, and the embryos are then directly transferred into the pseudopregnant females. Tissue-specic RNAi expression can be achieved using the RNA polymerase II promoter (42, 44). Additionally, the Cre-loxP conditional system and the tet-inducible system can be applied to the RNAi approach for spatiotemporal and reversible suppression of gene activity in vivo (41, 42, 45). The transgenic RNAi technology is particularly useful for dissecting in vivo gene function in a high-throughput manner. The limitations to this approach are (a) phenotype variation among the transgenic lines, depending on the RNAi expression level, copy number, and integration sites; (b) unexpected phenotypes due to the off-target effect of RNAi; and (c) the labor-intensive screening process of identifying the best shRNAs and transgenic lines.
Virus-Mediated Gene Delivery

DNA and RNA tumor viruses, such as adenoviruses and retroviruses, are powerful genetic tools for somatic cell gene transfer in mice (46). Virus-mediated somatic cell genetic modication differs from germ linemodication strategies in that alterations occur within a subset of the cells in the mouse and are not inherited unless they also occur in germ cells. Replication-decient recombinant adenovirus
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is commonly used to deliver oncogenes, dominant-negative tumor-suppressor genes, or Cre to mammalian cells in vivo and in vitro (47). The adenovirus expressing Cre is commonly used to generate mouse models of cancers (e.g., lung and ovarian cancers) when tissue-specic Cre mouse lines are not available (28, 48). However, the adenovirus does not integrate into the host chromosome and thus is unsuitable for stable gene expression. Retroviral and lentiviral vectors offer efcient, stable gene delivery to mammalian cells because they can integrate into the host genome for stable expression. Retroviruses have been extensively used to introduce oncogenes, shRNAs, Cre, and dominant-negative tumor-suppressor genes into mouse cells or tissues to model human cancers. For example, a mouse model of liver cancer was generated by genetically manipulating cultured embryonic liver progenitor cells with oncogene-containing retroviruses, then transplanting the cells into the livers of recipient mice (49). The usage of replication-competent avian retrovirus (RCAS), combined with tumor virus A (TVA) transgenic mice that express the avian retroviral receptor TVA in specic cell types, successfully yielded various mouse models of human cancers such as brain and ovarian cancers (50, 51). Additionally, retroviruses can be used in insertional mutagenesis screens to identify novel oncogenes and tumor-suppressor genes. The advantage of the virus-mediated approach is that it can closely mimic sporadic human cancers in which genetic alterations occur in a few initiating cells, rather than in all cells of a specic tissue. In addition, tumor multiplicity can be controlled by modulating virus titer and infection frequency. The disadvantages of this approach are (a) cross-contamination of neighboring tissues, particularly when the virus is directly injected into the mice; and (b) the low efciency of in vivo infection for some cell types, such as nondividing cells.
Chimeric Mice
Chimeras are useful in dening cellautonomous and non-cell-autonomous effects
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and for examining the roles of embryonic lethal genes in the later stages of development and adulthood (8, 32). The traditional approaches to generating chimeras are either the injection of mutant ES cells into wild-type blastocysts or the aggregation of mutant ES cells with wild-type host tetraploid embryos. Recently, a mosaic mouse model of lung cancer was developed by use of this approach (52). This method can generate a more clinically relevant mouse model because it can recapitulate the existence of a few cancer-initiating cells that are surrounded by normal stromal tissues. Disadvantages of this approach include difculty in generating mutant ES cells, controlling the localization and degree of ES cell contribution in a tissue of interest, and the inability to maintain the mouse lines through breeding.
Mosaic Analysis with Double Markers System

Most current mouse models have major limitations in modeling human cancers due to their inability to recapitulate the clonal nature of human cancer. The mosaic analysis with double markers (MADM) system overcomes this limitation by use of a strategy to manipulate genetic changes on the single-cell level (53). This system uses Cre-mediated interchromosomal recombination to simultaneously knock out a gene of interest while labeling clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric reporter cassettes are targeted into the same loci on homologous chromosomes, then transheterozygous mice are generated by breeding the two mouse lines. Upon Cre-mediated recombination at the G2 phase, two uorescent markers (red uorescent protein and green uorescent protein) are reconstituted to label wild-type, heterozygous, and homozygous mutant cells with red, yellow (double-labeled), and green, respectively (53). The labeling efciency and expression pattern of MADM can be controlled by using different tissue-specic Cre (53, 54). This system was successfully used for cell-autonomous lossof-function studies of the p27kip1 and NR2B
genes (55, 56). A slightly different strategy was used in a mosaic analysis of p53 mutant cells (57). Through the use of this strategy, two cassettes containing FRT sites and complementary halves of an Hprt selection cassette were targeted into chromosome 11, then the mice were bred with p53 mutant mice and ROSA26FLP mice. Unlike conventional p53 knockout mice, which predominantly develop sarcomas and lymphomas (58), the induced mitotic recombination of p53 in this model resulted in diverse tumor types, including epithelial malignancies that more closely mimicked human cancers. The differences in tumor incidence and spectrum between conventional knockout mice and mosaic mice underscore the importance of tumor setting, especially the interaction between a small number of tumor-initiating cells and the surrounding normal stroma. In this sense, the MADM system can be very useful in recapitulating sporadic human cancers. This system can also be useful in understanding the cell of origin of cancers through lineage tracing as well as in studying the roles of cell-autonomous and non-cell-autonomous mechanisms in tumor development. Additionally, when split marker genes are replaced with the GAL4/UAS system or the tet-inducible system, MADM can be used for gain-of-function studies in a small population of cells (53). The major disadvantages of this approach are (a) the limited availability of MADM-engineered mice other than for chromosomes 6 and 11; (b) the complicated breeding scheme to generate mice with uorescent markers, Cre, and a mutation on a specic chromosome of interest; and (c) the labeling efciency, which varies depending on the Cre lines.
MADM: mosaic analysis with double markers
FORWARD GENETICS APPROACHES

Thus far, we have discussed the current approaches to generating GEM models of cancers with dened genetic alterations. These approaches are based mainly on reverse genetics, which focus on gene function. In this section, we outline another set of approaches to
Environmental carcinogen test
UV
Cancer-gene discovery
GEM
Imaging
Figure 3
Therapeutics
Applications of genetically engineered mouse (GEM) models. GEM models can serve as powerful platforms for testing the carcinogenic effects of environmental factors, imaging tumors for longitudinal evaluation of treatment efcacy, identifying novel cancer genes, and discovering and testing anticancer drugs.
creating new pools of mouse models of cancers by using forward genetics. These phenotypedriven mouse models of cancers can serve as useful platforms in identifying cancer genes and testing anticancer drugs in preclinical studies (Figure 3).
Retroviral Insertional Mutagenesis

Retroviral insertional mutagenesis is a powerful tool used to identify novel oncogenes, tumorsuppressor genes, and cooperating mutations in tumor development. Slow-transforming retroviruses, such as murine leukemia virus (MuLV) and MMTV, have been widely used in genetic screens for mutations involved in the onset of tumorigenesis in mice (59, 60). These retroviruses do not carry an oncogene but can induce oncogenic mutations through insertion of a provirus into the host genome, which leads to tumor development. The provirus can be inserted into the upstream or promoter region of a cellular gene, which could result in
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MuLV: murine leukemia virus
higher levels of the endogenous gene transcript or generation of a chimeric transcript. In addition, the intragenic insertion of the provirus can cause premature termination of the gene transcript, resulting in gene activation or inactivation (59, 60). The proviral insertion sites can be identied by Southern analysis or polymerase chain reaction (PCR)-based methods. Many oncogenes, such as c-myc, Evi1, and Pim1, were identied through analyses of multiple retroviral integration sites in MuLV-induced T cell lymphomas and myeloid tumors from AKXD-23-recombinant inbred mice (6163). Tumor-suppressor genes are less frequently found than oncogenes, possibly because two mutations are often required to fully inactivate tumor-suppressor gene function, whereas a single mutation can be sufcient to activate an oncogene. Furthermore, retroviruses preferentially integrate near the transcription start sites of actively transcribed genes rather than in the intragenic region. This problem has been partially addressed by retroviral insertional
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mutagenesis in Blm-decient mice to induce a loss of heterozygosity in many chromosomal regions (64). This strategy resulted in the identication of a number of tumorsuppressor genes, including Rbl1, Rbl2, and Cdkn2c. The major limitation of retroviral insertional mutagenesis is the narrow tumor range induced by the retrovirus. MuLV and MMTV viruses induce mainly hematopoietic (T cell lymphoma) and mammary cancers (60). This problem has been addressed, in part, by use of the Moloney murine leukemia virus (MMLV) to infect E-myc transgenic mice to induce B cell lymphoma (65) or recombinant MMLV encoding the platelet-derived growth factor B chain to induce brain tumors (66). Additional disadvantages include the lack of ability of retroviruses to infect nondividing cells, inefcient infection in slowly replicating cells and tissues that have a basement membrane or mucin layer (59), and the difculty of revealing the integration effects on distant genes.
mutations can be induced, depending on the location of the ENU-induced point mutation and the type of amino acid change. ENU mutagenesis can create many different types of alleles of one gene, allowing for structure-function studies. In addition, the mutant mice can be bred with other mouse models of cancers with dened genetic alterations to identify cooperating oncogenic events. For example, ENU mutagenesis in Pim-1 transgenic mice revealed dosedependent involvement of c-myc and Ras during lymphomagenesis (11). The biggest challenge of ENU mutagenesis is to nd tumor-initiating mutations in the mouse genome, which requires the use of positional cloning. Positional cloning is an extremely laborious and timeconsuming process, but further development of high-throughput DNA-sequencing technologies should accelerate the process of mutation identication.
ENU: N-ethyl-Nnitrosourea Min: multiple intestinal neoplasia Genetic modier: a gene that alters the phenotypic expression of a nonallelic locus
Recombinant Inbred Mouse Strains

Inbred mouse strains, which show different tumor predisposition, can serve as models to study the complex genetic traits of human cancers. For example, BALB/c inbred mice are highly susceptible to MMTV-induced mammary tumors, whereas STS inbred mice are not (71), suggesting that there are interstrain differences in genetic modiers that confer tumor susceptibility and resistance. The identication of such modiers in specic types of cancers can improve our understanding of molecular mechanisms underlying different tumor predispositions among the human population. To identify tumor-susceptibility genes, two inbred mouse strains with high and low tumor susceptibility are intercrossed; then, the offspring mice are characterized by tumor phenotype (e.g., incidence, latency) and marker loci (e.g., microsatellites, singlenucleotide polymorphisms), which are polymorphic between the two parental strains. The correlation between the susceptibility phenotype and the marker distribution is used to map the susceptibility loci to specic chromosomal locations. Mapping can be facilitated by
N-Ethyl-N-Nitrosourea Mutagenesis
The alkylating agent N-ethyl-N-nitrosourea (ENU) is a powerful germ-line mutagen (67) that induces point mutations in a genome-wide manner, which facilitates the creation of mouse models that resemble human mutations in cancers (68). For ENU mutagenesis, inbred or transgenic male mice are treated with ENU and crossed with wild-type female mice to generate F1 offspring, which serve as founder mice for ENU-induced mutation phenotype screening (68). Phenotype-driven ENU screens have yielded mouse models of various human cancers. For example, Min (multiple intestinal neoplasia) mice, which are used as models to study human intestinal cancer, were created by ENU mutagenesis (69). Mutation analysis revealed that the Min phenotype was caused by a point mutation in the Apc gene (70). ENU mutagenesis provides a unique resource in which to study complex cancer traits, dissect gene functions, and identify oncogenes and cooperating genes. Loss-of-function and gain-of-function
SB: Sleeping Beauty transposon
a number of generated inbred mouse strains, such as recombinant inbred strains, recombinant congenic strains, chromosome substitution strains, genome-tagged mice, and advanced intercross lines (72). Through the use of these mouse strains, a large number of cancer-susceptibility loci have been mapped in the mouse genome, including loci that are involved in colon, intestinal, lung, mammary, skin, ovarian, liver, and hematopoietic cancers (72). These mouse strains were used to identify a number of cancer-susceptibility genes: Ptprj, a colon cancersusceptibility gene; Dnd1, a testicular germ cell tumor gene; and Sipa1, a mammary tumormetastasis gene (7375). The major disadvantages of the recombinant inbred approach are that it is extremely laborious, time consuming, and expensive because the generation of mouse lines for mapping requires extensive mouse breeding. Additionally, tumor analysis may be complicated by the restricted tumor spectrum, long latency, and incomplete penetrance.
Transposon System
Emerging transposon techniques have provided an alternative to insertional mutagenesis as a way of identifying novel cancer genes. Transposons are mobile genetic elements that harbor a cargo sequence anked by two inverted repeats/direct repeats (32, 59). The cargo sequence contains the 5 -long terminal repeat of the murine stem cell virus and a splice donor, as well as splice acceptors and polyadenylation sites in both orientations, and it therefore acts as a bidirectional gene trap and promoter to activate or inactivate cellular genes (76, 77). Once the transposase enzyme binds to two inverted repeat/direct repeat sites, it can mobilize the transposon within the genome through the cutand-paste mechanism. Currently, the Sleeping Beauty (SB) and piggyBac (PB) transposon systems are being used. The SB transposon system has been successfully used in insertional mutagenesis screens to identify known and novel oncogenes as well as cooperating cancer genes (7679). To use the transposon system in the
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mouse, two transgenic mouse lines need to be generatedone line expressing the transposon and the other line expressing the transposase. The transposase can be ubiquitously expressed by the CAGGS promoter or ROSA26 locus (76, 77). In addition, a Cre-mediated conditional SB system has been developed by targeting the loxP-STOP-loxP-SB transposase cassette into the ROSA26 locus (7880). In this system, tissue-specic transposase expression can be achieved by using tissue-specic Cre. Transposon insertional mutagenesis can be used as a complementary approach to retroviral insertional mutagenesis for cancer-gene discovery (59). First, the transposon shows a different insertional bias compared with transforming retroviruses. Second, the tissue-specic transposition allows for cancer-gene discovery in a broad range of cancer types. Third, spatial and temporal control over the transposition is possible when using tissue-specic or inducible Cre. Finally, the mutated genes can be easily identied by high-throughput PCR methods because the SB transposon acts as a tag. However, the SB transposonmediated approach has its limitations, including local hopping (preferential integration near the original integration site), a dramatically reduced transposition efciency by a large (>2-kb) cargo size, variable transposase expression in different target tissues, and a complicated tumor analysis due to transposition-induced genomic rearrangements from a sevenbase pair footprint that is left behind (59, 81, 82). Additionally, high-copy transposon integration is required to induce tumors (76, 77). Finally, this approach cannot model specic types of tumors and metastasis in spite of ubiquitous transposition (76, 77). However, these problems can be addressed by use of mutagenic SB transposon vectors harboring tissue-specic promoters (80), hyperactive SB transposase (83), or an alternative transposon, such as PB (8486). The PB transposon is more active; its integration into somatic cells and the germ line is more random; and it can carry up to 9.1 kb of cargo (85, 86). In addition, tight temporal control of transposition can be achieved using the inducible PB system (84).
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APPLICATIONS OF MOUSE MODELS OF CANCER

Mouse models are extremely useful tools for understanding tumor biology and have provided insights into the following questions: (a) What are the initiating genetic alterations in cancer development? (b) How do cancer genes cooperate in different stages of tumor progression? (c) What is the cell of origin in different types of tumors? (d ) Why do individuals show different tumor susceptibility? (e) What are the genetic modiers? ( f ) How do tumor cells grow and metastasize? ( g) Can environmental factors cause cancer? (h) How do tumor cells interact with neighboring normal stromal cells? (i ) What are the mechanisms underlying chemoresistance? ( j) What are the mechanisms underlying tumor dormancy and recurrence? (k) Which therapeutic strategies will work in certain types of cancer? (l ) How can we detect tumors in early stages of development? We highlight some applications in which mouse models are used to answer these questions.
sunburn in melanoma were identied in mouse studies (91, 92). The etiology of UV-induced basal cell carcinoma through the Patched gene mutation was replicated in UV-irradiated Patched heterozygous mice (93). Various chemicals have been implicated in the initiation and progression of tumors by use of mouse models. For example, the nature of the promoting agent phenobarbital was characterized in mouse models of liver cancer and was shown to promote clonal expansion of -catenin-mutated cells to form liver tumors (94).
BLI: bioluminescence imaging
In Vivo Imaging
The utility of mouse models for tumor biology and preclinical studies can be augmented with in vivo imaging techniques. Noninvasive tumor imaging allows for sequential measurements of different factors, including the effects of candidate therapeutic drugs. Various modalities have been developed for imaging mouse tumors, including micropositron emission tomography, single-photon emission computed tomography, magnetic resonance imaging, microcomputed tomography, bioluminescence imaging (BLI), whole-body uorescence imaging, intravital microscopy, and ultrasound (95). In addition to the development of imaging modalities, transgenic technologies have facilitated the generation of diverse biosensor reporter mice (96, 97). Reporter mice expressing luciferase, uorescent protein, or HSV-tk individually or in combination have been the most commonly generated because such mice can be used for BLI, uorescence imaging, and positron emission tomography imaging, respectively. Luciferase or uorescent protein can be fused with tissue-specic promoters, transcription factors, or responsive elements and subsequently introduced into the mouse genome by gene-targeting or transgenic approaches. These reporter mice can serve as biosensors to detect the activity of oncogenes or tumor-suppressor genes as well as to visualize specic processes during tumorigenesis in vivo (96, 97). For example, tumor-directed neoangiogenesis was traced in mice expressing a
Dening the Roles of Environmental Factors in Tumor Development

Environmental factors such as hormones, diet, UV, radiation, and chemicals have causal links to specic human cancers; however, it is unclear whether these factors can directly initiate or promote tumor development. One important application of mouse models is the ability to directly test the roles of environmental factors in tumor development, which is impossible or unethical to do in humans. For example, studies on the effects of oophorectomy and estrogen removal led to the discovery that tamoxifen inhibits the growth of mammary tumors in mice, facilitating its approval for the treatment of human breast cancer (87, 88). The role of caloric restriction in the reduction of tumor incidence was also demonstrated in C3H inbred strains that spontaneously develop hepatomas (89). Additionally, research on Min mice has shown the inuence of dietary fat on polyp phenotype (90). The causative roles of UV and
Oncogenomics: a new eld of genomics that applies highthroughput technologies to characterize genes associated with cancer; also known as cancer genomics
GFP reporter under the control of the vascular endothelial growth factor (VEGF) promoter (98), and hypoxia-inducible factor (HIF) activity was monitored in mice expressing the HIF-luciferase fusion protein (99). A combination of the reporter mice and mouse models of cancer can be used to monitor spatiotemporal tumor development. For example, transgenic mice that express luciferase under the control of the intermediate lobespecic POMC (proopiomelanocortin) promoter were bred with conditional Rb knockout mice in order to monitor pituitary tumor development in vivo using BLI (100). In this study, the same reporter mice also served as bioindicators of tumor response to chemotherapy. A combination of reporter mice, mouse models of cancers, and imaging technologies is expected to signicantly facilitate further testing of novel anticancer drugs. Additionally, reporter mice, which can track progenitor cells and their descendants, could be useful in identifying cell origins of cancers.
Cancer-Gene Discovery
Several high-throughput genomic technologies have been developed to assist in the discovery of cancer genes. These technologies include oligonucleotide and complementary DNA microarrays, multicolor uorescence in situ hybridization and spectral karyotyping, comparative genomic hybridization, and high-resolution genome-scanning representational oligonucleotide microarray analysis (59). However, genomic instability and the heterogeneity of human tumors hamper the identication of true cancer genes that drive tumorigenic processes. In many human cancers, the causative, driver mutations are masked by large deletions or amplications and by other, passenger mutations that do not contribute to the tumor phenotype. Therefore, efcient cancer-gene discovery requires strategies to distinguish causal genetic alterations from genomic noise and to prioritize cancer genes for further functional validation, so mouse models of cancers can be valuable tools for the discovery
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of new cancer genes. An important advantage of mouse models is that their tumors are initiated by a small number of dened genetic alterations, which produce focal oncogenomic proles for feasible, comparative genomic analysis. Cross-species comparative oncogenomics is a very powerful method to identify tumor-driving genes and assess their oncogenic capacity in the appropriate genetic context (101103). In this approach, oncogenomic proles from dened mouse tumors are compared with proles from their corresponding human tumors to lter and prioritize relevant genetic alterations and narrow down the number of candidate oncogenes within the overlapping genomic alterations (e.g., amplicons). For example, cross-species expression proling of human lung tumors and K-ras2 mouse models revealed an oncogenic K-ras2 expression signature in human lung cancer that was not identied in the analysis of human tumors alone (102). Cross-species comparative approaches also successfully identied NEDD9 as a mediator of melanoma metastasis (101) and cIAP1 and Yap as hepatocellular carcinoma oncogenes (49). These genomic approaches were combined with genetic screens that use shRNA libraries to identify new tumor-suppressor genes, such as Xpo4, DLC1, and Rad17 (103105).
Therapeutics
Recent advances in high-throughput screening have identied numerous potential molecular targets for drug discovery. Therefore, there is an urgent need to prioritize potential targets for further drug development and to test the efcacy of new anticancer drugs in clinically relevant and molecularly characterized mouse models. Increasing evidence shows that mouse models can be useful in evaluating the efcacy of novel anticancer drugs and predicting chemotherapeutic response. For example, transgenic mouse models of multistage pancreatic islet cell cancer (RIP1-Tag2 mice) and prostate cancer (known as TRAMP mice) have been used to test the efcacy of angiogenesis inhibitors (106, 107). These studies have
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also provided insight into antiangiogenic drugs, which may be most effective when targeted during specic stages of cancer. Studies of mouse models suggest that response to therapy can be dictated by the molecular and genetic makeup of the tumors. For example, one recent study demonstrated that a mouse model of AML can accurately predict therapy response in patients and that the p53 pathway is a key determinant of chemotherapy response in AML patients (108). Mouse models can be used to evaluate the efcacy of novel anticancer drugs as well as to investigate mechanisms underlying response, resistance, and toxicity prior to clinical trials (109). For example, preclinical studies of the farnesyl protein transferase inhibitor (FTI) in mouse models revealed that the efcacy of FTI was not due to its ability to inhibit hyperactive K-ras, but rather was due to so-called off-target activities (110). This nding explains why FTI failed to show activity in human cancers harboring mutant K-ras. Also, the resistance to anti-VEGF receptor therapy that appeared in the RIP1-Tag2 GEM model revealed broblast growth factor (FGF)-mediated VEGFindependent angiogenesis as the mechanism (111). Ventricular-specic ErbB2 knockout mice revealed the essential role of ErbB2 in preventing dilated cardiomyopathy and explaining the unexpected toxicity of a Her2/neu-blocking antibody in clinical trials (112). In addition, mouse models are useful in preclinical studies and for testing the synergistic effects of combination chemotherapy. One example is the mouse model of acute promyelocytic leukemia (APL). APL patients frequently have chromosomal translocations that fuse the retinoic acid receptor (RARA) gene to various partner genes including promyelocytic leukemia (PML) and promyelocytic leukemia zinc nger (PLZF). Similarly, transgenic mice expressing PML-RARA and PLZF-RARA fusion genes developed APL and responded similarly to APL patients to retinoic acid therapy (113). On the basis of this observation, new combination therapy with retinoic acid and arsenic trioxide was tested in these mouse models. Whereas combination therapy showed a synergistic effect
in PML-RARA mouse APL, it was not effective in PLZF-RARA mouse APL (114). These observations in mouse models led to the development of combination therapy for APL, which is currently in clinical trials. Importantly, the synergistic efcacy of combination therapy was observed only in the mouse models, not in in vitro studies (114), which underscores the importance of mouse models in drug testing.
FTI: farnesyl protein transferase inhibitor APL: acute promyelocytic leukemia
Biomarker Discovery
Mouse models can facilitate the discovery of tumor biomarkers for the diagnosis, prognosis, and monitoring of cancer progression and recurrence. Recent improvements in proteomic proling methods, based primarily on mass spectrometry, have allowed the detection and identication of peptides in blood and biological uids (115). However, biomarker discovery in patient specimens is challenging due to genetic and environmental heterogeneity among patients. In contrast, mouse models, with their genetically dened, homogeneous tumors, can be an efcient means for biomarker discovery. Serum proteomic approaches in mouse models of pancreatic cancer successfully identied serum proteomic proles that are detectable in mice with early-stage lesions (116) and uncovered ve potential biomarkers, which were able to detect early lesions in patients (117). Serum proteomic approaches were also used in mouse models of colon, breast, and ovarian cancers to identify potential biomarkers, such as cathepsins B and D in colon cancer; bulin-2 and osteopontin in breast cancer; and IGFBP2, TIMP-1, RARRES2, CD14, and GRN in ovarian cancer (118120).
LESSONS LEARNED FROM MOUSE MODELS

Studies of mouse models of cancer have provided valuable insights into the molecular mechanisms underlying tumor initiation, progression, metastasis, maintenance, and acquired
chemoresistance. Some examples of lessons that have been learned are described below.
Tumor microenvironment: comprises the normal cells, molecules, and blood vessels that surround and feed a tumor cell; also known as the stroma
1. Cancer genes often play major roles in normal physiology and development. The disruption or activation of tumorsuppressor genes and oncogenes in mice often results in embryonic lethality or severe tissue phenotypes (21, 22). In addition, important developmental pathways, such as the Wnt/ -catenin and sonic hedgehog signaling pathways, are aberrantly expressed in many tumors (121, 122). Therefore, knowledge of the normal development of a tissue is necessary to understand the aberrant development of cancer. 2. Embryonic lethality in tumor-suppressor gene knockout mice or oncogene knockout mice is often rescued by the inactivation of tumor-suppressor genes or oncogenes that function in the same, or functionally related, pathway. One example is the rescue of embryonic lethality of Brca1 knockout mice by loss of p53 function (123). This rescue indicates that the loss of p53 function is required for a cell to tolerate the loss of Brca1 function and may explain why a signicantly higher frequency of p53 mutations are found in patients with BRCA1-associated tumors than in patients with sporadic forms of these tumors (124). 3. Dosage effect on tumor-suppressor genes does not always correlate with increased tumor predisposition. According to the Knudson model (125), two hits are required to inactivate tumor-suppressor gene function. However, as demonstrated in p27 knockout mice, heterozygous mice develop tumors without the need for an additional loss or mutation of the other allele (126). 4. Multiple genetic alterations are required for tumor development. For example, in mouse models of ovarian cancer, multiple genetic alterations (i.e., loss of p53 and a combination of two oncogenic pathways) are required to induce tumors (50). Also,
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only certain combinations of genetic alterations cooperate in inducing tumorigenesis. For example, c-myc, but not Kras, Akt, or Her-2, cooperates with the loss of Brca1 and p53 in transforming mouse ovarian epithelial cells (127). Consistent with the cooperative role of c-myc, p53, and Brca1, tumors from patients with BRCA1 mutations are typically associated with p53 mutations and amplication of c-myc (124, 128). Certain tumors show preferential dependence on specic oncogenes and oncogenic signaling pathways (129). Mouse models have shown that certain tumors require continuous expression of specic oncogenes, such as H-ras, K-ras, and cmyc, for tumor maintenance (33, 36, 37). If such oncogene addiction exists in human cancers, more emphasis should be placed on pathway-specic treatment intervention. The functions of tumor-suppressor genes and oncogenes are context dependent. Several oncogenes play dual roles that either stimulate or suppress tumorigenesis, depending on their genetic context (101, 130, 131). Also, modier screening of inbred mouse strains supports the notion that genetic modiers (e.g., single-nucleotide polymorphisms in specic tumor loci) are responsible for different tumor susceptibilities (72). Tumor cells activate angiogenic, hypoxic, and metabolic pathways for survival and growth (132134). Moreover, one of the mechanisms by which tumor cells acquire chemoresistance is the activation of alternative survival pathways to compensate for the compromised pathway (e.g., tumor cells activate VEGF-independent angiogenesis mediated by FGF following VEGF2-targeted therapy) (111). Studies from stroma-specic mutant mice emphasize the importance of the tumor microenvironment in tumor development (135, 136). The tumor microenvironment contains various immune cells,
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growth factors, hormones, and tissuespecic factors. The selectivity of specic oncogenic mutations in different human tumors (e.g., K-ras mutation in colon and pancreatic cancer, H-ras mutation in tumors of stratied squamous epithelia, and N-ras mutation in melanoma and leukemia) (137) might be the result of the local environments that promote the outgrowth of the tumor-initiating cells with the specic mutation. 9. Cross-species comparison can be useful in identifying novel cancer genes and conserved regulatory elements (138). Considering that many important regulatory pathways are conserved among species, cross-species comparison could provide novel insights into how specic cancer genes or pathways are regulated.
Tumor biology
Cell of origin Sporadic somatic mutations Stochastic acquisition of genetic lesions Stepwise progression Metastasis Tumor microenvironment
Drug response Chemoresistance
Ideal GEM
Histopathology Expression profiling Genomic alterations In vivo imaging High-throughput screening
Figure 4 Ideal genetically engineered mouse (GEM) models. Criteria for the creation of ideal GEM models, which could serve as surrogates for research on human cancers.
LIMITATIONS AND CHALLENGES IN MODELING HUMAN CANCERS IN MICE

Ideal GEM models of human cancers need to fulll multiple criteria (Figure 4) (139). They should mimic the clonal origin of human tumors, tumor histopathology, and the multistage processes of tumorigenesis, including metastatic spread and recurrence after intervention. They should have similar multiple mutations in specic genes, gross chromosomal aberrations induced by genomic instability, and specic pathway alterations known to be involved in human cancers. Additionally, they should be suitable for high-throughput screening and testing of new tumor biomarkers and anticancer drugs, and their drug response should accurately predict the results of clinical trials. However, there are several limitations and challenges that impede the creation of ideal cancer models. These limitations and challenges are discussed below.
Species-Specic Differences
The most signicant challenge in mouse models of human cancers is the species-specic
differences (140). The mouse is different from the human in terms of size, life span, and organ morphology and physiology. One critical difference in the mouse is the activity of telomerase, which is largely inactive in adult human cells. Because most mouse cells have active telomerase, they tend to immortalize and transform more readily than do human cells. Thus, mouse tumors require fewer genetic alterations for malignant transformation than human tumors do. Telomerase activity in mice also prevents the modeling of genomic instability in human cancers. To accurately mimic human cancers, it may be necessary to inactivate telomerase in mouse models. For example, concomitant deletion of Terc and p53 in mice results in chromosomally unstable tumors that are more representative of human tumors (141). These species-specic differences also result in mouse tumors with a different histology and/or spectrum from human tumors. For example, Rb heterozygous mutant mice develop pituitary adenocarcinomas, unlike children with the Rb mutation, who develop retinoblastomas (19). Likewise, p53 null mice develop mainly sarcomas (58), whereas patients with Li-Fraumeni syndrome develop primarily carcinomas (142). Additionally, mouse models tend to develop
relatively few metastases, or they display metastases with different tissue specicity from human cancers, suggesting that the mechanisms underlying metastasis might differ between the species. Finally, differences in metabolic rate and pathways (e.g., the cytochrome P450 pathway for drug metabolism) (143) might result in a different drug response in mouse models. This is of particular concern when mouse models are used in drug testing and preclinical trials.
so-called hit-and-run strategy to allow for the introduction of multiple genetic alterations at different time points in a small number of cells.
Deciencies in Effective Drug Testing

Despite mouse models superiority in modeling human tumors, the value of mouse models in anticancer drug discovery and preclinical studies is still uncertain. As discussed above, the differences in drug metabolism and drug afnity to target proteins between mouse and humans indicate that mouse models might show different drug responses, thereby hampering the identication of the best drug for patients. Additionally, GEM models are not ideal for systematic drug testing in comparison with xenograft models. First, GEM models are more expensive and difcult to generate than xenograft models. Second, breeding mice to generate GEM models with multiple genetic alterations can be complicated. Third, from the perspective of a large-scale screening of drug candidates, it is very difcult to use GEM models because they develop tumors with long latency and variable penetrance. Fourth, the use of available GEM models is often restricted by intellectual property rights and patents (e.g., the OncoMouse R patent). Fifth, the majority of GEM models have not yet been evaluated in drug trials. Active interinstitutional efforts for validation and preclinical trials of currently available GEM models will be necessary in order to identify models that accurately predict drug response in human cancers. Further development and support for international resources for the generation and characterization of mouse models, such as repositories of targeted ES cell clones, mouse lines, and tumor tissues, as well as data banks with freely available and easily accessible information, will be necessary to take full advantage of mouse models in preclinical trials.
Limited Recapitulation of Genetic Alterations in Human Cancers

Considering the complexity of human tumor development, mouse models might be oversimplied. In many mouse models, genomic alterations typically occur in the germ line or in a large portion of somatic cells. In contrast, in human cancers germ-line mutations are quite rare, and most somatic mutations are stochastic. Thus, most mouse models are more representative of human cancerpredisposition syndromes rather than sporadic cancers. In addition, most mouse models focus on oncogenes and tumor-suppressor genes, omitting important aspects of de novo tumor development, such as the tumor microenvironment and the host immune system. Furthermore, most genetic alterations in mouse models result in the overexpression or deletion of certain genes, whereas in human cancers, genes typically contain point mutations. Due to a limited number of initiating genetic alterations, mouse tumors are typically more homogeneous than human tumors. This homogeneity can be benecial in dissecting the functions of a gene of interest. Additionally, analyses of gene-expression changes in homogeneous mouse models can help to identify biomarkers that have been obscured in heterogeneous human cancers. In contrast, homogeneity in mouse tumors can be an obstacle to modeling the heterogeneity of human cancers, especially in drug testing. To recapitulate sporadic, stochastic acquisition of multiple mutations in human cancers, it will be necessary to further rene technologies such as viral gene delivery, MADM, and the
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CONCLUSION
It is unclear whether the development of ideal mouse models of cancer is achievable. Considering the signicant species differences
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between mice and humans, mouse models are unlikely to replace research on human tumor samples, cell lines, and patients. Currently, there is no one best model in which to study cancer. Human cancer studies are very powerful for identifying putative cancer genes and testing anticancer drugs, but they suffer from heterogeneity and complexity, depend on correlation studies, and provide little insight
into the gene function and mechanisms of tumor development. In contrast, mouse models are very useful in studying gene function and the specic pathways that play a role in tumor progression; however, they are not the best models in which to test anticancer drugs. Therefore, combinatorial approaches that use multiple model systems are necessary to understand the complexity of human cancers.
SUMMARY POINTS
1. GEM models have signicantly increased our understanding of the molecular mechanisms underlying tumor initiation, progression, metastasis, and chemoresistance. 2. Gene-targeting and transgenic technologies allow for conditional and inducible control of gene expression in both the mouse germ line and somatic cells. 3. In mouse models, forward genetics approaches, such as retroviruses, transposons, and ENU, can effectively identify novel cancer genes. 4. Mouse models can serve as powerful platforms for the validation of gene functions, identication of novel cancer genes and tumor biomarkers, and anticancer drug testing. 5. There are several limitations in mouse modeling, such as species-specic differences, limited recapitulation of de novo human tumor development, and drug response. 6. The potential of mouse models can be maximized by further development of technologies that allow for more accurate recapitulation of the biological and clinical aspects of human cancers. Also, improvements in high-throughput screening and in vivo imaging will be necessary for efcient and cost-effective use of GEM models in preclinical trials.
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holding that might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Miklos Peterfy, Ruprecht Wiedemeyer, and Kristy J. Daniels for their critical reading of the review. Research in the Orsulic laboratory is supported by the following organizations: National Cancer Institute, R01 CA103924; Ovarian Cancer Research Fund; Sarcoma Foundation of America; Liddy Shriver Sarcoma Initiative; LMSarcoma Direct Research Foundation; Garber Foundation; and the Milken Family Foundation.
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Contents
Annual Review of Pathology: Mechanisms of Disease Volume 6, 2011
Starting in Immunology by Way of Immunopathology Emil R. Unanue p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 The Pathogenesis of Sepsis Deborah J. Stearns-Kurosawa, Marcin F. Osuchowski, Catherine Valentine, Shinichiro Kurosawa, and Daniel G. Remick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 19 EGFR Mutations and Lung Cancer Gilda da Cunha Santos, Frances A. Shepherd, and Ming Sound Tsao p p p p p p p p p p p p p p p p p p p p 49 Zebrash Models for Cancer Shu Liu and Steven D. Leach p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 71 Mouse Models of Cancer Dong-Joo Cheon and Sandra Orsulic p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Disorders of Bone Remodeling Xu Feng and Jay M. McDonald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 121 The Acute Respiratory Distress Syndrome: Pathogenesis and Treatment Michael A. Matthay and Rachel L. Zemans p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 147 The HIF Pathway and Erythrocytosis Frank S. Lee and Melanie J. Percy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 165 Parkinsons Disease: Genetics and Pathogenesis Joshua M. Shulman, Philip L. De Jager, and Mel B. Feany p p p p p p p p p p p p p p p p p p p p p p p p p p p p 193 Pathogenic Mechanisms of HIV Disease Susan Moir, Tae-Wook Chun, and Anthony S. Fauci p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 223 Pathogenesis of Myeloma Kenneth C. Anderson and Ruben D. Carrasco p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249 Alternative Macrophage Activation and Metabolism Justin I. Odegaard and Ajay Chawla p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 275
The Pathobiology of Arrhythmogenic Cardiomyopathy Jeffrey E. Saftz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 299 Mechanisms of Leukocyte Transendothelial Migration William A. Muller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 323 Retinoids, Retinoic Acid Receptors, and Cancer Xiao-Han Tang and Lorraine J. Gudas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 345 Biomedical Differences Between Human and Nonhuman Hominids: Potential Roles for Uniquely Human Aspects of Sialic Acid Biology Nissi M. Varki, Elizabeth Strobert, Edward J. Dick Jr., Kurt Benirschke, and Ajit Varki p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365 A Glimpse of Various Pathogenetic Mechanisms of Diabetic Nephropathy Yashpal S. Kanwar, Lin Sun, Ping Xie, Fu-you Liu, and Sheldon Chen p p p p p p p p p p p p p p p 395 Pathogenesis of Liver Fibrosis Virginia Hernandez-Gea and Scott L. Friedman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 425 Mesenchymal Stem Cells: Mechanisms of Inammation Nora G. Singer and Arnold I. Caplan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457 Molecular Genetics of Colorectal Cancer Eric R. Fearon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 479 The Pathogenesis of Systemic Sclerosis Tamiko R. Katsumoto, Michael L. Whiteld, and M. Kari Connolly p p p p p p p p p p p p p p p p p p p 509 Indexes Cumulative Index of Contributing Authors, Volumes 16 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 539 Cumulative Index of Chapter Titles, Volumes 16 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 542 Errata An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articles may be found at http://pathol.annualreviews.org
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Mouse Model Overview

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Mouse Model Overview

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ANNUAL REVIEWS

Mouse Models of Cancer

Need spatial and temporal control? No Yes

Need reversible change? No Yes Inducible system

Conditional (loxP-STOP-loxP) Inducible

Need sporadic change? No Conditional knockout Yes

Virus-mediated Cre delivery

A small number of cells

A specific group of tissue cells

Whole tissues and germ line

Mosaic analysis with double markers system (MADM)

Hit and run Head-to-head loxP Virus-mediated delivery

Conditional and inducible knockout Transgenic with a tissue-specific promoter

Knockout Transgenic with a ubiquitous promoter

GENETICALLY ENGINEERED MOUSE MODELS

GEM: genetically engineered mouse MMTV: mouse mammary tumor virus

BAC: bacterial articial chromosome

Conditional and Inducible Systems

AML: acute myeloid leukemia

www.annualreviews.org Mouse Models of Cancer

extremely low recombination frequency between distant chromosomal sites (38).

Virus-Mediated Gene Delivery

Mosaic Analysis with Double Markers System

MADM: mosaic analysis with double markers

FORWARD GENETICS APPROACHES

Environmental carcinogen test

Retroviral Insertional Mutagenesis

MuLV: murine leukemia virus

Recombinant Inbred Mouse Strains

SB: Sleeping Beauty transposon

APPLICATIONS OF MOUSE MODELS OF CANCER

BLI: bioluminescence imaging

Dening the Roles of Environmental Factors in Tumor Development

FTI: farnesyl protein transferase inhibitor APL: acute promyelocytic leukemia

LESSONS LEARNED FROM MOUSE MODELS

Drug response Chemoresistance

LIMITATIONS AND CHALLENGES IN MODELING HUMAN CANCERS IN MICE

Deciencies in Effective Drug Testing

Limited Recapitulation of Genetic Alterations in Human Cancers

www.annualreviews.org Mouse Models of Cancer

Annual Review of Pathology: Mechanisms of Disease Volume 6, 2011

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