Mouse Model Overview

PM06CH05-Orsulic ARI 4 December 2010 8:52

Mouse Models of CancerDong-Joo Cheon and Sandra OrsulicWomen’s Cancer Research Institute, Samuel Oschin Comprehensive Cancer Institute,Cedars-Sinai Medical Center, Los Angeles, California 90048; email: [email protected]

Annu. Rev. Pathol. Mech. Dis. 2011. 6:95–119

First published online as a Review in Advance onOctober 5, 2010

The Annual Review of Pathology: Mechanisms ofDisease is online at pathol.annualreviews.org

This article’s 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 significantly contributed toour understanding of cancer biology. They have proven to be usefulin validating gene functions, identifying novel cancer genes and tumorbiomarkers, gaining insight into the molecular and cellular mechanismsunderlying tumor initiation and multistage processes of tumorigenesis,and providing better clinical models in which to test novel therapeuticstrategies. However, mice still have significant limitations in modelinghuman cancer, including species-specific differences and inaccurate re-capitulation of de novo human tumor development. Future challengesin mouse modeling include the generation of clinically relevant mousemodels that recapitulate the molecular, cellular, and genomic events ofhuman cancers and clinical response as well as the development of tech-nologies that allow for efficient in vivo imaging and high-throughputscreening in mice.

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INTRODUCTIONCancers are thought to result from the accu-mulation of multiple genetic aberrations thattransform cells, which allows for their abnor-mal growth, proliferation, and metastasis. Dis-covery of these aberrations and understandinghow they contribute to the pathophysiology ofcancer are necessary for improvements in diag-nosis and therapy. More than any other modelsystem, mice have revolutionized our ability tostudy gene function in vivo and understand themolecular mechanisms of cancer pathogenesis.As a model system, mice have several impor-tant advantages over other mammalian models:(a) They are small in size; (b) they are inexpen-sive to maintain; (c) they reproduce rapidly andhave large litters; and (d ) they can be geneti-cally manipulated. Advances in the genetic ma-nipulation of mice have facilitated the introduc-tion of defined genetic alterations that can be

Gene of interest

Gain of functionLoss of function

Knockdown

RNAi

No Yes

No Yes

No Yes

No Yes

No Yes NoYes

• Transgenic

• Knockin

Knockout Dominant-negative Transgenic Knockin Virus-mediateddelivery

Need spatialand temporal

control?


control?

Need tissue-specificexpression?

Transgenicwith aubiquitouspromoter

Transgenicwith atissue-specificpromoter

Need reversiblechange?

• Conditional(loxP-STOP-loxP)

• Inducible

Conventionalknockin toendogenouslocus (e.g., point mutations)

Conventionalknockout

Need sporadicchange?

Conditionalknockout

Virus-mediatedCre delivery

Induciblesystem


control?

Figure 1A strategy for the generation of genetically engineered mouse (GEM) models of cancer. The flow chart shows the decision-makingprocess of selecting the correct approach to generating GEM models of cancers.

controlled temporally and spatially in order tofaithfully mimic the pathophysiology of humancancers.

Currently, numerous techniques for geneticmanipulation are available to mouse modelers.Selecting the correct set of techniques is animportant first step in generating mouse mod-els of cancers. In this review, we describe thecurrently available techniques, their advantagesand disadvantages, and the unique featuresthat must be considered in the planning phaseof mouse modeling (Figure 1). It is alsoimportant to understand the intended purposeof a specific mouse model. The use of a specificapproach (forward or reverse genetics) and thelevel of tissue involvement (a single cell, a smallnumber of cells, a whole tissue, or a wholeorganism) vary depending on the intended useof the mouse model, such as validation of a genefunction, identification of novel cancer genes

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Single cellA small number

of cellsA specific group

of tissue cellsWhole tissuesand germ line

Mosaic analysis with double

markers system(MADM)

Hit and run

Head-to-head loxP

Virus-mediateddelivery

Knockout

Transgenic witha ubiquitous

promoter

Conditional andinducible knockout

Transgenic with atissue-specific

promoter

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Figure 2Different 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.

GEM: geneticallyengineered mouse

MMTV: mousemammary tumor virus

and tumor biomarkers, or anticancer drugtesting (Figure 2). We introduce examplesof mouse model use in cancer research anddescribe the important lessons learned frommouse models. We also discuss the importantremaining challenges in generating idealmouse models of human cancers.

GENETICALLY ENGINEEREDMOUSE MODELS

The development of transgenic and gene-targeting technologies in mouse embryonicstem (ES) cells in the 1980s (1–5) facilitatedthe generation of genetically engineered mouse(GEM) models to study tumor biology throughthe manipulation of the mouse germ line. Themost common ways to generate mouse mod-els of cancers are to activate oncogenes orinactivate tumor-suppressor genes (or both)in vivo through the use of transgenic andgene-targeting approaches, such as knockoutsand knockins. Loss-of-function studies typi-cally employ knockout or conditional knock-out alleles, whereas gain-of-function studies usetransgenic, conditional transgenic, and knockinapproaches. Although the technical aspects of

generating genetically altered mice are dis-cussed in more detail in several reviews (6–8),we briefly highlight the main features of theseapproaches and their applications in generatingGEM models of cancer.

Transgenic Approaches

Transgenic mice are created by microinjectionof foreign DNA into the pronuclei of fertil-ized zygotes. Once introduced, the transgenesequences are integrated into random sites ofthe mouse genome with variable frequency.The first GEM models of human cancers weregenerated by overexpressing viral and cellularoncogenes in specific tissues (1, 2). The expres-sion of the transgene in certain types of tissuesat specific developmental stages depends on thenature of the promoter or regulatory elementused. Certain promoters, such as the mousemammary tumor virus (MMTV) and wheyacidic protein (WAP) promoters, are well char-acterized and are still actively used to generatemouse models of breast cancer (9, 10). Tumordevelopment in such transgenic mice indicatesin vivo oncogenic potential of a gene of interest.In addition, transgenic mice can be intercrossedwith other mouse lines or exposed to chemicals

www.annualreviews.org • Mouse Models of Cancer 97

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BAC: bacterialartificial chromosome

or retroviruses in order to identify cooperatinggenes during tumorigenesis (11, 12). A modifiedversion of this approach is mouse ES cell–basedtransgenesis in which mouse ES cells are eitherinfected with lentiviruses containing transgenes(13) or electroporated with transgene DNA(14).

The advantages of the transgenic approachare the straightforward methodology to assessin vivo tumorigenic functions of a gene and theshort time it takes to generate the mice in com-parison to the gene-targeting strategy. How-ever, this approach also has several disadvan-tages. The main disadvantage is the inabilityto control the level and pattern of transgeneexpression. Even with well-characterized pro-moters, the level and pattern of transgene ex-pression vary among the transgenic foundersbecause the copy number and the integrationsites of the transgene are random. Random in-tegration of the transgene is of particular con-cern because it can result in a lack of transgeneexpression due to positional effects or an un-expected phenotype resulting from secondaryeffects of transgene integration into sensitivegenomic sites. Another caveat to this approachis the limited availability of tissue-specific pro-moters. In terms of tumor biology, transgenictumor models deviate from human tumors intwo ways: (a) The transgene is integrated intothe mouse genome, where two copies of thewild-type alleles are present, whereas in humantumors, it is typical to have one wild-type andone mutant allele; and (b) because all tumor andstromal cells in the transgenic mice express thetransgene, the mouse model cannot recapitu-late the clonal and sporadic development of hu-man tumors. Several strategies have been devel-oped to address these limitations. The level andpattern of transgene expression can be partiallycontrolled through ES cell–based transgenesisand the screening of ES cells for appropriate ex-pression patterns and levels (14). The positionaleffect can be prevented by the use of insulators,which are DNA-sequence elements at geneboundaries that prevent the neighboring chro-matin environment from disrupting the pro-grammed pattern of expression of the enclosed

gene (15). In addition, the development of newtransgenic technology, such as site-specific in-tegration in ES cells (16) and single-copy trans-genesis through long interspersed element type1 (17), allows for control of the transgene copynumber.

One method to introduce large genomicfragments into the mouse genome is bacterialartificial chromosome (BAC) transgenesis.BAC transgenesis is typically used to rescue themutant phenotype and to express a transgeneunder the control of its endogenous promoterand cis elements. This approach has beenused to generate so-called humanized mice byreplacing a whole mouse genomic locus witha syntenic human genomic locus (18). Thecaveat to this approach is the possibility thatnoncoding regions and regulatory elementscould contribute to the phenotype. This is animportant issue, especially when human BACis introduced into the mouse genome.

Gene-Targeting Approaches

A specific endogenous locus of a gene can be dis-rupted or mutated or its expression pattern vi-sualized by gene targeting. The gene-targetingapproach is based on homologous recombina-tion in ES cells to replace endogenous ES cellchromatin with a targeting vector that disruptsthe allele (8). This approach involves multiplesteps that result in either deletions of the codingsequence of a gene (knockout) or the introduc-tion of exogenous sequences into the specificlocus (knockin) (7). In the knockout approach,a coding region of the gene of interest (typi-cally a few critical exons that are necessary forthe function of the gene) is deleted or replacedwith a neomycin-positive selection marker ora reporter gene cassette (e.g., lacZ or GFP),thereby creating a null allele. Several crucialtumor-suppressor genes, such as Rb (19), p53(20), and Brca1 (21, 22), have been disruptedin mice through the use of this technique. Suchknockout mice have significantly contributed toour understanding of the functions of tumor-suppressor genes during embryonic develop-ment and tumorigenesis. This technique is

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Dominant-negative:refers to a mutationwhose gene productadversely affects thenormal, wild-type genewithin the same cell

particularly useful for modeling heritabletumor syndromes. For example, p53 knock-out mice have provided models in which tostudy Li-Fraumeni syndrome, which is causedby germ-line mutations of p53 (20). The ma-jor disadvantage of this approach is the lack ofspatial and temporal control over the gene ofinterest. This is of particular concern becausedisruption of oncogenes or tumor-suppressorgenes in the mouse germ line often causes em-bryonic lethality (21, 22). Another disadvan-tage is the occasional occurrence of incompletegene disruption due to alternative splicing orthe presence of a cryptic promoter, which mayresult in a truncated protein that retains somebiological activity, possibly causing wild-type,hypomorph, or dominant-negative phenotypes.Even when the gene of interest is completelydisrupted, the model fails to mimic sporadicmultistep tumorigenesis because the initiatingmutation is present throughout the body andgerm line from the beginning of development.Additionally, the tumor spectrum in knockoutmice may vary depending on the mouse strain.Furthermore, studies of specific tumor typesmay be prevented due to the faster developmentof tumors in other tissues, which may result inprecocious mouse morbidity (e.g., lymphomain p53 knockout mice) (20).

The knockin approach is used to introduceexogenous sequences into the specific locusof a gene, including point mutations, loxPsites, reporter gene cassettes, and transgenes.This approach is used to test the oncogenicpotential of a mutated gene, visualize a geneof interest under the control of its endogenouspromoter and regulatory elements, and gen-erate a modified version of transgenic mice bytargeting a specific oncogene into a ubiquitouslocus (e.g., the ROSA26 locus) (23) or a specificlocus driving tissue-specific gene expression.For example, p53R271H , which is a mutationcommonly found in human cancer patients, wassuccessfully introduced into an endogenousp53 locus, resulting in p53 knockin mice witha different pattern of tumor development andmetastasis from p53 knockout mice (24). Thisapproach offers better models of human cancers

than does transgenesis because the introducedgene or mutation is regulated under the en-dogenous promoter and regulatory elements.Additionally, subtle genetic changes—such asmutations, small insertions, or deletions thatlead to the activation of proto-oncogenes, orthe generation of dominant-negative forms ofgenes—mimic genetic alterations in humancancers. The knockin approach can be furthermodified to include (a) conditional and in-ducible systems; (b) chromosomal engineeringthrough two gene-targeting events in ES cells;or (c) the so-called hit-and-run strategy, whichtakes advantage of spontaneous recombinationbetween duplicated genomic sequences at alow frequency during cell division (25). Thisstrategy is of particular interest because itcan recapitulate important aspects of humancancers, such as sporadic gene mutation andhost–tumor cell interaction. For example,when two nonfunctional K-ras gene copieswere introduced in tandem, the expression ofactivated K-ras occurred at a low frequencyas a result of spontaneous intrachromoso-mal recombination between homologoussequences. Mice generated by use of thisapproach were predisposed to a variety oftumors, predominantly lung adenocarcinomas(25).

Conditional and Inducible Systems

Conditional and inducible systems allow forthe induction of somatic mutations in a tissue-specific and time-controlled manner. The mostwidely used are (a) the Cre-loxP system; (b) theflippase (FLP)–flippase recognition target (FRT )system; (c) the inducible Cre (e.g., CreERT) sys-tem; and (d ) the tetracycline (tet)-inducible sys-tem. Recent mouse models use a combinationof these technologies to more accurately mimictumor onset and progression.

The most common conditional gene-expression strategy in the mouse is the Cre-loxP system. Cre recombinase mediates site-directed DNA recombination between two 34–base pair loxP sequences. The relative orienta-tion of the two loxP sites determines whether


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recombination results in excision, inversion, ortranslocation of host DNA (26). Two same-oriented loxP sites are introduced into the spe-cific gene locus through the knockin approachto generate either conditional knockout miceor conditional transgenic mice. Cre recombi-nase can be introduced by crossing these miceto transgenic mouse lines that express Cre in aspecific tissue or by administering a lentivirusor adenovirus that expresses Cre. Conditionalknockout mice are useful in uncovering theroles of genes that cause embryonic lethal-ity in conventional knockouts. For example,Brca1 knockout mice die during embryonic de-velopment, but Brca1 conditional mice—whenbred with WAP-Cre mice, which express Cre inthe mammary gland epithelia—develop mam-mary tumors (27). The Cre-loxP system canalso be used to conditionally activate an onco-gene. For example, conditional activation of K-ras in LSL-K-rasG12D mice (28) was achievedby targeting loxP-STOP (three tandem polyAsequences)-loxP-K-rasG12D into the endogenousK-ras locus through the knockin approach.Oncogenic K-ras can only be expressed afterCre-mediated removal of the STOP sequences.

The advantage of the Cre-loxP conditionalsystem is that gene function can be studied ina specific tissue or cell type at a specific timepoint, thereby allowing more accurate mod-eling of sporadic mutational events in a sub-set of cells. In this regard, virus-mediated Credelivery is complementary to Cre transgenicmice because it makes it possible to control theamount, time, and frequency of virus injectionfor the optimal Cre-mediated recombination.The major disadvantage of the conditional sys-tem is its irreversibility; once a deletion has oc-curred in a cell, all of its descendants will carrythe recombined allele, even if the promoter thatdrives Cre is no longer active in the descendants.This can be useful in cell-fate mapping but doesnot accurately mimic the roles of oncogenes indifferent stages of tumor progression. Cre ex-pression outside of the tissue of interest or at anearly developmental stage can be problematicbecause it might cause unexpected phenotypesor embryonic lethality. Reporter mouse strains,

such as the ROSA26-lacZ reporter (R26R) strain(29), allow for a thorough analysis of Cre ex-pression throughout development. In addition,this approach could possibly yield a hypomor-phic phenotype due to an incomplete deletionby Cre or an incorrect choice of critical exons.The limited availability of Cre mouse lines isanother caveat to this approach. Although thisproblem may be largely overcome by the us-age of lentivirus or adenovirus Cre, this alterna-tive strategy also poses several problems. De-pending on the tissue type, Cre delivery maybe restricted or may require survival surgery.Leakage problems are a significant issue be-cause the Cre virus can also infect neighbor-ing tissues, making tumor analysis more com-plicated. Additionally, a virus could cause a hostimmune response, depending on the dosage andfrequency of delivery. Finally, prolonged Creexpression may have adverse effects, possiblydue to recombination at pseudo-loxP sites in themouse genome (30). Such cytotoxic activity canbe overcome by a self-deleting lentiviral Cre ora cell-permeable Cre (30, 31).

Inducible systems allow for temporalcontrol over genetic changes. Currently, thereare two widely used types of inducible systems:(a) the tamoxifen-inducible system and (b) thetet-inducible system, which is also known asthe Tet-On/Tet-Off system. In the tamoxifen-inducible system, the ligand-binding domain ofa mutated estrogen receptor is fused to eitherCre recombinase (Cre-ERT or Cre-ERT2)or oncogene complementary DNA (32). Thefused protein can then be activated by ad-ministration of 4-hydroxytamoxifen (4-OHT)or inactivated by removal of 4-OHT. Fortissue-specific genetic changes, a tissue-specificpromoter and local injection of 4-OHT arecommonly used. Systems that are not onlyinducible but also reversible are especiallyuseful for studying the role of a gene of interestin tumor maintenance. For example, Pelengariset al. (33) generated tumor mice by adminis-tering 4-OHT topically to mice that expressedan inducible form of c-myc (c-mycERTM) underthe control of the human involucrin promoter.Sustained c-myc activation in adult suprabasal

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AML: acute myeloidleukemia

epidermis was sufficient to induce papillo-matosis; however, this premalignant lesionregressed upon deactivation of c-myc. Themajor advantage of this approach is the abilityto control gene expression in a time-dependentmanner. The major disadvantage of this systemis the toxicity of tamoxifen and the fusionprotein. Excess amounts of tamoxifen can belethal or can cause damage to certain tissues,such as the uterus (34). In addition, the Cre-ERfusion protein is toxic in the hematopoieticsystem (35). Also, in the case of Cre-ERT, Creexpression without 4-OHT (leakiness) or weakCre expression in a few cells after 4-OHTtreatment (poor inducibility) can be problem-atic. However, this characteristic can be usefulin mimicking sporadic genetic alterations inhuman tumors, which are thought to occur in afew cells rather than in entire groups of cells andtissues.

The tetracyclin (tet)-inducible system ismost commonly used to switch gene expressionon and off. It is a binary system composed ofthe tet transactivator (tTA)/reverse tTA (rtTA)and the operator sequences of the tet operon(tetO). In the Tet-Off system, tTA cannot bindto the tetO sequences located upstream of thetarget transgene in the presence of tet, therebyturning off transgene expression. The Tet-Onsystem works in the opposite way: rtTA bindsthe tetO sequences and turns on transgene ex-pression only in the presence of tet (32). Tetor doxycycline (a less toxic version of tet) canbe injected into mice or simply added to theirdrinking water. Tissue specificity is achievedby tissue-specific promoters that drive tTA orrtTA expression. This approach is very usefulin studying the roles of sustained expression ofoncogenes in tumor maintenance. Inactivationof mutant Ras by the withdrawal of doxycyclineresults in clinical and histological regressionof melanoma and lung cancers, which demon-strates the role of Ras in tumor maintenance (36,37). The main advantage of this inducible sys-tem is the ability to repeatedly turn transgeneexpression on and off. The major disadvantageis the need to generate two transgenic mouse

lines: one expressing tTA/rtTA and the otherexpressing the tetO oncogene.

Chromosome Engineering

Many human cancers have various chromo-somal abnormalities, such as large deletions,inversions, and translocations. As such, an im-portant aspect of tumor modeling is the recapit-ulation of genomic aberrations. Chromosomeengineering allows for controlled generationof chromosomal abnormalities through the useof ES cell–mediated gene targeting and theCre-loxP system (38). In this approach, two loxPsites are sequentially introduced into two lociin the ES cell genome. The doubly targeted EScells are exposed to Cre to induce recombina-tion between the loxP sites and to generate therearranged chromosome. ES cell clones withthe desired rearranged chromosome are thenmicroinjected into blastocysts to generate themice. This approach allows nonhomologouschromosome segments to be recombined,thereby creating various chromosomal re-arrangements, such as deletion, duplication,inversion, or translocation, depending on thechromosomal location (cis or trans) and relativeorientation of the two loxP sites (38). This strat-egy was successfully used to replicate the t(8;21)translocation found in human acute myeloidleukemia (AML) (39). More recently, Bagchiet al. (40) used chromosome engineering toidentify a novel tumor-suppressor gene, Chd5,by generating mouse strains with a deletion(df ) or duplication (dp) of the syntenic regionto human 1p36, which is frequently deletedin various human cancers. The result of the dfheterozygosity was increased proliferation andspontaneous tumor development, whereas thedp heterozygosity caused the opposite effect.A short hairpin RNA (shRNA)-mediatedproliferation-suppression screening amongthe candidate genes in that genomic regionidentified Chd5 as a novel tumor-suppressorgene. Disadvantages of this approach includethe need to generate double-targeted ES cellswith two independent targeting events and the


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Cell-autonomous:describes a genetictrait in which onlymutant cells exhibitthe mutant phenotype

Non-cell-autonomous:describes a genetictrait in which mutantcells cause other cellsto exhibit a mutantphenotype

extremely low recombination frequencybetween distant chromosomal sites (38).

RNA Interference

In vivo RNA interference (RNAi) is an al-ternative to gene targeting in ES cells and isused to rapidly assess the consequences of sup-pressing gene activity. For a stable knockdownin mammalian cells, plasmid-based RNAi iscommonly used to target and silence specificgenes by shRNA. To generate RNAi trans-genic mice, RNAi vectors can be introducedvia microinjection, electroporation, or viral in-fection into ES cells or zygotes (41–45). TheRNAi-transduced ES cells are subsequentlyinjected into blastocysts or aggregated withtetraploid embryos, and the embryos are thendirectly transferred into the pseudopregnant fe-males. Tissue-specific RNAi expression can beachieved using the RNA polymerase II pro-moter (42, 44). Additionally, the Cre-loxP con-ditional system and the tet-inducible system canbe applied to the RNAi approach for spatiotem-poral and reversible suppression of gene activ-ity in vivo (41, 42, 45). The transgenic RNAitechnology is particularly useful for dissect-ing in vivo gene function in a high-throughputmanner. The limitations to this approach are(a) phenotype variation among the transgeniclines, depending on the RNAi expression level,copy number, and integration sites; (b) unex-pected phenotypes due to the off-target effectof RNAi; and (c) the labor-intensive screen-ing process of identifying the best shRNAs andtransgenic lines.

Virus-Mediated Gene Delivery

DNA and RNA tumor viruses, such as aden-oviruses and retroviruses, are powerful genetictools for somatic cell gene transfer in mice(46). Virus-mediated somatic cell genetic mod-ification differs from germ line–modificationstrategies in that alterations occur within asubset of the cells in the mouse and are notinherited unless they also occur in germ cells.Replication-deficient recombinant adenovirus

is commonly used to deliver oncogenes,dominant-negative tumor-suppressor genes,or Cre to mammalian cells in vivo and invitro (47). The adenovirus expressing Cre iscommonly used to generate mouse models ofcancers (e.g., lung and ovarian cancers) whentissue-specific Cre mouse lines are not available(28, 48). However, the adenovirus does notintegrate into the host chromosome and thusis unsuitable for stable gene expression. Retro-viral and lentiviral vectors offer efficient, stablegene delivery to mammalian cells because theycan integrate into the host genome for stableexpression. Retroviruses have been extensivelyused to introduce oncogenes, shRNAs, Cre, anddominant-negative tumor-suppressor genesinto mouse cells or tissues to model human can-cers. For example, a mouse model of liver cancerwas generated by genetically manipulatingcultured embryonic liver progenitor cells withoncogene-containing retroviruses, then trans-planting the cells into the livers of recipientmice (49). The usage of replication-competentavian retrovirus (RCAS), combined with tumorvirus A (TVA) transgenic mice that express theavian retroviral receptor TVA in specific celltypes, successfully yielded various mouse mod-els of human cancers such as brain and ovariancancers (50, 51). Additionally, retroviruses canbe used in insertional mutagenesis screens toidentify novel oncogenes and tumor-suppressorgenes. The advantage of the virus-mediated ap-proach is that it can closely mimic sporadic hu-man cancers in which genetic alterations occurin a few initiating cells, rather than in all cells ofa specific tissue. In addition, tumor multiplicitycan be controlled by modulating virus titerand infection frequency. The disadvantagesof this approach are (a) cross-contaminationof neighboring tissues, particularly when thevirus is directly injected into the mice; and (b)the low efficiency of in vivo infection for somecell types, such as nondividing cells.

Chimeric Mice

Chimeras are useful in defining cell-autonomous and non-cell-autonomous effects


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MADM: mosaicanalysis with doublemarkers

and for examining the roles of embryonic lethalgenes in the later stages of development andadulthood (8, 32). The traditional approachesto generating chimeras are either the injectionof mutant ES cells into wild-type blastocystsor the aggregation of mutant ES cells withwild-type host tetraploid embryos. Recently,a mosaic mouse model of lung cancer wasdeveloped by use of this approach (52). Thismethod can generate a more clinically relevantmouse model because it can recapitulate theexistence of a few cancer-initiating cells thatare surrounded by normal stromal tissues. Dis-advantages of this approach include difficultyin generating mutant ES cells, controlling thelocalization and degree of ES cell contributionin a tissue of interest, and the inability tomaintain the mouse lines through breeding.

Mosaic Analysis with DoubleMarkers System

Most current mouse models have major limita-tions in modeling human cancers due to theirinability to recapitulate the clonal nature ofhuman cancer. The mosaic analysis with dou-ble markers (MADM) system overcomes thislimitation by use of a strategy to manipulategenetic changes on the single-cell level (53).This system uses Cre-mediated interchromo-somal recombination to simultaneously knockout a gene of interest while labeling clones ofsomatic cells or isolated single cells in vivo.Two reciprocally chimeric reporter cassettesare targeted into the same loci on homologouschromosomes, then transheterozygous mice aregenerated by breeding the two mouse lines.Upon Cre-mediated recombination at the G2phase, two fluorescent markers (red fluorescentprotein and green fluorescent protein) are re-constituted to label wild-type, heterozygous,and homozygous mutant cells with red, yellow(double-labeled), and green, respectively (53).The labeling efficiency and expression patternof MADM can be controlled by using differ-ent tissue-specific Cre (53, 54). This systemwas successfully used for cell-autonomous loss-of-function studies of the p27kip1 and NR2B

genes (55, 56). A slightly different strategy wasused in a mosaic analysis of p53 mutant cells(57). Through the use of this strategy, two cas-settes containing FRT sites and complemen-tary halves of an Hprt selection cassette weretargeted into chromosome 11, then the micewere bred with p53 mutant mice and ROSA26-FLP mice. Unlike conventional p53 knockoutmice, which predominantly develop sarcomasand lymphomas (58), the induced mitotic re-combination of p53 in this model resulted indiverse tumor types, including epithelial ma-lignancies that more closely mimicked humancancers. The differences in tumor incidence andspectrum between conventional knockout miceand mosaic mice underscore the importanceof tumor setting, especially the interaction be-tween a small number of tumor-initiating cellsand the surrounding normal stroma. In thissense, the MADM system can be very use-ful in recapitulating sporadic human cancers.This system can also be useful in understand-ing the cell of origin of cancers through lin-eage tracing as well as in studying the rolesof cell-autonomous and non-cell-autonomousmechanisms in tumor development. Addition-ally, when split marker genes are replaced withthe GAL4/UAS system or the tet-inducible sys-tem, MADM can be used for gain-of-functionstudies in a small population of cells (53). Themajor disadvantages of this approach are (a) thelimited availability of MADM-engineered miceother than for chromosomes 6 and 11; (b) thecomplicated breeding scheme to generate micewith fluorescent markers, Cre, and a mutationon a specific chromosome of interest; and (c) thelabeling efficiency, which varies depending onthe Cre lines.

FORWARD GENETICSAPPROACHES

Thus far, we have discussed the currentapproaches to generating GEM models ofcancers with defined genetic alterations.These approaches are based mainly on reversegenetics, which focus on gene function. In thissection, we outline another set of approaches to


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GEM

TherapeuticsImaging

Cancer-gene discoveryEnvironmentalcarcinogen test

UV

0

Figure 3Applications of genetically engineered mouse (GEM) models. GEM models can serve as powerful platformsfor testing the carcinogenic effects of environmental factors, imaging tumors for longitudinal evaluation oftreatment efficacy, identifying novel cancer genes, and discovering and testing anticancer drugs.

MuLV: murineleukemia virus

creating new pools of mouse models of cancersby using forward genetics. These phenotype-driven mouse models of cancers can serve asuseful platforms in identifying cancer genesand testing anticancer drugs in preclinicalstudies (Figure 3).

Retroviral Insertional Mutagenesis

Retroviral insertional mutagenesis is a powerfultool used to identify novel oncogenes, tumor-suppressor genes, and cooperating mutationsin tumor development. Slow-transformingretroviruses, such as murine leukemia virus(MuLV) and MMTV, have been widely usedin genetic screens for mutations involved in theonset of tumorigenesis in mice (59, 60). Theseretroviruses do not carry an oncogene but caninduce oncogenic mutations through insertionof a provirus into the host genome, whichleads to tumor development. The proviruscan be inserted into the upstream or promoterregion of a cellular gene, which could result in

higher levels of the endogenous gene transcriptor generation of a chimeric transcript. In ad-dition, the intragenic insertion of the proviruscan cause premature termination of the genetranscript, resulting in gene activation or in-activation (59, 60). The proviral insertion sitescan be identified by Southern analysis or poly-merase chain reaction (PCR)-based methods.Many oncogenes, such as c-myc, Evi1, and Pim1,were identified through analyses of multipleretroviral integration sites in MuLV-inducedT cell lymphomas and myeloid tumors fromAKXD-23-recombinant inbred mice (61–63).Tumor-suppressor genes are less frequentlyfound than oncogenes, possibly because twomutations are often required to fully inactivatetumor-suppressor gene function, whereas asingle mutation can be sufficient to activate anoncogene. Furthermore, retroviruses preferen-tially integrate near the transcription start sitesof actively transcribed genes rather than inthe intragenic region. This problem has beenpartially addressed by retroviral insertional


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ENU: N-ethyl-N-nitrosourea

Min: multiple intestinalneoplasia

Genetic modifier:a gene that alters thephenotypic expressionof a nonallelic locus

mutagenesis in Blm-deficient mice to inducea loss of heterozygosity in many chromo-somal regions (64). This strategy resultedin the identification of a number of tumor-suppressor genes, including Rbl1, Rbl2, andCdkn2c. The major limitation of retroviralinsertional mutagenesis is the narrow tumorrange induced by the retrovirus. MuLV andMMTV viruses induce mainly hematopoietic(T cell lymphoma) and mammary cancers (60).This problem has been addressed, in part, byuse of the Moloney murine leukemia virus(MMLV) to infect Eμ-myc transgenic mice toinduce B cell lymphoma (65) or recombinantMMLV encoding the platelet-derived growthfactor B chain to induce brain tumors (66).Additional disadvantages include the lack ofability of retroviruses to infect nondividingcells, inefficient infection in slowly replicatingcells and tissues that have a basement mem-brane or mucin layer (59), and the difficultyof revealing the integration effects on distantgenes.

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-widemanner, which facilitates the creation of mousemodels that resemble human mutations in can-cers (68). For ENU mutagenesis, inbred ortransgenic male mice are treated with ENU andcrossed with wild-type female mice to gener-ate F1 offspring, which serve as founder micefor ENU-induced mutation phenotype screen-ing (68). Phenotype-driven ENU screens haveyielded mouse models of various human can-cers. For example, Min (multiple intestinal neo-plasia) mice, which are used as models to studyhuman intestinal cancer, were created by ENUmutagenesis (69). Mutation analysis revealedthat the Min phenotype was caused by a pointmutation in the Apc gene (70). ENU mutage-nesis provides a unique resource in which tostudy complex cancer traits, dissect gene func-tions, and identify oncogenes and cooperatinggenes. Loss-of-function and gain-of-function

mutations can be induced, depending on the lo-cation of the ENU-induced point mutation andthe type of amino acid change. ENU mutagen-esis can create many different types of alleles ofone gene, allowing for structure-function stud-ies. In addition, the mutant mice can be bredwith other mouse models of cancers with de-fined genetic alterations to identify cooperatingoncogenic events. For example, ENU mutage-nesis in Pim-1 transgenic mice revealed dose-dependent involvement of c-myc and Ras duringlymphomagenesis (11). The biggest challengeof ENU mutagenesis is to find tumor-initiatingmutations in the mouse genome, which re-quires the use of positional cloning. Positionalcloning is an extremely laborious and time-consuming process, but further development ofhigh-throughput DNA-sequencing technolo-gies should accelerate the process of mutationidentification.

Recombinant Inbred Mouse Strains

Inbred mouse strains, which show differenttumor predisposition, can serve as models tostudy the complex genetic traits of human can-cers. For example, BALB/c inbred mice arehighly susceptible to MMTV-induced mam-mary tumors, whereas STS inbred mice are not(71), suggesting that there are interstrain dif-ferences in genetic modifiers that confer tu-mor susceptibility and resistance. The iden-tification of such modifiers in specific typesof cancers can improve our understandingof molecular mechanisms underlying differ-ent tumor predispositions among the humanpopulation. To identify tumor-susceptibilitygenes, two inbred mouse strains with highand low tumor susceptibility are intercrossed;then, the offspring mice are characterizedby tumor phenotype (e.g., incidence, latency)and marker loci (e.g., microsatellites, single-nucleotide polymorphisms), which are poly-morphic between the two parental strains. Thecorrelation between the susceptibility pheno-type and the marker distribution is used tomap the susceptibility loci to specific chromo-somal locations. Mapping can be facilitated by


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SB: Sleeping Beautytransposon

a number of generated inbred mouse strains,such as recombinant inbred strains, recom-binant congenic strains, chromosome substi-tution strains, genome-tagged mice, and ad-vanced intercross lines (72). Through the useof these mouse strains, a large number ofcancer-susceptibility loci have been mapped inthe mouse genome, including loci that are in-volved in colon, intestinal, lung, mammary,skin, ovarian, liver, and hematopoietic cancers(72). These mouse strains were used to iden-tify a number of cancer-susceptibility genes:Ptprj, a colon cancer–susceptibility gene; Dnd1,a testicular germ cell tumor gene; and Sipa1, amammary tumor–metastasis gene (73–75). Themajor disadvantages of the recombinant inbredapproach are that it is extremely laborious, timeconsuming, and expensive because the genera-tion of mouse lines for mapping requires exten-sive mouse breeding. Additionally, tumor anal-ysis may be complicated by the restricted tumorspectrum, long latency, and incomplete pene-trance.

Transposon System

Emerging transposon techniques have pro-vided an alternative to insertional mutagenesisas a way of identifying novel cancer genes.Transposons are mobile genetic elements thatharbor a cargo sequence flanked by two invertedrepeats/direct repeats (32, 59). The cargo se-quence contains the 5′-long terminal repeat ofthe murine stem cell virus and a splice donor,as well as splice acceptors and polyadenylationsites in both orientations, and it therefore actsas a bidirectional gene trap and promoter to ac-tivate or inactivate cellular genes (76, 77). Oncethe transposase enzyme binds to two invertedrepeat/direct repeat sites, it can mobilize thetransposon within the genome through the cut-and-paste mechanism. Currently, the SleepingBeauty (SB) and piggyBac (PB) transposon sys-tems are being used. The SB transposon systemhas been successfully used in insertional mu-tagenesis screens to identify known and noveloncogenes as well as cooperating cancer genes(76–79). To use the transposon system in the

mouse, two transgenic mouse lines need to begenerated—one line expressing the transposonand the other line expressing the transposase.The transposase can be ubiquitously expressedby the CAGGS promoter or ROSA26 locus (76,77). In addition, a Cre-mediated conditionalSB system has been developed by targetingthe loxP-STOP-loxP-SB transposase cassetteinto the ROSA26 locus (78–80). In this system,tissue-specific transposase expression can beachieved by using tissue-specific Cre.

Transposon insertional mutagenesis can beused as a complementary approach to retroviralinsertional mutagenesis for cancer-gene discov-ery (59). First, the transposon shows a differentinsertional bias compared with transformingretroviruses. Second, the tissue-specific trans-position allows for cancer-gene discovery in abroad range of cancer types. Third, spatial andtemporal control over the transposition is possi-ble when using tissue-specific or inducible Cre.Finally, the mutated genes can be easily iden-tified by high-throughput PCR methods be-cause the SB transposon acts as a tag. How-ever, the SB transposon–mediated approach hasits limitations, including local hopping (pref-erential integration near the original integra-tion site), a dramatically reduced transpositionefficiency by a large (>2-kb) cargo size, vari-able transposase expression in different targettissues, and a complicated tumor analysis dueto transposition-induced genomic rearrange-ments from a seven–base pair footprint that isleft behind (59, 81, 82). Additionally, high-copytransposon integration is required to induce tu-mors (76, 77). Finally, this approach cannotmodel specific types of tumors and metastasis inspite of ubiquitous transposition (76, 77). How-ever, these problems can be addressed by useof mutagenic SB transposon vectors harboringtissue-specific promoters (80), hyperactive SBtransposase (83), or an alternative transposon,such as PB (84–86). The PB transposon is moreactive; its integration into somatic cells and thegerm line is more random; and it can carry up to9.1 kb of cargo (85, 86). In addition, tight tem-poral control of transposition can be achievedusing the inducible PB system (84).


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BLI: bioluminescenceimaging

APPLICATIONS OF MOUSEMODELS OF CANCER

Mouse models are extremely useful tools for un-derstanding tumor biology and have providedinsights into the following questions: (a) Whatare the initiating genetic alterations in cancerdevelopment? (b) How do cancer genes coop-erate in different stages of tumor progression?(c) What is the cell of origin in different typesof tumors? (d ) Why do individuals show differ-ent tumor susceptibility? (e) What are the ge-netic modifiers? ( f ) How do tumor cells growand metastasize? ( g) Can environmental fac-tors cause cancer? (h) How do tumor cells in-teract with neighboring normal stromal cells?(i ) What are the mechanisms underlyingchemoresistance? ( j) What are the mechanismsunderlying tumor dormancy and recurrence?(k) Which therapeutic strategies will work incertain types of cancer? (l ) How can we de-tect tumors in early stages of development? Wehighlight some applications in which mousemodels are used to answer these questions.

Defining the Roles of EnvironmentalFactors in Tumor Development

Environmental factors such as hormones, diet,UV, radiation, and chemicals have causal linksto specific human cancers; however, it is unclearwhether these factors can directly initiate orpromote tumor development. One importantapplication of mouse models is the ability todirectly test the roles of environmental factorsin tumor development, which is impossible orunethical to do in humans. For example, studieson the effects of oophorectomy and estrogenremoval led to the discovery that tamoxifeninhibits the growth of mammary tumors inmice, facilitating its approval for the treatmentof human breast cancer (87, 88). The role ofcaloric restriction in the reduction of tumor in-cidence was also demonstrated in C3H inbredstrains that spontaneously develop hepatomas(89). Additionally, research on Min mice hasshown the influence of dietary fat on polypphenotype (90). The causative roles of UV and

sunburn in melanoma were identified in mousestudies (91, 92). The etiology of UV-inducedbasal cell carcinoma through the Patched genemutation was replicated in UV-irradiatedPatched heterozygous mice (93). Various chem-icals have been implicated in the initiation andprogression of tumors by use of mouse models.For example, the nature of the promotingagent phenobarbital was characterized inmouse models of liver cancer and was shown topromote clonal expansion of β-catenin-mutatedcells to form liver tumors (94).

In Vivo Imaging

The utility of mouse models for tumor biologyand preclinical studies can be augmented within vivo imaging techniques. Noninvasive tumorimaging allows for sequential measurementsof different factors, including the effects ofcandidate therapeutic drugs. Various modal-ities have been developed for imaging mousetumors, including micro–positron emission to-mography, single-photon emission computedtomography, magnetic resonance imaging,microcomputed tomography, bioluminescenceimaging (BLI), whole-body fluorescence imag-ing, intravital microscopy, and ultrasound (95).

In addition to the development of imagingmodalities, transgenic technologies havefacilitated the generation of diverse “biosen-sor” reporter mice (96, 97). Reporter miceexpressing luciferase, fluorescent protein, orHSV-tk individually or in combination havebeen the most commonly generated becausesuch mice can be used for BLI, fluorescenceimaging, and positron emission tomographyimaging, respectively. Luciferase or fluores-cent protein can be fused with tissue-specificpromoters, transcription factors, or responsiveelements and subsequently introduced into themouse genome by gene-targeting or transgenicapproaches. These reporter mice can serve asbiosensors to detect the activity of oncogenes ortumor-suppressor genes as well as to visualizespecific processes during tumorigenesis in vivo(96, 97). For example, tumor-directed neoan-giogenesis was traced in mice expressing a


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Oncogenomics: anew field of genomicsthat applies high-throughputtechnologies tocharacterize genesassociated with cancer;also known as cancergenomics

GFP reporter under the control of the vascularendothelial growth factor (VEGF) promoter(98), and hypoxia-inducible factor (HIF)activity was monitored in mice expressing theHIF-luciferase fusion protein (99). A combina-tion of the reporter mice and mouse models ofcancer can be used to monitor spatiotemporaltumor development. For example, transgenicmice that express luciferase under the controlof the intermediate lobe–specific POMC (pro-opiomelanocortin) promoter were bred withconditional Rb knockout mice in order to mon-itor pituitary tumor development in vivo usingBLI (100). In this study, the same reporter micealso served as bioindicators of tumor responseto chemotherapy. A combination of reportermice, mouse models of cancers, and imag-ing technologies is expected to significantlyfacilitate further testing of novel anticancerdrugs. Additionally, reporter mice, which cantrack progenitor cells and their descendants,could be useful in identifying cell origins ofcancers.

Cancer-Gene Discovery

Several high-throughput genomic technolo-gies have been developed to assist in thediscovery of cancer genes. These technologiesinclude oligonucleotide and complementaryDNA microarrays, multicolor fluorescence insitu hybridization and spectral karyotyping,comparative genomic hybridization, andhigh-resolution genome-scanning represen-tational oligonucleotide microarray analysis(59). However, genomic instability and theheterogeneity of human tumors hamper theidentification of true cancer genes that drive tu-morigenic processes. In many human cancers,the causative, “driver” mutations are masked bylarge deletions or amplifications and by other,“passenger” mutations that do not contributeto the tumor phenotype. Therefore, efficientcancer-gene discovery requires strategies todistinguish causal genetic alterations from ge-nomic noise and to prioritize cancer genes forfurther functional validation, so mouse modelsof cancers can be valuable tools for the discovery

of new cancer genes. An important advantageof mouse models is that their tumors areinitiated by a small number of defined geneticalterations, which produce focal oncogenomicprofiles for feasible, comparative genomicanalysis. Cross-species comparative oncoge-nomics is a very powerful method to identifytumor-driving genes and assess their oncogeniccapacity in the appropriate genetic context(101–103). In this approach, oncogenomic pro-files from defined mouse tumors are comparedwith profiles from their corresponding humantumors to filter and prioritize relevant geneticalterations and narrow down the number ofcandidate oncogenes within the overlappinggenomic alterations (e.g., amplicons). Forexample, cross-species expression profiling ofhuman lung tumors and K-ras2 mouse modelsrevealed an oncogenic K-ras2 expression signa-ture in human lung cancer that was not identi-fied in the analysis of human tumors alone (102).Cross-species comparative approaches alsosuccessfully identified NEDD9 as a mediator ofmelanoma metastasis (101) and cIAP1 and Yapas hepatocellular carcinoma oncogenes (49).These genomic approaches were combinedwith genetic screens that use shRNA librariesto identify new tumor-suppressor genes, suchas Xpo4, DLC1, and Rad17 (103–105).

Therapeutics

Recent advances in high-throughput screen-ing have identified numerous potential molecu-lar targets for drug discovery. Therefore, thereis an urgent need to prioritize potential tar-gets for further drug development and to testthe efficacy of new anticancer drugs in clin-ically relevant and molecularly characterizedmouse models. Increasing evidence shows thatmouse models can be useful in evaluating theefficacy of novel anticancer drugs and predict-ing chemotherapeutic response. For example,transgenic mouse models of multistage pan-creatic islet cell cancer (RIP1-Tag2 mice) andprostate cancer (known as TRAMP mice) havebeen used to test the efficacy of angiogene-sis inhibitors (106, 107). These studies have


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FTI: farnesyl proteintransferase inhibitor

APL: acutepromyelocyticleukemia

also provided insight into antiangiogenic drugs,which may be most effective when targeted dur-ing specific stages of cancer. Studies of mousemodels suggest that response to therapy can bedictated by the molecular and genetic makeupof the tumors. For example, one recent studydemonstrated that a mouse model of AML canaccurately predict therapy response in patientsand that the p53 pathway is a key determinant ofchemotherapy response in AML patients (108).

Mouse models can be used to evaluate theefficacy of novel anticancer drugs as well as toinvestigate mechanisms underlying response,resistance, and toxicity prior to clinical trials(109). For example, preclinical studies of thefarnesyl protein transferase inhibitor (FTI) inmouse models revealed that the efficacy of FTIwas not due to its ability to inhibit hyperactiveK-ras, but rather was due to so-called off-targetactivities (110). This finding explains whyFTI failed to show activity in human cancersharboring mutant K-ras. Also, the resistanceto anti-VEGF receptor therapy that appearedin the RIP1-Tag2 GEM model revealed fibro-blast growth factor (FGF)-mediated VEGF-independent angiogenesis as the mechanism(111). Ventricular-specific ErbB2 knockoutmice revealed the essential role of ErbB2 in pre-venting dilated cardiomyopathy and explainingthe unexpected toxicity of a Her2/neu-blockingantibody in clinical trials (112).

In addition, mouse models are useful in pre-clinical studies and for testing the synergisticeffects of combination chemotherapy. One ex-ample is the mouse model of acute promyelo-cytic leukemia (APL). APL patients frequentlyhave chromosomal translocations that fuse theretinoic acid receptor α (RARA) gene to variouspartner genes including promyelocytic leukemia(PML) and promyelocytic leukemia zinc finger(PLZF). Similarly, transgenic mice expressingPML-RARA and PLZF-RARA fusion genes de-veloped APL and responded similarly to APLpatients to retinoic acid therapy (113). On thebasis of this observation, new combination ther-apy with retinoic acid and arsenic trioxide wastested in these mouse models. Whereas com-bination therapy showed a synergistic effect

in PML-RARA mouse APL, it was not effec-tive in PLZF-RARA mouse APL (114). Theseobservations in mouse models led to the de-velopment of combination therapy for APL,which is currently in clinical trials. Importantly,the synergistic efficacy of combination ther-apy was observed only in the mouse models,not in in vitro studies (114), which under-scores the importance of mouse models in drugtesting.

Biomarker Discovery

Mouse models can facilitate the discovery of tu-mor biomarkers for the diagnosis, prognosis,and monitoring of cancer progression and re-currence. Recent improvements in proteomicprofiling methods, based primarily on massspectrometry, have allowed the detection andidentification of peptides in blood and biologi-cal fluids (115). However, biomarker discoveryin patient specimens is challenging due to ge-netic and environmental heterogeneity amongpatients. In contrast, mouse models, with theirgenetically defined, homogeneous tumors, canbe an efficient means for biomarker discovery.Serum proteomic approaches in mouse mod-els of pancreatic cancer successfully identifiedserum proteomic profiles that are detectablein mice with early-stage lesions (116) and un-covered five potential biomarkers, which wereable to detect early lesions in patients (117).Serum proteomic approaches were also used inmouse models of colon, breast, and ovarian can-cers to identify potential biomarkers, such ascathepsins B and D in colon cancer; fibulin-2and osteopontin in breast cancer; and IGFBP2,TIMP-1, RARRES2, CD14, and GRN in ovar-ian cancer (118–120).

LESSONS LEARNED FROMMOUSE MODELS

Studies of mouse models of cancer have pro-vided valuable insights into the molecularmechanisms underlying tumor initiation, pro-gression, metastasis, maintenance, and acquired


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Tumormicroenvironment:comprises the normalcells, molecules, andblood vessels thatsurround and feed atumor cell; also knownas the stroma

chemoresistance. Some examples of lessons thathave been learned are described below.

1. Cancer genes often play major rolesin normal physiology and development.The disruption or activation of tumor-suppressor genes and oncogenes in miceoften results in embryonic lethality or se-vere tissue phenotypes (21, 22). In addi-tion, important developmental pathways,such as the Wnt/β-catenin and sonichedgehog signaling pathways, are aber-rantly expressed in many tumors (121,122). Therefore, knowledge of the nor-mal development of a tissue is necessaryto understand the aberrant developmentof cancer.

2. Embryonic lethality in tumor-suppressorgene knockout mice or oncogeneknockout mice is often rescued by theinactivation of tumor-suppressor genesor oncogenes that function in the same,or functionally related, pathway. One ex-ample is the rescue of embryonic lethalityof Brca1 knockout mice by loss of p53function (123). This rescue indicates thatthe loss of p53 function is required for acell to tolerate the loss of Brca1 functionand may explain why a significantlyhigher frequency of p53 mutations arefound in patients with BRCA1-associatedtumors than in patients with sporadicforms of these tumors (124).

3. Dosage effect on tumor-suppressor genesdoes not always correlate with increasedtumor predisposition. According to theKnudson model (125), two hits are re-quired to inactivate tumor-suppressorgene function. However, as demonstratedin p27 knockout mice, heterozygous micedevelop tumors without the need for anadditional loss or mutation of the otherallele (126).

4. Multiple genetic alterations are requiredfor tumor development. For example, inmouse models of ovarian cancer, multiplegenetic alterations (i.e., loss of p53 and acombination of two oncogenic pathways)are required to induce tumors (50). Also,

only certain combinations of genetic al-terations cooperate in inducing tumori-genesis. For example, c-myc, but not K-ras, Akt, or Her-2, cooperates with theloss of Brca1 and p53 in transformingmouse ovarian epithelial cells (127). Con-sistent with the cooperative role of c-myc,p53, and Brca1, tumors from patients withBRCA1 mutations are typically associatedwith p53 mutations and amplification ofc-myc (124, 128).

5. Certain tumors show preferential depen-dence on specific oncogenes and onco-genic signaling pathways (129). Mousemodels have shown that certain tumorsrequire continuous expression of specificoncogenes, such as H-ras, K-ras, and c-myc, for tumor maintenance (33, 36, 37).If such “oncogene addiction” exists in hu-man cancers, more emphasis should beplaced on pathway-specific treatment in-tervention.

6. The functions of tumor-suppressor genesand oncogenes are context dependent.Several oncogenes play dual roles thateither stimulate or suppress tumorigen-esis, depending on their genetic context(101, 130, 131). Also, modifier screeningof inbred mouse strains supports thenotion that genetic modifiers (e.g.,single-nucleotide polymorphisms inspecific tumor loci) are responsible fordifferent tumor susceptibilities (72).

7. Tumor cells activate angiogenic, hypoxic,and metabolic pathways for survival andgrowth (132–134). Moreover, one of themechanisms by which tumor cells acquirechemoresistance is the activation of al-ternative survival pathways to compen-sate for the compromised pathway (e.g.,tumor cells activate VEGF-independentangiogenesis mediated by FGF followingVEGF2-targeted therapy) (111).

8. Studies from stroma-specific mutant miceemphasize the importance of the tu-mor microenvironment in tumor devel-opment (135, 136). The tumor microen-vironment contains various immune cells,


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growth factors, hormones, and tissue-specific factors. The selectivity of spe-cific oncogenic mutations in different hu-man tumors (e.g., K-ras mutation in colonand pancreatic cancer, H-ras mutationin tumors of stratified squamous epithe-lia, and N-ras mutation in melanoma andleukemia) (137) might be the result of thelocal environments that promote the out-growth of the tumor-initiating cells withthe specific mutation.

9. Cross-species comparison can be useful inidentifying novel cancer genes and con-served regulatory elements (138). Con-sidering that many important regulatorypathways are conserved among species,cross-species comparison could providenovel insights into how specific cancergenes or pathways are regulated.

LIMITATIONS ANDCHALLENGES IN MODELINGHUMAN CANCERS IN MICE

Ideal GEM models of human cancers needto fulfill multiple criteria (Figure 4) (139).They should mimic the clonal origin of hu-man tumors, tumor histopathology, and themultistage processes of tumorigenesis, includ-ing metastatic spread and recurrence after in-tervention. They should have similar multiplemutations in specific genes, gross chromoso-mal aberrations induced by genomic instabil-ity, and specific pathway alterations known tobe involved in human cancers. Additionally,they should be suitable for high-throughputscreening and testing of new tumor biomark-ers and anticancer drugs, and their drug re-sponse should accurately predict the results ofclinical trials. However, there are several limi-tations and challenges that impede the creationof ideal cancer models. These limitations andchallenges are discussed below.

Species-Specific Differences

The most significant challenge in mousemodels of human cancers is the species-specific

Tumor biology

• Cell of origin

• Sporadic somatic mutations

• Stochastic acquisition of genetic lesions

• Stepwise progression

• Metastasis

• Tumor microenvironment

Drug response

Chemoresistance

In vivo imaging

High-throughput screening

Histopathology

Expression profiling

Genomic alterations

IdealGEM

Figure 4Ideal genetically engineered mouse (GEM) models. Criteria for the creation ofideal GEM models, which could serve as surrogates for research on humancancers.

differences (140). The mouse is differentfrom the human in terms of size, life span,and organ morphology and physiology. Onecritical difference in the mouse is the activityof telomerase, which is largely inactive in adulthuman cells. Because most mouse cells haveactive telomerase, they tend to immortalize andtransform more readily than do human cells.Thus, mouse tumors require fewer geneticalterations for malignant transformation thanhuman tumors do. Telomerase activity in micealso prevents the modeling of genomic insta-bility in human cancers. To accurately mimichuman cancers, it may be necessary to inacti-vate telomerase in mouse models. For example,concomitant deletion of Terc and p53 in miceresults in chromosomally unstable tumors thatare more representative of human tumors (141).These species-specific differences also result inmouse tumors with a different histology and/orspectrum from human tumors. For example,Rb heterozygous mutant mice develop pituitaryadenocarcinomas, unlike children with the Rbmutation, who develop retinoblastomas (19).Likewise, p53 null mice develop mainly sarco-mas (58), whereas patients with Li-Fraumenisyndrome develop primarily carcinomas (142).Additionally, mouse models tend to develop


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relatively few metastases, or they displaymetastases with different tissue specificity fromhuman cancers, suggesting that the mechanismsunderlying metastasis might differ between thespecies. Finally, differences in metabolic rateand pathways (e.g., the cytochrome P450 path-way for drug metabolism) (143) might result ina different drug response in mouse models. Thisis of particular concern when mouse modelsare used in drug testing and preclinical trials.

Limited Recapitulation of GeneticAlterations in Human Cancers

Considering the complexity of human tumordevelopment, mouse models might be over-simplified. In many mouse models, genomicalterations typically occur in the germ line orin a large portion of somatic cells. In contrast,in human cancers germ-line mutations arequite rare, and most somatic mutations arestochastic. Thus, most mouse models are morerepresentative of human cancer–predispositionsyndromes rather than sporadic cancers. In ad-dition, most mouse models focus on oncogenesand tumor-suppressor genes, omitting impor-tant aspects of de novo tumor development,such as the tumor microenvironment andthe host immune system. Furthermore, mostgenetic alterations in mouse models result inthe overexpression or deletion of certain genes,whereas in human cancers, genes typically con-tain point mutations. Due to a limited numberof initiating genetic alterations, mouse tumorsare typically more homogeneous than humantumors. This homogeneity can be beneficial indissecting the functions of a gene of interest.Additionally, analyses of gene-expressionchanges in homogeneous mouse models canhelp to identify biomarkers that have beenobscured in heterogeneous human cancers. Incontrast, homogeneity in mouse tumors canbe an obstacle to modeling the heterogeneityof human cancers, especially in drug testing.To recapitulate sporadic, stochastic acquisitionof multiple mutations in human cancers, itwill be necessary to further refine technologiessuch as viral gene delivery, MADM, and the

so-called hit-and-run strategy to allow for theintroduction of multiple genetic alterations atdifferent time points in a small number of cells.

Deficiencies in Effective Drug Testing

Despite mouse models’ superiority in modelinghuman tumors, the value of mouse models inanticancer drug discovery and preclinical stud-ies is still uncertain. As discussed above, the dif-ferences in drug metabolism and drug affinity totarget proteins between mouse and humans in-dicate that mouse models might show differentdrug responses, thereby hampering the iden-tification of the best drug for patients. Addi-tionally, GEM models are not ideal for system-atic drug testing in comparison with xenograftmodels. First, GEM models are more expensiveand difficult to generate than xenograft mod-els. Second, breeding mice to generate GEMmodels with multiple genetic alterations can becomplicated. Third, from the perspective of alarge-scale screening of drug candidates, it isvery difficult to use GEM models because theydevelop tumors with long latency and variablepenetrance. Fourth, the use of available GEMmodels is often restricted by intellectual prop-erty rights and patents (e.g., the OncoMouse R©

patent). Fifth, the majority of GEM modelshave not yet been evaluated in drug trials. Ac-tive interinstitutional efforts for validation andpreclinical trials of currently available GEMmodels will be necessary in order to identifymodels that accurately predict drug responsein human cancers. Further development andsupport for international resources for the gen-eration and characterization of mouse models,such as repositories of targeted ES cell clones,mouse lines, and tumor tissues, as well as databanks with freely available and easily accessi-ble information, will be necessary to take fulladvantage of mouse models in preclinical trials.

CONCLUSION

It is unclear whether the development ofideal mouse models of cancer is achievable.Considering the significant species differences


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between mice and humans, mouse models areunlikely to replace research on human tumorsamples, cell lines, and patients. Currently,there is no one best model in which to studycancer. Human cancer studies are very pow-erful for identifying putative cancer genesand testing anticancer drugs, but they sufferfrom heterogeneity and complexity, depend oncorrelation studies, and provide little insight

into the gene function and mechanisms oftumor development. In contrast, mouse modelsare very useful in studying gene function andthe specific pathways that play a role in tumorprogression; however, they are not the bestmodels in which to test anticancer drugs.Therefore, combinatorial approaches thatuse multiple model systems are necessary tounderstand the complexity of human cancers.

SUMMARY POINTS

1. GEM models have significantly increased our understanding of the molecular mecha-nisms underlying tumor initiation, progression, metastasis, and chemoresistance.

2. Gene-targeting and transgenic technologies allow for conditional and inducible controlof gene expression in both the mouse germ line and somatic cells.

3. In mouse models, forward genetics approaches, such as retroviruses, transposons, andENU, can effectively identify novel cancer genes.

4. Mouse models can serve as powerful platforms for the validation of gene functions,identification of novel cancer genes and tumor biomarkers, and anticancer drug testing.

5. There are several limitations in mouse modeling, such as species-specific 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 technologiesthat allow for more accurate recapitulation of the biological and clinical aspects of humancancers. Also, improvements in high-throughput screening and in vivo imaging will benecessary for efficient and cost-effective use of GEM models in preclinical trials.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holding thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Miklos Peterfy, Ruprecht Wiedemeyer, and Kristy J. Daniels for their critical readingof the review. Research in the Orsulic laboratory is supported by the following organizations:National Cancer Institute, R01 CA103924; Ovarian Cancer Research Fund; Sarcoma Foundationof America; Liddy Shriver Sarcoma Initiative; LMSarcoma Direct Research Foundation; GarberFoundation; and the Milken Family Foundation.

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PM06-FrontMatter ARI 1 December 2010 11:14

Annual Review ofPathology:Mechanisms ofDisease

Volume 6, 2011Contents

Starting in Immunology by Way of ImmunopathologyEmil R. Unanue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Pathogenesis of SepsisDeborah J. Stearns-Kurosawa, Marcin F. Osuchowski, Catherine Valentine,

Shinichiro Kurosawa, and Daniel G. Remick � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �19

EGFR Mutations and Lung CancerGilda da Cunha Santos, Frances A. Shepherd, and Ming Sound Tsao � � � � � � � � � � � � � � � � � � � �49

Zebrafish Models for CancerShu Liu and Steven D. Leach � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Mouse Models of CancerDong-Joo Cheon and Sandra Orsulic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �95

Disorders of Bone RemodelingXu Feng and Jay M. McDonald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 121

The Acute Respiratory Distress Syndrome: Pathogenesisand TreatmentMichael A. Matthay and Rachel L. Zemans � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 147

The HIF Pathway and ErythrocytosisFrank S. Lee and Melanie J. Percy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 165

Parkinson’s Disease: Genetics and PathogenesisJoshua M. Shulman, Philip L. De Jager, and Mel B. Feany � � � � � � � � � � � � � � � � � � � � � � � � � � � � 193

Pathogenic Mechanisms of HIV DiseaseSusan Moir, Tae-Wook Chun, and Anthony S. Fauci � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 223

Pathogenesis of MyelomaKenneth C. Anderson and Ruben D. Carrasco � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 249

Alternative Macrophage Activation and MetabolismJustin I. Odegaard and Ajay Chawla � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 275

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PM06-FrontMatter ARI 1 December 2010 11:14

The Pathobiology of Arrhythmogenic CardiomyopathyJeffrey E. Saffitz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

Mechanisms of Leukocyte Transendothelial MigrationWilliam A. Muller � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 323

Retinoids, Retinoic Acid Receptors, and CancerXiao-Han Tang and Lorraine J. Gudas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 345

Biomedical Differences Between Human and Nonhuman Hominids:Potential Roles for Uniquely Human Aspects of Sialic Acid BiologyNissi M. Varki, Elizabeth Strobert, Edward J. Dick Jr., Kurt Benirschke,

and Ajit Varki � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 365

A Glimpse of Various Pathogenetic Mechanismsof Diabetic NephropathyYashpal S. Kanwar, Lin Sun, Ping Xie, Fu-you Liu, and Sheldon Chen � � � � � � � � � � � � � � � 395

Pathogenesis of Liver FibrosisVirginia Hernandez-Gea and Scott L. Friedman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 425

Mesenchymal Stem Cells: Mechanisms of InflammationNora G. Singer and Arnold I. Caplan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

Molecular Genetics of Colorectal CancerEric R. Fearon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 479

The Pathogenesis of Systemic SclerosisTamiko R. Katsumoto, Michael L. Whitfield, and M. Kari Connolly � � � � � � � � � � � � � � � � � � � 509

Indexes

Cumulative Index of Contributing Authors, Volumes 1–6 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Cumulative Index of Chapter Titles, Volumes 1–6 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 542

Errata

An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articlesmay be found at http://pathol.annualreviews.org

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Documents

Mouse Model Overview