Maximizing mouse cancer models

REVIEWS Maximizing mouse cancer models Kristopher K. Frese and David A. Tuveson

Abstract | Animal models of cancer provide an alternative means to determine the causes of and treatments for malignancy, thus representing a resource of immense potential for cancer medicine. The sophistication of modelling cancer in mice has increased to the extent that investigators can both observe and manipulate a complex disease process in a manner impossible to perform in patients. However, owing to limitations in model design and technology development, and the surprising underuse of existing models, only now are we realising the full potential of mouse models of cancer and what new approaches are needed to derive the maximum value for cancer patients from this investment. Xenograft Tumour tissue or cell lines from one species propagated in immunodeficient mice in ectopic or orthotopic sites.

Genetically engineered mice (GEM). Mice harbouring genetic modifications designed to express either exogenous or endogenous mutated genes. In cancer modelling, these are frequently oncogenes or tumour-suppressor genes.

Cell autonomous A trait engendered only in cells harbouring the mutation. In the case of a carcinoma, a cell autonomous function occurs only in the mutant tumour epithelial cells.

Non-cell autonomous A trait engendered in cells that do not harbour the mutation. In the case of a carcinoma, a non-cell autonomous function occurs in stromal, immune and endothelial cells. Cambridge Research Institute, Cancer Research UK and University of Cambridge Department of Oncology, Cambridge, CB2 0RE, UK. Correspondence to D.A.T. e‑mail: david.tuveson@ cancer.org.uk doi:10.1038/nrc2192 Published online 9 August 2007

Cancer represents an increasing cause of morbidity and mortality throughout the world as health advances con‑ tinue to extend the life spans of our populations. Although our basic understanding of cancer has increased consid‑ erably since 1971, when United States President Richard Nixon initiated the ‘War on Cancer’, our ability to trans‑ late this knowledge into a health benefit for patients has been restricted to certain malignancies and often only temporarily. Importantly, specific hypotheses developed from our knowledge of cancer biology can be tested in increasingly complex model systems ranging from cell culture to genetically engineered mouse models, and such investigations should prove invaluable in discover‑ ing new methodologies for the detection, management and treatment of cancer in humans.

The evolution of cancer modelling The laboratory mouse (Mus musculus) is one of the best model systems for cancer investigations owing to various factors including its small size and propensity to breed in captivity, lifespan of 3 years, extensive physi‑ ological and molecular similarities to humans, and an entirely sequenced genome. M. musculus cancer models have progressed through several phases of increasing complexity, including xenograft tumours derived from tumour cell lines or explants, chemical and viral car‑ cinogens, and several variations of genetically engineered mice (GEM). Each approach has its own advantages and disadvantages, and it is important to choose the most appropriate system for particular questions. Tissue culture and ‘animal culture’ The establishment of cell lines from human and animal tumours is largely responsible for our early progress in cancer research. For example, these studies led to the

nature reviews | cancer

description and ultimate discovery of transforming genes, thereby laying the groundwork for the field of cancer biology and cell biology. However, many of our initial observations are now being re‑evaluated owing to our recent appreciation of cancer as a complex disease with intricate interactions between transformed cells that harbour oncogenic mutations (commonly referred to as the cell autonomous compartment) and surrounding non-cell autonomous constituents, such as normal cells, stromal cells and immune cells1. Indeed, several facets of tumorigenesis, including angiogenesis and metastasis, are not possible to assess in cell culture. Improvements in tissue sampling, genomics and biostatistics have enabled the direct characterization of primary human tumours; however, these analyses are limited and still do not take into account the contributions of the entire body. The development of xenograft models enabled the rapid and facile in vivo assessment of tumour tissue and cell lines in immunocompromised mice2. Indeed, patient-specific models have recently been proposed as a means to prospectively personalize treatment regimens3,4. However, several crucial differences exist when comparing tumour xenografts with patientderived specimens or autochthonous murine tumours, including the derangement of the normal tumour architecture and diminished genetic heterogeneity that is inherent to autochthonous tumours. Crucial features of the tumour microenvironment that are altered or lost in xenograft tumours include nearby normal tissues, stro‑ mal cells, vasculature and lymphatic circulation, and immune cells5,6. The cell-autonomous feature that is most often misrepresented in tumour xenografts is the expansion of a certain clonal constituent of polyclonal tumours owing to the selective pressures of cell culture or tissue explantation7–9. These differences in tumour volume 7 | September 2007 | 645

© 2007 Nature Publishing Group

REVIEWS At a glance • The laboratory mouse (Mus musculus) is one of the best model systems for investigations of cancer biology in vivo, ranging from basic models such as xenograft tumours derived from tumour cell lines or explants, to highly complex genetically engineered mice (GEM). • We suggest that xenografts should no longer be referred to as mouse cancer models. Xenografts represent an intermediate step between cell culture and mouse cancer models, and could be more accurately termed ‘animal culture’. • GEM can be classified as either transgenic or endogenous. This distinction is not merely semantic but is highly relevant, because the type of GEM can determine the experimental outcome in certain situations. • Transgenic GEM are mutant mice that express oncogenes or dominant-negative tumour-suppressor genes (TSGs) in a non-physiological manner owing to ectopic promoter and enhancer elements. Advantages of transgenic GEM include the ability to reversibly control target-gene expression with exogenous ligands. One disadvantage is that it might be difficult to elicit the exquisite control necessary to express oncogenes at physiological levels. • Endogenous GEM represent mutant mice that lose the expression of TSGs or express dominant-negative TSGs or oncogenes from their native promoters through the use of knockout and knockin technology. Conditional GEM models rely on the use of site-specific recombinases, such as Cre, to control the spatiotemporal mutation of the mouse genome. The use of these conditional models will prove to be key in addressing important molecular and therapeutic questions. • Modern GEM are poised to explore facets of cancer biology and medicine that are difficult or impossible to pursue clinically. However, all GEM described so far have certain shortcomings in mimicking human malignancy. Several issues (such as humanizing mice) and practical considerations concerning GEM will need to be addressed in order to meet our objectives.

cells and the microenvironment are probably relevant in the therapeutic evaluation of compounds, as, fre‑ quently, anti-neoplastics with demonstrable activity in xenografts are ineffective in the clinical setting10, and some evidence suggests that the converse is also true11. Indeed, we would suggest that xenografts should no longer be referred to as mouse cancer models. Rather, xenografts are an intermediate step between cell culture and mouse cancer models, and could be more accurately termed ‘animal culture’. Autochthonous tumour An endogenous or in situ tumour that evolves from normal cells of a tumourbearing animal. This is in contrast to animal cultures in which exogenous tumour cells are implanted into a nontumour-bearing animal.

Tumour microenvironment The stroma and supporting milieu surrounding the tumour that consists of fibroblasts, immune cells and endothelial cells.

Oncogene addiction The hypothesis that tumours arising as a result of a particular oncogenic lesion are exquisitely dependent on continued expression of that oncogene.

Environmentally-induced cancer models Certain strains of mice develop cancer either spontane‑ ously12 or following various ‘environmental’ exposures, including radiation13, chemicals14, pathogenic viruses15 and microbial flora16. These models have collectively been useful for various genetic and preclinical stud‑ ies, including the identification of oncogenes and tumour-suppressor genes (TSGs), the mapping of tumour susceptibility traits, commonly referred to as modifier genes, and the assessment of the carcinogenic or chemopreventative effects of various compounds. However, such models develop a restricted subset of tumour types and grades with incomplete penetrance and variable latency. These significant limitations prompted the development of new technologies to provide mouse cancer models that accurately reflect the common forms of human cancer and that allow the systematic investigation of tumour genetics and gene–environment interactions.

646 | September 2007 | volume 7

Genetically engineered models GEM are the most sophisticated animal models of human cancer, and many now exist that accurately mimic the pathophysiological and molecular features of human malignancies (TABLE 1). Despite the diverse tech‑ nical approaches used17, GEM can be simply classified as either transgenic or endogenous. This distinction is not merely semantic but is highly relevant, because the type of GEM can determine the experimental outcome in certain situations. Transgenic GEM. Mutant mice that express oncogenes or dominant-negative tumour-suppressor genes in a non-physiological manner owing to ectopic promoter and enhancer elements are broadly termed transgenic GEM18 (FIG. 1). In its simplest form, this describes the classical transgenic mice generated by pronuclear injec‑ tion of cDNA constructs that contain promoter elements designed to restrict tissue tropism. Transgenic GEM reca‑ pitulate the genetic features of translocated or amplified proto-oncogenes, and such mice have been extremely informative in establishing and confirming the basic ten‑ ets of cancer biology and in laying the groundwork for the development of more refined GEM. Transgenic GEM can be produced by the direct injection of fertilized oocytes or through gene targeting and lentiviral transduction in embryonic stem cells. However, the promoter fragments typically used represent the minimal sequence absolutely required, and do not necessarily accurately recapitulate the exquisite control conveyed by endogenous regula‑ tory elements19. Indeed, many transcription factor and microRNA binding sites are present within untranslated regions of a gene20, and would therefore not be present in a typical transgene. Furthermore, transgenes often integrate as large concatamers that invariably promote overexpression. Owing to the random nature of trans‑ gene integration, chromosomal positional effects can result in variegation and incomplete penetrance, thus potentially confounding results21; although insertion of the transgene into a well-characterized euchromatic locus such as Rosa26 can overcome this limitation. In addition, this approach relies on the availability of appro‑ priate promoters to obtain the desired effect (BOX 1). Therefore, both expression levels and cell tropism may not completely reflect that of the endogenous gene. An approach that attempts to address some of these issues is the transgenic introduction of episomal artificial chro‑ mosomes that harbour oncogenes to create multiple or single-copy transgenic mice22. Nonetheless, the artificial chromosomes are often integrated and expression levels may not be predictable23. Advantages of transgenic GEM include the ability to reversibly control target-gene expression with exogenous ligands, such as doxycycline24 or interferon25 (FIG. 1). The most widely used system takes advantage of the tetracy‑ cline operon. Further increasing its usefulness, this system has been engineered so that tetracycline, or its non-toxic analogue doxycycline, can either promote or inhibit tran‑ scription as determined by tissue-specific expression of the tetracycline-regulated transcription factors. These systems have been used to study the concept of oncogene addiction26 www.nature.com/reviews/cancer


REVIEWS Table 1 | Examples of genetically engineered mouse models that recapitulate human solid cancers Organ

Histopathology

Genetics

Lung

Adenocarcinoma

Kras55

Squamous cell carcinoma

NA

Large cell carcinoma

NA

Small cell carcinoma

Rb1;Trp53 (REF. 167)

Polypoid adenocarcinoma

Kras;Apc168

Hereditary non-polyposis carcinoma

Msh6169

Ductal carcinoma

Brca2;Trp53 (REF. 170)

Lobular carcinoma

Cdh1;Trp53 (REF. 171)

Ductal adenocarcinoma

Kras; Cdkn2a172, Kras;Trp53 (REF. 173)

Mucinous cystic neoplasm

Kras;Dpc4 (REF. 96)

Intraductal papillary mucinous neoplasia

NA

Prostate

Prostate carcinoma

Pten174, Pten;Nkx.1 (REF. 175), Rb1;Trp53 (REF. 176)

Liver

Hepatocellular carcinoma

Apc177, Myc;Trp53 (REF. 178), Myc;TGFA179

Ovary

Endometrioid carcinoma

Kras;Pten180, Apc;Pten181

Serous carcinoma

NA

Mucinous carcinoma

NA


Pten;Dpc4 (REF. 182); Ccnd1;Trp53 (REF. 183)

Colon Breast Pancreas

Oesophagus

Adenocarcinoma

NA

Bladder

Transitional cell carcinoma

Hras184

Kidney

Renal cell carcinoma

Apc;Trp53 (REF. 185)

Brain

Astrocytoma

Pten;Rb1 (REF. 186)

Glioblastoma

Nf1;Trp53 (REF. 198)

Stomach

Gastric carcinoma

Wnt;Ptgs2;Ptges188

Skin

Melanoma

HRAS;Ink4a27


Xpd*189

* Requires exposure to UVB light. NA, none available.

(and the feasibility of treating different cancers with thera‑ peutics that target crucial oncogenes) by demonstrating a requirement for Hras, Kras, Myc, Bcr–Abl and Erbb2 (also known as Neu) in the maintenance of melanoma27, lung adenocarcinoma28, acute myeloid leukaemia and T-cell lymphoma29, B-cell lymphoma/leukaemia30 and breast cancer31, respectively. One limitation of this system is that it may be difficult to elicit the exquisite control necessary to express oncogenes at physiological levels, an impor‑ tant consideration as many oncogenes cause toxic effects including senescence32 or apoptosis33 when overexpressed in vivo. Oestrogen receptor fusion proteins represent an additional mechanism by which oncogene or TSG function can be inducibly regulated (FIG. 1). In this case, the fusion protein is translated, but remains inac‑ tive in the cytoplasm because the oestrogen receptor (ER) ligand-binding domain causes misfolding and sequestration by heat shock proteins. On treatment with Tamoxifen, the ER domain is released by the heat shock proteins and the fusion protein can translocate into the nucleus and rapidly mediate its effects. This technique has been used to demonstrate a requirement for MYC in tumour maintenance 34 and, conversely, nature reviews | cancer

that restoration of p53 function can promote tumour regression35,36. Although this line of experimentation has only been used for nuclear proteins in mouse mod‑ els, success in cell culture indicates that it is possible to similarly regulate cytoplasmic proteins. These alleles can prove very powerful because they may be expressed from either ectopic or endogenous promoters, and function can be easily temporally regulated; however, extensive characterization is required to demonstrate that the activated fusion protein behaves exactly like the activated wild-type protein. Although most transgenic GEM are propagated by vertical transmission, two versions arise somatically and thereby minimize the time and resource investment required to generate new mouse models — estimated to be at least 12 months and US$100,000 in some studies37. One approach is the use of recombinant avian sarcoma leukosis virus (ASLV) to deliver cDNA constructs into murine tissues that express the ASLV receptor TVA. This system offers the possibility of simultaneously tar‑ geting one organ with various oncogenes (or dominantnegative TSGs), and has been used to generate models of glioblastoma38, ovarian39, pancreatic40 and liver can‑ cer 41. This system suffers from non-physiological volume 7 | September 2007 | 647


REVIEWS a Constitutive transgenes

b Tetracycline-inducible

TSP

Oncogene

Tet on

TSP

rTta

tetO Pro

Tg

Tet off

TSP

tTa

tetO Pro

Tg

or

TSP

DN TSG

+ Dox

tetO Pro

Tg

tetO Pro

Tg

transgenes

Oncogene ERT

Onco -ERT

+ Tam

HSP

TSP

HSP

c Tamoxifen-regulatable proteins

+ Dox

Onco -ERT

Figure 1 | Transgenic GEM. Constitutive (a) or inducible (b) transgenes represent the first generation of genetically Nature Reviews |(Tet) Cancer engineered mice (GEM) in which gene expression is controlled by an exogenous promoter. The tetracycline on or off systems are shown. The transgene is only expressed in the presence (Tet on) or absence (Tet off) of doxycycline (Dox). Transgene expression can also be regulated at the protein level (c). The active form of tamoxifen (4-hydroxytamoxifen;Tam) is used to regulate protein activity through modifying the interaction of the oestrogen receptor fusion protein (onc-ERT) with heat shock protein (HSP) chaperones. In the absence of Tam, onco-ERT is inactive. In the presence of Tam onco-ERT no longer interacts with HSP and is functional. DN TSG, dominantnegative tumour-suppressor gene; rTta, tetracycline-regulatable transcription factors; Tg, transgene; TSP, tissuespecific promoter.

Loss of heterozygosity Mutation or ‘loss’ of the remaining wild-type allele in a heterozygous GEM.

Haploinsufficiency A phenotypic state that results from loss of one functional allele of any given gene in diploid cells. Sometimes also called allelic insufficiency.

Conditional models GEM that rely on site-specific recombinase systems to engender gene expression in a spatially and/or temporally restricted manner.

Site-specific recombinase (SSR). An enzyme, such as bacterial Cre, that catalyses recombination between two specific inverted repeat sequences (such as LoxP).

CreERT A general term for a tamoxifen-inducible Cre recombinase in which the Cre cDNA is fused to the oestrogen receptor ligand-binding domain. This fusion protein is sequestered in the cytoplasm by heat shock proteins until exposure to Tamoxifen promotes nuclear translocation.

expression of delivered genes as well as the inability to express oncogenes in non-dividing cells. Although the latter has been overcome by pseudotyping a len‑ tivirus with the ASLV envelope protein 42, this has not been shown in vivo. A second method of somatic mutation through retrovirus insertional mutagenesis strategies have been used to identify genes involved in tumorigenesis in a non-biased manner43. Many alleles described to date are presumptive proto-oncogenes and TSGs that are aberrantly expressed or disrupted owing to retroviral insertion, accompanied by whole genome losses and gains. Endogenous GEM. Endogenous GEM represent mutant mice that lose the expression of TSGs or express domi‑ nant-negative TSGs or oncogenes from their native pro‑ moters18 (FIG. 2). Gene ‘knockout’ approaches entail the replacement of endogenous embryonic stem cell chroma‑ tin by a targeting vector that disrupts this allele, and were the first methods used to generate such mice44. Germline biallelic disruptions of TSGs often cause embryonic lethality, identifying previously unrecognized roles of these genes in normal murine development. Therefore, heterozygous mutant mice are often used to deter‑ mine the tumorigenic potential of TSG mutant alleles, with either the somatic loss of heterozygosity (LOH) or haploinsufficiency identified as crucial mediators of tumorigenesis. Alternatively, embryonic lethality can be bypassed in chimeric mice using embryonic stem cells that harbour homozygous TSG disruptions to gener‑ ate novel GEM45. Although these models were initially designed to model human disease, this has often proven difficult. In part, this is due to the fact that, unlike most human cancers in which somatic mutations promote malignancies in the context of a normal organ, the germline mutations in GEM are constitutively present


throughout the mouse. This can lead to developmental defects, undesirable effects outside tissues of interest, or compensation from related gene products or those in the same pathway46–48. Therefore, in terms of model‑ ling human disease these germline mutations are more useful in studying familial cancer diseases such as the Li‑Fraumeni syndrome. Unfortunately, many TSG mutant GEM commonly develop mesodermal-derived tumours rather than the carcinomas that frequently afflict human patients. Despite these caveats, these models represented an essential method to functionally characterize individual genes, before the introduction of RNA interference approaches. The advent of conditional models heralded a key advance in the generation of cancer-prone GEM. These models rely on the use of site-specific recombinases (SSR) to control the spatiotemporal mutation of the genome. The most common conditional system uses the bacteriophage Cre-Lox system in which Cre recombinase recognizes a pair of inverted repeat DNA elements, or LoxP sites, and catalyses recombination resulting in deletion or inversion of the intervening sequence49. Further temporal control can be achieved with a ligand-regulatable SSR such as CreERT, in which the recombinase remains inactive before induction. Conditional inactivation of a TSG is achieved by flanking exons of interest with LoxP sites arranged in the same orientation. Exposure to the SSR excises these exons, resulting in a non-functional or unstable gene product (FIG. 2). Ideal conditional alleles exhibit normal gene expression following the intronic insertion of LoxP sites, and in this setting conditional alleles can be har‑ boured either heterozygously or homozygously and have no effect on mouse development. Endogenous GEM that express oncogenes in the mouse germline are infrequently described50,51, reflect‑ ing the paucity of such mutations in familial cancer www.nature.com/reviews/cancer


REVIEWS Box 1 | Tissue-specific promoters Tissue-specificity in conditional models is determined by Cre transgenes expressed from tissue-specific promoters. Unfortunately, these promoters are often not exclusively active in the cell or tissue of interest, or in the desired kinetic fashion. For example, the two primary Cre alleles used to study pancreatic cancer are expressed from the promoters of the Pdx1 homeobox transcription factor and the Ptf1a (also known as p48) helix-loop-helix transcription factor, both of which are active in the pancreatic progenitor compartment153, 154. However, Pdx1 is also expressed in the developing foregut (stomach and duodenum), and Ptf1a is active in the brain155. Use of either allele results in extra-pancreatic phenotypes that could potentially confound results156. One potential solution to overcome this limitation is the construction of a ‘promoter overlap’ model in which a requirement for transcription from multiple promoters engenders the desired specificity. Alternatively, the discovery of new promoters active in pancreatic niches and by analogy other tissues should be encouraged to take full advantage of conditional technology in the generation of genetically engineered mice (GEM).

LoxStopLox A LoxP-flanked sequence, often containing an antibiotic resistance marker, that prevents expression of a gene when placed between the promoter and coding exons.

Latent allele An endogenous GEM strategy in which a stochastic recombination event is required to activate expression of the oncogenic allele. This is one of the few endogenous GEM that does not require a site-specific recombinase.

syndromes. Rather, GEM52 and patients53 that express certain oncogenes in their germline have profound developmental or neoplastic sequelae. Therefore, endog‑ enous GEM are usually conditional alleles constructed by the insertion of a transcriptional and translational LoxStopLox ‘stop’ cassette between the promoter and first coding exon of the oncogenic allele54,55 (FIG. 2). On expression of an active SSR, the stop cassette is excised and the mutant oncogene is subsequently expressed. Stop cassettes can be used in the context of transgenic GEM; however, the multiple LoxP sites present in the transgene concatamer might promote chromosomal breakage56. In addition to focal mutations or deletions of genes, several cancers are caused by chromosomal transloca‑ tions, deletions or inversions57. In some cases, the junc‑ tions of these translocations are known and the resulting fusion gene products are well-characterized58. These represent an additional mechanism that can be modelled using conditional approaches. Chromosomal deletions can be modelled in an analogous fashion, as conditional TSGs, except LoxP sites are distally located on the same chromosome59. Chromosomal inversions are similarly modelled, except the LoxP sites are oriented facing each other60 (FIG. 2). To overcome the possibility of re‑inversion back to wild type, it is possible to either temporally control Cre expression or use heterospecific mutant LoxP sites that primarily favour recombination in one direction61. Chromosomal translocations, although less efficient than intrachromosomal rearrangements, can be accomplished by Cre-mediated recombination owing to the insertion of a single LoxP site at each breakpoint62. Latent alleles are particularly elegant mouse models that use a variation on the hit-and-run strategy in which the ‘run’ occurs in vivo as a result of unequal sister chro‑ matid exchange or intrachromasomal recombination. This strategy was used to express oncogenic Kras and Ctnnb1 (which encodes β‑catenin) alleles from their endogenous promoters, and resulted in lung adeno‑ carcinoma63 or intestinal metaplasia64, respectively. The stochastic nature of these models represents a notable advantage over other systems because lesions arise in a manner more similar to human cancers. In addition, because the activation event is mediated by somatic recombination, it should occur in all tissues and does not


require the use of a tissue-specific SSR mouse. However, using this endogenous GEM strategy, it is currently not possible to modify the onset of tumour formation or the tumour spectrum. Whereas many endogenous GEM correspond to spe‑ cific TSGs or oncogenes implicated in human cancer, there is also a class of GEM that incorporate alleles that may not be directly mutated in cancer but are thought to have a role in genome stability, and thereby might indi‑ rectly influence the development of cancer. A dramatic example of this is the Terc knockout mouse that lacks the RNA subunit of telomerase and is cancer prone in various tissues65, particularly in a Trp53 deficient back‑ ground66. In addition, genes may be specifically control‑ led constitutively, conditionally or inducibly by other methods that influence mRNA stability or translation, such as RNA interference67,68 or microRNA expression69. An important consideration with such approaches is that the efficiency of mRNA destruction or translational modulation might be incomplete, and additional genes may be influenced. Recently, forward genetic systems were described that represent a new class of endogenous and transgenic GEM. These GEM are based on a bipartite transposon system consisting of a specific transposase and concatam‑ ers of mutagenic transposons that either aberrantly direct the expression of proto-oncogenes or disrupt TSGs70,71. Furthermore, transposon systems can be either broadly active or restricted to particular tissues depending on the promoters used to construct the transposons. The choice of GEM can have a profound influence on the model generated. For example, elastase-KRASG12D mutant mice predictably develop pancreatic acinar dys‑ plasia and neoplasia, as the elastase promoter is confined to acinar and centroacinar cells, and would thereby restrict the expression of the mutant Kras cDNA to this compartment72. In stark contrast, LSL-KRASG12V;elastasetTA;tet-o-Cre mice develop pancreatic ductal carcinoma, and not acinar carcinoma73. In this model, endogenous KrasG12V expression initiates in exocrine pancreatic cells that are elastase positive, but the development of the appropriate cell lineage that gives rise to ductal pancre‑ atic cancer is not otherwise restricted or predetermined. Indeed, this latter model has led to the hypothesis that the centroacinar cell may be the cell of origin for duc‑ tal pancreatic cancer. Therefore, it will be of interest to carefully compare the transgenic and endogenous GEM representing other cancers to determine how closely each accurately reflects the cognate human malignancy. A comparison of currently available GEM reveals that endogenous GEM are more likely to show the entire spectrum of disease, including early preinvasive lesions that are proposed to evolve into frank carcinoma (TABLE 2).

Current challenges and potential solutions Modern GEM are poised to explore facets of cancer biology and medicine that are difficult or impossible to pursue clinically. This optimism is largely due to recent progress in genetic engineering, tumour biology and biomedical technology. However, all GEM described volume 7 | September 2007 | 649


REVIEWS a Allele name

Targeted locus

Germline knockout

1

Neo

3

null

1

2

3

wild type

Germline knockin

1

2*

3

mutant

1

2

3

wild type

b Allele name

Allele prior to Cre

Allele after Cre

Conditional knockout

1

2

3

wild type

1

1

2

3

wild type

1

Conditional LoxStopLox

1

2*

3

null

1

2

3

wild type

Conditional minigene

1

2-3

2* 3

wild type

1

1

2

3

wild type

1

Conditional exon inverter

1

3

wild type

1

2

3

wild type

1

2*

Conditional genomic translocator

1

2

2

3

null

2

3

wild type

1

2*

3

mutant

1

2

3

wild type

2*

3

mutant

3

wild type

3

mutant

2

3

wild type

2

2*

1

2

3

+

1

B

C

mutant

A

B

C

+

A

2

3

mutant

Figure 2 | Endogenous GEM. a | Germline endogenous GEMs. Germline alleles are expressed in every cell throughout Nature Reviews | Cancer the mouse. Knockout alleles replace one or more exons with a selectable marker resulting in a null allele, whereas knockin alleles insert the desired mutation into an exon. b | Conditional endogenous GEMs. Conditional alleles are used to express mutations in specific tissues and rely on site-specific recombinases such as Cre to induce mutations. More recent alleles (conditional minigene and inverter) are capable of more accurately recapitulating the genetics of human cancers. Arrows depict promoters, boxes depict exons, blue arrowheads depict LoxP sites, and asterisks denote mutations. The phenotype of the allele is given to the right of each diagram.

to date also exhibit certain shortcomings in mimicking human malignancy. Therefore, several issues and practi‑ cal considerations (BOX 2) concerning GEM will need to be addressed in order to meet our objectives.

Human cancer genetics in mice As GEM are designed to model human cancers, it is important that the genetics of human cancer guide the generation of mouse models. Failure to adhere to this simple principle can waste both time and money. For example, although farnesyltransferase inhibitors (FTIs) showed promising results in transgenic GEM that har‑ boured Hras oncogenes75, the failure of FTIs in clinical trials74 might reflect the decreased effectiveness of FTIs towards the inhibition of the KRAS oncoprotein that is more commonly present in human malignancies76,77. It is also possible that different mutations in the same gene might elicit slightly different phenotypes. Therefore, careful attention to the exact oncogenic lesion must be considered when designing mouse models. It is also important to note that the mutation of a pathway rather than a specific gene does not necessar‑ ily reflect an accurate model. For example, mutations 650 | September 2007 | volume 7

in PTEN (phosphatase and tensin homologue), but not PIK3CA (phosphatidylinositol 3-kinase, catalytic, a polypeptide), are frequently associated with prostate cancer78. Although PI3K activation and PTEN inactiva‑ tion both promote Akt kinase activity, the pleiotropic functions of these proteins might confound interpreta‑ tion of data based on so‑called ‘pathway models’. In light of this, it will be interesting to determine whether the recently discovered role for nuclear PTEN in maintain‑ ing genomic integrity79, a function not modelled by activating PI3K mutations, has a role in prostate cancer progression. In addition to incorporating the most relevant muta‑ tions, it is also important to consider the limitations of the system used. For example, generation of an endogenous conditional oncogenic allele by placing a stop cassette between the promoter and coding sequence will render mice functionally heterozygous for that gene before expression of Cre (FIG. 2), thereby exposing the mouse to potential effects of haploinsufficiency. Relevant to conditional models that use endogenous oncogenic Kras GEM, several groups have unveiled a tumour‑suppressor role for the wild-type Kras allele80,81. Therefore, it is www.nature.com/reviews/cancer


REVIEWS Table 2 | Modelling pre-invasive lesions in transgenic and endogenous GEM Neoplasm

Precancerous lesion

Transgenic GEM ref.

Endogenous GEM ref.

Lung cancer

Atypical adenomatous hyperplasia

28

55

Colon cancer

Intestinal polyps

190

191

Breast cancer

Ductal carcinoma in situ

192

170

Lobular carcinoma in situ

NA

NA

Pancreatic cancer

Pancreatic intraepithelial neoplasia

NA

193

Prostate cancer

Prostatic intraepithelial neoplasia

194

195

Liver cancer

Adenomatous hyperplastic foci

196

NA

Ovarian cancer

Surface epithelial hyperplasia

NA

180

Oesophageal cancer

Barrett’s oesophagus

NA

NA

Bladder cancer

Urothelial hyperplasia

184

NA

Kidney cancer

Renal cysts

NA

197

Brain cancer

Low-grade glioma

38

198

Gastric cancer

Intestinal metaplasia

199

64

Melanoma

Nevi

NA

NA

Squamous cell cancer

Papillomatosis

NA

200

GEM, genetically engineered mouse; NA, none available.

Hypomorphic allele A mutation conveying decreased activity, either through reduced expression or partial loss of function.

Neomorphic allele A mutation conveying a novel activity not present in the wild type protein.

Adeno-associated virus (AAV) Small DNA viruses often used for gene therapy due to their broad host range and the ability to infect non-dividing cells.

possible that mice heterozygous for wild-type Kras (that is, before Cre expression) show increased susceptibility to neoplasia. It is also worth noting that tissues of the mouse that are not exposed to Cre remain heterozygous for these alleles, thus potentially affecting the noncell autonomous contribution to carcinogenesis. One method to overcome this limitation uses LoxP-flanked minigene cassettes that express the wild-type allele before Cre-mediated recombination, and the mutant version following Cre activation82,83 (FIG. 2). Although there are no obvious detrimental effects from this approach84, use of cDNA minigenes (and their corresponding absence of introns and regulatory sequences contained therein) could theoretically perturb endogenous gene-expression mechanisms. An ideal system would use a conditional allele consisting of a completely wild-type allele that is converted to the mutant form in the presence of Cre. Although this has not yet been achieved, a recent clever attempt placed an inverted mutant exon upstream of the corresponding wild-type exon such that, in the absence of Cre, the wild-type exon would be used82 (FIG. 2). The inverted mutant and wild-type exons were flanked by heterospecific LoxP sites such that Cre-mediated inver‑ sion would flip the cassette and lead to the expression of the mutant gene. Technical limitations prevented the success of this system; however, similar strategies have been successfully used in analogous systems85,86. Conditional deletions of TSGs are superior to con‑ stitutive knockouts because the presence of LoxP sites flanking exons of interest do not affect gene expression


before Cre expression; however, perturbation of TSG function in human tumours can also occur through point mutations that either generate a hypomorphic protein or result in a frameshift and subsequent truncation. This mutant protein could result in partial loss of function, and is therefore not equivalent to the absence of wildtype protein. In the case of conditional Trp53 models, there is a clear difference between the tumour spectrum elicited by conditional deletion versus conditional mutation of Trp53 (REF. 87), with the greater incidence of carcinoma in the conditionally expressed mutants more closely modelling human cancers. One hypothesis to explain this different phenotype is that point mutation of Trp53 elicits a gain-of-function that is not possible in the conditionally deleted mutant88. Therefore, human can‑ cer-associated point mutations representing hypomorphic or neomorphic alleles might more accurately phenocopy human disease. To date, introduction of conditional point mutations have only been achieved by a strategy analogous to that used for conditional oncogenes, and are therefore subject to the same pitfalls. However, the aforementioned inverter technology represents a potential solution to this problem.

Limiting the cancer-initiating population Although human cancers are thought to develop from a single mutated cell in the context of a relatively normal organ, the oncogenic event in many GEM is initiated simultaneously throughout the organ, creating a nonphysiological situation in which the pre-neoplastic cell does not evolve in the context of a normal microenvi‑ ronment. Therefore, new efforts are needed to restrict the mutagenic events to fewer cells. Current attempts to limit the cancer-initiating population in conditional models rely on restricted expression of the active SSR. For instance, Cre has been delivered by infection with a replication-deficient Cre-expressing adenovirus, and infection with low viral titres can limit recombination to a few cells resulting in hyperplasia in the context of surrounding normal cells55. Although some tissues dem‑ onstrate resistance to adenovirus infection, the develop‑ ment of lentiviral and adeno-associated virus (AAV)-Cre viruses have partially overcome this obstacle89. However, injection of Cre-expressing viruses into specific organs can require difficult surgical techniques and may limit the use of this approach. An alternative approach is to genetically encode a spatiotemporally-regulatable SSR. These can be regulated either at the transcriptional level through the use of inducible promoters24,25 or at the post-translational level by generating Tamoxifeninducible fusion proteins90. In principle these SSR are either not expressed or remain dormant until exposure to ligand, and low doses of inducing agent might pro‑ vide a stochastic method of activating the SSR in a small percentage of cells. In practice it has proven difficult to determine suitable doses, and these SSR are universally leaky, possessing measurable activity even in the absence of ligand91. More refined Tamoxifen-inducible SSRs dis‑ play increased responsiveness to ligand and decreased (albeit measurable) leakiness92. Although leakiness can result in the sporadic initiation of carcinogenesis, this volume 7 | September 2007 | 651


REVIEWS Box 2 | Additional practical considerations in GEM Analysis of genetically engineered mice (GEM) on different mouse strains that present with different biological predispositions represents a method of assessing the roles of different polymorphisms similar to those encountered in the human population. However, the use of different embryonic stem cells by individual groups necessitates backcrossing onto pure backgrounds for rigorous comparison of different mutations. Such backcrosses take years to complete and are not fiscally possible for many labs. Microsatellite-based ‘rapid backcrossing’ can accelerate this process; however, this technique remains prohibitively expensive. We would therefore urge that the mousemodelling community come to a consensus on one or a few embryonic stem cell lines in an effort to standardize the field and eliminate the need for costly backcrossing. As models increase in complexity, typically greater than two mutant alleles must be present to elicit the desired phenotype. Genotyping for these alleles can quickly become unwieldy and take up a large amount of time and money for the investigator. With improvements in high-throughput robotics and sequencing technology, outsourcing of genotyping is now feasible. Increased demand should continue to make this option both efficient and fiscally sensible.

system is not easily controllable and is subject to much variability in tumour burden and latency. An intriguing system was recently developed in which Cre is regulated at both the transcriptional and post-translational levels, resulting in practically no detectable background recom‑ bination91. Unfortunately, none of these systems is ideally suitable for tight control of stochastic recombination in a single cell. Perhaps the most accurate model is the previ‑ ously mentioned latent allele; however, there is currently no method to either control tissue specificity or assess recombination frequency. A recent hypothesis posits that tumour-initiating or cancer stem cells might be sufficient to maintain the tumour cell population despite representing a small fraction of total tumour cells93. GEM should enable the identification and characterization of such cancer stem cells through the construction of specific SSR alleles that define the lineage of different cell types that make up the organ.

Recapitulating the kinetics of carcinogenesis Both molecular and epidemiological data indicate that carcinogenesis is a multistep process requiring an esti‑ mated five to six mutant alleles to promote tumour pro‑ gression94. Compound mutant mice are now frequently used to study the synergistic effects of multiple mutations on carcinogenesis. Although these experiments clearly show that tumours appear more quickly in compound mutant mice, simultaneous initiation of all oncogenic lesions promotes rapid tumour progression in a manner inconsistent with the evolution of human cancers. For instance, several recent manuscripts describe condi‑ tional models for pancreatic cancer in which disruption of a TSG in the context of an oncogenic Kras mutation exacerbated tumorigenesis95,96. Although these genes are simultaneously mutated in the mouse models, in human pancreatic cancer KRAS mutations are considered an early initiating event and mutation of CDKN2A, DPC4 and TP53 are thought to occur relatively late in carcino‑ genesis97. There can be little doubt that tumour evolution is artificially pre-determined when these mutations are simultaneously initiated. Indeed, it has recently been pro‑ posed that, whereas late-stage DPC4 mutations promote 652 | September 2007 | volume 7

the progression of pancreatic ductal adenocarcinoma, simultaneous activation of KRAS and mutation of DPC4 in the developing pancreas promotes mucinous cystic neoplasia, a histologically distinct and clinically relevant form of pancreatic cancer96. This concern also applies to monoalleleic as opposed to biallelic deletion of TSGs. These issues can be addressed with conditional endogenous GEM that use several SSR. For example, the Cre/LoxP system can be used for the initiating mutation and subsequent mutations can be regulated by the FLP/FRT system98 (BOX 3)(FIG. 3).

Humanized mice One of the major criticisms of modelling cancer in mice is that the biology of humans and mice is simply too disparate, and that mouse models cannot recapitulate aspects of human disease 99. For example, germline knockout of Nf2 results primarily in osteosarcomas in mice100, whereas kindreds afflicted with neurofibroma‑ tosis type 2 develop schwannomas 101. This finding was used to suggest that inherent differences between humans and mice limited the use of mice to model can‑ cer. However, subsequent conditional biallelic deletion of Nf2 in mouse schwann cells promoted a phenotype reminiscent of neurofibromatosis type 2 in humans102. Similarly, other discrepancies between mice and humans can potentially be addressed through systematic experimentation (FIG. 4). Gene structure and regulatory elements. Although orthologous genes are often highly conserved, signifi‑ cant differences do exist between the mouse and human genomes that might limit the ability to model human cancer. Therefore, one mechanism of humanization is the insertion of human genes into the mouse genome. This approach is frequently addressed through knockin of a human cDNA into the corresponding murine genomic locus103,104; however, these models cannot assess the contri‑ bution of non-coding elements. To overcome this caveat, several groups have used bacterial artificial chromosome transgenes that encompass over 100 kb of sequence surrounding a gene; however, these large constructs are subject to chromosomal positional effect and frequently rearrange23. It has recently been shown that syntenic replacement of the mouse α globin locus with 120 kb of human sequence encoding a mutated α globin locus resulted in a model that more accurately recapitulates α thalassemia than the corresponding knockin mutation in the mouse gene105. This method, termed recombinasemediated genomic replacement, could theoretically be used to replace hundreds to thousands of kilobases of mouse genomic sequence with syntenic human sequence and analyse the contribution of non-coding mutations that affect oncogene or TSG expression. Telomeres, telomerase and genomic instability. A fun‑ damental difference between mouse and human cells is their telomere length: human telomeres are typically 5–10 kb in length, whereas telomeres in M. musculus are over 40 kb long106. This difference in telomere length www.nature.com/reviews/cancer


REVIEWS gene, Rtel110. The main Rtel transcripts from the two strains appear to differ by only 18 nucleotides at the 3′ end on the coding sequence, thus making it theoreti‑ cally possible to humanize M. musculus by knocking in the M. spretus Rtel allele.

TSP–Cre

TSP–FLPERT + Tam

TSP–FLPERT + Tam TSP–FLPERT – Tam

FLPERT -induced regression

Uninduced FLPERT

FLPERT-induced cooperation

Figure 3 | Sequential mutations in mouse cancer models. The initial oncogenic lesion Nature Reviews | Cancer in a conditional GEM is induced by tissue-specific expression of Cre recombinase. A second genetic event controlled by a ligand-acitvatable FLP recombinase can be used to initiate either tumour regression (that is, through the deletion of the initiating oncogene or induction of a short hairpin RNA) or tumour progression (that is, through deletion of a tumour-suppressor gene). This system can be used to test the effectiveness of therapeutic agents on different stages of carcinogenesis or against specific genetic lesions. Tam, Tamoxifen; TSP, tissue-specific promoter.

Xenobiotic receptors A family of enzymes involved in the metabolism of drugs by sensing the presence of a drug and initiating a response.

Cytochrome P450 A family of metabolic enzymes responsible for detoxifying and modifying drugs and other foreign compounds.

is correlated with the limited or absent telomerase expression in most somatic human cells107, as opposed to persistent telomerase expression in mouse tissues108. As telomeres maintain chromosomal integrity and tel‑ omere dysfunction has an important role in oncogen‑ esis, the replacement of murine telomere components with human may be an important consideration to better recapitulate the neoplastic process. In fact, the Terc-deficient mice previously described must be bred for several generations before they show critically short telomeres and consequential neoplastic morbidity109. An alternative approach to humanizing M. musculus telomeres is to backcross these mice onto the M. spretus background, as M. spretus telomeres are 5–15 kb long108. However, the genetics of M. spretus are much less understood, the backcrossing is laborious, and M. spretus husbandry is challenging. Interestingly, the locus that appears to regulate telomere length in M. musculus and M. spretus has recently been mapped to a specific

Box 3 | Alternatives to Cre Most conditional alleles currently used rely on the bacteriophage Cre recombinase. Although Cre is considered a site-specific recombinase, several lines of in vitro data indicate that Cre cleaves multiple sequences in the genome157–159, and thus represents a potentially mutagenic event. Furthermore, more complex models will soon require the use of more than one recombinase to achieve all genetic manipulations required. It is therefore necessary to develop and characterize alternatives to Cre. The yeast-derived FLP-FRT system, the most common alternative to Cre, has been optimized and its efficiency in mouse models approaches that of Cre160,161. In addition, several other enzymes such as φC31 integrase and sleeping beauty transposase represent further options. In vitro experiments demonstrating the low toxicity and high efficiency162 of φC31 and sleeping beauty make them attractive candidates as new tools in mouse modelling.


Metabolism. With the increasing interest in using GEM as preclinical models for therapeutics, it is important to consider both the relevance of the target as well as the pharmacokinetics of the drugs. Documented spe‑ cies-specific differences in xenobiotic metabolism make it imperative that every effort be made to humanize drug metabolizing enzymes so that these studies can be accurately extrapolated to humans111. The rates of drug absorption, distribution, metabolism and excretion, all of which might differ substantially between humans and mice, can affect the target-tissue concentration. In most cases, differences in xenobiotic receptor (XR) and cytochrome P450 (CYP) expression levels, tissue distribution, enzymatic activities, substrate preferences and ligand affinities lead to dramatically different responses in mice and men112. Therefore, several GEM have been gener‑ ated in which the human XR or CYP genes are expressed in mice that lack the corresponding murine gene113,114. Although there are over 15 mouse strains humanized for metabolic enzymes, of particular interest are those for CYP2D6 (REF. 115) and CYP3A4 (REF. 116), two enzymes that metabolize over 70% of currently available drugs117. Extensive characterization has shown that these mice have metabolic properties more similar to humans than wild-type control mice. To our knowledge, these strains have not been used in autochthonous-tumour-bearing mice treated with chemotherapeutic or targeted agents, although in principle this could easily be accomplished by crossing existing strains. Immune system. Both the adaptive and innate immune response are crucial modulators of tumorigenesis through a complex interplay between inflammation and immunosurveillance that promote and inhibit tumour growth, respectively118,119. These intricacies make it impossible to accurately model tumorigenesis with ani‑ mal culture models in immunodeficient mice. To date, humanizing the murine immune system has consisted of ablating the endogenous immune system followed by engraftment with human immune cells120; however, species-specific differences in both the adaptive121 and innate122,123 immune systems can confound interpreta‑ tions. Humanization attempts that rely on transgenic expression of human immunoreceptor genes124,125 have primarily been performed in otherwise wild-type mice expressing the homologous murine receptors that might heterodimerize with the human gene products. With the advent of recombinase-mediated genomic replacement, it might now be possible to knock in the human loci for the entire T-cell and B-cell receptors that span hundreds of kilobases. In an analogous fashion, specific major his‑ tocompatability complexes (MHCs) can be introduced into mice that lack endogenous MHC genes 126, thus making it theoretically possible to reconstitute the major components of the human immune system. volume 7 | September 2007 | 653


REVIEWS

Genomic instability

Biomarkers

Gene expression

Metabolism

Immune system

Figure 4 | Hallmarks of humanized mouse models. Currently available technologies can be used to generate GEM that contain entire human genes. Such models can be Nature Reviews | Cancer used to humanize metabolic enzymes, glycosylation enzymes, telomere structure and the immune system, which will prove useful in biomarker discovery as well as preclinical testing of therapeutics.

Glycosylation. It is pertinent to note that differential expression and activities of glycosylating enzymes in humans and mice could significantly alter protein dis‑ tribution and function127,128. Highly glycosylated anti‑ gens such as CA19-9 (pancreatic, gastric and colonic cancer)129, CA15-3 and CA27.29 (breast cancer)130 and CA125 (ovarian cancer)131 are frequently used as cancer biomarkers; however, species-specific differences in glycosylating enzymes prevent the synthesis of similar epitopes in mice owing to the lack of the Lewis sialyA antigen. Therefore, a method to generate similar biomar‑ kers in mice could be to provide the fucosyl transferase that is deficient in mice132.

Modelling the microenvironment GEM are particularly suited to explore the role of inflammation and the stromal microenvironment in carcinogenesis. Many epidemiological studies suggest a link between chronic inflammation and cancer risk, as exemplified by associations between gastric cancer and colonization with Helicobacter pylori133, inflammatory bowel disease and colorectal cancer134, cirrhosis and liver cancer135, and familial pancreatitis and pancreatic cancer136. Indeed, GEM have confirmed the importance of chronic pancreatitis in pancreatic cancer73, and have demonstrated a role for microbial flora in intestinal tum‑ origenesis137. In addition, several studies have identified a role for the innate138 and acquired139 immune systems in promoting cancer using GEM that either lack specific 654 | September 2007 | volume 7

proteases, such as matrix metalloproteinase 9 (MMP9), or that lack specific immune effector cell function, respectively. These GEM can now be more stringently evaluated with additional mutant alleles and pharmaco‑ logical agents to determine what cell types and pathways are required to stimulate tumorigenesis. Stromal cells, particularly fibroblasts, have also been implicated in carcinogenesis through studies that identified paracrine growth stimulatory proper‑ ties 140,141. Indeed, the discovery of genetic 142,143 and epigenetic 144 mutations in stromal cells obtained from clinical samples has led to the hypothesis that the microenvironment co‑evolves with transformed epithelial cells in carcinomas. GEM offer the ability to characterize such interactions functionally, and several notable studies have already revealed a role for mutation of the transforming growth factor-β (TGFβ) pathway 201 and Trp53 (REF. 145) in modulating the tumour-stimulating properties of stromal cells. These intriguing results should prompt groups to actively mutate stromal cells in autochthonous tumour models as a means of initiating or promoting tumorigenesis. This could be achieved by combining multiple SSR sys‑ tems, one to mutate the cell autonomous compartment and the other to mutate the non-cell autonomous com‑ partment. Depending on the SSR allele used, it might be possible to mutate fibroblasts, invading immune cells or even normal epithelial cells.

Detecting disease With the exception of patients with a familial cancer predisposition, most people are diagnosed with cancer after seeking medical attention. Unfortunately, this is not possible when working with laboratory animals, and scientists often recognize morbidity after the dis‑ ease has progressed considerably. At present, most researchers rely on daily health checks that consist of cursory examination of the cage for such characteristics as anti-social behaviour and lack of grooming. As any experienced mouse modeller knows, mouse behaviour changes throughout the day, making it challenging to differentiate between early signs of morbidity and vari‑ ations in normal behaviour. One interesting possibility would be the merging of techniques used by behavioural biologists with cancer biology. For example, radio fre‑ quency emitters that are now used to track large mouse colonies could be exploited to keep track of a mouse’s movement throughout the day and night. Any early indications of lethargy or social separation could be indicative of illness. A much more sophisticated means of tracking early disease is the use of non-invasive imaging techniques. Two basic ways of imaging tumours are technologicallybased methods (such as magnetic resonance imaging (MRI), positron emission tomography (PET) and ultra‑ sound) that are used in both humans and mice, and genetically-based methods (such as bioluminescence and fluorescence) that are only available in model systems146 (BOX 4). Such approaches are needed to comprehensively investigate the natural history of tumorigenesis in GEM that possess variable penetrance and latency. www.nature.com/reviews/cancer


REVIEWS Box 4 | Non-invasive imaging of autochthonous tumours Current genetically engineered mice (GEM) use reporters such as green fluorescent protein (GFP) or luciferase, the expression of which is controlled by the same techniques used for conditional or inducible models. For example, a conditional luciferase allele can be activated at the same time and place as a conditional oncogene, and therefore marks every descendent cell163. Unfortunately, as most Cre alleles are typically activated in many cells throughout the organ, a bioluminescent signal that is indicative of Cre activity rather than initiation of carcinogenesis leads to a low signal/noise ratio. Therefore, one must rely on a relative increase in signal owing to increased cellularity and extrapolate the presence of a tumour. A much more desirable system would turn on bioluminescence under specific conditions, such as tumour initiation or progression. This could potentially be achieved through thorough characterization of key events in specific steps of carcinogenesis, such as the transcription of a novel gene. Versions of this concept have already been published in which an E2F‑, hypoxia-inducible factor 1-α (HIF1α)-, and nuclear factor κB (NFκB)-responsive luciferase reporters were used to image actively proliferating164, hypoxic165 or stressed166 cells, respectively. Similar reporters could be generated that are responsive to Akt, mitogen-activated protein kinase, Notch, sonic hedgehog, Wnt and other key pathways associated with specific cancers. In this fashion, it will be possible to assess the effects of specific pathways on tumour progression. Furthermore, these reporters will be useful in assessing drug efficacy more accurately than relying on bulk tumour volume.

Conclusion — medical milestones for models Although mouse cancer models represent a tremendous resource to evaluate the mechanistic underpinnings of malignancy, we feel that the overall goal of mouse mod‑ elling should be refocused to alleviate human cancer suf‑ fering by positively affecting the detection and treatment of cancer in patients. One aim of this challenge is to determine what fea‑ tures of tumour biology are relevant therapeutic targets besides the oncogenes previously identified from patient specimens. Target discovery typically takes place in a controlled environment such as cell culture and is then validated using in vivo systems; however, one might argue that this methodology is inherently flawed because the initial screen is performed in an unrepresentative setting. A new technology that should enable this endeavour is transposon-mediated forward genetics. 1. 2.

3.

4. 5.

6.

7.

8.

Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000). Kendall, S. D., Adam, S. J. & Counter, C. M. Genetically engineered human cancer models utilizing mammalian transgene expression. Cell Cycle 5, 1074–1079 (2006). Lock, R. B. et al. The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse. Blood 99, 4100–4108 (2002). Rubio-Viqueira, B. et al. An in vivo platform for translational drug development in pancreatic cancer. Clin. Cancer Res 12, 4652–4661 (2006). Sikder, H. et al. Disruption of Id1 reveals major differences in angiogenesis between transplanted and autochthonous tumors. Cancer Cell 4, 291–299 (2003). Becher, O. J. & Holland, E. C. Genetically engineered models have advantages over xenografts for preclinical studies. Cancer Res. 66, 3355–3358 (2006). De Both, N. J., Vermey, M., Groen, N., Dinjens, W. N. & Bosman, F. T. Clonal growth of colorectal-carcinoma cell lines transplanted to nude mice. Int. J. Cancer 72, 1137–1141 (1997). Staroselsky, A. N. et al. The use of molecular genetic markers to demonstrate the effect of organ environment

9. 10.

11.

12. 13.

14.

15.

Different versions of forward genetic screens can be incorporated into any currently existing conditional model to identify oncogenes or TSGs that cooperate with sensitized genetic backgrounds during carcino‑ genesis. Genes whose deletion or inhibition promotes cell death in tumours also need to be identified. Such genes are interesting because they represent excellent drug targets for either the cell autonomous or non-cell autonomous compartment. These screens are inherently quite difficult, as the inhibition or deletion of the target of interest will kill the tumour cell and thus make it dif‑ ficult to recover any information; however, there is little doubt that many targets are missed because they are only assessed in cell culture. In addition, the most accurate and accepted GEM should be evaluated in preclinical therapeutic schemes with currently available and experimental therapies. There are many considerations for conducting such studies properly, including an appropriate physical infrastructure and trained personnel, a stable finan‑ cial arrangement that is challenging to attain in most academic centres, and an engaged legal affairs office to address the significant intellectual property issues147,148. A pressing question to address first is whether autoch‑ thonous GEM demonstrate superior predictive capa‑ bility to animal culture models, and these results will greatly clarify whether GEM will be useful in the private sector for drug development. In support of this idea, two recent studies demonstrated that GEM based on the genetics of human lung adenocarcinoma develop histopathologically similar disease that responds to therapeutic agents currently in use in the clinic149,150. Although these studies lend credence to the use of these GEM for preclinical applications, even more exciting are the promising results from a preclinical study on a mouse model for acute promyelocytic leukaemia (APL)151 that have since translated into an effective new treatment for APL152. Thus, at long last, the tremendous potential of cancer modelling in mice is beginning to help win the war on cancer.

on clonal dominance in a human renal-cell carcinoma grown in nude mice. Int. J. Cancer 51, 130–138 (1992). Kang, Y. et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3, 537–549 (2003). Johnson, J. I. et al. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br. J. Cancer 84, 1424–1431 (2001). Fiebig, H. H., Berger, D. P., Winterhalter, B. R. & Plowman, J. In vitro and in vivo evaluation of US‑NCI compounds in human tumor xenografts. Cancer Treat. Rev. 17, 109–117 (1990). Hardisty, J. F. Factors influencing laboratory animal spontaneous tumor profiles. Toxicol. Pathol. 13, 95–104 (1985). van Kranen, H. J. et al. Frequent p53 alterations but low incidence of ras mutations in UV‑B-induced skin tumors of hairless mice. Carcinogenesis 16, 1141–1147 (1995). Balmain, A. & Pragnell, I. B. Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene. Nature 303, 72–74 (1983). Cardiff, R. D. & Kenney, N. Mouse mammary tumor biology: a short history. Adv. Cancer Res. 98, 53–116 (2007).


16. Enno, A. et al. MALToma-like lesions in the murine gastric mucosa after long-term infection with Helicobacter felis. A mouse model of Helicobacter pylori-induced gastric lymphoma. Am. J. Pathol. 147, 217–222 (1995). 17. Jonkers, J. & Berns, A. Conditional mouse models of sporadic cancer. Nature Rev. Cancer 2, 251–265 (2002). 18. Tuveson, D. A. & Jacks, T. Technologically advanced cancer modeling in mice. Curr. Opin. Genet. Dev. 12, 105–110 (2002). 19. Gannon, M., Gamer, L. W. & Wright, C. V. Regulatory regions driving developmental and tissue-specific expression of the essential pancreatic gene pdx1. Dev. Biol. 238, 185–201 (2001). 20. Mattick, J. S. & Makunin, I. V. Non-coding RNA. Hum. Mol. Genet. 15 Spec. No 1, R17–R29 (2006). 21. Robertson, G. et al. Position-dependent variegation of globin transgene expression in mice. Proc. Natl Acad. Sci. USA 92, 5371–5375 (1995). 22. Palomo, C., Zou, X., Nicholson, I. C., Butzler, C. & Bruggemann, M. B‑cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu). Cancer Res. 59, 5625–5628 (1999). 23. Kaufman, R. M., Pham, C. T. & Ley, T. J. Transgenic analysis of a 100-kb human b-globin cluster-containing

volume 7 | September 2007 | 655 © 2007 Nature Publishing Group

REVIEWS

24.

25. 26. 27.

28.

29. 30. 31.

32.

33. 34. 35. 36. 37. 38.

39. 40.

41.

42.

43.

44. 45. 46.

DNA fragment propagated as a bacterial artificial chromosome. Blood 94, 3178–3184 (1999). Schonig, K., Schwenk, F., Rajewsky, K. & Bujard, H. Stringent doxycycline dependent control of CRE recombinase in vivo. Nucleic Acids Res. 30, e134 (2002). Kuhn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995). Weinstein, I. B. Cancer. Addiction to oncogenes — the Achilles heal of cancer. Science 297, 63–64 (2002). Chin, L. et al. Essential role for oncogenic Ras in tumour maintenance. Nature 400, 468–472 (1999). The first mouse solid tumour models to experimentally demonstrate a requirement for oncogene expression in tumour maintenance. Fisher, G. H. et al. Induction and apoptotic regression of lung adenocarcinomas by regulation of a K‑Ras transgene in the presence and absence of tumor suppressor genes. Genes Dev. 15, 3249–3262 (2001). Felsher, D. W. & Bishop, J. M. Reversible tumorigenesis by MYC in hematopoietic lineages. Mol. Cell 4, 199–207 (1999). Huettner, C. S., Zhang, P., Van Etten, R. A. & Tenen, D. G. Reversibility of acute B‑cell leukaemia induced by BCR-ABL1. Nature Genet. 24, 57–60 (2000). Moody, S. E. et al. Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis. Cancer Cell 2, 451–461 (2002). Sarkisian, C. J. et al. Dose-dependent oncogeneinduced senescence in vivo and its evasion during mammary tumorigenesis. Nature Cell Biol. 9, 493–505 (2007). This paper demonstrates that the tetracycline system can be used to modulate ectopic HrasG12V levels in vivo. The authors find that maximal HrasG12V transgene expression causes mammary gland proliferative arrest (premature senescence), whereas low level HrasG12V expression promotes proliferation. It also raises the concern that inducible ectopic oncogenes (and constitutive ectopic oncogenes) may inadvertently cause a nonphysiological toxic response in cells in vivo. Blakely, C. M. et al. Developmental stage determines the effects of MYC in the mammary epithelium. Development 132, 1147–1160 (2005). Blyth, K. et al. Sensitivity to myc-induced apoptosis is retained in spontaneous and transplanted lymphomas of CD2-mycER mice. Oncogene 19, 773–782 (2000). Martins, C. P., Brown-Swigart, L. & Evan, G. I. Modeling the therapeutic efficacy of p53 restoration in tumors. Cell 127, 1323–1334 (2006). Ventura, A. et al. Restoration of p53 function leads to tumour regression in vivo. Nature 445, 661–665 (2007). Rusk, N. Making mice at high speed. Nature Methods 4, 196–197 (2007). Holland, E. C., Hively, W. P., DePinho, R. A. & Varmus, H. E. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev. 12, 3675–3685 (1998). Orsulic, S. et al. Induction of ovarian cancer by defined multiple genetic changes in a mouse model system. Cancer Cell 1, 53–62 (2002). Lewis, B. C., Klimstra, D. S. & Varmus, H. E. The c‑myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer. Genes Dev. 17, 3127–3138 (2003). Lewis, B. C. et al. The absence of p53 promotes metastasis in a novel somatic mouse model for hepatocellular carcinoma. Mol. Cell Biol. 25, 1228–1237 (2005). Lewis, B. C., Chinnasamy, N., Morgan, R. A. & Varmus, H. E. Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector. J. Virol. 75, 9339–9344 (2001). Theodorou, V. et al. MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer. Nature Genet. 39, 759–769 (2007). Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryoderived stem cells. Cell 51, 503–512 (1987). Cichowski, K. et al. Mouse models of tumor development in neurofibromatosis type 1. Science 286, 2172–2176 (1999). Clarke, A. R. et al. Requirement for a functional Rb‑1 gene in murine development. Nature 359, 328–330 (1992).

47. Jacks, T. et al. Effects of an Rb mutation in the mouse. Nature 359, 295–300 (1992). 48. Lee, E. Y. et al. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359, 288–294 (1992). 49. Lakso, M. et al. Targeted oncogene activation by sitespecific recombination in transgenic mice. Proc. Natl Acad. Sci. USA 89, 6232–6236 (1992). 50. Rubin, B. P. et al. A knock-in mouse model of gastrointestinal stromal tumor harboring kit K641E. Cancer Res. 65, 6631–6639 (2005). 51. Sotillo, R. et al. Invasive melanoma in Cdk4-targeted mice. Proc. Natl Acad. Sci. USA 98, 13312–13317 (2001). 52. Tuveson, D. A. et al. Endogenous oncogenic K‑ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 5, 375–387 (2004). 53. Schubbert, S. et al. Germline KRAS mutations cause Noonan syndrome. Nature Genet. 38, 331–336 (2006). 54. de Alboran, I. M. et al. Analysis of C‑MYC function in normal cells via conditional gene-targeted mutation. Immunity 14, 45–55 (2001). 55. Jackson, E. L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K‑ras. Genes Dev. 15, 3243–3248 (2001). 56. Lewandoski, M. & Martin, G. R. Cre-mediated chromosome loss in mice. Nature Genet. 17, 223–225 (1997). 57. Dewald, G. W., Noel, P., Dahl, R. J. & Spurbeck, J. L. Chromosome abnormalities in malignant hematologic disorders. Mayo Clin. Proc. 60, 675–689 (1985). 58. Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nature Rev. Cancer 7, 233–245 (2007). 59. Smith, A. J., Xian, J., Richardson, M., Johnstone, K. A. & Rabbitts, P. H. Cre-loxP chromosome engineering of a targeted deletion in the mouse corresponding to the 3p21. 3 region of homozygous loss in human tumours. Oncogene 21, 4521–4529 (2002). 60. Kmita, M., Kondo, T. & Duboule, D. Targeted inversion of a polar silencer within the HoxD complex re‑allocates domains of enhancer sharing. Nature Genet. 26, 451–454 (2000). 61. Langer, S. J., Ghafoori, A. P., Byrd, M. & Leinwand, L. A genetic screen identifies novel non-compatible loxP sites. Nucleic Acids Res. 30, 3067–3077 (2002). 62. Forster, A. et al. Engineering de novo reciprocal chromosomal translocations associated with Mll to replicate primary events of human cancer. Cancer Cell 3, 449–458 (2003). 63. Johnson, L. et al. Somatic activation of the K‑ras oncogene causes early onset lung cancer in mice. Nature 410, 1111–1116 (2001). Describes the generation of a stochastic model for lung cancer that accurately mimics the mutation of a single cell in the context of normal surrounding tissue. 64. Coste, I., Freund, J. N., Spaderna, S., Brabletz, T. & Renno, T. Precancerous lesions upon sporadic activation of b-catenin in mice. Gastroenterology 132, 1299–1308 (2007). 65. Rudolph, K. L. et al. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701–712 (1999). 66. Chin, L. et al. p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell 97, 527–538 (1999). References 65 and 66 demonstrate that mutation of telomerase predisposes mice to cancers that exhibit genomic instability, a hallmark of human cancers. 67. Seibler, J. et al. Reversible gene knockdown in mice using a tight, inducible shRNA expression system. Nucleic Acids Res. 35, e54 (2007). 68. Xue, W. et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445, 656–660 (2007). 69. Kumar, M. S., Lu, J., Mercer, K. L., Golub, T. R. & Jacks, T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nature Genet. 39, 673–677 (2007). 70. Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J. & Largaespada, D. A. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276 (2005). 71. Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G. & Jenkins, N. A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221–226 (2005).


72.

73.

74.

75.

76. 77.

78. 79. 80.

81. 82.

83. 84.

85.

86.

87. 88.

89.

90.

91.

92.

References 70 and 71 report the use of a genetically encoded transposon-based mutagenesis system to perform forward genetic screens in mice. Grippo, P. J., Nowlin, P. S., Demeure, M. J., Longnecker, D. S. & Sandgren, E. P. Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice. Cancer Res. 63, 2016–2019 (2003). Guerra, C. et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K‑Ras oncogenes in adult mice. Cancer Cell 11, 291–302 (2007). Cohen, S. J. et al. Phase II and pharmacodynamic study of the farnesyltransferase inhibitor R115777 as initial therapy in patients with metastatic pancreatic adenocarcinoma. J. Clin. Oncol. 21, 1301–1306 (2003). Kohl, N. E. et al. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nature Med. 1, 792–797 (1995). Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res. 49, 4682–4689 (1989). Lerner, E. C., Qian, Y., Hamilton, A. D. & Sebti, S. M. Disruption of oncogenic K‑Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor. J. Biol. Chem. 270, 26770–26773 (1995). Majumder, P. K. & Sellers, W. R. Akt-regulated pathways in prostate cancer. Oncogene 24, 7465–7474 (2005). Shen, W. H. et al. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell 128, 157–170 (2007). James, R. M. et al. K‑ras proto-oncogene exhibits tumor suppressor activity as its absence promotes tumorigenesis in murine teratomas. Mol. Cancer Res. 1, 820–825 (2003). Zhang, Z. et al. Wildtype Kras2 can inhibit lung carcinogenesis in mice. Nature Genet. 29, 25–33 (2001). Bayascas, J. R., Sakamoto, K., Armit, L., Arthur, J. S. & Alessi, D. R. Evaluation of approaches to generation of tissue-specific knock-in mice. J. Biol. Chem. 281, 28772–28781 (2006). Forster, A. et al. The invertor knock-in conditional chromosomal translocation mimic. Nature Methods 2, 27–30 (2005). Dankort, D. et al. A new mouse model to explore the initiation, progression, and therapy of BRAFV600Einduced lung tumors. Genes Dev. 21, 379–384 (2007). An excellent example of a conditional oncogene mouse model in which the animal is functionally wild type before induction of the mutation. Oberdoerffer, P., Otipoby, K. L., Maruyama, M. & Rajewsky, K. Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/ lox71. Nucleic Acids Res. 31, e140 (2003). Zhang, Z. & Lutz, B. Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Res. 30, e90 (2002). Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li‑Fraumeni syndrome. Cell 119, 847–860 (2004). Song, H., Hollstein, M. & Xu, Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nature Cell Biol. 9, 573–580 (2007). Ahmed, B. Y. et al. Efficient delivery of Crerecombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors. BMC Neurosci. 5, 4 (2004). Vooijs, M., Jonkers, J. & Berns, A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2, 292–297 (2001). Kemp, R. et al. Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity. Nucleic Acids Res. 32, e92 (2004). A mouse model in which Cre activity is regulated at both the transcriptional and translational level, therefore preventing the background recombination associated with other inducible Cre models. Hameyer, D. et al. Toxicity of ligand-dependant Crerecombinases and generation of a conditional Credeleter mouse allowing mosaic recombination in peripheral tissues. Physiol. Genomics 12 June 2007 (doi:10. 1152/physiolgenomics. 00019. 2007)

www.nature.com/reviews/cancer © 2007 Nature Publishing Group

REVIEWS 93. Bjerkvig, R., Tysnes, B. B., Aboody, K. S., Najbauer, J. & Terzis, A. J. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nature Rev. Cancer 5, 899–904 (2005). 94. Peto, R. in Origins of Human Cancer 1403–1428 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1977) (eds Hiett, H. H., Watson, J. D & Winsten, J. A.). 95. Ijichi, H. et al. Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression. Genes Dev. 20, 3147–3160 (2006). 96. Izeradjene, K. et al. Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell 11, 229–243 (2007). 97. Hruban, R. H., Wilentz, R. E. & Kern, S. E. Genetic progression in the pancreatic ducts. Am. J. Pathol. 156, 1821–1825 (2000). 98. Kondo, S. et al. Efficient sequential gene regulation via FLP-and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells. Microbiol. Immunol. 50, 831–843 (2006). 99. Rangarajan, A. & Weinberg, R. A. Opinion: Comparative biology of mouse versus human cells: modelling human cancer in mice. Nature Rev. Cancer 3, 952–959 (2003). 100. McClatchey, A. I. et al. Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev. 12, 1121–1133 (1998). 101. Trofatter, J. A. et al. A novel moesin-, ezrin-, radixinlike gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 72, 791–800 (1993). 102. Giovannini, M. et al. Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2. Genes Dev. 14, 1617–1630 (2000). 103. Liu, K. et al. Recombinase-mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels. Genesis 44, 556–564 (2006). 104. Zhuang, Y., Barndt, R. J., Pan, L., Kelley, R. & Dai, M. Functional replacement of the mouse E2A gene with a human HEB cDNA. Mol. Cell Biol. 18, 3340–3349 (1998). 105. Wallace, H. A. et al. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell 128, 197–209 (2007). An example of recombinase-mediated cassette exchange in which a large human genomic region was introduced into the syntenic mouse locus to elicit a disease phenotype that accurately mimics the human condition. 106. Kipling, D. & Cooke, H. J. Hypervariable ultra-long telomeres in mice. Nature 347, 400–402 (1990). 107. Harley, C. B., Futcher, A. B. & Greider, C. W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990). 108. Prowse, K. R. & Greider, C. W. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc. Natl Acad. Sci. USA 92, 4818–4822 (1995). 109. Lee, H. W. et al. Essential role of mouse telomerase in highly proliferative organs. Nature 392, 569–574 (1998). 110. Ding, H. et al. Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell 117, 873–886 (2004). 111. Martignoni, M., Groothuis, G. M. & de Kanter, R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin. Drug Metab. Toxicol. 2, 875–894 (2006). 112. Gonzalez, F. J. & Kimura, S. Study of P450 function using gene knockout and transgenic mice. Arch. Biochem. Biophys. 409, 153–158 (2003). 113. Gonzalez, F. J. Cytochrome P450 humanised mice. Hum. Genomics 1, 300–306 (2004). 114. Xie, W. & Evans, R. M. Pharmaceutical use of mouse models humanized for the xenobiotic receptor. Drug Discov. Today 7, 509–515 (2002). 115. Corchero, J. et al. The CYP2D6 humanized mouse: effect of the human CYP2D6 transgene and HNF4α on the disposition of debrisoquine in the mouse. Mol. Pharmacol. 60, 1260–1267 (2001). 116. Granvil, C. P. et al. Expression of the human CYP3A4 gene in the small intestine of transgenic mice: in vitro metabolism and pharmacokinetics of midazolam. Drug Metab. Dispos. 31, 548–558 (2003).

117. Gonzalez, F. J. & Yu, A. M. Cytochrome P450 and xenobiotic receptor humanized mice. Annu. Rev. Pharmacol. Toxicol. 46, 41–64 (2006). An excellent review of currently available transgenic mice expressing human metabolic enzymes. 118. Bui, J. D. & Schreiber, R. D. Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? Curr. Opin. Immunol. 19, 203–208 (2007). 119. de Visser, K. E., Eichten, A. & Coussens, L. M. Paradoxical roles of the immune system during cancer development. Nature Rev. Cancer 6, 24–37 (2006). 120. Shultz, L. D., Ishikawa, F. & Greiner, D. L. Humanized mice in translational biomedical research. Nature Rev. Immunol. 7, 118–130 (2007). 121. Chang, C. H., Fodor, W. L. & Flavell, R. A. Reactivation of a major histocompatibility complex class II gene in mouse plasmacytoma cells and mouse T cells. J. Exp. Med. 176, 1465–1469 (1992). 122. Rehli, M. Of mice and men: species variations of Tolllike receptor expression. Trends Immunol. 23, 375–8 (2002). 123. Hayakawa, Y., Huntington, N. D., Nutt, S. L. & Smyth, M. J. Functional subsets of mouse natural killer cells. Immunol. Rev. 214, 47–55 (2006). 124. Chen, Z. et al. A 320-kilobase artificial chromosome encoding the human HLA DR3-DQ2 MHC haplotype confers HLA restriction in transgenic mice. J. Immunol. 168, 3050–3056 (2002). 125. Chen, Z. et al. Humanized transgenic mice expressing HLA DR4-DQ3 haplotype: reconstitution of phenotype and HLA-restricted T‑cell responses. Tissue Antigens 68, 210–219 (2006). 126. Madsen, L. et al. Mice lacking all conventional MHC class II genes. Proc. Natl Acad. Sci. USA 96, 10338–10343 (1999). 127. Kobata, A. The third chains of living organisms‑a trail of glycobiology that started from the third floor of building 4 in NIH. Arch. Biochem. Biophys. 426, 107–121 (2004). 128. Raju, T. S., Briggs, J. B., Borge, S. M. & Jones, A. J. Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10, 477–486 (2000). 129. Carpelan-Holmstrom, M., Louhimo, J., Stenman, U. H., Alfthan, H. & Haglund, C. CEA, CA 19–9 and CA 72–4 improve the diagnostic accuracy in gastrointestinal cancers. Anticancer Res. 22, 2311–2316 (2002). 130. Gion, M. et al. CA27. 29: a valuable marker for breast cancer management. A confirmatory multicentric study on 603 cases. Eur. J. Cancer 37, 355–363 (2001). 131. Gadducci, A. et al. Serum half-life of CA 125 during early chemotherapy as an independent prognostic variable for patients with advanced epithelial ovarian cancer: results of a multicentric Italian study. Gynecol. Oncol. 58, 42–47 (1995). 132. Falk, P. G., Bry, L., Holgersson, J. & Gordon, J. I. Expression of a human alpha-1, 3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of Leb-containing glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection. Proc. Natl Acad. Sci. USA 92, 1515–1519 (1995). 133. Houghton, J. & Wang, T. C. Helicobacter pylori and gastric cancer: a new paradigm for inflammationassociated epithelial cancers. Gastroenterology 128, 1567–1578 (2005). 134. Itzkowitz, S. H. & Yio, X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G7–G17 (2004). 135. Seitz, H. K. & Stickel, F. Risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress. Biol. Chem. 387, 349–360 (2006). 136. Jura, N., Archer, H. & Bar-Sagi, D. Chronic pancreatitis, pancreatic adenocarcinoma and the black box in‑between. Cell Res. 15, 72–77 (2005). 137. Engle, S. J. et al. Elimination of colon cancer in germfree transforming growth factor b 1‑deficient mice. Cancer Res. 62, 6362–6366 (2002). 138. Coussens, L. M., Tinkle, C. L., Hanahan, D. & Werb, Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103, 481–490 (2000).


139. de Visser, K. E., Korets, L. V. & Coussens, L. M. De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 7, 411–423 (2005). 140. Olumi, A. F. et al. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 59, 5002–5011 (1999). 141. Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF‑1/CXCL12 secretion. Cell 121, 335–348 (2005). 142. Kurose, K. et al. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas. Nature Genet. 32, 355–357 (2002). 143. Fukino, K., Shen, L., Patocs, A., Mutter, G. L. & Eng, C. Genomic instability within tumor stroma and clinicopathological characteristics of sporadic primary invasive breast carcinoma. JAMA 297, 2103–2111 (2007). 144. Hu, M. et al. Distinct epigenetic changes in the stromal cells of breast cancers. Nature Genet. 37, 899–905 (2005). 145. Hill, R., Song, Y., Cardiff, R. D. & Van Dyke, T. Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell 123, 1001–1011 (2005). 146. Lyons, S. K. Advances in imaging mouse tumour models in vivo. J. Pathol. 205, 194–205 (2005). 147. Olive, K. P. & Tuveson, D. A. The use of targeted mouse models for preclinical testing of novel cancer therapeutics. Clin. Cancer Res. 12, 5277–5287 (2006). 148. Sharpless, N. E. & Depinho, R. A. The mighty mouse: genetically engineered mouse models in cancer drug development. Nature Rev. Drug Discov. 5, 741–754 (2006). 149. Ji, H. et al. The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell 9, 485–495 (2006). 150. Politi, K. et al. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to downregulation of the receptors. Genes Dev. 20, 1496–1510 (2006). References 149 and 150 demonstrate that mouse cancer models with relevant mutations respond accordingly to currently available therapies. 151. Lallemand-Breitenbach, V. et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043–1052 (1999). A seminal publication in which therapeutic intervention in a mouse cancer model led to the successful development of an arsenic trioxide as a treatment for childhood acute promyelocytic leukaemia. 152. Soignet, S. & Maslak, P. Therapy of acute promyelocytic leukemia. Adv. Pharmacol. 51, 35–58 (2004). 153. Gu, G., Dubauskaite, J. & Melton, D. A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447–2457 (2002). 154. Kawaguchi, Y. et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nature Genet. 32, 128–134 (2002). 155. Obata, J. et al. p48 subunit of mouse PTF1 binds to RBP-Jκ/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos. Genes Cells 6, 345–360 (2001). 156. Bardeesy, N. et al. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev. 20, 3130–3146 (2006). 157. Loonstra, A. et al. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc. Natl Acad. Sci. USA 98, 9209–9214 (2001). 158. Pfeifer, A., Brandon, E. P., Kootstra, N., Gage, F. H. & Verma, I. M. Delivery of the Cre recombinase by a selfdeleting lentiviral vector: efficient gene targeting in vivo. Proc. Natl Acad. Sci. USA 98, 11450–11455 (2001). 159. Schmidt, E. E., Taylor, D. S., Prigge, J. R., Barnett, S. & Capecchi, M. R. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl Acad. Sci. USA 97, 13702–13707 (2000). 160. Raymond, C. S. & Soriano, P. High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS ONE 2, e162 (2007).

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REVIEWS 161. Rodriguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nature Genet. 25, 139–140 (2000). 162. Ehrhardt, A., Xu, H., Huang, Z., Engler, J. A. & Kay, M. A. A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase. Mol. Ther. 11, 695–706 (2005). 163. Safran, M. et al. Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination. Mol. Imaging 2, 297–302 (2003). 164. Uhrbom, L., Nerio, E. & Holland, E. C. Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nature Med. 10, 1257–1260 (2004). 165. Safran, M. et al. Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production. Proc. Natl Acad. Sci. USA 103, 105–110 (2006). 166. Carlsen, H., Moskaug, J. O., Fromm, S. H. & Blomhoff, R. In vivo imaging of NF‑κB activity. J. Immunol. 168, 1441–1446 (2002). 167. Meuwissen, R. et al. Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell 4, 181–189 (2003). 168. Sansom, O. J. et al. Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K‑ras oncogene in vivo. Proc. Natl Acad. Sci. USA 103, 14122–14127 (2006). 169. Edelmann, W. et al. Mutation in the mismatch repair gene Msh6 causes cancer susceptibility. Cell 91, 467–477 (1997). 170. Jonkers, J. et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nature Genet. 29, 418–425 (2001). 171. Derksen, P. W. et al. Somatic inactivation of E‑cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 10, 437–449 (2006). 172. Aguirre, A. J. et al. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev. 17, 3112–3126 (2003). 173. Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005). 174. Wang, S. et al. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4, 209–221 (2003). 175. Kim, M. J. et al. Cooperativity of Nkx3. 1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc. Natl Acad. Sci. USA 99, 2884–2889 (2002). 176. Zhou, Z. et al. Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer. Cancer Res. 66, 7889–7898 (2006).

177. Colnot, S. et al. Liver-targeted disruption of Apc in mice activates β-catenin signaling and leads to hepatocellular carcinomas. Proc. Natl Acad. Sci. USA 101, 17216–17221 (2004). 178. Zender, L. et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125, 1253–1267 (2006). 179. Santoni-Rugiu, E., Nagy, P., Jensen, M. R., Factor, V. M. & Thorgeirsson, S. S. Evolution of neoplastic development in the liver of transgenic mice co‑expressing c‑myc and transforming growth factor-α. Am. J. Pathol. 149, 407–428 (1996). 180. Dinulescu, D. M. et al. Role of K‑ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer. Nature Med. 11, 63–70 (2005). 181. Wu, R. et al. Mouse model of human ovarian endometrioid adenocarcinoma based on somatic defects in the Wnt/β-catenin and PI3K/Pten signaling pathways. Cancer Cell 11, 321–333 (2007). 182. Teng, Y. et al. Synergistic function of Smad4 and PTEN in suppressing forestomach squamous cell carcinoma in the mouse. Cancer Res. 66, 6972–6981 (2006). 183. Opitz, O. G. et al. A mouse model of human oralesophageal cancer. J. Clin. Invest. 110, 761–769 (2002). 184. Mo, L. et al. Hyperactivation of Ha‑ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis. J. Clin. Invest. 117, 314–325 (2007). 185. Sansom, O. J., Griffiths, D. F., Reed, K. R., Winton, D. J. & Clarke, A. R. Apc deficiency predisposes to renal carcinoma in the mouse. Oncogene 24, 8205–8210 (2005). 186. Xiao, A. et al. Somatic induction of Pten loss in a preclinical astrocytoma model reveals major roles in disease progression and avenues for target discovery and validation. Cancer Res. 65, 5172–5180 (2005). 187. Shih, A. H. et al. Dose-dependent effects of plateletderived growth factor‑B on glial tumorigenesis. Cancer Res. 64, 4783–4789 (2004). 188. Oshima, H. et al. Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway. Gastroenterology 131, 1086–1095 (2006). 189. Andressoo, J. O. et al. An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria. Cancer Cell 10, 121–132 (2006). 190. Haramis, A. P. et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004). 191. Su, L. K. et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256, 668–670 (1992). 192. Schulze-Garg, C., Lohler, J., Gocht, A. & Deppert, W. A transgenic mouse model for the ductal carcinoma in situ (DCIS) of the mammary gland. Oncogene 19, 1028–1037 (2000).


193. Hingorani, S. R. et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437–450 (2003). 194. Kelavkar, U. P., Parwani, A. V., Shappell, S. B. & Martin, W. D. Conditional expression of human 15‑lipoxygenase-1 in mouse prostate induces prostatic intraepithelial neoplasia: the FLiMP mouse model. Neoplasia 8, 510–522 (2006). 195. Abdulkadir, S. A. et al. Conditional loss of Nkx3. 1 in adult mice induces prostatic intraepithelial neoplasia. Mol. Cell Biol. 22, 1495–1503 (2002). 196. Conner, E. A. et al. Dual functions of E2F‑1 in a transgenic mouse model of liver carcinogenesis. Oncogene 19, 5054–5062 (2000). 197. Rankin, E. B., Tomaszewski, J. E. & Haase, V. H. Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res. 66, 2576–2583 (2006). 198. Reilly, K. M., Loisel, D. A., Bronson, R. T., McLaughlin, M. E. & Jacks, T. Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nature Genet. 26, 109–113 (2000). 199. Silberg, D. G. et al. Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice. Gastroenterology 122, 689–696 (2002). 200. Pelengaris, S., Littlewood, T., Khan, M., Elia, G. & Evan, G. Reversible activation of c‑Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol. Cell 3, 565–577 (1999). 201. Bhowmick, N. A. et al. TGF-b signalling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848–851 (2004).

Acknowledgements

We thank members of the Tuveson lab for helpful comments on the manuscript. Work is supported by US National Institutes of Health grants 1F32CA123887-01 (K.K.F.), CA101973 (D.A.T.), CA111292 (D.A.T.), CA084291 (D.A.T.), CA105490 (D.A.T.) and the Lustgarten Foundation for Pancreatic Cancer Research (D.A.T.). D.A.T. is a group leader of Cancer Research UK.

Competing interest statement

The authors declare no competing financial interests.

DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene Abl | Bcr | CDKN2A | CYP2D6 | CYP3A4 | DPC4 | ER | Erbb2 | Hras | Kras | MMP9 | Myc | Nf2 | p53 | PIK3CA | PTEN | Rtel | Terc | TGFβ National Cancer Institute: http://www.cancer.gov

FURTHER INFORMATION Author’s homepage: http://science.cancerresearchuk.org/ research/loc/cambridge/ccri/tuvesond/ ?view=CRI&source=research

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