Aug 9, 2007 - Nixon initiated the 'War on Cancer', our ability to transâ ... realising the full potential of mouse models of cancer and what new approaches are ...
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
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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
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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.
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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
© 2007 Nature Publishing Group
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
Squamous cell carcinoma
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
Squamous cell carcinoma
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
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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
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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
© 2007 Nature Publishing Group
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
nature reviews | cancer
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
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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
© 2007 Nature Publishing Group
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
nature reviews | cancer
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
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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
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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.
nature reviews | cancer
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
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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
© 2007 Nature Publishing Group
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.
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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.
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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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