Engineering cancer resistance in mice

Peter Klatt and Manuel Serrano1

Spanish National Centre of Biotechnology, Department of Immunology and Oncology, Campus de Cantoblanco, Madrid E-28049, Spain

1 To whom correspondence should be addressed Email: mserrano{at}cnb.uam.es


    Abstract
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 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
Research on cancer has benefited enormously from the genetic manipulation of mice. Until recently, most of the emphasis has been put on tailoring genetic alterations to accelerate tumorigenesis or to recapitulate particular aspects of the tumorigenic process. The goal of engineering mice with an increased resistance to cancer is a novel aspect that is of importance to understand cancer susceptibility and to validate therapeutic and chemopreventive strategies. Here, we review the different mouse models described to date that manifest a ‘cancer resistance’ phenotype, with a particular emphasis on the molecular basis of the resistance and on the associated phenotypes that may have a negative impact on health.

Abbreviations: AP-1, activator protein-1; BMP, bone morphogenetic proteins; COX, cyclooxygenase isozyme; DNMTs, DNA methyl-transferase isozymes; IGF-1, insulin-like growth factor-1; Min, multiple intestinal neoplasia; Mom-1, modifier of Min-1; NO, nitric oxide; NOS-2, NO synthase; PLA2 s, phospholipase A2 enzymes; TGF-ß, transforming growth factor-ß; TNF, tumour necrosis factor.


    Introduction
 Top
 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
Genetic manipulation of the laboratory mouse is the most powerful tool for the validation of hypothesis, regulatory pathways, and therapeutic approaches in cancer research. There is an exponentially growing number of genetically modified mouse strains modelling the complex, and still incompletely understood, network of events causing cancer. The overwhelming majority of the genetic manipulations introduced into mice to study cancer have the goal of favouring tumorigenesis, which is achieved in increasingly sophisticated ways to closely recapitulate specific human tumours (reviewed in refs 14). However, the reciprocal question of whether it is possible to confer cancer resistance to a living organism has remained largely unexplored until recently. Only a few genetically manipulated mice have been generated with the specific purpose of increasing cancer resistance. Mice generated with other intentions have also unveiled ‘cancer resistance’ phenotypes. In total, there are over 20 mouse models in which increased cancer resistance of one type or another has been observed. These mouse models of ‘cancer resistance’ constitute the focus of this review and are summarized in Table I.


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Table I. Genetically engineered mice with cancer resistance phenotypes

 
Phenotypes of cancer resistance have been achieved after manipulating a variety of cellular and organismal systems, which include pathways related to inflammation, maintenance of genomic integrity, cell proliferation and programmed cell death (Figure 1). The various mouse models are discussed below, together with a brief description of the most relevant phenotypes and a discussion of the molecular basis of these phenotypes. We believe that the development and characterization of mouse models for ‘cancer resistance’ will be an important avenue for the validation of anticancer strategies, including genetic manipulation of cells ex vivo previous to auto-transplantation and chemoprevention, and also will serve to understand the genetic basis of cancer susceptibility.



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Fig. 1. Cellular and organismal systems known to affect cancer resistance.

 

    Inflammation
 Top
 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
Phospholipases A2
Phospholipase A2 enzymes (PLA2 s) catalyse the release of free fatty acids and lysophospholipids from phosphoglycerides. Two main functions have been attributed to phospholipases A2 (reviewed in ref. 5). On one hand, secreted PLA2 s, such as Pla2g2, are thought to participate in the degradation of phospholipids in the gastrointestinal tract, and could conceivably have a bactericidal activity. On the other hand, cytosolic PLA2 s, such as Pla2g4, play an important role in the production of precursors for inflammatory reactions, particularly, arachidonic acid.

The implication of Pla2g2 in cancer resistance goes back to the identification by Moser et al. (6) of a mouse mutation named Min’ (for multiple intestinal neoplasia) that causes a phenotype closely resembling familial adenomatous polyposis (FAP). The Min mutation is now known to affect the mouse homologue of the human APC tumour suppressor gene (7). Mice heterozygous for the Min mutation (hereafter referred to as Min/+) develop numerous intestinal and colonic adenomas similar in morphology to the adenomas seen in FAP (homozygous Min embryos die in utero). Interestingly, the number of intestinal tumours in ApcMin/+ mice is strongly affected by the genetic background and, in particular, the AKR genetic background was found to contain a strong modifier gene that suppressed tumorigenesis induced by the Min allele. The postulated modifying gene, called Mom-1 (modifier of Min-1), was mapped to the distal portion of mouse chromosome 4 (8). A subsequent study showed that the gene for secretory type II PLA2 (Pla2g2) maps to the same region that contains Mom-1, and displays complete concordance between allele type and tumour susceptibility (9). The groups of Dove and Lander generated a transgenic mouse carrying a functional allele of the Pla2g2 gene on a Min/+ susceptible background and found that the transgene partially rescued the effects of the Min allele (10). However, the potency of the Pla2g2 transgene was weaker than predicted and later work has provided evidence for the existence of an, as yet, unidentified gene in the Mom-1 region that, in addition to Pla2g2, might contribute to tumour resistance (11). The relevance of these findings for human tumour biology was confirmed very recently by a study demonstrating a significant positive correlation between Pla2g2 expression levels and survival of gastric cancer patients (12). Despite its evident relevance, the molecular mechanisms by which Pla2g2 affects tumour growth remain obscure. It has been proposed that the tumour suppressor activity of Pla2g2 might be related to its antibacterial activity that distinguishes this enzyme from the other PLA2 isozymes (reviewed in ref. 13).

In the case of the cytosolic phospholipase Pla2g4, the effects on tumorigenesis are exactly the opposite. Indeed, cytosolic Pla2g4 is thought to provide to the cells arachidonic acid, which in turn is the substrate used by the cyclooxygenase-2 isozyme (COX-2) to produce prostaglandins. COX-2 inhibitors, including non-steroidal anti-inflammatory drugs, are known for their antitumorigenic effect (14). Accordingly, inhibition of Pla2g4 should lead to a decrease of prostaglandin production and, as a consequence, should impair tumorigenesis. In support of this model, Lander's laboratory generated mice with a deletion of the Pla2g4 gene in the context of the Min/+ susceptible background (15). Ablation of Pla2g4 dramatically lowered prostaglandin levels in various tissues and, on the Min/+ background, results in an 83% reduction in tumour number. Apart from conferring a cancer resistant phenotype, Pla2g4 ablation in mice was shown to be protective in models of neurotoxic and pro-inflammatory challenges (reviewed in ref. 16), but causes pregnancy failures due to a retarded feto-placental development (17).

Cyclooxygenase
A major mechanism for the regulation of prostaglandin synthesis occurs at the level of COX, also known as prostaglandin-endoperoxide synthase, which catalyses the first rate-limiting step in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been identified: constitutively expressed COX-1 and mitogen-inducible COX-2. Both COX-1 and COX-2 are activated during inflammatory processes, and deregulated expression of COX-2, but not of COX-1, is associated with malignant lesions (reviewed in ref. 18). In keeping with a causal link between COX-2 expression and cancer, specific inhibition of COX-2 has emerged as a promising anti-inflammatory therapeutic approach to block tumour progression (14). Genetic evidence further supports the idea that ablation of COX-2 protects against cancer. Knocking out COX-2 on the background of a mouse strain carrying a truncated form of the Apc gene (Apc{Delta}716), a model similar to the popular Min/+ mouse (see above), was shown to reduce dramatically the number and size of intestinal polyps (19). The impact of COX-2 on tumour induction in the intestine was further corroborated by the observation that treating Apc{Delta}716/+ mice with a selective COX-2 inhibitor successfully suppressed colorectal polyposis. Later work showed that ablation of COX-2 abolished the formation and growth of teratocarcinomas produced by injection of embryonic stem cells into syngeneic mice (20), and reduced chemically induced skin carcinogenesis (21). Together, these studies strongly support the idea that COX-2 inhibition intercepts a central event in carcinogenesis. However, this concept has been challenged by a recent report describing the phenotype of a transgenic mouse overexpressing COX-2 under the control of the human keratin-14 promoter (22). These mice developed skin tumours at a much lower frequency than their wild-type littermates (3 versus 93%, respectively) when exposed to chemical carcinogenesis. Thus, for reasons that remain to be investigated, instead of promoting tumour formation, in this particular experimental setting skin-targeted COX-2 overexpression had a pronounced antitumorigenic effect.

Despite the absence of previous data pointing to a role of COX-1 in tumorigenesis, a recent study has addressed the role of COX-1 in intestinal cancer and skin carcinogenesis (21,23). These authors showed that disruption of COX-1 suppressed both polyp formation and skin papillomas by ~80%, an effect similar to that observed with COX-2-deficient mice. These data modify the view on the predominant impact of COX-2 on tumorigenesis and rather suggest comparable contributions of both COX isozymes to the generation of neoplastic lesions.

Protection against cancer by COX ablation comes at a high price. COX-2-deficient mice display various anomalies such as renal dysplasia, severe nephropathy and susceptibility to peritonitis (24). In addition, they exhibit multiple defects in ovulation, fertilization, implantation, decidualization (25) and, importantly, a dramatically increased risk of post-natal mortality due to problems in the closure of the ductus arteriosus (26). The phenotype of COX-1-deficient animals is less severe and includes a mild decrease in platelet aggregation as well as a moderately impaired inflammatory response to arachidonic acid (26).

Nitric oxide synthase
Generation of the gaseous radical nitric oxide (NO) by the cytokine-inducible isoform of NO synthase (iNOS or NOS-2), has emerged as a major signalling pathway in infection and inflammatory processes (reviewed in ref. 27). A causal link between carcinogenesis and elevated NO production has been suggested by various authors (reviewed in ref. 28). To support this hypothesis, NOS-2 knockout mice were crossed with Min/+ mice. As anticipated and similar to pharmacological inhibition of NO production, ablation of NOS-2 resulted in a substantial decrease in intestinal adenoma formation (29). However, a similarly designed study concluded that lack of NOS-2 accelerates intestinal tumorigenesis (30). In favour of the anti-neoplastic effect of NOS-2 inhibition, a subsequent report showed that genetic disruption of NOS-2 decreased urethane-induced lung tumour multiplicity by ~80% (31). A further level of complexity derives from the fact that NOS-2 ablation increases susceptibility to bacterial and viral pathogens (reviewed in ref. 32), which in turn could favour tumorigenesis. At present, therefore, the cancer resistance phenotype of NOS-2 null mice remains to be clarified (see also ref. 28).

Tumour necrosis factor
The signalling cascade induced by tumour necrosis factor (TNF), either through TNF-{alpha} derived from activated monocytes or TNF-ß secreted by mitogen-stimulated T-lymphocytes, has emerged as a major mediator of inflammation (33). The role of TNF in carcinogenesis is complex because acute administration of TNF has pronounced anticancer effects, whereas chronic upregulation of TNF signalling promotes tumorigenesis and metastasis (reviewed in ref. 34). Detailed studies on mice deficient for TNF-{alpha} in various models of carcinogen-induced skin tumour formation showed that ablation of TNF-{alpha} confers resistance to the development of benign and malignant skin tumours (up to 90% reduction in tumour number) (35,36). The tumour preventive effect of TNF-{alpha} ablation could be partially recapitulated (~50% reduction in skin tumours) with mice deficient for the monocyte chemoattractant protein-1 (MCP-1), a major downstream target of TNF-{alpha} (36).

The view that TNF signalling confers cancer resistance has been further reinforced by a more recent study exploring the relationship between TNF-triggered stem cell proliferation and liver carcinogenesis in mice deficient for either the type 1 or type 2 TNF receptor (37). The authors of this study subjected these mice to a carcinogenic diet (choline-deficient and ethionine-supplemented), a treatment considered to provoke liver carcinogenesis by aberrantly inducing the proliferation of hepatic stem cells. They found that in TNF receptor type 1 knockout mice, stem cell proliferation was substantially impaired and tumorigenesis was reduced. Of note, in this model, inhibition of TNF receptor type 2 had no appreciable effect on liver carcinogenesis. Together, these studies provide solid evidence that ablation of TNF signalling confers a cancer resistant phenotype. However, mice deficient in TNF signalling are considerably less protected against infections (38,39) and are susceptible to neuronal damage (40).

T-cell activation
Activated T-helper cells are able to induce or suppress inflammatory reactions. The choice between these alternatives depends on the balance between the two major subsets of T-helper cells, namely Th1 and Th2, which are characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation (reviewed in ref. 41). To investigate the role of Ag-specific T-cell activation in carcinogenesis, Beissert et al. (42) disrupted the B7/CD28-CTLA4 signalling pathway in the skin by overexpressing the high affinity CD28/CTLA-4 antagonist CTLA41 g under the control of the keratin-14 promoter. Chronic exposure of these mice to UV resulted in a significantly reduced number of skin tumours as compared with wild-type animals. Detailed analysis of the immune response in these animals indicated an impairment of the Th2 (anti-inflammatory) responses and, consequently, a relative increase in Th1 (inflammatory responses). This result does not support the general concept that decreasing inflammation enhances tumour suppression. However, more studies in different tumour types are necessary to clarify the impact of T cell-mediated responses on tumorigenesis.

Considering all the previous mouse models, it is possible to propose that, in general, a decrease in inflammatory responses is associated with a lower susceptibility to cancer development (Figure 1).


    Genomic integrity
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 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
DNA methyltransferase
In mammalian genomes, the complex methylation pattern of DNA is established and maintained by the action of a family of DNA methyl-transferase isozymes (DNMTs; reviewed in ref. 43). Aberrant de novo methylation of 5-cytosines at CpG islands located in transcriptionally active promoter regions has emerged as a highly relevant mechanism for cancer. Hypermethylation of genes involved in cell cycle control, apoptosis and DNA repair is tightly linked to tumorigenesis. In particular, in the case of colorectal cancer, there is suggestive evidence for a causal relation between silencing of tumour suppressor genes through promoter hypermethylation and tumorigenesis (reviewed in ref. 44). To support this hypothesis, Laird et al. (45) crossed the Min/+ mouse strain to animals carrying a targeted heterozygous deficiency in the gene coding for the DNA methyltransferase Dnmt1. This particular Dnmt is thought to be responsible for the maintenance of the genomic methylation pattern in adult somatic cells and also of the aberrant methylation of promoters during tumorigenesis. The double mutant progeny (Dnmt1+/-; ApcMin/+) developed 60% fewer intestinal adenomas than the Min/+ animals carrying two intact copies of Dnmt1. Further reduction of DNA methylation by treatment of the double mutants with a demethylating drug completely eliminated tumorigenesis. Subsequent studies using hypomorphic alleles of Dnmt1 confirmed and extended these findings (46,47). Based on similar genetic strategy, Trinh et al. (48) studied cancer susceptibility in mice doubly deficient for Dnmt1 and for the DNA mismatch repair gene Mlh1 (Dnmt1+/-; Mlh1-/-). Mlh1-deficient mice are prone to develop intestinal cancers as well as invasive T- and B-cell lymphomas. Surprisingly, while the protective effect of reduced Dnmt1 activity against intestinal neoplasia could be confirmed in this model, lymphoma frequencies were elevated from 21 to 90% in the same Dnmt1 mutants. This study highlights the importance of studying cancer resistance in different tumour models. Despite this concern, the above data strongly support the idea that it is feasible to confer cancer resistance to the colon by inhibiting Dnmt1.

Telomerase
The extensive proliferation needed for the formation of tumours requires mechanisms that overcome the proliferation block associated with progressive telomere erosion (reviewed in ref. 49). In line with this idea, accumulating evidence suggests that telomerase activity is critical to both oncogene-induced cell transformation in vitro and carcinogenesis in vivo (reviewed in ref. 50). To test the hypothesis that inhibition of telomerase may limit the growth of cancer cells by triggering critical telomere shortening and cell death, Blasco's group (51) subjected mice deficient for Terc RNA, the gene coding for the RNA component of telomerase, to an experimental protocol of multi-stage skin carcinogenesis. The authors found that Terc knockout mice with short telomeres were remarkably resistant to the development of skin papillomas, thus confirming that suppression of telomerase activity, at least in this experimental setting, may confer cancer resistance to mice. Similarly, Terc-/- mice were found to be relatively resistant to spontaneous tumorigenesis in cancer-prone mice, such as INK4A/ARF-/-(52) and Min/+ (53) mice. In other aspects, however, the chromosomal instability provoked by ablation of telomerase is not beneficial for the animal as evident from a substantially decreased life span due to multiple premature aging-like organ failure and reduced stress tolerance (reviewed in ref. 54).

Poly(ADP-ribose)polymerase-1
ADP-ribosylation is a posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a pivotal role in DNA repair and recovery of the cell from DNA damage. Ablation of poly(ADP-ribose) polymerase (Parp-1) in mice resulted in severe chromosomal instability, a pronounced defect in the base excision repair of DNA, abrogation of pro-inflammatory processes dependent on NF-{kappa}B signalling and a reduction in cancer susceptibility (reviewed in ref. 55). Importantly, despite their reduced capacity to cope with environmental and endogenous genotoxic agents, Parp-1 knockout mice did not develop cancer. In an attempt to link Parp-1 deficiency to increased cancer resistance, the group of de Murcia generated mice doubly deficient for Parp-1 and p53. They found that this genetic manipulation prolonged tumour-free survival of the double mutants by 50% as compared with p53-/- mice that were wild-type for Parp-1 (56). In addition, ras-transformed Parp-1-/- p53-/- fibroblasts, in contrast to Parp-1 expressing cells, were not tumorigenic when injected into nude mice. In the light of the multiple pathways linked to Parp-1 (see above), the mechanisms by which Parp-1 ablation intercepts tumorigenesis remain to be further defined. Nevertheless, it is tempting to speculate that the absence of Parp-1 decreases the survival of DNA-damaged cells, thus disfavouring the appearance of aberrant clones. Additionally, the reduced inflammatory responses of Parp-1-deficient mice could also contribute to cancer resistance (see above).

Methylguanine-DNA methyltransferase
O(6)-alkylguanine is the major mutagenic lesion in DNA induced by environmental and endogenous mutagens, and leads to G to A mutations. Chronic exposure of animals to N- alkylated nitrosamines and derivatives such as N-methyl-N-nitrosourea (MNU) is widely used as a standard in vivo carcinogenesis protocol generating a variety of malignant alterations including lymphomas, mammary tumours and bladder carcinomas. This class of mutagenic DNA adducts are removed by the ubiquitous and unique repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). To study the functional importance of MGMT in cancer prevention, human MGMT was introduced into the mouse germ line as a classical transgene. Mice overexpressing human MGMT in the thymus were found to be protected from developing thymic lymphomas after exposure to MNU (57). A number of subsequent studies from the Gerson laboratory extended these findings demonstrating resistance to both spontaneous and chemically induced tumorigenesis in transgenic mice overexpressing human or bacterial MGMT in a variety of target tissues (5864). More recent work from this group (65) showed that the MGMT transgene partially rescues the cancer-prone phenotype of low dose MNU-treated mice either deficient in the DNA mismatch repair gene Pms2 or with a reduced gene dosage of p53 (p53+/-). The general concept, that transgenic expression of the DNA repair gene MGMT confers cancer resistance, received further support by the analysis of mice with transferrin promoter-driven human MGMT overxpression in brain and liver (66). These transgenics were generated on the C3HeB/FeJ background, an inbred mouse strain prone to develop spontaneous liver tumours. MGMT overexpression in C3HeB/FeJ mice significantly reduced the occurrence of age-related spontaneous hepatocellular carcinoma, and most of the few tumours arising in the transgenic animals were found to be deficient in MGMT protein. Importantly, the general resistance to cancer conferred by MGMT overexpression resulted in increased average longevity due to a decrease in age-related spontaneous cancer (67,68). Together, the data demonstrate that upregulation of DNA repair mechanisms confers cancer resistance.

Summarizing this section, we conclude that cancer resistance can be conferred by manipulations of genomic integrity in the following directions: by difficulting genetic or epigenetic aberrations (Dnmt1, MGMT), by difficulting the permanent stabilization of telomeres (Terc) and by difficulting the survival of damaged cells (Parp-1).


    Cell proliferation and cell death
 Top
 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
AP-1
The numerous biological functions of the activator protein-1 (AP-1) family of transcription factors include cellular proliferation, oncogenic transformation, and programmed cell death (reviewed in ref. 69). AP-1 comprises a group of related dimeric basic region-leucine zipper proteins that belong to the JUN, FOS, MAF and ATF subfamilies. Accumulating evidence suggests that the AP-1 subunit c-Jun is crucial for proper regulation of cell cycle progression through its impact on both the transcriptional upregulation of cyclin D1 and the transcriptional repression of the cell cycle inhibitor p21Cip1. In addition, the requirement for c-Jun to promote oncogenic transformation in a variety of experimental settings suggests a role for this AP-1 subunit in tumorigenesis. Studies on the potential implication of c-Jun in tumorigenesis were precluded due to embryonic lethality of the null mutation (70). Thus, Young et al. (71) generated a transgenic mouse in which the human keratin-14 promoter directed the expression of a dominant negative mutant of c-Jun to the basal cells of the epidermis. Expression of the transgene blunted transactivation of the c-Jun promoter by TPA but, interestingly, did not inhibit TPA-induced expression of a subset of putative AP-1 target genes. Accordingly, no effect of the transgene on TPA-induced hyperproliferation in the epidermis could be detected. Surprisingly, despite the lack of anti-proliferative effects, expression of the dominant negative c-Jun mutant dramatically inhibited TPA-induced papilloma formation (by ~80%). The data fit well with previously reported findings from the laboratory of Spiegelman (72) reporting the resistance of mice deficient for the AP-1 subunit c-Fos to malignant progression of skin tumours in a similarly designed multi-stage skin carcinogenesis model. These authors showed that, upon treatment with TPA, c-Fos knockout mice carrying a v-Hras transgene were able to develop benign tumours with similar kinetics and relative incidence as wild-type animals. However, c-Fos-deficient papillomas quickly became very dry and hyperkeratotic, taking on an elongated, horny appearance. While wild-type papillomas eventually progressed into malignant tumours, c-Fos-deficient lesions failed to undergo malignant conversion. Together the data suggest that suppression of the c-Jun/c-Fos members of the AP-1 family of transcription factors confers skin cancer resistance to mice, despite its numerous prejudicial effects on the animal's health, at least in the case of c-Fos ablation (73,74) (see Table I).

Transforming growth factor-ß
Transforming growth factor-ß (TGF-ß) is a member of a family of dimeric polypeptide growth factors implicated in the regulation of cellular proliferation and differentiation. Alterations in TGF-ß signalling have been associated with a variety of diseases including cancer (reviewed in ref. 75). Accordingly, mice transgenic for TGF-ß display multiple alterations including severe fibrosis in liver and kidney, glomerular disease, loss of renal function, hydronephrosis, lipodystrophy-like syndrome, deregulated dental physiology as well as multiple metabolic and endocrine abnormalities (76,77). Notwithstanding, the potent growth inhibitory potential of TGF-ß stimulated various trials to engineer cancer resistance in mice. For example, transgenic mice overexpressing the type I isoform of TGF-ß (TGF-ß1) under the control of the mouse mammary tumour virus (MMTV) promoter/enhancer exhibited mammary ductal hypoplasia and were almost completely protected against both spontaneous and chemically induced mammary tumours (78). Similarly, MMTV-TGF-{alpha} transgenic mice, a model for the development of spontaneous mammary tumours in humans, were rendered resistant by MMTV-driven overexpression of TGF-ß1 (78). In support of a suppressive role for the TGF-ßl signalling pathway, chemically induced tumorigenesis in the mammary gland and lung was greatly enhanced by introduction of a dominant-negative mutant of the type II receptor for TGF-ß (79). Recent work supports the idea that transgenic overexpression of TGF-ß blocks progression of premalignant epithelium to malignant tumours by limiting the proliferative potential of epithelial stem cells (80). However, the notion that TGF-ß1 confers cancer resistance should be taken with caution. Previous work carefully exploring the function of TGF-ß1 in murine skin carcinogenesis showed that, during long-term exposure to skin carcinogens, keratinocyte-targeted overexpression of TGF-ß1 initially suppressed the induction of benign tumours but, with ongoing treatment, enhanced malignant progression (81).

Bone morphogenetic protein-6
Bone morphogenetic proteins (BMPs) belong to the TGF-ß superfamily, and their activity is related to a series of developmental processes including chemotaxis, proliferation and differentiation, which finally result in the transient formation of cartilage and the production of bone (reviewed in ref. 82). Recently, the BMP isoform BMP-6 has emerged as a potential player in the regulation of epidermal cell proliferation and suggested to be implicated in tumorigenesis (see refs 83,84 and references cited therein). Wach et al. (83) tested the putative tumour-promoting effect of BMP-6 in vivo by generating a transgenic mouse model overexpressing BMP-6 in the epidermis. In line with the predicted pro-mitogenic role of BMP-6, these mice displayed increased mitotic indexes which was the basis for the observed spontaneous psoriatic lesions (85). However, despite epidermal hyper-proliferation, BMP-6 transgenics showed a reduced incidence of chemically induced benign tumours and an impaired progression to malignant skin tumours. The authors provided experimental evidence that these apparently paradoxical findings could be accounted for by a BMP-6 induced increment of apoptosis as well as transcriptional downregulation of tumour-promoting AP-1 family members (83).

Protein kinase C{delta}
Protein kinase C{delta} (PKC{delta}) belongs to the PKC subfamily of the ‘novel’ PKCs and is involved in B-cell signalling as well as in the regulation of growth, apoptosis and differentiation of a variety of cell types (reviewed in ref. 86). Generation of transgenic mice overexpressing PKC{delta} in the epidermis under control of the human keratin-14 promoter revealed the potential of this protein kinase to confer cancer resistance (87). The authors of this study subjected PKC{delta} transgenics to chemical carcinogenesis of the skin and found that both tumour initiation and promotion were significantly suppressed (~70 and 50%, respectively). In addition, in PKC{delta} transgenics, progression to carcinoma was 37% (females) and 7% (males), while identically treated wild-type mice developed skin cancer at a much higher frequency (78 and 45%, respectively).

Bcl-2
The B-cell lymphoma-2 protein (Bcl-2) has been established as one of the key inhibitors of apoptosis due to its capacity to impede caspase activation (reviewed in ref. 88). Ablation of Bcl-2 was found to be associated with growth retardation, reduced life span, hair greying, polycystic kidney disease and lymphocytopenia (89). More recently, the emerging concept that cancer requires suppression of apoptosis has converted Bcl-2 into an interesting target for cancer therapy (90). Genetic evidence for the beficial effect of Bcl-2 ablation in tumorigenesis comes from attempts to identify modifier genes that determine individual susceptibility to Raf oncogene-induced lung adenomas (91). In this study, tumour-prone mice with lung-targeted expression of a constitutively active form of c-Raf kinase were crossed with Bcl-2 knockouts. Control mice overexpressing c-Raf displayed elevated levels of Raf signalling exclusively in pneumocyte type II cells and developed cuboid cell adenomas with short latency and 100% incidence. In contrast, c-Raf overexpression in mice lacking Bcl-2 caused a 6–8-fold delay in tumor incidence.

Tumor suppressor p53
The tumor suppressor protein p53 is of paramount importance in sensing different types of stress such as DNA damage, critical telomere shortening, hypoxia, nucleotide depletion, aberrant growth signalling, and excessive generation of reactive oxygen species. The p53 protein integrates these different types of stress through a variety of potentially interacting but distinct pathways and translates them into cell cycle arrest or, alternatively, apoptosis (reviewed in ref. 92). Previous attempts to generate mouse models globally overexpressing p53, either by introducing extra copies of p53 under the control of a heterologous promoter or by eliminating mdm2, the protein that targets p53 for degradation, did not allow the study of the cancer-protective role of p53 because constitutively high levels of p53 in these mice led to early embryonic lethality (reviewed in ref. 93). In keeping with the detrimental effects of enhanced p53 function at the organismal level, restriction of p53 overexpression to a particular cell type was shown to result in atrophy and premature functional degeneration of the target tissue (9496). In contrast with these negative results, three recent mouse models with moderately elevated and, in one case, properly regulated p53 expression provide the long-sought proof of the principle that boosting p53 confers cancer resistance.

The first of these models was generated in the laboratory of Donehower as a result of an aberrant gene-targeting event in murine embryonic stem cells (97), which eliminates the first six exons and, as a consequence, almost two thirds of the p53 protein. The deleted region includes functionally important regions at the N-terminus of p53, which are essential for its transactivation activity, sequence-specific DNA binding, and regulation through interaction with mdm2. Importantly, the deletion includes a large genomic region upstream of p53 (~630 kb) that codes for at least 30 genes and puts the remaining five exons of p53 under control of an unknown promoter. The obtained mutant allele was termed the ‘m’ allele and the mouse strain carrying this mutation was designated the ‘p53+/m mouse’. The C-terminal p53 fragment expressed from the ‘m’ allele was proposed to interact with wild-type p53 and, in agreement with previous reports on the stimulatory effects of peptides derived from the C-terminus of p53, it appears to marginally enhance transactivation and DNA binding activity of the wild-type protein. Importantly, the p53+/m mice exhibit enhanced resistance to spontaneous tumours as compared with their p53+/- and p53+/+ littermates. Over the course of their lifespan, >80% of the p53+/- and approximately half of the p53+/+ (wild-type) mice developed overt, life-threatening tumours while almost all of the p53+/m mice survived tumour free. Apart from conferring tumour resistance, this mutation was found to have unexpected side-effects such as reduced longevity, osteoporosis, lordokyphosis, skin thinning, delayed wound healing, hair greying, impaired hair regrowth, reduced size, loss of body mass due to muscle atrophy and loss of adipose tissue, generalized organ atrophy and diminished stress tolerance. In summary, these p53+/m mice displayed the complete phenotypic spectrum of premature aging. The obvious extrapolation from these results was that p53-mediated tumour suppression inevitably comes at a price: accelerated aging (93,98,99).

In support of this hypothesis, Scrable presented preliminary data on a mouse model carrying a truncated copy of the p53 gene in heterozygosis with full-length p53 (see: http://sageke.sciencemag.org/cgi/content/abstract/sageke;2002/40/nw139). Of note, this genetic manipulation of one endogenous p53 copy does not modify adjacent genes. The truncated p53 gene product, termed p44, lacks the domain required for its degradation through its interaction with mdm2 but retains the region required for heterotetramerization with p44 and p53. It is conceivable that such a mixed p44/p53 quartet is extraordinarily stable resulting in an enhanced activation of p53. p44/p53 mice are viable and do not develop tumours but show signs of premature aging, such as loss of muscle mass and decreased fertility. In addition, p44/p53 mice are only half normal size, and cells explanted from p44-carrying embryos proliferate slowly. In contrast with this growth-retarded phenotype, cells from these animals turn up the growth-promoting insulin-like growth factor-1 (IGF-1) pathway as they contain large amounts of IGF-1 receptor and accumulate activated Akt, a downstream target of IGF-1. At the moment, it remains unclear which are the molecular mechanisms that link p44 to IGF-1 signalling. This mouse model constitutes one further piece of evidence backing up the emerging hypothesis that enhanced p53 necessarily leads to premature aging. However, it is important to emphasize that in the two preceding mouse models, p53+/m and p44/p53, the p53 protein produced by the wild-type allele is presumably subject to a degree of constitutive activation. Thus, it can be speculated that these mutations in p53 mimic to some extent a chronic exposure to stress, and this, in addition to provide cancer resistance, could also constitute the basis of the premature aging phenotype.

In contrast with these findings, a ‘super-p53’ mouse model recently generated in our laboratory (100), demonstrates that increased p53 function and cancer resistance can be uncoupled from premature aging. ‘Super-p53’ mice were generated by introducing supernumerary copies of the p53 gene in the form of large genomic transgenes using bacterial artificial chromosomes (BACs). These BACs contain the entire p53 gene and, importantly, >100 kb surrounding genomic sequence. Thus, one can assume that the transgene contains the entire set of regulatory DNA sequences that constitute the genomic landscape required for proper regulation of p53 expression. We could demonstrate that the p53 transgenic allele when present in a p53-null genetic background, in fact, behaves as a functional replica of the endogenous gene. ‘Super p53’ mice, carrying one or two transgenic p53 alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage and are significantly protected from cancer when compared to normal mice. Finally, in contrast to previously reported mice with constitutively active p53, ‘super p53’ mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control, i.e. is not constitutively active but is ‘super’-activated only in response to the appropriate stimuli. In conclusion, this model provides the proof of the principle that cancer resistance can be enhanced in a global manner by a simple genetic modification and in the absence of undesirable effects.

To summarize this section, it can be concluded that manipulations that decrease proliferation or increase apoptosis have the potential to confer cancer resistance, and this can be achieved in some instances in the absence of deleterious effects for the organism.


    Concluding remarks
 Top
 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 
During the past years, it has become clear that cancer resistance is a flexible trait that can be enhanced by genetic manipulations. Many, but not all, of these manipulations are associated with secondary deleterious effects. Understanding cancer resistance and its global consequences on the organism is of paramount importance, because it provides the basis for designing chemopreventive strategies. This has been successfully achieved in the case of COX-2 inhibitors, even in humans affected by familial adenomatous polyposis (reviewed in ref. 101). The generation of mouse models resistant to cancer is providing more targets that remain to be investigated in the context of chemoprevention.


    Acknowledgments
 
This work was supported by the European Union and the Spanish Ministry of Science and Technology. The Department of Immunology and Oncology, at the CNB, was founded and is supported by a consortium between the CSIC and Pharmacia Corporation.


    References
 Top
 Abstract
 Introduction
 Inflammation
 Genomic integrity
 Cell proliferation and cell...
 Concluding remarks
 References
 

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Received February 5, 2003; accepted March 25, 2003.