The role of murine models of prostate cancer in drug target discovery and validation

Review

10.1517/17460440903049308 © 2009 Informa UK Ltd ISSN 1746-0441 879All rights reserved: reproduction in whole or in part not permitted

TheroleofmurinemodelsofprostatecancerindrugtargetdiscoveryandvalidationImran Ahmad†, Owen J Sansom & Hing Y LeungThe Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, G61 6BD, UK

Background: Mice provide us with an excellent preclinical model of human prostate cancer. They have expanded our understanding of the molecular pathways involved in prostate carcinogenesis as well as allowing us to explore both novel and traditional treatment regimes based on the molecular profile of these lesions. Continuing refinement of the transgenic prostate models has proven challenging since no one model seems to represent the entire continuum of the disease, thus currently limiting its applicability to the human condition. This platform may potentially have major impact in validation of drug targeting specific biological process of prostate carcinogenesis, supple-menting (or even replacing) many of the current in vitro and in vivo assays with the in vivo environment that transgenic prostate models provide. Objective/method: This review focuses primarily on the current state of murine model systems as a preclinical therapeutic platform for the treatment of prostate cancer, as well as hope for the future of the field. Conclusion: Much of the work in the drug discovery field has been done with the PTEN-/- and TRAMP models of prostate cancer. Despite their limitations they have con-tributed much to our understanding of the pathophysiology of the disease. There is, however, a need for transgenic models that better reflect the stepwise progression found in the human condition. We feel that they will prove to be invaluable as a preclinical platform regarding efficacy and tolerability of various anticancer agents, which ultimately allows us to translate these findings to the clinical setting to prognosticate and ultimately render cancer patients disease-free.

Keywords: drug discovery, GFP, mouse models, prostate cancer, PTEN

Expert Opin. Drug Discov. (2009) 4(8):879-888

1. Introduction

Prostate cancer (CaP) is a significant health problem worldwide. It is the most common cancer among men in North America, and second only to lung cancer as a cause of cancer-related deaths in men. The development of CaP represents a stepwise process, arising from ‘multiple genetic hits’ in tumour suppressor genes and oncogenes that lead from transformation of benign prostatic epithelium to prostatic intraepithelial neoplasia (PIN), with progression, to locally invasive and then hormone-resistant disease, ultimately leading to advanced disease signified with metastasis [1].

The mouse provides us with an excellent preclinical model of human CaP. In the past we have achieved this with xenograft transplants consisting of either primary or genetically altered cell lines derived from localised and metastatic disease. Although mice do not spontaneously develop CaP, the transgenic models have provided new tools to study prostate carcinogenesis [2]. They have expanded our understanding of the molecular pathways involved in prostate carcinogenesis

1. Introduction

2. Animal models of prostate

cancer

3. Clinical applications

4. Assessment of treatment

response by in vivo imaging

5. Role of transgenic mice in drug

discovery

6. Expert opinion

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Theroleofmurinemodelsofprostatecancerindrugtargetdiscoveryandvalidation

880 ExpertOpin.DrugDiscov.(2009) 4(8)

as well as allowing us to explore both novel and traditional treatment regimes based on the molecular profile of these lesions. Continuing refinement of the transgenic prostate models has proven challenging as no one model seems to represent the entire continuum of the disease, thus cur-rently limiting its applicability to the human condition. However, as these models continue to develop at an increas-ing rate, the expectation is rising that they will eventually recreate the molecular, physiological and clinico-pathological features of human CaP. This platform may potentially have a major impact in the validation drugs that target specific biological processes of prostate carcinogenesis, supplementing (or even replacing) many of the current in vitro and in vivo assays with the in vivo environment that transgenic prostate models provide.

Thus, this review will focus primarily on the current status of murine model systems as a preclinical therapeutic platform for the treatment of CaP, as well as hopes for the future of the field. Currently, many drugs in development are very potent in in vitro and in vivo models, but fail in clinical trials [3]. It is hoped that transgenic models will be more representative of clinical patterns and may allow more accurate assessment of new drugs in development that focus on specific biological targets.

2. Animalmodelsofprostatecancer

2.1 RatRats are one of the few species that develop this disease spontaneously and have been explored as one of the early models of CaP [4]. The best-defined model is the Dunning rat model that shows well-differentiated, non-metastatic and slow-growing tumours [5]. It mimics human disease in the respect that it is initially hormone dependent, becoming even-tually hormone resistant. Further refinements by researchers of this model have led to rats that develop highly metastatic tumours, which spread to lymph nodes and lung [6].

Another model, the Lobund–Wistar (LW) rat, shows two stages of prostate tumourigenesis. The early premalignant stage is testosterone-dependent with tumours regressing following withdrawal of androgen [7]. However, the late testosterone- independent stage results in highly aggressive tumour formation, which does not respond to testosterone deprivation.

2.2 CanineProstate cancer often develops spontaneously in canines, one of the few animals in which this is the case. However, the tumours differ from those in humans in that they often lack a functional androgen receptor and thus when diagnosed are already androgen independent, limiting their use in drug research for human disease. However, dogs do develop bone metastasis, which is a very common feature in human CaP, representing a key advantage for the assessment of bone-directed regimes [8]. Despite this, there are numerous drawbacks including cost and care [9].

2.3 MouseMost previous preclinical studies in the mouse system have used xenografts. They are easy to apply, but suffer many drawbacks including their poor confidence in predicting in vivo efficacy in formal clinical trials [10]. It is likely that many factors are at play. This may be owing to the location of the xenograft, the environment (which is especially important given the role of the stroma in prostate carcinogenesis) and ultimately the cell line selected.

Transgenic mouse models show de novo tumour development, with an intact microenvironment, and often show distant metastasis with time, thus providing clinically relevant in vivo environment to develop and evaluate novel drugs. Primary cells can be harvested from the tumours (and metastasis) allowing rapid in vitro assessment of drug action as well as providing the information regarding the mechanistic nature of the drug.

The prostate of the human and the mouse share many similarities but do have significant differences that must be considered when one attempts to make conclusions across species [2]. The peripheral zone is where 70% of human CaP arise, and in the mouse the comparable area is the dorsolateral prostate. However, this is based on histological descriptions rather than on molecular characteristics [11]. Transgenic murine models also have their drawbacks: time and resources must be invested and generation and maintenance of the mice is expensive. They are unpredictable and especially when investigating new pathways or genetic defects, they may succumb before CaP actu-ally develops, if indeed it does develop. The genetic background can also have a modifying influence on the formation and/or progression of CaP [12]. Therefore, meticulous care must be taken when initially describing the genetic background and breeding regimes involved in creating any novel transgenic model [11].

These first murine models used prostate-specific expression of viral oncogenes, namely, the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Using the short probasin (a gene expressed specifically in the murine prostate epithe-lium; -426/+28 bp Probasin (PB)) promoter to express the SV40 early genes (T/t antigen), the terminally differentiated tall columnar epithelial cells of the prostate were targeted [13,14]. The male mice developed locally invasive disease that became metastatic. High-grade PIN was evident by 12 weeks, with metastasis (predominantly to lung and lymph nodes) by 30 weeks. Given this was the first effective model of CaP, much characterisation of this strain has been carried out. Subsequently, much of the preclinical drug research has been done on this platform (see later). However, in humans, viral antigens are not implicated in clinical CaP, and their effects are pleiotrophic (i.e., affect several phenotypic traits). The tumours tend to show an extensive neuroendocrine differentia-tion that is not seen in early CaP in humans [15-17].Given these inherent flaws with the TRAMP model, researchers were motivated to create models that recapitulated relevant genetic events in human CaP better. Strong prostate-specific promot-ers such as probasin or PSA (prostate-specific antigen, another organ-specific gene) linked to the Cre (causes

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ExpertOpin.DrugDiscov.(2009) 4(8) 881

recombination)-IoxP (locus of X over P1) system were devel-oped to drive target gene expression [4,18]. Several different target genes have been manipulated to achieve either a gain- or a loss-of-function; the target genes include cell-cycle regulators, steroid hormone receptors (androgen/oestrogen receptors), oncogenes, tumour-suppressor genes, homeobox genes, growth factors and their receptors and regulators of apoptosis [2].

The ‘perfect’ transgenic model of CaP would fulfil several criteria: it would mirror the genetic and molecular charac-teristics of CaP, have a short latency and almost complete penetrance, be simple to create and easy to use and would have the capacity to allow assessment of tumour burden and treatment response (e.g., in vivo imaging).

Thus far, the PTEN (phosphatase and tensin homologue deleted on chromosome 10) null CaP mouse is the most successful and widely used Cre-loxP prostate model. PTEN is a tumour suppressor gene that is frequently mutated in a variety of spontaneous cancers including CaP. It is a negative regulator of the PI3K/AKT pathway and it is reported that loss or mutation occurs in ∼ 30% of primary CaP [19] and in 63% of metastatic tumours [20]. The loss of PTEN has been shown to be embryonically lethal [21]; Wang showed that prostate-targeted conditional loss of one allele (PTEN+/-) is associated with high-grade PIN with incomplete penetrance after a long latency but invasive adenocarcinomas do not develop [22]. Complete PTEN loss (PTEN-/-) results in invasive carcinoma with lymphovascular infiltration within 12 weeks, which progressed to lung metastasis. This condi-tional PTEN null mouse represents the first animal model in which deletion of a single endogenous gene leads to metastatic CaP. Androgen ablation (resulting in castrated levels of cir-culating androgens) in these mice has revealed that the cancer cells do respond to treatment, but that these cells continue to proliferate even after androgen withdrawal, eventually developing into hormone-resistant CaP. Drawbacks of this model are that not all clinical prostate tumours have PTEN loss, signifying the fact that several genetic ‘hits’ are likely to be required to drive carcinogenesis to invasive tumour and metastasis in human. Also, while bone metastases are commonly observed in clinical disease [23], the PTEN null mouse model does not replicate such lesions, despite evidence of bone remodelling activity in the mature PTEN null mice.

While transgenic mouse models represent powerful tools for the study of CaP, they presume involvement of individual genes for development of the disease. However, as human CaP seems to be far more complicated from a genetic standpoint, these models may represent an oversimplification of the resulting pathophysiology. This is why further models that better mimic these genetic lesions found in humans are essential for us to better understand the disease process.

3. Clinicalapplications

One of the major applications of modelling human CaP in mice is to develop relevant models for the evaluation of new

targeted therapies. So far, most of the work carried out has used the TRAMP model. It is hoped that the formal application of more current murine transgenic prostate models will be informative and reliable (Table 1).

3.1 Chemotherapeutics/chemopreventionGenistein, the predominant phytoestrogen in soy food, has previously shown effects as a chemopreventative agent against hormone dependent and independent CaP in the TRAMP model [24]. Incidences of poorly differentiated prostate tumours decreased by 50 and 35% in the hormone dependent and independent groups, respectively. This preclinical model highlighted the importance of the androgen receptor and growth factor signalling pathways in the mechanisms of genistein.

Resveratrol, another polyphenol, is found in grape derivatives. In vitro studies have demonstrated its ability to halt carcinogenesis at the initiation, promotion and progression stage [25]. Harper and colleagues demonstrated an over sevenfold reduction in poorly differentiated prostate adeno-carcinoma using resveratrol in the diet of TRAMP mice [26]. These mice demonstrated decreases in the growth factor IGF-1, downregulation of downstream effector pERK 1/2 and an increase in the tumour suppressor ER-β. Another group demon-strated that oral grape seed extract in the TRAMP mice inhibited CaP growth and progression by means of a strong suppres-sion of cell cycle progression, cell proliferation and an increase in apoptosis [27]. This is thought to be owing to a decrease in expression of cyclins and cell-dependent kinases (Cdks).

Green tea polyphenols have also been shown to reduce the incidence of CaP and subsequent metastasis in the TRAMP model [28]. It was also demonstrated that there was an increase in apoptosis of CaP cells compared with the water-fed controls. Earlier work by the same group identified that in the TRAMP model ornithine decarboxylase (ODC) activity as well as protein expression was markedly higher in their prostates compared to controls [29]. As a result they investigated the role of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, against CaP. Results showed a significant decrease in weight and volume, as well as ODC enzyme activity in the dorsolateral prostate. None of the DFMO-fed TRAMP mice had any distant metastases to lymph nodes and lungs. The protein expression of E-cadherin and alpha- and beta-catenin was found to be restored in DFMO-fed animals. Interestingly, it has been shown that their chempreventative effect depends on the stage of the disease in the TRAMP mouse, effective only when the predominate disease is PIN [30].

All-trans retinoic acid (ATRA) has also been shown to slow prostate tumour cell proliferation, induce apoptosis and block the emergence of the neuroendocrine phenotype (which is often associated with advanced and aggressive disease) [31]. Similarly, antioxidant treatment (Vitamin E, selenium, lyco-pene) in the Long Probasin-Tag (LADY) model reduced the incidence of invasive CaP fourfold and increased survival in these mice [32].

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Nexrutine® (Next Pharamceuticals, California, USA), a commercially available herbal extract, is derived from the bark of the Phellodendron amurense tree and contains isoquinoline alkaloids, phenolic compounds and flavone glycosides [33]. Kumar et al. showed that this compound significantly suppressed the development of palpable tumours and progression of cancer in the TRAMP model, along with reduced expression of AKT, cAMP response element binding protein (CERB) and cyclin D1 in the prostate [34].

The bacterial macrolide rapamycin has been shown to inhibit the protein, mammalian target of rapamycin (mTOR) kinase, which is a downstream effector of the PI3K/AKT pathway, critically implicated in CaP [35]. As a result Majumder and colleagues used a cohort of transgenic mice that overexpressed AKT and treated them with the mTOR inhibitor RAD001 [36]. It was found that expansion of malignant cells in this model was mTOR dependent and its inhibition led to the complete reversal of the PIN phenotype in the prostate through apoptosis of epithelial cells.

An exciting study carried out by Abate-Shen’s group looked at the role of dual activation of both AKT/mTOR and MAPK pathways in the development of androgen-independent CaP [37]. They developed a Nkx3.1:PTEN mutant transgenic line that developed hormone refractory tumours after castration.

Table1.Transgenicmiceusedindrugresearch.

Treatment Mousemodel Ref.

Rampimcin TRAMP [35]

Rapamycin and PD0235901

Probasin PTEN;Nkx3.1

[37]

NSAIDs: Celecoxib TRAMP [38-42]

Polyphenols: geinstein, revesterol

TRAMP [24-26]

DFMO TRAMP [30]

ATRA TRAMP [31]

Silibinin TRAMP [45-49]

Oral grape seed extract TRAMP [27]

Antioxidant treatment TRAMP [32]

Antiandrogens: flutamide/tamoxifen

TRAMP [43-44]

Neamine MPAKT [50-52]

Nexrutine TRAMP [33-34]

CDLA-4 antibodies TRAMP [53-54]

Tumour vaccination Probasin PTEN [55]

2-fluoroadenine TRAMP [56]

Endostatin/angiostatin TRAMP [57]

ATRA: All-trans retinoic acid; DFMO: Alpha-difluoromethylornithine;

MPAKT: murine prostate-restricted AKT; PTEN: Phosphatase and tensin

homologue deleted on chromosome 10; TRAMP: Transgenic adenocarcinoma

of the mouse prostate model.

Combination treatment using rapamycin and PD0235901 (an MEK1 inhibitor) slowed down the development of androgen independent prostate cancer (AIPC), with a 3.3-fold reduction in prostate weight (p = 0.012) and 3.4-fold reduction in cellular proliferation (p = 0.018). This represents one of the first studies to use a prostate targeted transgenic model in the evaluation of combinational drug therapy.

NSAIDs have been demonstrated to reduce tumour progression in TRAMP mice. Wechter et al. showed that E-7869 (R-flurbiprofen) reduced the incidence of primary prostatic tumour and metastasis [38]. Celecoxib (Celebrex) has similar effects, reducing the formation of primary and meta-static tumours [39]. Animals with established tumours also showed increased survival on a celecoxib regime. Further studies revealed a dose-dependent response for celecoxib’s preventa-tive effects [40]. The exact mechanism of action is uncertain, possibly owing to its effects on COX-2 enzyme activity or mRNA levels [41]. Recently, OSU03012, an orally bioavail-able celecoxib-derived PDK1 inhibitor (but COX-2 inactive), has been tested in the TRAMP model [42]. PDK1 inhibits activation of AKT, and its use reduced the severity of prostatic intraepithelial neoplasia lesions and prostatic weight. However, there was no significant decrease in the incidence of cancer (both primary and metastasis).

Traditional drug treatments such as the anti-androgens have also been used in the TRAMP model. Flutamide and (tamoxifen derivative) toremifene were assessed in the TRAMP model [43,44]. Tumours from high-dose flutamide-treated animals were more differentiated and retained much of the normal glandular archi-tecture than those of the placebo group, in which the tumours were predominantly poorly differentiated. Toremifene was more effective than flutamide [43]. Incidence of HGPIN was reduced and the animals lived longer than placebo-treated animals. By 33 weeks of age, 100% of the placebo-treated animals had developed palpable tumours and died (or were killed), whereas 60% of the toremifene-treated animals remained tumour-free.

Using the TRAMP model, Raina and colleagues have shown the stage-specific inhibition of primary prostatic tumour growth as well as the protective effects against angiogenesis and late stage metastasis of Silibinin [45-47]. Silibinin is a flavonolignan isolated from milk thistle seeds and is well-known for its hepatoprotective activity [48]. Mechanistically it is thought to have many anticancer effects including induc-tion of a differentiated morphology, reduction of PSA and induction of cell cycle arrest accompanied by an increase in cyclin-dependent kinase (Cdk) inhibitors [49].

Angiogenin (ANG) is a 14-kDa angiogenic ribonuclease that is shown to be upregulated in human CaP [50]. Mouse ANG is the most significantly upregulated gene in AKT-induced PIN in the murine prostate-restricted AKT (MPAKT) [51]. Ibaragi and colleagues found that using the ANG inhibitor naemine in the MPAKT mouse prevented the AKT-induced PIN formation, as well as fully reversing developed PIN in these mice [52]. This was accompanied by a decrease in rRNA synthesis, cell

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proliferation and angiogenesis, with a corresponding increase in epithelial apoptosis.

3.2 ImmunotherapyThere is a paucity of data regarding immunotherapy in vivo, and these studies have tended to use the TRAMP model.

Hurwitz and colleagues reported that antibodies targeting CTLA-4, an inhibitory receptor on T cells, can be effective at inducing regression of subcutaneous transplantable murine tumours [53,54]. They then evaluated the effects of combining CTLA-4 blockade and an irradiated tumour cell vaccine (GM-CSF-expressing vaccine) in primary prostate tumour formation in the TRAMP mice [54]. Treatment was delivered subcutaneously and resulted in a significant reduction in tumour incidence (15 versus 75% control) with lower tumour grades (peak severity 3.9 versus 5.5 control, p = .0009). Their prostates also revealed significant accumulation of inflammatory cells in inter-ductal spaces in treated prostate.

Recently Haga et al. vaccinated non-tumour bearing littermates of the PTEN null mouse with their adenocarcinoma cell lines [55]. These mice went on to show significant protective effect against subsequent tumour challenge. Intra-tumour adoptive transfer of effector cells resulted in significant growth inhibition of pre-established prostate tumours in vivo. These experiments demonstrate the potential to investigate tumour cell vaccination and adoptive immunotherapeutic strategies in transgenic systems.

3.3 GenetherapyMartiniello-Wilks and colleagues used TRAMP mice treated with intra-prostatic injections of OAdV220, an ovine adenovirus expressing purine nucleoside phosphorylase (PNP) gene under the control of the Rous sarcoma virus promoter [56]. This was followed by systemic fludarabine treatment. Purine nucleoside phosphorylase is an Escherichia coli enzyme that can convert the systemically administered pro-drug fludarabine phosphate to the toxic metabolite 2-fluoroadenine (2-FA). Subsequently, 2-FA acts to inhibit DNA, RNA and protein synthesis. Their results showed that PNP-gene-directed enzyme pro-drug therapy delivered by the ovine adenovirus vector caused significant suppression of CaP progression (significant reductions in prostate weight) and increased survival (n = 9, 67% alive in treatment group versus 30% in cohort group). They also limited TRAMP animals to a treatment window of 25 – 33 weeks of age to allow recovery from treatment and to allow survival to the study endpoint.

Anti-angiogenic therapy is showing real signs of promise as an adjuvant therapy that overcomes some of the limitations of conventional therapies. Sustained endostatin and angiostatin therapy has shown effects at different stages of tumour development in the TRAMP model [57]. The treatment was delivered by means of a gene transfer method, using the recombinant adeno-associated virus (rAAV) 6 vector to deliver systemically stable levels of the agent. Results show remarkable survival only after the drug was given at early

stages, before the onset of high-grade neoplasia, compared with when the treatment was given for invasive cancer. It arrested the progression of moderately differentiated carcinoma to the poorly differentiated stage and metastasis. Immuno-histochemistry revealed significantly lower endothelial cell proliferation and increased tumour cell apoptosis.

4. Assessmentoftreatmentresponsebyin vivoimaging

In vivo imaging of tumours has allowed one to follow disease progression as well as the effects of therapeutic interventions in the murine model system [58]. The two most widely available molecules for in vivo imaging are bioluminescent enzyme firefly luciferase and green fluorescent protein (GFP). The latter allows one to directly monitor cells, whereas the former only allows one to view derived pseudolayered images. Visualisation of GFP does not require the injection of substrate. Despite this, luciferase is more sensitive and allows one to simultaneously monitor several mice (whereas GFP requires multiple excitation sources for several mice) [59].

One of the most useful techniques applies bioluminescent imaging (BLI) to noninvasively monitor the growth of luciferase-expressing carcinoma cells in vivo. Recent trans-genic models were developed to express firefly luciferase specifically in the prostate in an androgen-dependent fashion [60-62]. The combi-nation of the luciferase tech-nology with the TRAMP model provides a useful means to follow the progression of in vivo tumour development and development of metastasis [61].

Liao et al. have recently reported the combination of a PB-Cre4 PTEN-/- mutant with conditional luciferase and enhanced GFP reporter line [63]. In this model, the recom-bination mechanism that inactivates the PTEN alleles also activates the reporter gene. Surgical castration resulted in a reduced bioluminescence signal and tumour regression. In these animals emergence of androgen-independent cancer can also be detected using BLI (7 – 28 weeks following castration), in addition to monitoring growth, regression or relapse. Figure 1 shows the PTEN-/- mouse developing a clear GFP signal as imaged on the OV100 microscope (Olympus).

The use of an enhanced GFP (EGFP) technique provides an opportunity to locate or assess tumour bulk and isolate populations of cancer cells from tissues via fluorescence-based technologies [64]. Although by intra-tibial injection of GFP labelled CaP cells, it has been demonstrated that treat-ment response with pamidronate can be accurately followed up by GFP imaging of the skeleton [65].

MRI assessment of the TRAMP mouse prostate is shown to be precise and reproducible, and allows excellent volumetric determination of the extent of regression and monitoring of treatment over time [66]. More recently, the TRAMP model has also been applied successfully in a proof-of-principle experiment using in vivo MR spectro-scopic (MRS) studies with non-proton nuclei (13C) [67].

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Elevated 13C lactate was observed in both primary and metastatic tumours.

An interesting study using diffusion weighted MRI (DWI) in the cryptdin-2 model of CaP allowed identification of tumours < 1 mm in diameter. This is owing to the twofold difference in apparent diffusion coefficient between benign and malignant prostatic tissue (p < 0.00001) [68].

Following intra-tibial injection of GFP labelled CaP cells, Burton et al. [65] demonstrated that treatment response with pamidronate could be followed up.

5. Roleoftransgenicmiceindrugdiscovery

Murine models can be used at many stages of the drug discovery pipeline, whether it be further characterising disease biology to allow compound design, identification of novel biomarkers or preclini-cal testing of drug efficacy (Figure 2). It is thought that in the field of preclinical testing of drug efficacy that these transgenic models will prove most useful. The ability to test a compound’s efficacy in a system that had de novo CaP development with an intact organ microenvironment may improve the chances of success in progressing novel drugs from Phase I/II to Phase III trials. In addition in vivo transgenic models have much potential in facilitating the identification of relevant biomark-ers in subsequent clinical trials. As outlined earlier there are many criteria that have to be filled to give us the ‘ideal’ CaP model. Unfortunately, these criteria may be at odds with one another; it may be ideal to have a model that rapidly develops cancer, but does this represent the human condition, especially when considering the microenvironment and the impact of further random genetic events? This is especially pertinent in CaP, which is a heterogeneous multifocal disease. In reality,

we may never achieve the ideal transgenic model, but a more effective approach would be to use several mice that reflect the full spectrum of genetic and molecular variability seen in CaP. Indeed even the simplest models that show subversion of single oncogenic pathways could be of use especially when testing agents to those specific pathways.

Cancer development not only involves somatic mutations, the consequences of which can be robustly tested in murine models, but also copies number variations and epigenetic events, which are much more difficult to reconstitute in a mouse model.

6. Expertopinion

Murine models have provided invaluable data about the molecular pathways involved in CaP. They have allowed preclinical trials of chemo-preventative, immunotherapy and gene transfer agents. More recently, technology has allowed in vivo techniques for monitoring growth, regression and relapse of prostate tumours to treatment. As further novel genetic mutations are explained it is vital we model future transgenic mouse models on the basis of new insights of prostate carcinogenesis. Other than with the TRAMP model there is a paucity of data about therapeutic trials in murine models. As newer models mimic human prostate tumouri-genesis more closely, they will offer attractive platforms for on which these chemoprophylatic trials can be based.

Cre-loxP systems, which are not only tissue-specific but inducible in a time-specific fashion, have been available for some time now. Ratnacaram et al. generated a PSA-Cre-ERT2 that expresses the tamoxifen-dependent Cre-ERT2 recombinase selectively in prostatic epithelium [69]. This has the advantage

A. B. C.

Figure1.GFP imaging in livemouseat9monthsofage. Wildtype (A) and PB-Cre4 PTEN+/- (B) reveal no detectable signal. In comparison PB-Cre4 PTEN-/- (C) shows evidence of a prostatic mass (arrow).

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of being able to target fully differentiated adult prostatic epithelial cells as well as influencing the number of genetically recombined cells. This early model, however, suffers from long latency and incomplete penetrance to CaP. Recently, Acevedo and colleagues described an inducible FGFR1 prostate model, which was activated by chemical inducers of dimerization (CID) [70]. This led to prostatic adenocarci-noma in a synchronous and stepwise fashion. Interestingly, the development of PIN was completely reversible on withdrawal of CID; however, CaP lesions became FGFR1 independent. The ultimate aim of these inducible models is that they would mimic the multi-step tumourigenesis of human CaP more closely. These models that carry out both temporal and spatial gene inactivation will become the gold standard for transgenic mice models, allowing us to study the role of mutation reversal on cancer progression as well as ultimate survival.

At the time of writing of this article no model as yet demonstrated metastasis to the skeletal system. The race to achieve this is on as a transgenic mouse that demonstrates this would be invaluable in preclinical therapeutic models. Why this

Targetvalidation

Hit to lead andADMET analysis

Leadoptimisation

Preclinicalin in vivo efficacy

Clinicaltrails

Assay design andhigh throughputscreening (HTS)

Figure2.Theroleoftransgenicmiceindrugdiscovery.ADMET:Absorption, distribution, excretion, metabolism,toxicity.

has not been achieved remains to be seen. It is not known yet whether the murine platform represents fundamental pathophysiological differences (e.g., pelvic venous drainage), or whether the genetic defects created as per requirement in current transgenic models are not sufficient to initiate bone metastasis, and ‘additional’ events are required.

There is also a great need for further promoters that are prostate-specific, but androgen independent. This will allow careful study of the effects of castration and antiandrogen treatments on oncogenes-related tumours, in isolation from the effect of oncogene inactivation secondary to transgene silencing in models generated with androgen-sensitive promoters.

Much work has recently been done with positron-emission tomography imaging of murine xenograph models of CaP, suggesting a variety of tracers are useful in this setting [71-73]. However, as mentioned, this system does not reflect human prostate tumourigenesis as closely as the transgenic lines and one would envisage the next step would be to carry out feasibility studies in transgenic lines.

In future effects of free radicals, ageing and infection should be modelled [41]. Recently, there has been interest in the use of bacteria to deliver specific molecular antitumour therapeutics in in vivo murine prostate tumours [74,75]. Cur-rently, this has only been used in orthotopic models, using human cancer cells, but certainly the next stage would be to evaluate this technology in transgenic mouse models.

In summary murine models will continue to provide us with invaluable information about tumour initiation and progression, identification of molecular markers and further elucidation of molecular pathways. We feel that they will prove to be invaluable as a preclinical platform regarding efficacy and tolerability of various anticancer agents, which ultimately allows us to translate these findings to the clinical setting to prognosticate and ultimately render cancer patients disease-free.

Prostate tumourigenesis in humans require multiple mutations, in a stepwise fashion. Newer murine models will allow us to switch on and off genes in a prostate-specific manner at different time periods and in different sequences, enabling us to better understand what is occurring in the human. This in turn will allow better targeted treatment at a molecular level, initially trailed on the murine platform, before being translated to human treatments.

Declarationofinterest

The authors state no conflict of interest and have received no payment in preparation of this manuscript.

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AffiliationImran Ahmad†, Owen J Sansom & Hing Y Leung†Author for correspondenceThe Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, G61 6BD, UK Tel: +441413303984; Fax: +441419426521; E-mail: [email protected]

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