Download Genetically Engineered Mouse Models of Prostate Cancer

european urology supplements 7 (2008) 566–575
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Genetically Engineered Mouse Models of Prostate Cancer
Martijn C. Nawijn a,*, Andreas M. Bergman a, Henk G. van der Poel b
a
b
Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands
Department of Urology, Netherlands Cancer Institute, Amsterdam, The Netherlands
Article info
Abstract
Keywords:
Knockout
Molecular genetics
Mouse cancer model
Preclinical testing
Prostate cancer
Transgene
Objectives: Mouse models of prostate cancer are used to test the contribution of individual genes to the transformation process, evaluate the
collaboration between multiple genetic lesions observed in a single
tumour, and perform preclinical intervention studies in prostate cancer
research.
Methods: Mouse models for human prostate cancer are generated
through genetic engineering of the mouse germline, introducing lesions
that reflect those observed in human prostate cancer specimens. The
optimal mouse model accurately reflects the pathogenesis of the disease
including the sporadic nature of the initiating insult, the identity of the
genetic lesions accumulated throughout the transformation process, the
hormone dependency of the malignant cells, the incidence and tissue
specificity of metastatic lesions, and the responses to therapeutic intervention.
Results: Although this ultimate goal has not yet been reached, the
currently existing mouse models for prostate cancer have yielded important insights. These mostly relate to the contribution of individual genes
and the mechanism of oncogene collaboration in the early stages of the
disease. Modelling metastatic and hormone-refractory prostate cancer,
however, remains a major challenge.
Conclusions: Mouse models have made an invaluable contribution in
identifying the genetic lesions involved in high-grade prostatic intraepithelial neoplasia lesions and locally invasive prostate cancer. Most
mouse models are less accurate in modelling the progression to metastatic disease. Moreover, most mouse models for prostate cancer do
not facilitate analysis of hormone-refractory prostate cancer, although
this would constitute the most valuable contribution to preclinical
testing of novel therapeutic intervention strategies for the human
disease.
# 2008 European Association of Urology and European Board of Urology. Published by
Elsevier B.V. All rights reserved.
* Corresponding author. Lab Allergology and Pulmonary Medicine, Section Medical Biology,
Department of Pathology and Laboratory Medicine, University Medical Center Groningen/
UMCG, University of Groningen, Hanzeplein 1, PO Box 30.001, 9700 RB Groningen,
The Netherlands. Tel. +31 503610998; Fax: +31 503619007.
E-mail address: [email protected], [email protected] (M.C. Nawijn).
1569-9056/$ – see front matter # 2008 European Association of Urology and European Board of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eursup.2008.01.019
european urology supplements 7 (2008) 566–575
1.
Introduction
1.1.
The pathophysiology of prostate cancer
Prostate cancer is the most common malignancy in
men and represents the second-leading cause of
cancer-related male deaths in the Western world [1].
The development of prostate cancer advances
through a series of distinct stages ranging from
atypical hyperplasia, a commonly found early
lesion, to metastatic disease present in bone, lung,
and liver in advanced stages of the disease. Benign
prostatic hyperplasia (BPH) is characterised by a
major proliferation of stromal cells. In contrast,
prostatic intraepithelial neoplasia (PIN) is characterised by foci of dysplastic ductal and acinar cells
and high-grade PIN (HGPIN) is thought to comprise
the first stage of invasive prostate adenocarcinoma,
which usually occurs in the transition zone of the
prostate. The formation of PIN lesions is a common
event and is detected in up to one third of all men
over age 45 yr [2]. Still, most of these lesions do not
progress to clinically detectable tumours. HGPIN
lesions are thought to progress to invasive adenocarcinoma, locally confined and locally advanced
tumours, and eventually to metastatic prostate
cancer [3]. Prostate cancer biopsies usually contain
multiple independent sites of cancerous and HGPIN
lesions. Interestingly, independent lesions found in
a single specimen are often genetically heterogeneous, supporting the concept that prostate cancer
originates as a multifocal disease [4]. On radical
prostatectomy a biochemical relapse, detected by a
rise in prostate-specific antigen (PSA) levels, is
observed in about 30% of the cases [5]. Although
metastatic prostate cancer can initially be treated by
androgen-ablation therapy, the disease invariably
relapses as a highly aggressive androgen-independent prostate cancer, which cannot be cured with
current treatment modalities [6].
The heterogeneity of prostate cancer has precluded unambiguous identification of the critical
genetic lesions contributing to the disease. Several
genomic lesions are frequently present in prostate
cancer samples, including chromosomal losses at
8p, 10q, 13q, and 17p, as well as gain of 8q and
chromosomes 7 and X [3,7]. Loss of 8p21 is
considered an early event because it is found with
a similar, high frequency in PIN lesions and
advanced cancer. The homeobox gene NKX3.1 maps
to the critical 8p21 genomic region [8]. Loss of 10q23
is found in 30% of primary prostate cancers and up
to 65% of metastatic tumour samples, implicating it
as a later event than loss of 8p21 [3,9,10]. The tumour
suppressor PTEN, one of the most frequently deleted
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genes in a variety of human malignancies, maps to
the minimally deleted 10q23 chromosomal region
[11,12]. The amplified genomic regions contain
genes such as MYC (8q) and AR (X chromosome),
as well as numerous other genes. Although these
kinds of associative studies lend some support to the
notion that gain or loss of the individual candidate
genes might induce or contribute to the process of
malignant transformation of prostate epithelial
cells, formal proof of such a hypothesis requires
further experimental validation. Since the 1980s,
mouse genetic engineering, allowing a wide variety
of genetic modifications, has taken centre stage in
these kinds of mechanistic studies. In fact, the
pioneering work on mouse knockout technology has
been awarded the 2007 Nobel Prize in Physiology or
Medicine [13]. To date, genetic modelling of the
mouse has come to provide an invaluable resource
for the detailed characterisation of the molecular
events driving the cellular transformation process in
prostate and many other cancers.
1.2.
Mouse models for prostate cancer
The study of prostate cancer in humans is limited by
the ability to perform mechanistic studies with
regard to the contribution of individual genetic
lesions to the transformation process of prostate
epithelial cells. Moreover, the evaluation of novel
therapeutic strategies in patients requires large
cohort studies, which are difficult to control for
disease stage, genetic heterogeneity, diet, age, and
other risk factors for prostate cancer [14]. In the
mouse model, experimental control over both
environmental factors and the genetic lesions
applied to the prostate epithelial cells allows for a
far more detailed analysis of the mechanisms
underlying prostate cancer development as well as
the preclinical evaluation of novel therapeutic
strategies, thereby facilitating the design of targeted
approaches for further clinical studies.
Initially, the mouse was used as a xenograft
model for in vivo analyses of human prostate cell
lines. However, this approach largely neglects the
complexity of cancer as a disease with intricate
interactions between the transformed cells and the
surrounding resident cells in normal tissue, the
stromal cells, the endothelial cells, and the immune
cells that all play an critical role in the pathogenesis
of the disease [15]. Especially with regard to the
therapeutic evaluation of the antineoplastic activity
of (novel) compounds, xenograft studies frequently
fail as a preclinical approach [16]. Therefore, these
xenograft models are no longer referred to or
considered to be true mouse cancer models, but
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instead are considered to represent an intermediate
step between cell culture and an animal model and
relevant only for the evaluation of certain cell
autonomous features of clonal cell lines [15]. In
contrast, mouse models for prostate cancer are
characterised by the occurrence of endogenous
prostate tumours that evolve from normal cells.
Here, we will focus on transgenic mouse models of
prostate cancer, in which relevant genetic lesions
have been introduced into the mouse germline to
drive the transformation process of the prostate
epithelial cells.
The first transgenic mouse prostate cancer
models
displaying
autochthonous
tumours
depended on prostate-specific overexpression of
the SV40 small T and large T antigens (such as the
TRAMP model) or SV40 large T antigen only (such as
the LADY model) [17]. These models have the
drawback of using a very strong transforming agent
(SV40 large T inactivates both the p53 and the Rb
tumour suppressor proteins), inducing an oncogenic process that does not accurately recapitulate
the pathogenesis of the human disease. Moreover,
these models have several other shortcomings,
such as the strong neuroendocrine component seen
in advanced stages of the disease in the TRAMP
model, which preclude their use as preclinical
models for the evaluation of therapeutic treatments. We will therefore not discuss these SV40
large T-driven models in this update, but focus
instead on mouse cancer models using defined
genetic lesions as observed in human prostate
cancer specimens. We will discuss the current
approach towards modelling prostate cancer in
the mouse, present some of the data derived from
these models with relevance to the pathogenesis or
treatment of prostate cancer, and discuss the
limitations of these mouse models.
lesions, and (3) the preclinical evaluation of therapeutic
intervention strategies.
2.1.
Genetic manipulation of the mouse germline has facilitated
the generation of mutant mouse strains carrying engineered
genomic lesions (Fig. 1). These lesions can result in the loss of a
single gene from the germline (knockout mouse strains;
Fig. 1B) or the insertion of multiple additional copies of a gene
into the germline (transgenic mouse strains; Fig. 1C). In
transgenic mice, the expression of the transgene is usually
driven by a cell- or tissue-specific promoter sequence,
resulting in expression of the transgene during a certain
developmental stage or in a specific cell type. In addition,
single genes can be replaced by specific mutant alleles to
generate allelic series of a certain gene, or by unrelated
sequences to generate single-copy transgenic strains (knockin mouse strains). Finally, to allow experimental control over
the loss of gene expression or induction of transgene
expression, conditionally mutant mice have been generated
using the Cre recombinase [19]. In conditional knockout mice,
the gene of interest is placed in between loxP sequences, but
remains present and active in the germline (Fig. 1D). The
conditional allele can then be removed by Cre-mediated
recombination, mediating the fusion of the two loxP
sequences with excision of the intervening sequences. By
applying a Cre transgene, which acts in a tissue-specific
fashion (such as specifically in the prostate epithelium), it is
possible to induce loss of the tumour suppressor in a specific
tissue or during a specific developmental stage [19]. Conditional transgenic mice carry a transgene, which will only be
expressed on Cre-mediated removal of a conditional STOP
sequence, which abrogates the translation of the transgene
from its mRNA [19]. All of these approaches have been used to
study the contribution of single genes to prostate cancer in the
mouse. In this approach, over-expression of relevant oncogenes can be modelled in transgenic mouse strains, whilst loss
of a tumour suppressor gene can be modelled by generating a
(conditional) knockout strain (Fig. 1).
2.2.
2.
The contribution of single genes to prostate cancer
Oncogenes
Methods and results
In general, cancer is thought to develop through a series of
genetic and epigenetic changes driving the transformation of
normal cells into malignant cancer cells [18]. In sporadic
cancer, the initiating lesion affects a single cell in an otherwise
normal tissue environment [19]. The mouse model for human
prostate cancer needs to reflect the pathogenesis of the
human disease accurately, including the sporadic nature of
the initiating insult, the identity of the genetic lesions
accumulated throughout the transformation process, the
hormone dependency of the malignant cells, the incidence
and tissue specificity of metastatic lesions, and the responses
to therapeutic intervention.
To date, mouse models for prostate cancer have been used
to study (1) the contribution of single genes to the transformation process, (2) the collaboration between multiple genetic
As stated above, several genes (including MYC, NKX3.1, and
AR) have been found to be frequently amplified in human
prostate cancer and have been proposed to contribute to the
transformation process. The causal relationship between
over-expression of a given oncogene and the induction or
progression of a cancerous lesion can only be formally
addressed using genetically engineered mouse models. To
study the specific contribution of an individual oncogene to
prostate cancer, the gene of interest is over-expressed in
prostate epithelial cells using a transgenic cassette that drives
tissue-specific expression in the mouse (Fig. 1A). To direct
transgene expression to the mouse prostate, regulatory
sequences derived from the rat minimal probasin promoter
containing two androgen-responsive elements are commonly
used [17]. This transgenic cassette has been modified by the
inclusion of two additional androgen-responsive elements to
european urology supplements 7 (2008) 566–575
Fig. 1 – Genetic engineering of the mouse germline. Most
genes present in the mouse genome will contain several
exons, indicated as open rectangles, spaced by the
noncoding intervening sequences known as introns. Gene
expression is controlled by regulatory sequences
positioned upstream of the transcription initiation site
(both enhancer and promoter sequences), in the intronic
sequences or downstream of the last exon (depicted
schematically as the black arrow). (B) Single gene
knockouts are generated by replacing most of the coding
sequences, or a critical part of the coding sequence, by
unrelated sequences. Expression from the endogenous
locus will result in a nonfunctional protein. The knockout
allele is often referred to as a null allele. (C) Multicopy
transgenics are generated by genomic insertion of
multiple copies of a transgene, usually the cDNA sequence
of the gene of interest (indicated as the fused open
rectangles) placed under transcriptional regulation of a
generic or cell-type specific promoter sequence (indicated
by the grey arrow). Transgene integration occurs at a
random position in the genome, and copy numbers vary
between individual founder mice, allowing for the
selection of high- and low-copy transgenic strains. (D)
Conditional knockout strains are generated by the
introduction of loxP sequences (indicated as solid
triangles) at opposite ends of the gene of interests.
Expression (usually transgenic) of the Cre recombinase
will result in fusion of the two loxP sites, with excision of
the intervening sequences, in this case most of the coding
sequence of the gene. Hence, a conditional knockout allele
allows the normal developmental expression pattern of
the gene up to the moment when Cre recombinase is
introduced into the (somatic) cells. Depending on the
expression pattern of the Cre transgene, this approach
allows the generation of tissue-specific knockouts. (E)
Conditional transgenic strains are generated by the
introduction of a conditional STOP cassette upstream of
the coding sequences of the transgene of interest. The
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increase specificity and expression levels [20]. In so-called
reporter mouse models, the activity of a Cre recombinase
transgene driven by these regulatory sequences was confined
to the secretory epithelium of the prostate, underscoring the
specificity of this particular transgenic cassette [21]. Using this
cassette it was tested whether the prostate-specific overexpression of the MYC oncogene would, in fact, induce
prostate cancer in the mouse. Indeed, MYC over-expression
was sufficient to drive transformation of murine prostate
epithelial cells resulting in invasive prostate carcinoma [22]. In
a similar approach, activated Akt (which is observed in human
prostate cancers with loss of PTEN) expressed in a prostatespecific fashion was shown to be sufficient for inducing PIN
lesions in the mouse prostate (mPIN), but not invasive prostate
carcinoma [23].
The drawback of this conventional approach is that most, if
not all, of the prostate epithelial cells will express high levels
of the transgene, which does not accurately reflect the
pathogenesis of human cancer, where the lesion will occur
in a single cell within a normal tissue environment. Hence,
these models often do not accurately reflect the pathophysiology of the human disease. Despite this shortcoming, this
approach has been successfully used to experimentally
confirm the critical role of several oncogenes in driving the
transformation of prostate epithelial cells [17].
2.3.
Tumour suppressor genes
In addition to gene amplification, loss of certain genomic
regions is also frequently observed in human prostate cancer
specimens, which can contribute to loss of expression of
critical tumour suppressor genes. In addition to genomic
deletion, epigenetic silencing also contributes to loss of
tumour suppressor activity during transformation. In mouse
models, loss of a tumour suppressor gene can be mimicked by
deletion of the gene of interest from the germline (Fig. 1B). In
case of p27Kip1, for instance, it was shown that p27-deficient
mice displayed mild hyperplasia of the prostate epithelium,
but no mPIN lesions or more advanced stages of the disease
[24]. Also, in NKX3.1-deficient mice, hyperplasia and atypical
dysplasia were found in the prostate gland, but again, more
advanced stages of the disease were not observed [25]. Finally,
p53-deficient mice, which are predisposed to developing
various types of tumours, do not develop any lesions in the
prostate [26], indicating that loss of p53 does not predispose to
prostate cancer.
Unfortunately, homozygous deletion of critical tumour
suppressor genes from the germline often results in early
embryonic lethality, precluding the analysis of the role of the
tumour suppressor gene to the transformation process. To
circumvent these limitations, gene loss can be achieved by
prostate-specific deletion of a conditional allele (Fig. 1D). Using
STOP cassette abrogates translation from the mRNA and
precludes expression of the transgene. Introduction of Cre
recombinase results in removal of the STOP cassette and
thereby induces expression of the transgene. The
expression pattern of the Cre recombinase determines the
tissue-specific activation of the transgene.
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european urology supplements 7 (2008) 566–575
this approach to generate mutant mice with a prostatespecific deletion of the PTEN tumour suppressor, the critical
role of PTEN loss in inducing prostate cancer was shown [27].
2.4.
The collaboration between multiple genetic lesions
The availability of genetically engineered mouse models
allows for the direct evaluation of the functional interactions
between individual genetic lesions in driving the transformation process. This is especially relevant for prostate cancer,
because biopsies usually present with multiple independent
sites of cancerous and HGPIN lesions [4], precluding the
straightforward identification of the critical genetic lesions
driving the transformation process. Independent lesions
found in a single specimen are often genetically heterogeneous, supporting the concept that prostate cancer originates
as a multifocal disease. Hence, multiple genetic lesions found
in a single specimen do not necessarily collaborate in the
transformation process, but might merely represent independent cancerous lesions present within the specimen. In
compound mutant mouse models, the interaction between
discrete genetic lesions can be evaluated experimentally. The
power of the mouse model to disentangle the complex
interactions between individual genetic lesions can be nicely
illustrated by the studies on the roles of the tumour
suppressors PTEN and p53 in prostate cancer.
The tumour suppressors p53 and PTEN are mutated or not
expressed in a wide range of human malignancies. Functionally, the two proteins act in distinct cellular pathways. The p53
pathway integrates a variety of cellular stress signals and
responds by inducing cell cycle arrest, cellular senescence, or
apoptosis [28]. Using mouse models, it has been shown that
loss of p53 as a single genetic lesion does not induce any
prostate pathology [26], which could be interpreted to
disqualify p53 as a relevant tumour suppressor in prostate
cancer. However, the role of the p53 pathway in prostate
cancer became apparent only after concomitant loss of PTEN.
PTEN counteracts phosphatidylinositol 30 -kinase (PI3-K)
signalling, by reverting the second messenger phosphoinositol-3,4,5-trisphosphate (PIP3; produced by PI3-K activity) into
PIP2, which does not activate downstream signalling. PI3-K
activity regulates the Akt kinase as well as other downstream
pathways relevant to cellular survival and proliferation [29]. In
prostate cancer, loss of PTEN facilitates the progression
towards androgen-independent disease [30,31]. In human
prostate cancer specimens, PTEN deletions are among the
most frequent lesions. Despite this high frequency of
chromosomal losses at the PTEN locus in primary prostate
tumours, homozygous loss of PTEN is relatively rare and
strongly correlates with advanced stage tumours [32,33]. Full
loss of PTEN protein expression is observed in 20–30% of
prostate tumour metastases [32,34].
In mice, loss of PTEN also induces prostate tumours. Pten
heterozygous mice are prone to develop malignancies of
various histologic origins [35,36]. In prostates of Pten heterozygous mice, mPIN induction is observed with long latency
and at incomplete penetrance [37,36]. Interestingly, Pten
heterozygous tumours do not frequently lose expression of
the Pten wild-type allele [37], mirroring the situation found in
human tumours [32,33].
To analyse in detail the effect of Pten gene dosage on the
transformation of the prostate epithelium in mice, a so-called
Pten hypomorphic allele was generated. The hypomorphic
allele produces lower levels of the wild-type PTEN protein than
the germline allele. This allows the analysis of the effects of a
wide range of PTEN dosages on cellular transformation in the
prostate. Mice heterozygous for one null allele and one
hypomorphic Pten allele (resulting in lower PTEN levels than
observed in mice heterozygous for one null and one wild-type
Pten allele), developed massive mPIN and invasive prostate
cancer with intermediate latency and at full penetrance [37].
Still, residual PTEN protein expression was retained in the
lesions of these mice [37], indicating that remnant PTEN
activity might, in fact, be beneficial for tumour growth. In
support of this, recent studies in mouse models show that full
loss of Pten leads to oncogene-induced senescence in a p19Arfdependent fashion [38], counteracting the outgrowth of
malignant tumours in PTEN-deficient prostates. Apparently,
loss of heterozygosity (LOH) of the wild-type allele in Pten
heterozygous mice likely requires inactivating lesions in the
p19Arf/p53/p21 pathway. Indeed, concomitant loss of Pten and
p53 causes aggressive tumour growth in the mouse prostate
and a lack of induction of oncogene-induced senescence [38].
Because p53-deficient mice do not develop any prostate
pathology [26], these data indicate that PTEN loss precedes
p53 loss in prostate cancer, although p53 loss is required for
LOH of PTEN, which facilitates hormone-refractory disease.
In addition to the analysis of the functional interactions
between two genetic lesions present within the transformed
cells, the mouse model of prostate cancer has also been used
to characterise the contribution of genetic lesions in the
stromal compartment of the tumour to the progression of the
disease. During the transformation process the interactions
between the cancerous cells and the surrounding tissue
constituents (non-transformed tissue cells, stromal cells
and immune cells) govern critical processes such as angiogenesis and metastasis. The progression of cancerous lesions to a
fully malignant phenotype is therefore also dependent on the
interactions with the tumour microenvironment. Interestingly, in mouse models of prostate cancer it has recently been
observed that the stromal compartment of the tumour can
acquire specific genetic lesions independently from the cancer
cells, underscoring the critical role of the stromal cells to
tumour progression and the strong selective pressure on the
stromal cells during this process [39].
2.5.
The preclinical evaluation of therapeutic intervention
strategies
Whereas mouse models have been useful in dissecting the
specific contribution of individual genes to the transformation process, and in charting the functional interactions
between multiple genetic lesions commonly found in a single
tumour, the availability of genetically engineered mouse
strains closely mimicking the genetic make-up of human
prostate tumours holds the promise of not only modelling the
pathogenesis of human prostate cancer but also of evaluating
the efficiency of various therapeutic treatment regimens in
tumours with a specific genetic fingerprint. As such, validated
mouse models of human prostate cancer could facilitate
european urology supplements 7 (2008) 566–575
preclinical testing to optimise treatment regimens for very
specific tumour types.
The value of mouse models of prostate cancer for
preclinical testing can be further enhanced by the availability
of noninvasive imaging techniques, allowing the longitudinal
monitoring of the responses of individual tumours to a certain
therapeutic treatment regimen. Several approaches to small
animal imaging are available, such as magnetic resonance
imaging, computed tomography, positron emission tomography, single-photon emission computed tomography, and
bioluminescence imaging [40]. Imaging of the mouse prostatic
lobes is technically challenging, given their small size, the
complex anatomy, and the proximity of the bladder, where
specific imaging probes can accumulate. Hence, most progress
in imaging prostate cancer in the mouse has been made using
bioluminescence imaging techniques. In this approach, a
bioluminescent reporter transgene is expressed in a prostatespecific manner [41] or as a conditional reporter, activated only
after Cre-mediated recombination and thereby specific for
cells carrying an engineered genetic mutation requiring Cre
activity [42]. The latter approach has facilitated the detection
of recurrent tumour growth after castration of mice with
prostate-specific deletion of PTEN [43].
Unfortunately, most currently available mouse models
have not been used extensively to study advanced prostate
cancer (see below), precluding the application of mouse
models for the design of novel treatment regiments for
metastatic or hormone-refractory disease, which by and large
pose the major clinical problems associated with prostate
cancer. Notwithstanding these limitations, mouse models
carrying a genetic lesion that is sufficient to induce cellular
transformation, the formation of mPIN lesions and locally
invasive carcinoma are useful to explore therapeutic intervention aimed at disarming prostate cancer cells carrying that
specific genetic lesion. In fact, cancer cells are thought to
remain, at least in part, dependent on the activities induced by
their primary lesion, a phenomenon called oncogene addiction [44]. Therefore, identifying a potentially effective therapeutic approach acting on cancer cells carrying a very specific
genetic lesion might prove to be a fruitful approach for
targeting well-characterised subsets of prostate cancer. This
approach has proven its validity in treating chronic myeloid
leukaemia with a specific inhibitor (imatinib mesylate) of the
tyrosine kinase ABL, where complete regression of advanced
tumours is observed, and recurrent disease invariably carries
mutations in the BCR-ABL oncogene rendering the kinase
insensitive to the drug [45].
With respect to prostate cancer, however, this approach
might be less straightforward given the multifocal nature of
the disease and the presence of genetically heterogeneous
independent lesions found in a single specimen [4]. The
clinical efficacy of a therapeutic approach targeting an
individual pathway can therefore be expected to be limited.
Nevertheless, specific approaches aimed at disarming the
frequently activated PI3K/Akt pathway (see above) have been
evaluated in mouse models for Akt-driven prostate cancer,
and given the central role for the PTEN tumour suppressor as
an early lesion in a large fraction of prostate cancers, these
data are relevant to the treatment of human disease. As
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discussed above, the PTEN tumour suppressor negatively
regulates the PI3-K pathway, thereby limiting the activation of
Akt and other downstream pathways. This genetic lesion has
been modelled by prostate-specific deletion of PTEN, resulting
in invasive and metastatic disease [27], and by prostatespecific over-expression of an activated form of the downstream Akt kinase [23], inducing mPIN lesions but not invasive
carcinoma. The latter model has been used to evaluate the
response of these lesions to inhibition of mammalian target of
rapamycin (mTOR), which is placed even further downstream
in this signalling cascade and the levels of which are elevated
in human tumours with loss of PTEN or over-expression of Akt
[46]. This treatment regimen induced complete regression of
the mPIN lesions and seemed to largely affect HIF-1a–driven
transcriptional programme in the transformed epithelial cells
[47]. These data are supportive of further studies in the mouse
model on the inhibition of the PI3-K–activated pathways,
especially given the assumed role for PTEN loss in setting the
stage for progression to hormone-refractory disease [30,31].
The increased severity of disease in the mouse models driven
by loss of PTEN as compared to the activated Akt-driven model
argues for the use of the former in such studies and the
evaluation of alternative, more upstream kinases as a target
for intervention.
3.
Discussion
3.1.
Limitations of the transgenic mouse model
The mouse model of prostate cancer offers a wide
range of experimental possibilities and has been
invaluable in dissecting the critical pathways governing the early stages of prostate cancer. Nevertheless, transgenic mouse models of prostate cancer
faces several shortcomings. The mouse and human
prostate are anatomically dissimilar, complicating
the interpretation of data derived from a mouse
model. In addition, mice do not spontaneously
develop prostate cancer, and it appears that the
engineered genetic lesion requires a certain degree
of robustness to allow the model to mimic most of
the characteristics of the human disease. Finally,
numerous mouse models depend on androgendriven transcriptional cassettes to establish prostate-specific over-expression of the transgenes.
3.2.
Anatomic differences between the prostate of
man and mouse
Historically, the key anatomic differences between
the murine and the human prostate have led to
concerns about the validity of the mouse model [48].
The human prostate is a single lobular structure
surrounding the urethra at the base of the bladder.
In contrast, the mouse prostate consists of three
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distinct lobes, which are arranged circumferentially
at the basis of the bladder [48,49]. Both mouse and
human prostate are composed of similar epithelial
cell types (columnar secretory epithelial cells, basal
cells, and neuroendocrine cells), although the ratios
are different. Also, the mouse prostate has a very
modest stromal component. In the mouse, the
dorsal and lateral lobes of the prostate share a
ductal system and are thought to be most similar to
the human peripheral zone of the prostate, where
most carcinomas arise. In contrast, the mouse
anterior prostate is considered to be analogous to
the human central zone, which is rarely a site for
neoplastic transformation [49]. In human prostate
cancer, the contribution of neuroendocrine cells to
advanced stages of the disease is limited, even
though they can be present in significant numbers
in prostate adenocarcinoma. Hence, mouse models
that display preferential transformation of neuroendocrine cells (such as the SV40 T antigen-driven
models) or in which tumour outgrowth preferentially occurs in the anterior prostate (such as in some
Pten-deficient models) are less informative for the
pathogenesis of human disease. Consequently,
efforts have been made to standardise the pathologic grading of mouse prostate cancer lesions [50],
and expert pathologic evaluation of the lesions
found in newly generated models with reference to
this Bar Harbor classification system is a prerequisite for the accurate interpretation of the observed
phenotype.
3.3.
Limitations to inducing advanced prostate cancer in
the mouse
The mouse does not spontaneously develop benign
or malignant prostate pathology. This is in contrast
to the human situation, where in elderly men benign
proliferations such as nodular hyperplasia have a
nearly 100% prevalence and prostate cancer can be
found at high frequencies during autopsy. In fact,
prostate cancer is a very chronic disease. PIN lesions
can already be found in the prostates of men in their
twenties, whereas clinically detectable prostate
cancer is usually not observed before the sixth
decade of life. This chronic nature of the progression
of the disease cannot be accurately modelled in the
mouse, due to its limited lifespan and the logistical
restraints of experimental research. These drawbacks are not a major issue for the study of the early
stages of the disease, such as atypical hyperplasia,
PIN lesions, and progression to microinvasive
carcinoma, and mouse models have confirmed the
critical roles of MYC over-expression and loss of
PTEN and NKX3.1 in these early stages. However,
most of these models do not progress beyond the
stage of invasive carcinoma, which has severely
hampered the study of advanced disease [17]. In fact,
very few mouse models of prostate cancer display
metastatic disease to the relevant target organs
(bone, liver, and lung). The clinical problems
associated with prostate cancer, however, are
largely restricted to advanced disease. Whereas
locally invasive prostate cancer can be efficiently
treated by a surgical approach, metastatic disease
present at the time of surgery is largely responsible
for the remission of disease, and subsequent
androgen-ablation therapy offers only temporary
relieve. As such, mouse models of prostate cancer
will only significantly contribute to the design of
novel therapeutic treatment regimens when they
accurately model metastatic disease, facilitating the
study of androgen-ablation therapy.
This brings us to a final shortcoming in a number
of the currently available mouse models. To direct
the expression of a transgene to the prostatic
secretory epithelium, numerous models have used
regulatory sequences driving prostate-specific gene
transcription. Unfortunately, these regulatory
sequences usually contain several androgenresponsive elements, rendering the expression
levels of the transgene androgen dependent. Consequently, transgene expression levels drop dramatically on castration (to mimic androgen-ablation
therapy), thereby inducing massive death of the
cancer cells and remission of the tumour. These
direct effects preclude the use of these transgenic
mouse models to study the responses of the
transformed cells to androgen ablation, and the
mechanisms contributing to the outgrowth of
hormone-refractory prostate cancer. In mouse
models using prostate-specific deletion of conditional tumour suppressor genes (Fig. 1B) or prostatespecific activation of conditional oncogenes
(expressed from generic promoters; Fig. 1C) using
Cre-lox technology, these problems can be avoided
because only the Cre transgene will depend on
androgen-driven transcription.
4.
Future directions
Mouse models for prostate cancer have made major
contributions to the characterisation of the individual roles of distinct genetic lesions in the transformation process. However, most of these data bear
relevance only to the early stages of the disease
including invasive but not metastatic lesions. The
clinical practice, however, could benefit considerably from more detailed knowledge concerning the
european urology supplements 7 (2008) 566–575
mechanisms of metastatic disease and the inevitable hormone-refractory stages. Therefore, modelling advanced prostate cancer using genetically
engineered mice will require models allowing both
the combination of multiple individual genetic
lesions (for advanced stages of disease) and noninvasive imaging techniques to efficiently monitor
metastatic spread of the primary tumour. Moreover,
the pathways under scrutiny should include those
uniquely involved in advanced prostate cancer, such
as over-expression of the polycomb group proteins
BMI1 and EZH2.
An alternative approach to study advanced
prostate cancer could be the orthotopic transplantation of cells carrying the genetic lesions required for
the early stages, in combination with an in vitro
manipulation of these cells to introduce additional
genetic lesions required for metastatic disease. Such
orthotopic transplantation models, however, are
also faced with certain technical complications,
such as the size and the complex anatomy of the
target organ and the requirement for the correct
stromal compartment to facilitate advanced stages
of the disease.
The main goals for mouse models of prostate
cancer remain the validation of the specific contributions of individual genetic lesions observed in
human disease and the preclinical testing of novel
approaches to therapeutic intervention. In both
instances, the main challenge lies in the accurate
modelling of advances stages of the disease,
specifically, metastatic lesions in lung, bone, and
liver and hormone-refractory disease.
5.
Conclusions
The mouse model has allowed the critical testing of
the causal relationship between gene loss or gene
over-expression and the induction of prostate
cancer. Especially for the early stages of the disease,
the contribution of individual lesions, such as loss of
PTEN or p53 and gain of MYC has been well
characterised using these mouse cancer models.
Moreover, the collaboration between some of these
genetic lesions and their mechanistic basis of action
has been dissected into great detail. However, the
progress in modelling metastatic disease as well as
hormone-refractory prostate cancer is limited.
Current models can be used to evaluate therapeutic
approaches directed against the activities conferred
on the cancer cells by the single experimentally
induced genetic lesion. However, to further preclinical research where it really counts—in the area
of metastatic disease and hormone-refractory pros-
573
tate cancer—a further investment in generating
mouse models for advanced prostate cancer will be
required.
Conflicts of interest
The authors have nothing to disclose. M.C.N. was
supported by the MUGEN Network of Excellence.
A.M.B. was supported the Dutch Cancer Society.
Acknowledgements
The authors wish to thank Dr. Renée van Amerongen for critical review of the manuscript. M.C.N. was
supported by the MUGEN Network of Excellence.
A.M.B. was supported by the Dutch Cancer Society.
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CME questions
4. Noninvasive imaging of mouse prostate tumours
has been used to detect recurrent disease after
androgen ablation. The imaging technique allowing such analyses is:
A. Magnetic resonance imaging (MRI).
B. Single-photon emission computed tomography (SPECT).
C. Bioluminescence imaging (BLI).
D. Positron emission tomography (PET).
Please visit www.eu-acme.org/europeanurology
to answer these EU-ACME questions on-line. The
EU-ACME credits will then be attributed automatically.
1. Mouse models for prostate cancer allow experimental control over:
A. The genetic lesions leading to prostate cancer.
B. The environmental factors contributing to
prostate cancer.
C. Both the genetic lesions and environmental
factors in prostate cancer.
D. The progression to hormone-refractory prostate cancer.
2. Mouse models of prostate cancer over-expressing
oncogenes in a prostate-specific fashion:
A. Progress to locally invasive cancer.
B. Progress to metastatic disease.
C. Accurately model sporadic prostate cancer.
D. Have oncogene expression in most prostate
epithelial cells.
3. The part of the mouse prostate most closely
resembling the peripheral zone of the human
prostate is:
A. The dorsal and lateral lobes.
B. The ventral lobe.
C. The lateral lobe.
D. The lateral and ventral lobes.
5. Progression of prostate cancer to androgenindependent disease can be modelled in genetically engineered mice. The mouse model allowing
such analyses is:
A. A conditional knock-out for PTEN in combination with prostate-specific Cre transgene.
B. A prostate-specific MYC transgene in combination with prostate-specific Cre transgene.
C. A conditional knock-out for p53 in combination with a prostate-specific MYC transgene.
D. A conditional knock-out for p53 in combination with a prostate-specific Cre transgene.
6. In mouse models of prostate cancer, loss of p53:
A. Induces invasive prostate cancer in the
absence of other genetic lesions.
B. Does not induce any prostate pathology,
irrespective of other genetic lesions.
C. Induces hormone-refractory disease only after
full loss of PTEN.
D. Prevents oncogene-induced senescence induced by full loss of PTEN.