- Email: [email protected]
Tumor suppressor mutations in mice: the next generation
304
Tumor suppressor mutations Andrea Mouse
I
strains
genetically
carrying
suppressor
have and will be identified
however,
it is clear that future
on a new generation existing
tumor
and Tyler Jacks? mutations
mimic familial forms of human
suppressors
mouse;
McClatchey*
models,
of experiments
cancer.
to tumorigenesis
designed
drug therapies.
New tumor
and mutated investigation
aimed
in the will focus
at improving
and using them to delineate
pathways
in mice: the next generation
the molecular
and to test the value of rationally
Addresses *Massachusetts General Hospital Cancer Center and Harvard Medical School Department of Pathology, Building 149, 13th Street, Charlestown, Massachusetts 02129, USA; e-mail: [email protected] tDepartment of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; e-mail: [email protected] Current Opinion in Genetics
& Development
1998, 8:304-310
http://biomednet.com/elecref/0959437X00800304 0 Current Biology Ltd ISSN 0959-437X Abbreviations APC adenomatous polyposis coli BCC basal cell carcinoma CDK cyclin-dependent kinase Hh Hedgehog LOH loss of heterozygosity MEF mouse embryonic fibroblast retinoblastoma protein PPb PtC patched Shh Sonic hedgehog
of the
mouse
as a genetically
oncogenes
in every
cell of a particular
suppressors identified are cancer prone but human counterparts these differences are our understanding of and more completely otherwise.
to date have been reported; most many do not faithfully model their (reviewed in [Z]). Explanations for emerging and may ultimately drive tumorigenic pathways more rapidly than would have been possible
The past year has witnessed exciting progress in the development and analysis of several mouse tumor suppressor models. Bridges between embryogenesis and tumorigenesis have been dramatically strengthened with the identification of critical developmental functions for individual tumor suppressor genes. Primary cell cultures derived from mutant mice have again proven to be a powerful reagent for characterizing the molecular function of tumor suppressor proteins. Importantly, further genetic manipulation and fine tuning of existing models has
It
mutations
has grown enormously over the past decade. Although the impact of this technology has been felt in many research areas, the field of cancer research may claim some of the greatest and most prolonged benefits. Genetically manipulated mice are being developed as tools for the study of the molecular, developmental and cell biological functions of genes mutated in human cancer and as mammalian models of human cancer that become logical and suitable vehicles in which to test rational cancer therapies. Manipulation of the tumor suppressor class of genes in the mouse provides a straightforward route toward modeling human cancer. In humans, individuals with various familial cancer syndromes inherit a mutant allele of a given tumor suppressor gene mutation, which causes predisposition to the development of certain cancers. Loss of the remaining wild-type allele occurs somatically, initiating tumor formation [l]. The generation of mice carrying a heterozygous mutation in an individual tumor suppressor gene mimics this situation genetically, allowing the
by the practice of tumor suppressor
in the mouse
is evident that most not evolve to suppress control cellular proliferation system
a situation
a few familial cancer syndromes. mutations in nearly all of the tumor
Patched
manipulable
tissue,
that is seen in only Mouse strains carrying
begun (Figure 1). This is exemplified conditional targeting to focus individual mutations spatially and temporally.
Introduction Use
development of subsequent mutations to occur randomly. This is in contrast to transgenic mice that overexpress
tumor suppressor genes did tumorigenesis but rather to in certain contexts. Essential
developmental functions for nearly every tumor suppressor identified have been revealed via the generation of mice homozygous for an individual mutation. In contrast, Patched
tumor (Ptc)
suppressor gene protein was well
known as a component of an important developmental signaling pathway in Drosoph’la prior to its identification as a tumor suppressor. Ptc is a multiple pass trdnsmembrdne protein that is negatively regulated by the secreted Hedgehog (Hh) protein [3-S]. Ptc, in turn, negatively regulates the expression of several target genes, including the Wnt family member wing/m, the TGF-B family member Decapeatapkgic (Dpp) and Ptc itself. This pathway is responsible for several crucial patterning events during Drosoph’la development. In 1996, positional cloning of the gene responsible for basal cell nevus syndrome (BCNS) identified the human Ptc homologue [6,7]. BCNS patients exhibit a number of symptoms, including skeletal abnormalities and the predisposition to develop cancer-predominantly basal cell carcinomas (BCCs) of the skin-but also childhood medulloblastoma and meningioma (reviewed in [S]). The autosomal dominant nature of BCNS inheritance and de-
Tumor
Figure
suppressor
mutations
in mice
McClatchey
305
and Jacks
1
The study of tumor suppressor
Mosaic
mutations
in mice moves to a new level
Tumor DNA, ”
analysis
mRNA
Genetic
background
Y Therapeutic testing Current Opinion m Genetics & Development
New methodologies
are being
used to refine tumor
suppressor
mutant
mouse
models
and to use them to define
the molecular
pathways
to
tumongenesls.
tection of loss of heterozygosity (LOH) for genetic markers in the vicinity of the BCNS locus in BCNS-associated and sporadic tumors were indicative of a tumor suppressor gene. This was confirmed by evidence for the somatic inactivation of Ptc in both sporadic and BCNS-associated BCCs and medulloblastoma, forging a link between an important developmental signal transduction pathway and tumorigenesis [6,7,9-121. Mouse models of Ptc function in development and tumorigenesis soon followed. Reasoning that the sequential repressive actions of Hh and Ptc indicated that loss of Ptc-as in BCNS tumors -might be equivalent to the overexpression of Hh, Oro et a/. overexpressed Sonic hedgehog (Shh; one of several mammalian Hh homologues) in the mouse epidermis [13”]. Indeed, transgenic embryos developed many BCC-like hyperproliferations of the epidermis shortly after epidermal formation (at embryonic day [E] 18.5). S/z/&transgenics also develop skeletal abnormalities similar to those of BCNS patients, despite the apparently restricted epidermal expression
of Shh by the well characterized keratin 14 promoter, suggesting that Shh was able to access and affect internal tissues too. The discovery that overexpression of Shh was sufficient for the development of BCCs prompted the search for and identification of (presumably activating) mutations tumors [ 13**].
in Shh itself in human
BCNS-related
Targeted disruption of the murine Ptc homologue reveals additional information about its role in murine development and tumorigenesis [14”]. Ptc-null embryos exhibit gross abnormalities by E8.0, including failed neural tube closure and abnormal cardiac development. The expression patterns of S/h and Ptc in wild-type embryos and of other markers in Ptd- embryos are consistent with a model in which Ptc is required in the dorsal neural tube to block ventral cell fate determination induced by Shh produced ventrally in the notochord. In the absence of Ptc, Shh induces a ventral cell fate throughout the neural tube apparently leading to failed neural tube closure. These observations dovetail nicely with a body of work
306
Genetics
of
disease
attributing
a critical
patterning
of several
role species
for Shh
in the
(reviewed
dorsal-ventral
information
in [15,16]).
cell lines prevalent
from melanoma
kindreds,
primary
Importantly, a significant percentage of Pt& mice develop cerebellar tumors that are similar to the childhood medulloblastomas seen in a small but significant percentage of BCNS patients [ 14**]. Paradoxically, mutation of Ptc and loss of Ptc function in these tumors should lead to derepression of Ptc and elevation of Ptc mRNA
affect both pl6iNK4A and pZ9ARF and inactivate p16INK4A exclusively [25,26].
levels. Although the presence or absence of the remaining wild-type Ptc allele was not examined here, the expression of a /ucZ transgene included in the targeting vector was markedly higher in the tumors than in the surrounding tissues and could be used to detect early nonpathologic lesions. Alternatively, this could also reflect either activation or overexpression of .S& or some unrecognized pathway.
p16/nk+‘a/‘l9A?f null mice C57BL6/J x 129/Sv x SJL
Interestingly, Ptc+- mice do not develop BCCs, suggesting that loss of the wild-type Ptc allele may be rate limiting in this tissue; alternatively, the context of the surrounding tissue-embryological or mature -may play an important role in the development of mouse BCCs.
The p151nk4b/pl61nk4a/plgArf Originally
identified
kinase
and
attempt
to model
some
pl6/NK4A-associated
in the mouse targeted inactivating both pI6lflk40
appear
to
tumor
the deletion of and pJ9urf [27**].
are viable-at least on a mixed genetic background-and ex-
hibit signs of extramedullary hematopoiesis, suggesting that plblfl~4a/p/PAR~ may function to negatively regulate proliferation of the hematopoietic lineage. In principle, this could be related to the requirement for pRb function in the hematopoietic lineage during development [28-301. Importantly, pl6~n~4~/pl9A~-null animals are cancer prone, primarily developing subcutaneous fibrosarcomas and lymphomas at an average of 7.25 months of age, a latency accelerated by carcinogen treatment. Neither pituitary tumors, which develop at high frequency in Rb+/- mice [29], nor melanoma develop in pl6/n~4a/p19Ad++l- or -I[27**].
inhibitor
able to prevent cell cycle progression, p161NKq* was celebrated as the product of the tumor suppressor gene mapped to the familial melanoma susceptibility locus [ 17,181. At the Gl+S cell cycle transition, cyclin-dependent kinases (CDK4 or CDK6) regulatory subunits phosphorylate
first
susceptibility exon 2 thus
mice
locus
as a cyclin-dependent
The
tumors
now suggests that pZS’NK48 mutations are not in human cancer; most mutations of this locus
bound to D-type cyclin the retinoblastoma tu-
mor suppressor protein (pRb), derepressing its associated E2F transcriptional activity and leading to cell cycle progression (reviewed in [19,20]). pl6*NK4A disrupts this transition by preventing the interaction between CDK4 and cyclin D (hence the inhibitor of kinase 4 [INK4A] designation). In human cancers, mutational desensitization of CDK4 to the inhibitory effects of p16*nk4~, loss of pl6lnk42, overexpression of cyclin D, or inactivation pRb can be found in mutual exclusion, documenting importance of this pathway in tumor formation.
of the
In fact, molecular proof that pl6L$‘Kkd functions as a tumor suppressor has been the subject of much debate despite the plausibility of such a model. The scramble to identify pf6IlvKJA mutations in melanoma and other tumor types led to the realizations thatp/6LNKdA mutations are more common in tumor cell lines than in primary tumors and that the pi6/h’K4A locus was often grossly affected by large deletions or genomic rearrangements (reviewed in [Zl]). Careful examination of the pl6’*‘K4A locus revealed the presence of two additional open reading frames (ORFs), one encoding a protein with amino acid and functional similarity to pl6’.vK4A dubbed plSNK4fi [Z-24]. Th e second ORF begins upstream of p16LNK4A at a unique exon (lb) which is spliced to pZ6INK4A exons 2 and 3 and utilizes an alternative frame to encode a protein with no known homology, dubbed pl9AKF (alternative reading frame). A wealth of mutational
In an effort to develop an accurate model for melanoma, Chin et al. [31**] have reported dramatic support for cooperativity between oncogenic ras and loss ofpf6/nfi4alpl9Arf in tumorigenesis in viva. Overexpression of activated human H-ras (T24) specifically in mouse melanocytes leads to hyperproliferation of these cells but not to their transformation [32]. Mice carrying a similar melanocytespecific H-rusc~z~’ transgene on a pl6In~4alpl9W-null background, however, readily develop multiple cutaneous and ocular tumors that model human melanoma [31”].
p7SARF-specific mutation Although mutational, molecular and conceptual evidence favored the idea that loss of pl6lNK‘fA and not plWRl was relevant to tumor formation, this assumption was dramatically challenged with the report that the phenotype of pI9ARF-nuII, pJ6’flb4a-expressing mice (and cell lines, see below) is nearly indistinguishable from that of pZ6/n~4u/pl9Arf_null mice (and cell lines; [33”]). These mice carry a homozygous mutation in p/9ARp-specific exon 1; normal levels of functionally active p16*nk4a protein were expressed in pl9ARF-null fibroblasts. Barring some poorly understood genomic peculiarity, these results suggest that plqAl
Tumor suppressor mutations in mice McClatchey and Jacks
ways converge
and contribute
to tumor
formation.
Whereas
wild-type MEFs undergo limited population doublings in culture before entering into senescence, plbl1~k~“/pl9hrf-, pl9Arf- and p.53-deficient MEFs readily escape cellular senescence and are easily immortalized [27”,33”-35”]. Levels of pl61NKq.A. p19Alil’ and ~53 are elevated upon senescence and spontaneously immortalized fibroblasts often exhibit loss of either plf;l~~‘~4~/pZ9~1K”‘ or ~53 sequences but rarely both [33”,36-381. Spontancously immortalizated plUAti+/MEFs lose the wild-type pl9W allele but often retain both plSl”fi4” and ~5.1, indicating that loss of p19 ArfaIone can relieve a scnescencc block [33”]. A relationship between pl9Arf and ~53 is suggested by the apparently mutually exclusive nature of pl9Arf and p.53 loss during immortalization. Moreover, introduction of pl9Arf mto MEFs leads to growth arrest which requires ~53 function [33**]. This concept is dramatically supported by the recent discovery that pl9*111’ physically interacts with and functionally inhibits MDMZ, a negative regulator of ~53 [34”,35”]. MDMZ, which is often amplified during tumorigenesis, normally functions to promote ubiquitin-mediated degradation of ~53. p19AIil’ appears to promote degradation of h,lDh12 through an as yet unidentified pathway, stabilization of ~53. This relationship may the observation that ~53 mutations are rarely
leading to also explain identified in
human melanoma; the p53 pathway may bc altered by a different mechanism, perhaps through frequent mutation of the ~16 ‘ij’KdA/l/pl9AK”’ locus. Together, these observations suggest that thep~6/“~~~~/pl9,\Iflocus physically links the critical control.
pathways
of Rb-
and
p53-mediated
growth
In addition, MEFs lacking pl6ln!+Wpl9Arf, pl@f or ~53 can be transformed by oncogenic T(IS alone [33”]. Transformation of wild-type hlEFs normally requires collaboration between ras and another oncogene (reviewed in [39]). The observation that prolonged expression of oncogenic ms in wild-type MEFs leads to premature senescence which is relieved by loss of pl6Infi~~(rlpf9Aff or p53 suggests that senescence may reflect action taken by cells to prevent further cell division in the face of oncogenic mutation [40**]. This important series of suppressor mutant animals
experiments and their
utilizing tumor derivative primary
cell lines has linked the classically studied behaviour of cells in culture to the process of tumorigenesis. Moreover, these studies have delineated a functional pathway to cellular growth control that provides an explanation for why mutations in ras, p16/flk4a/p/9AIf and ~53 are found in so many human cancers.
The future: refinement mouse models
and utilization
of
As soon as the utility of targeted mutagenesis in the mouse was recognized and exploited, certain limitations also became apparent. The early developmental lethality
caused
by some
homozygous
tumor
suppressor
307
mutations
prevents easy establishment of primary cell lines for study. Phenotypic differences between mice and men represent obstacles to the translation of information between the two species. For heterozygous mutant mice, differences in tumor types suggest that the action of compensatory pathways in the relevant cell types may differ in mice and humans. Alternatively, differences in the genomic configuration of mice and humans may affect the rate of loss of the wild-type allele or the incidence of additional genomic alterations necessary for tumor formation. hlosaic
analysis
is a powerful
means
analysis of a null phenotype and of species-specific differences. For example,
of extending exploring although
the these loss of
either the Nf2 or Smad_I/Dpc4 tumor suppressors leads to embryonic failure prior to gastrulation, chimeric embryos composed of wild-type cells and of &Z-expressing null cells (for Nfz or 5’~2u&/Dpr4) have revealed later stages of development requiring the Nf2 or Smad4/Dpc4 function ([41’,42’]; AI hlcClatchey, T Jacks, unpublished observations). In addition, primary cell lines can be isolated from chimeric embryos on the basis of their resistance to selectable markers present within the targeted allele ([43*]; AI hlcClatchey, use of chimeric
‘I’ Jacks, analysis
unpublished observations). The to focus on a particular lineage
has also been demonstrated. For example, Rag-deficient blastocyst complementation analysis was used to assess the ability of Rb-deficient cells to contribute to B- and T-cell lineages in chimeras otherwise composed of Rag-deficient cells that cannot become mature B- and T-cells [44]. Finally, partial contribution of tumor suppressor null cells to adult animals can address directly whether the loss of the wild-type allele is rate limiting for tumor formation (T Jacks, unpublished observations). Despite the utility of chimeric analysis, the ability to control the expression of a targeting event both temporally and spatially would allow further refinement of these models. LJse of the bacteriophage-based Cre-/oxP system for the conditional deletion of a particular (tumor suppressor) gene is a powerful way to achieve this end [45]. This system was used recently to conditionally inactivate the murine adenomatous polyposis coli tumor suppressor gene (Apr) [46**]. Mutations in APC are responsible for familial adenomatous polyposis coli in humans, an inherited form of cancer predisposing the development of hundreds of colorectal polyps, a fraction of which are likely to progress to malignancy [47-49]. Both chemically-induced and targeted Apt mutant mouse strains have been studied [50-531. Heterozygous Apt-mutant mice mimic their human counterparts remarkably well but develop many polyps of the small intestine rather than in the colon; also, the lesions in the mouse rarely progress to malignancy. Moreover, homozygous mutant embryos fail prior to gastrulation, precluding an analysis of the effects of a homozygous Apt mutation in adult animals. In order to refine this model, Shibata et al. [46”]
308
Genetics
of disease
generated
and tumor
/oxP sites
in the mouse system at the forefront of our endeavor to understand and intervene in the development of human cancer.
mice carrying a targeted allele that features flanking exon 14 (a so-called ‘floxed’ allele). Although homozygous ‘floxed’ mice are phenotypically normal, colonic infection with a recombinant adenovirus expressing the Cre recombinase caused somatic deletion of exon 14 in some cells and the development of numerous colonic polyps, some of which progressed to malignancy [46**]. This study sets the stage for the use of conditional targeting to study the ramifications of tumor suppressor loss in any fetal or adult tissue. Delivery of Cre activity can also be achieved via transgenic mice expressing Cre in a tissue-specific manner. Moreover, the use of several well characterized inducible systems may be concomitantly used for temporal control of Cre recombinase expression (reviewed in [54*]). Interbreeding of cancer-prone mouse strains provides a way to investigate the potential for cooperativity between cancer-predisposing mutations and to delineate mutational pathways to tumorigenesis. In addition, genomic tools are now available for the identification of other regions of the genome that are frequently lost or amplified during the etiology of a particular tumor. Genetic and environmental modification of cancer susceptibility is evident in the tremendous variability of cancer incidence and severity within the general human population and within cancer-prone families. Historical documentation of the variable susceptibility of different inbred II~OLISC strains to cancer development, together with advancing progress toward mapping the mouse genome, suggests that the mouse will be a system amenable to the identification of genetic loci that modify cancer predisposition. This was heralded in 1993 with the genetic mapping of a locus that strongly modified the tumor predisposition of mice carrying the APChlill mutation [SS]. A candidate gene was subsequently identified as a secretory type II phospholipase (Pla?s) [56]. Recently, the candidacy of PlaZs has been strengthened greatly through transgenic analysis [57”], validating the approach of identifying quantitative trait loci in the mouse. Many laboratories are now using this approach to identify loci that modify cancer susceptibility or progression in a number of mouse models.
Conclusions The
powerful
established
and
emerging
tools
place
the
study
of tumor
Acknowledgements WCwould like to thank Jeff Scttleman
for valuable
manuscript.
to apologize
In addition.
we would
like
suppressors
comments
regarding
co our colleagues
the
whose
may not have been cited because of space limitations. AI McClatchey is a recipient of a Career Award from rhe Burroughs Wellcome Fund.
work
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40. ..
Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW: Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and pi 6’NKds. Cell 1997, 88:593-602. Although it is a long-held observation that primary cells undergo a permanent G1 arrest or cellular senescence after a limited number of doublings in culture, the physiological relevance of this behavior is not clear. This paper suggests that oncogenic ras induces cellular senescence that is overcome by loss of either the p53 or pl6lnk4a tumor suppressor, suggesting that senescence may be a cellular response to oncogenic activation that prevents further cell division. McClatchey Al, Saotome I, Ramesh V, Gusella JF, Jacks T: The Nf2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation. Genes Dev 1996, 11 :I 253-l 265. See annotation [42*].
41. .
Sirard C, de la Pompa JL, Elia A, ltie A, Mirtsos C, Cheung A, Hahn S, Wakeham A, Schwartz L, Kern SE et al.: The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev 1997, 12:107-l 19. This report and [41’] describe the use of /acZ-marked, tumor suppressor null cells to generate chimeras in which the null lineage can be calorimetrically traced. This type of analysis can be used to bypass the embryonic lethality of complete homozygosity and investigate the requirement for a particular tumor suppressor gene at later stages of development, to examine the consequences of tumor suppressor deficiency in adult tissues or to generate pure populations of tumor suppressor null primary cells (see [43’]). 42. .
43. .
Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ: Radiation-induced cell cycle arrest compromised by ~21 deficiency. Nature 1996, 377:552-557. The results described in this paper utilize the selection of tumor suppressor null primary fibroblasts from chimeric embryos on the basis of their neomycinresistance. 44.
Chen J, Gorman JR, Stewart V, Williams B, Jacks T, Alt F: Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells. Curr Biol 1993, 3:405-413.
45.
Gu H, Marth JD, Orban PC, Mossman H, Rajewsky K: Deletion of a DNA polymerase gene segment in T cells using cell typespecific gene targeting. Science 1994, 265:103-l 06.
310
Genetics
of disease
46. ..
Shibata H, Toyama K, Shioya H, Ito M, Hirota M, Hasegawa S, Matsumoto H, Takano H, Akiyama T, Toyoshima K et a/.: Rapid colorectal adenoma formation initiated by conditional targeting of the Apt gene. Science 1997, 278:120-l 23. A description of the first example of the conditional targeting of a tumor suppressor gene. Adenoviral-expressing Cre was delivered directly to colonic tissue, resulting in tissue-specific deletion of a /oxP-flanked allele of the APC tumor suppressor gene. 47.
Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A, Koyama K, Utsunomiya J, Baba S, Hedge P: Mutations of chromosome 5q21 genes in FAP and colorectal cancer. Science 1991, 253:665-669.
48.
Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, Joslyn G, Stevens J, Spirio L, Robertson M et a/.: Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991, 66:589-600.
49.
Joslyn G, Carlson M, Thliveris A, Albertsen H, Gelbert L, Samozitz W, Groden J, Stevens J, Spirio L, Robertson M et a/.: The identification of deletion mutations and three new genes at the familial polyposis locus. Cell 1991, 66:601-613.
50.
Moser AR, Pitot HC, Dove WF: A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990, 247:322-324.
51.
Su L-K, Luongo caused Science
52.
Fodde R, Edelmann W, Yang K, van Leeuwen C, Carlson C, Renault B, Breukel C, Alt E, Lipkin M, Khan PM, Kucherlapati R: A targeted chain termination in the mouse Apt gene results
Kinzler KW, Vogelstein B, Preislnger AC, Moser AR, C, Gould K, Dove WF: Multiple intestinal neoplasia by a mutation in the murine homolog of the APC gene. 1992, 256:668-670.
in multiple intestinal tumors. Proc Nat/ Acad Sci USA 1994, 91:896g-8973. 53.
Oshima M, Oshima H, Kitagawa K, Kobayashi M, ltakura C, Taketo M: Loss of Apt heterorygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apt gene. Proc Nat/ Acad Sci USA 1995, 92:44824486.
54. .
Saez E, No D, West A, Evans RM: Inducible gene expression in mammalian cells and transgenic mice. Curr Opin Biotechnol 1997, 8:608-616. This is an excellent review describing the advantages and disadvantages of several mammalian inducible gene expression systems as well as their potential utilization in vitro and in viva. 55.
Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo C, Borenstein N, Dove WF: Genetic identification of Mom-l, a major modifier locus affecting min-induced intestinal neoplasia in the mouse. Cell 1993, 75:631-639.
56.
MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD, Buchberg AM: The secretory phospholipase A2 gene is a candidate for the Mom7 locus, a major modifier of Ap&i”induced intestinal neoplasia. Cell 1995, 81:957-966.
57. ..
Cormier RT, Hong KH, Halberg RB, Hawkins TL, Richardson P, Mulherkar R, Dove WF, Lander ES: Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis. Nat Genet 1997, 17:88-92. The P/a& gene encoding a secretory hospholipase is a candidate modifier of intestinal tumor phenotype in APC Mp. ‘n mutant mice. This paper describes the recapitulation of this modification by transgenic expression of a PlaZscontaining cosmid on an otherwise sensitive genetic background, strongly supporting the candidacy of Plals as the modifier.
Tumor suppressor mutations Andrea Mouse
I
strains
genetically
carrying
suppressor
have and will be identified
however,
it is clear that future
on a new generation existing
tumor
and Tyler Jacks? mutations
mimic familial forms of human
suppressors
mouse;
McClatchey*
models,
of experiments
cancer.
to tumorigenesis
designed
drug therapies.
New tumor
and mutated investigation
aimed
in the will focus
at improving
and using them to delineate
pathways
in mice: the next generation
the molecular
and to test the value of rationally
Addresses *Massachusetts General Hospital Cancer Center and Harvard Medical School Department of Pathology, Building 149, 13th Street, Charlestown, Massachusetts 02129, USA; e-mail: [email protected] tDepartment of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; e-mail: [email protected] Current Opinion in Genetics
& Development
1998, 8:304-310
http://biomednet.com/elecref/0959437X00800304 0 Current Biology Ltd ISSN 0959-437X Abbreviations APC adenomatous polyposis coli BCC basal cell carcinoma CDK cyclin-dependent kinase Hh Hedgehog LOH loss of heterozygosity MEF mouse embryonic fibroblast retinoblastoma protein PPb PtC patched Shh Sonic hedgehog
of the
mouse
as a genetically
oncogenes
in every
cell of a particular
suppressors identified are cancer prone but human counterparts these differences are our understanding of and more completely otherwise.
to date have been reported; most many do not faithfully model their (reviewed in [Z]). Explanations for emerging and may ultimately drive tumorigenic pathways more rapidly than would have been possible
The past year has witnessed exciting progress in the development and analysis of several mouse tumor suppressor models. Bridges between embryogenesis and tumorigenesis have been dramatically strengthened with the identification of critical developmental functions for individual tumor suppressor genes. Primary cell cultures derived from mutant mice have again proven to be a powerful reagent for characterizing the molecular function of tumor suppressor proteins. Importantly, further genetic manipulation and fine tuning of existing models has
It
mutations
has grown enormously over the past decade. Although the impact of this technology has been felt in many research areas, the field of cancer research may claim some of the greatest and most prolonged benefits. Genetically manipulated mice are being developed as tools for the study of the molecular, developmental and cell biological functions of genes mutated in human cancer and as mammalian models of human cancer that become logical and suitable vehicles in which to test rational cancer therapies. Manipulation of the tumor suppressor class of genes in the mouse provides a straightforward route toward modeling human cancer. In humans, individuals with various familial cancer syndromes inherit a mutant allele of a given tumor suppressor gene mutation, which causes predisposition to the development of certain cancers. Loss of the remaining wild-type allele occurs somatically, initiating tumor formation [l]. The generation of mice carrying a heterozygous mutation in an individual tumor suppressor gene mimics this situation genetically, allowing the
by the practice of tumor suppressor
in the mouse
is evident that most not evolve to suppress control cellular proliferation system
a situation
a few familial cancer syndromes. mutations in nearly all of the tumor
Patched
manipulable
tissue,
that is seen in only Mouse strains carrying
begun (Figure 1). This is exemplified conditional targeting to focus individual mutations spatially and temporally.
Introduction Use
development of subsequent mutations to occur randomly. This is in contrast to transgenic mice that overexpress
tumor suppressor genes did tumorigenesis but rather to in certain contexts. Essential
developmental functions for nearly every tumor suppressor identified have been revealed via the generation of mice homozygous for an individual mutation. In contrast, Patched
tumor (Ptc)
suppressor gene protein was well
known as a component of an important developmental signaling pathway in Drosoph’la prior to its identification as a tumor suppressor. Ptc is a multiple pass trdnsmembrdne protein that is negatively regulated by the secreted Hedgehog (Hh) protein [3-S]. Ptc, in turn, negatively regulates the expression of several target genes, including the Wnt family member wing/m, the TGF-B family member Decapeatapkgic (Dpp) and Ptc itself. This pathway is responsible for several crucial patterning events during Drosoph’la development. In 1996, positional cloning of the gene responsible for basal cell nevus syndrome (BCNS) identified the human Ptc homologue [6,7]. BCNS patients exhibit a number of symptoms, including skeletal abnormalities and the predisposition to develop cancer-predominantly basal cell carcinomas (BCCs) of the skin-but also childhood medulloblastoma and meningioma (reviewed in [S]). The autosomal dominant nature of BCNS inheritance and de-
Tumor
Figure
suppressor
mutations
in mice
McClatchey
305
and Jacks
1
The study of tumor suppressor
Mosaic
mutations
in mice moves to a new level
Tumor DNA, ”
analysis
mRNA
Genetic
background
Y Therapeutic testing Current Opinion m Genetics & Development
New methodologies
are being
used to refine tumor
suppressor
mutant
mouse
models
and to use them to define
the molecular
pathways
to
tumongenesls.
tection of loss of heterozygosity (LOH) for genetic markers in the vicinity of the BCNS locus in BCNS-associated and sporadic tumors were indicative of a tumor suppressor gene. This was confirmed by evidence for the somatic inactivation of Ptc in both sporadic and BCNS-associated BCCs and medulloblastoma, forging a link between an important developmental signal transduction pathway and tumorigenesis [6,7,9-121. Mouse models of Ptc function in development and tumorigenesis soon followed. Reasoning that the sequential repressive actions of Hh and Ptc indicated that loss of Ptc-as in BCNS tumors -might be equivalent to the overexpression of Hh, Oro et a/. overexpressed Sonic hedgehog (Shh; one of several mammalian Hh homologues) in the mouse epidermis [13”]. Indeed, transgenic embryos developed many BCC-like hyperproliferations of the epidermis shortly after epidermal formation (at embryonic day [E] 18.5). S/z/&transgenics also develop skeletal abnormalities similar to those of BCNS patients, despite the apparently restricted epidermal expression
of Shh by the well characterized keratin 14 promoter, suggesting that Shh was able to access and affect internal tissues too. The discovery that overexpression of Shh was sufficient for the development of BCCs prompted the search for and identification of (presumably activating) mutations tumors [ 13**].
in Shh itself in human
BCNS-related
Targeted disruption of the murine Ptc homologue reveals additional information about its role in murine development and tumorigenesis [14”]. Ptc-null embryos exhibit gross abnormalities by E8.0, including failed neural tube closure and abnormal cardiac development. The expression patterns of S/h and Ptc in wild-type embryos and of other markers in Ptd- embryos are consistent with a model in which Ptc is required in the dorsal neural tube to block ventral cell fate determination induced by Shh produced ventrally in the notochord. In the absence of Ptc, Shh induces a ventral cell fate throughout the neural tube apparently leading to failed neural tube closure. These observations dovetail nicely with a body of work
306
Genetics
of
disease
attributing
a critical
patterning
of several
role species
for Shh
in the
(reviewed
dorsal-ventral
information
in [15,16]).
cell lines prevalent
from melanoma
kindreds,
primary
Importantly, a significant percentage of Pt& mice develop cerebellar tumors that are similar to the childhood medulloblastomas seen in a small but significant percentage of BCNS patients [ 14**]. Paradoxically, mutation of Ptc and loss of Ptc function in these tumors should lead to derepression of Ptc and elevation of Ptc mRNA
affect both pl6iNK4A and pZ9ARF and inactivate p16INK4A exclusively [25,26].
levels. Although the presence or absence of the remaining wild-type Ptc allele was not examined here, the expression of a /ucZ transgene included in the targeting vector was markedly higher in the tumors than in the surrounding tissues and could be used to detect early nonpathologic lesions. Alternatively, this could also reflect either activation or overexpression of .S& or some unrecognized pathway.
p16/nk+‘a/‘l9A?f null mice C57BL6/J x 129/Sv x SJL
Interestingly, Ptc+- mice do not develop BCCs, suggesting that loss of the wild-type Ptc allele may be rate limiting in this tissue; alternatively, the context of the surrounding tissue-embryological or mature -may play an important role in the development of mouse BCCs.
The p151nk4b/pl61nk4a/plgArf Originally
identified
kinase
and
attempt
to model
some
pl6/NK4A-associated
in the mouse targeted inactivating both pI6lflk40
appear
to
tumor
the deletion of and pJ9urf [27**].
are viable-at least on a mixed genetic background-and ex-
hibit signs of extramedullary hematopoiesis, suggesting that plblfl~4a/p/PAR~ may function to negatively regulate proliferation of the hematopoietic lineage. In principle, this could be related to the requirement for pRb function in the hematopoietic lineage during development [28-301. Importantly, pl6~n~4~/pl9A~-null animals are cancer prone, primarily developing subcutaneous fibrosarcomas and lymphomas at an average of 7.25 months of age, a latency accelerated by carcinogen treatment. Neither pituitary tumors, which develop at high frequency in Rb+/- mice [29], nor melanoma develop in pl6/n~4a/p19Ad++l- or -I[27**].
inhibitor
able to prevent cell cycle progression, p161NKq* was celebrated as the product of the tumor suppressor gene mapped to the familial melanoma susceptibility locus [ 17,181. At the Gl+S cell cycle transition, cyclin-dependent kinases (CDK4 or CDK6) regulatory subunits phosphorylate
first
susceptibility exon 2 thus
mice
locus
as a cyclin-dependent
The
tumors
now suggests that pZS’NK48 mutations are not in human cancer; most mutations of this locus
bound to D-type cyclin the retinoblastoma tu-
mor suppressor protein (pRb), derepressing its associated E2F transcriptional activity and leading to cell cycle progression (reviewed in [19,20]). pl6*NK4A disrupts this transition by preventing the interaction between CDK4 and cyclin D (hence the inhibitor of kinase 4 [INK4A] designation). In human cancers, mutational desensitization of CDK4 to the inhibitory effects of p16*nk4~, loss of pl6lnk42, overexpression of cyclin D, or inactivation pRb can be found in mutual exclusion, documenting importance of this pathway in tumor formation.
of the
In fact, molecular proof that pl6L$‘Kkd functions as a tumor suppressor has been the subject of much debate despite the plausibility of such a model. The scramble to identify pf6IlvKJA mutations in melanoma and other tumor types led to the realizations thatp/6LNKdA mutations are more common in tumor cell lines than in primary tumors and that the pi6/h’K4A locus was often grossly affected by large deletions or genomic rearrangements (reviewed in [Zl]). Careful examination of the pl6’*‘K4A locus revealed the presence of two additional open reading frames (ORFs), one encoding a protein with amino acid and functional similarity to pl6’.vK4A dubbed plSNK4fi [Z-24]. Th e second ORF begins upstream of p16LNK4A at a unique exon (lb) which is spliced to pZ6INK4A exons 2 and 3 and utilizes an alternative frame to encode a protein with no known homology, dubbed pl9AKF (alternative reading frame). A wealth of mutational
In an effort to develop an accurate model for melanoma, Chin et al. [31**] have reported dramatic support for cooperativity between oncogenic ras and loss ofpf6/nfi4alpl9Arf in tumorigenesis in viva. Overexpression of activated human H-ras (T24) specifically in mouse melanocytes leads to hyperproliferation of these cells but not to their transformation [32]. Mice carrying a similar melanocytespecific H-rusc~z~’ transgene on a pl6In~4alpl9W-null background, however, readily develop multiple cutaneous and ocular tumors that model human melanoma [31”].
p7SARF-specific mutation Although mutational, molecular and conceptual evidence favored the idea that loss of pl6lNK‘fA and not plWRl was relevant to tumor formation, this assumption was dramatically challenged with the report that the phenotype of pI9ARF-nuII, pJ6’flb4a-expressing mice (and cell lines, see below) is nearly indistinguishable from that of pZ6/n~4u/pl9Arf_null mice (and cell lines; [33”]). These mice carry a homozygous mutation in p/9ARp-specific exon 1; normal levels of functionally active p16*nk4a protein were expressed in pl9ARF-null fibroblasts. Barring some poorly understood genomic peculiarity, these results suggest that plqAl
Tumor suppressor mutations in mice McClatchey and Jacks
ways converge
and contribute
to tumor
formation.
Whereas
wild-type MEFs undergo limited population doublings in culture before entering into senescence, plbl1~k~“/pl9hrf-, pl9Arf- and p.53-deficient MEFs readily escape cellular senescence and are easily immortalized [27”,33”-35”]. Levels of pl61NKq.A. p19Alil’ and ~53 are elevated upon senescence and spontaneously immortalized fibroblasts often exhibit loss of either plf;l~~‘~4~/pZ9~1K”‘ or ~53 sequences but rarely both [33”,36-381. Spontancously immortalizated plUAti+/MEFs lose the wild-type pl9W allele but often retain both plSl”fi4” and ~5.1, indicating that loss of p19 ArfaIone can relieve a scnescencc block [33”]. A relationship between pl9Arf and ~53 is suggested by the apparently mutually exclusive nature of pl9Arf and p.53 loss during immortalization. Moreover, introduction of pl9Arf mto MEFs leads to growth arrest which requires ~53 function [33**]. This concept is dramatically supported by the recent discovery that pl9*111’ physically interacts with and functionally inhibits MDMZ, a negative regulator of ~53 [34”,35”]. MDMZ, which is often amplified during tumorigenesis, normally functions to promote ubiquitin-mediated degradation of ~53. p19AIil’ appears to promote degradation of h,lDh12 through an as yet unidentified pathway, stabilization of ~53. This relationship may the observation that ~53 mutations are rarely
leading to also explain identified in
human melanoma; the p53 pathway may bc altered by a different mechanism, perhaps through frequent mutation of the ~16 ‘ij’KdA/l/pl9AK”’ locus. Together, these observations suggest that thep~6/“~~~~/pl9,\Iflocus physically links the critical control.
pathways
of Rb-
and
p53-mediated
growth
In addition, MEFs lacking pl6ln!+Wpl9Arf, pl@f or ~53 can be transformed by oncogenic T(IS alone [33”]. Transformation of wild-type hlEFs normally requires collaboration between ras and another oncogene (reviewed in [39]). The observation that prolonged expression of oncogenic ms in wild-type MEFs leads to premature senescence which is relieved by loss of pl6Infi~~(rlpf9Aff or p53 suggests that senescence may reflect action taken by cells to prevent further cell division in the face of oncogenic mutation [40**]. This important series of suppressor mutant animals
experiments and their
utilizing tumor derivative primary
cell lines has linked the classically studied behaviour of cells in culture to the process of tumorigenesis. Moreover, these studies have delineated a functional pathway to cellular growth control that provides an explanation for why mutations in ras, p16/flk4a/p/9AIf and ~53 are found in so many human cancers.
The future: refinement mouse models
and utilization
of
As soon as the utility of targeted mutagenesis in the mouse was recognized and exploited, certain limitations also became apparent. The early developmental lethality
caused
by some
homozygous
tumor
suppressor
307
mutations
prevents easy establishment of primary cell lines for study. Phenotypic differences between mice and men represent obstacles to the translation of information between the two species. For heterozygous mutant mice, differences in tumor types suggest that the action of compensatory pathways in the relevant cell types may differ in mice and humans. Alternatively, differences in the genomic configuration of mice and humans may affect the rate of loss of the wild-type allele or the incidence of additional genomic alterations necessary for tumor formation. hlosaic
analysis
is a powerful
means
analysis of a null phenotype and of species-specific differences. For example,
of extending exploring although
the these loss of
either the Nf2 or Smad_I/Dpc4 tumor suppressors leads to embryonic failure prior to gastrulation, chimeric embryos composed of wild-type cells and of &Z-expressing null cells (for Nfz or 5’~2u&/Dpr4) have revealed later stages of development requiring the Nf2 or Smad4/Dpc4 function ([41’,42’]; AI hlcClatchey, T Jacks, unpublished observations). In addition, primary cell lines can be isolated from chimeric embryos on the basis of their resistance to selectable markers present within the targeted allele ([43*]; AI hlcClatchey, use of chimeric
‘I’ Jacks, analysis
unpublished observations). The to focus on a particular lineage
has also been demonstrated. For example, Rag-deficient blastocyst complementation analysis was used to assess the ability of Rb-deficient cells to contribute to B- and T-cell lineages in chimeras otherwise composed of Rag-deficient cells that cannot become mature B- and T-cells [44]. Finally, partial contribution of tumor suppressor null cells to adult animals can address directly whether the loss of the wild-type allele is rate limiting for tumor formation (T Jacks, unpublished observations). Despite the utility of chimeric analysis, the ability to control the expression of a targeting event both temporally and spatially would allow further refinement of these models. LJse of the bacteriophage-based Cre-/oxP system for the conditional deletion of a particular (tumor suppressor) gene is a powerful way to achieve this end [45]. This system was used recently to conditionally inactivate the murine adenomatous polyposis coli tumor suppressor gene (Apr) [46**]. Mutations in APC are responsible for familial adenomatous polyposis coli in humans, an inherited form of cancer predisposing the development of hundreds of colorectal polyps, a fraction of which are likely to progress to malignancy [47-49]. Both chemically-induced and targeted Apt mutant mouse strains have been studied [50-531. Heterozygous Apt-mutant mice mimic their human counterparts remarkably well but develop many polyps of the small intestine rather than in the colon; also, the lesions in the mouse rarely progress to malignancy. Moreover, homozygous mutant embryos fail prior to gastrulation, precluding an analysis of the effects of a homozygous Apt mutation in adult animals. In order to refine this model, Shibata et al. [46”]
308
Genetics
of disease
generated
and tumor
/oxP sites
in the mouse system at the forefront of our endeavor to understand and intervene in the development of human cancer.
mice carrying a targeted allele that features flanking exon 14 (a so-called ‘floxed’ allele). Although homozygous ‘floxed’ mice are phenotypically normal, colonic infection with a recombinant adenovirus expressing the Cre recombinase caused somatic deletion of exon 14 in some cells and the development of numerous colonic polyps, some of which progressed to malignancy [46**]. This study sets the stage for the use of conditional targeting to study the ramifications of tumor suppressor loss in any fetal or adult tissue. Delivery of Cre activity can also be achieved via transgenic mice expressing Cre in a tissue-specific manner. Moreover, the use of several well characterized inducible systems may be concomitantly used for temporal control of Cre recombinase expression (reviewed in [54*]). Interbreeding of cancer-prone mouse strains provides a way to investigate the potential for cooperativity between cancer-predisposing mutations and to delineate mutational pathways to tumorigenesis. In addition, genomic tools are now available for the identification of other regions of the genome that are frequently lost or amplified during the etiology of a particular tumor. Genetic and environmental modification of cancer susceptibility is evident in the tremendous variability of cancer incidence and severity within the general human population and within cancer-prone families. Historical documentation of the variable susceptibility of different inbred II~OLISC strains to cancer development, together with advancing progress toward mapping the mouse genome, suggests that the mouse will be a system amenable to the identification of genetic loci that modify cancer predisposition. This was heralded in 1993 with the genetic mapping of a locus that strongly modified the tumor predisposition of mice carrying the APChlill mutation [SS]. A candidate gene was subsequently identified as a secretory type II phospholipase (Pla?s) [56]. Recently, the candidacy of PlaZs has been strengthened greatly through transgenic analysis [57”], validating the approach of identifying quantitative trait loci in the mouse. Many laboratories are now using this approach to identify loci that modify cancer susceptibility or progression in a number of mouse models.
Conclusions The
powerful
established
and
emerging
tools
place
the
study
of tumor
Acknowledgements WCwould like to thank Jeff Scttleman
for valuable
manuscript.
to apologize
In addition.
we would
like
suppressors
comments
regarding
co our colleagues
the
whose
may not have been cited because of space limitations. AI McClatchey is a recipient of a Career Award from rhe Burroughs Wellcome Fund.
work
References and recommended
reading
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