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Mouse Models of BRCA1 and BRCA2 Deficiency

Mouse Models of BRCA1 and BRCA2 Deficiency

(2006) 25, 5885–5897 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects

B Evers and J Jonkers

Division of Molecular Biology, The Netherlands Institute, Amsterdam, The Netherlands

Germline in BRCA1 and BRCA2 are respon- risk of up to 80% for developing cancer and sible for a large proportion of hereditary breast and also display increased risk for (B40% ovarian . Soon after the identification of both for BRCA1 carriers and B20% for BRCA2 in the mid-1990s, investigators set out to develop carriers, respectively) (Narod and Foulkes, 2004). In mouse models for the associated disease. Whereas most breast cancers arising in BRCA1/2 carriers, conventional Brca1 and Brca2 mouse mutants did not inactivation of the wild-type allele has occurred by reveal a strong phenotype in a heterozygous setting, means of , thus abolishing normal most homozygous mutations caused embryonic lethality. expression (Smith et al., 1992; Collins et al., Consequently, development of mouse models for BRCA- 1995). BRCA1 tumors show a rather uniform tumor associated tumorigenesis required the generation of tissue- type of high-grade invasive ductal carcinomas, which are specific conditional knockout animals. In this review, we usually estrogen (ER) and HER2/neu negative give an overview of the conventional and the conditional (Chappuis et al., 2000; Phillips, 2000). These properties mouse models of BRCA1 and BRCA2 deficiency gener- are reminiscent of ‘triple-negative’ (ER-, progesterone ated over the last decade, as well as the contribution receptor (PR)- and HER2/neu-negative) basal-like of these models to our understanding of the biological sporadic breast cancers (Livasy et al., 2006). Indeed, and molecular functions of BRCA1 and BRCA2. The cluster analysis of expression data from human most advanced mouse models for BRCA1- and BRCA2- breast cancers revealed strong similarity between associated tumorigenesis mimic human disease to the BRCA1-mutated tumors and basal-like breast cancers extent that they can be used in studies addressing (Sorlie et al., 2003). BRCA2 deficient tumors, on the clinically relevant questions. These models will help to other hand, show larger variety in histological classifica- resolve yet unanswered questions and to translate our tion and are not easily distinguished from the overall increasing knowledge of BRCA1 and BRCA2 biology into spectrum of sporadic tumors. While parity in the general clinical practice. population has been associated with decreased breast Oncogene (2006) 25, 5885–5897. doi:10.1038/sj.onc.1209871 cancer risk, parity per se was not found to influence risk in BRCA1 mutation carriers, although multiparity did Keywords: BRCA1; BRCA2; ; tumor seem to protect as well. For BRCA2, data suggest suppressor; mouse model that increasing parity is associated with increased risk of breast cancer. BRCA2-mutation carriers also have an increased risk of developing pregnancy-associated breast cancer (Cullinane et al., 2005).

Introduction

Breast cancer is the most common malignancy in Conventional Brca1 and Brca2 mutants women of the Western world, affecting up to 10% of the female population (Alberg and Helzlsouer, 1997). Following identification of the BRCA1 and BRCA2 An estimated 5% of all breast cancers is ascribed to breast cancer genes in humans, investigators sought hereditary predisposition. Intensive research in the early to recapitulate the effects of these genetic lesions 1990s has led to the identification of two major breast in mouse models. Several groups have generated a cancer susceptibility genes, BRCA1 (Futreal et al., 1994; range of conventional Brca1 and Brca2 knockout mice Miki et al., 1994) and BRCA2 (Wooster et al., 1995; with mutations in different portions of the genes (see Tavtigian et al., 1996). Individuals carrying mutations in Figures 1 and 2). Somewhat disappointingly, none of one allele of either BRCA1 or BRCA2 have a lifetime these mouse mutants showed a strong tumor predispos- ing phenotype in a heterozygous setting. When bred to homozygosity, most Brca1 and Brca2 mouse mutants Correspondence: Dr J Jonkers, Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, displayed severe embryonic lethal phenotypes. Although The Netherlands. these traits have hindered in vivo analysis of tumor E-mail: [email protected] suppressor functions of Brca1 and Brca2, conventional Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5886

Figure 1 Conventional and conditional Brca1 mutant alleles generated to date. Exon compositions for the predicted splice products are indicated, including the Brca1-D11 splice variant and the – for mice putative – Brca1-IRIS product. A w indicates that the predicted transcript contains a premature stop, resulting from a frame shift. In that case only the exons encoding the (predicted) truncated protein product are indicated. The bottom panel shows all BRCA1 splice variants known to exist in humans and mice. BRCA1 lacks exon 4, which had been erroneously identified during the initial cloning of the BRCA1 gene (Miki et al., 1994). EL: embryonic lethality day.

mouse mutants have provided valuable insight into their observation that Brca-deficiency results in chromosomal biological roles. instability and increased sensitivity to DNA-damaging agents in cultured mouse cells (Sharan et al., 1997; Patel Functional clues et al., 1998; Shen et al., 1998; Tutt et al., 1999) and in Soon after the generation of Brca1 and Brca2 mouse living animals (Tutt et al., 2002). These phenotypes were mutants, it was acknowledged that the embryonic subsequently attributed to defects in repair of double- lethality of Brca1-deficient mice closely resembled the strand breaks (DSBs) by phenotype of mouse mutants of Rad51, a homologue (HR) (Moynahan et al., 1999, 2001; Xia et al., 2001). of the bacterial RecA protein known to be involved Whereas initial studies in Brca1 and Brca2 mouse in DNA damage repair (Lim and Hasty, 1996). mutants have highlighted similarities between both Subsequently, Rad51, Brca1 and Brca2 were proteins, other research efforts have also revealed clear shown to have similar expression patterns during mouse differences (Venkitaraman, 2002). BRCA1 appears to embryogenesis (Lane et al., 1995; Marquis et al., 1995; be more of a signal integrator, linking together sensors Sharan et al., 1997; Blackshear et al., 1998) and during and response mechanisms of several types of DNA cell-cycle progression (Rajan et al., 1996; Chen et al., damage. In contrast, BRCA2 is thought to be more 1997). In addition, Brca1, Brca2 and Rad51 were found directly involved in -directed DSB repair, as it to co-localize in nuclear dot patterns (Scully et al., 1997; mediates the formation of a RAD51-DNA nucleopro- Chen et al., 1998) and to interact with each other tein filament that catalyzes strand invasion during HR (Scully et al., 1997; Sharan et al., 1997; Chen et al., (Pellegrini and Venkitaraman, 2004). 1998), suggesting a role for the BRCA proteins in For BRCA1, a wealth of data describes many DNA-damage repair. This notion was supported by the interactions with other proteins, as well as many

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5887

Figure 2 Conventional and conditional Brca2 mutant alleles generated to date. The exon composition of the predicted splice product from the mutant allele is indicated. A w indicates that the predicted transcript contains a premature stop, resulting from a frame shift. In that case only the exons encoding the (predicted) truncated protein product are indicated. BRCrepeats that are still encoded by the predicted transcript are indicated. EL: embryonic lethality day. functions besides the involvement in the maintenance of defect (Hakem et al., 1996; Liu et al., 1996; Ludwig et al., genetic stability. Thus, apart from DNA repair, func- 1997; Suzuki et al., 1997). This striking observation tions have been described for BRCA1 in all phases of introduced a paradox in the biology of BRCA1 and the (Deng, 2006), (Lane, 2004) BRCA2. Whereas BRCA-deficient tumor cells proliferate and DNA decatenation (Lou et al., 2005). Studies with the rapidly in situ, cells in developing Brca-deficient embryos Brca1 mouse mutants – or primary cells derived thereof – suffer from a proliferation defect. Although this paradox have revealed several distinct activities of Brca1, including is still unresolved, part of it might be explained by its potential roles in the G2–M cell cycle and spindle the genetic interactions between BRCA1/2 and the assembly checkpoints (Xu et al., 1999b; Wang et al., pathway. Growth-arrested Brca1- and Brca2-deficient 2004a), maintenance of telomere integrity (McPherson embryos showed activation of the Cdkn1a gene encoding et al., 2006) and transcriptional repression of unsynapsed the cyclin-dependent kinase (cdk) inhibitor , a p53 chromosomal regions during (Turner et al., 2004). downstream target known to function in the G1–S cell Interesting with respect to the gender specificity observed cycle checkpoint (Hakem et al., 1996; Suzuki et al., 1997). in BRCA-associated cancers is the reported role for To investigate the role of p53 and p21 in Brca-associated BRCA1 in maintenance of X- inactivation proliferation arrest, compound mutant animals lacking (Ganesan et al., 2002). Recent studies have suggested that both Brca1/2 and p53 or p21 were produced by also BRCA2 may, in addition to its role in HR, have other cross-breeding (Hakem et al., 1997; Ludwig et al., 1997). functions in for example transcriptional regulation (Shin Whereas embryonic survival of Brca1 mutant embryos and Verma, 2003). Notably, primary mouse embryonic was partially rescued in a p53- or p21-deficient back- fibroblasts derived from hypomorphic Brca2 mutants have ground, rescue of Brca2 knockout animals by disruption provided important clues regarding possible roles of Brca2 of p53 was less clear. Together, these results indicate in stabilization of stalled DNA replication forks (Lomo- that in addition to p53 other factors play a role in the nosov et al., 2003) and cytokinesis (Daniels et al., 2004). proliferation defect of Brca1- or Brca2-deficient embryos.

The BRCA paradox Genotype–phenotype correlations The developmental failure of both Brca1- and Brca2- As shown in Figures 1 and 2, the various Brca1 deficient animals was mainly ascribed to a proliferation and Brca2 mouse mutants display large phenotypic

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5888 variation. Viability and life span differ considerably Alternative splice products between models, as did the rescue of embryonic lethality An important and often underappreciated aspect that on a p53-null background. Indeed, p53-nullizygosity has complicated proper interpretation of the Brca1 results in extensive phenotypic rescue of hypomorphic mutant models, is the fact that Brca1 transcripts are Brca1 mutants (Cressman et al., 1999; Xu et al., 2001; subject to alternative splicing. As a result of this, Cao et al., 2003), but only delays embryonic lethality of interpretations of targeted mutations in Brca1 should Brca1-null mutants (Hakem et al., 1997; Ludwig et al., not only take into account the effects on the full-length 1997). Apart from differences in targeted mutations in transcript, but also on all possible splice variants. Brca1 or Brca2, genetic background differences between For humans, several alternative splice products have the various Brca1 and Brca2 mutant mouse strains are been described (Lu et al., 1996; Thakur et al., 1997; likely to play an important role in the phenotypic Wilson et al., 1997; Xu et al., 1997; Elshamy and differences between the published models. Livingston, 2004; Fortin et al., 2005). Of all splice variants, only the BRCA1-D11 and BRCA1-IRIS Truncated proteins variants have been functionally analyzed to date (Huber Both Brca1 mouse mutants that are predicted to fail to et al., 2001; Elshamy and Livingston, 2004). The Brca1- produce Brca1 protein (i.e. Brca1ex2, Brca15À6) die around D11 splice variant is conserved in mice (Mixon et al., E7.5–9.5 when bred to homozygosity (see Figure 1). 2000), whereas existence of a rodent Brca1-IRIS splice Embryonic lethality in these Brca1null models is char- variant is still undetermined. acterized by reduced cell proliferation without signs of Several Brca1 mutations that encode truncated increased apoptosis (Hakem et al., 1996). Embryonic versions of the full-length protein could, at least in lethality is also observed in the Brca11700T model, carrying theory, still lead to the production of Brca1-D11 splice a C-terminal truncating mutation that removes the variants (see Figure 1), and the increased viability of second BRCT repeat (Hohenstein et al., 2001). Com- some of the homozygous mouse mutants strongly pared to the Brca1null models, however, homozygous suggests that these alternative splice products have a Brca11700T/1700T embryos show a delayed embryonic biological function. Indeed, similar to full-length Brca1, lethality marked by continued cell proliferation and Brca1-D11 is expressed in a cell-cycle regulated manner differentiation until an apoptotic response is activated and localizes to discrete nuclear foci, despite the fact around E9.5–10.5. These data suggest that C-terminal that this splice product lacks the nuclear localization truncating Brca1 mutations may have different effects on signals of full-length Brca1. DNA damage-induced normal cell function than Brca1-null mutations. Brca1 phosphorylation and Rad51 focus formation, on Phenotypic differences are also found for the different the other hand, were severely impaired in cells that only Brca2 mutations generated to date (see Figure 2). Based express Brca1-D11 (Huber et al., 2001). on these differences, it was previously suggested that Also Brca1-IRIS may have distinct biological activ- embryos carrying Brca2 mutations that did not encode ities, as it is capable of stimulating DNA synthesis, any BRCrepeat could not survive, whereas embryos presumably through its interaction with prereplication carrying Brca2 mutations that encoded at least three complexes (Elshamy and Livingston, 2004). The fact BRCrepeats were partially viable (Connor et al., 1997; that full-length BRCA1 and BRCA1-IRIS are expressed Friedman et al., 1998; McAllister et al., 2002). This, from different promoters raises the interesting possibi- however, appears not to be the case for the embryonic lity that functions of Brca1 towards tumor suppression lethal Brca2tm1Mhun mutation, which encodes a truncated and embryonic development might reside on different protein with four BRCrepeats (Yan et al., 2004). locations in the gene. Indeed, the only Brca1 Although several Brca1 and Brca2 mutations have mutant with an intact hypothetical Brca1-IRIS coding been described as resulting in truncating alleles (Figures unit does not suffer from a proliferation defect, yet dies 1 and 2), in most cases the presence of a truncated at E10.5 due to apoptosis (Hohenstein et al., 2001). protein has not been demonstrated. Some of these Further investigation of these exciting new aspects of mutations could be effectively null alleles if the mRNA BRCA1 biology will help to dissect the different or protein is unstable. Nevertheless, the large pheno- functions exerted by distinct BRCA1 splice variants. typic variation of the various mouse mutants predicts that also different human BRCA1 or BRCA2 germline Genetic background effects mutations will have different effects on normal cell Several lines of evidence point towards a strong function and tumor predisposition. Additional mouse influence of modifier genes on survival of Brca1- and models mimicking defined germline mutations are Brca2-deficient animals. Embryonic survival of a lethal needed to address this important issue. The first Brca2 mutation increased significantly from embryonic ‘humanized’ mouse models have already been generated day E8.5 on a 129 background to E10.5 on a BALBc via introduction of a BACtransgene containing the background (Bennett et al., 2000), and postnatal human BRCA1 gene into homozygous Brca1 knock- viability of two truncating Brca2 mutations was signi- outs. Whereas embryonic lethality was completely ficantly increased for homozygous mutant mice gener- rescued by wild-type BRCA1 (Chandler et al., 2001), ated in mixed 129/B6/DBA or 129/MF1 backgrounds no rescue was observed for a human BRCA1C64G allele compared to 129/B10 or 129/129 genetic backgrounds with a missense mutation in the RING finger domain (Connor et al., 1997; Friedman et al., 1998). Likewise, (Yang et al., 2003). embryonic lethality of a hypomorphic Brca1 mutation

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5889 (which still encodes for Brca1-D11) was completely via somatic delivery – results in tissue-specific and/ rescued when the mixed 129/B6 mice were backcrossed or time-controlled inactivation of conditional tumor onto 129/Sv or outcrossed using MF1 (Ludwig et al., suppressor genes. 2001a). Obviously, it would be interesting to perform backcross experiments in combination with whole- Targeting Cre expression to mammaryepithelium scanning studies in the mouse, in order to Several promoters have been used to achieve mammary map and eventually identify modifier genes that affect specific Cre expression, all with their own advantages embryonic lethality of Brca1 and Brca2 mutants. and disadvantages. Wagner and co-workers published Provided that similar mechanisms mediate viability of the creation of transgenic mice expressing Cre under the Brca-deficient embryos and survival of BRCA-deficient control of the whey acidic protein (WAP) tumor cells, such genes might form interesting ther- as well as the mouse mammary tumor virus long apeutic targets. terminal repeat (MMTV) promoter (Wagner et al., 1997), whereas Selbert et al. (1998) chose the ovine Tumor predisposition b-lactoglobulin promoter to drive Cre expression. An Whereas most Brca1 and Brca2 mouse mutants were important limitation of these three mammary gland- embryonic lethal when bred to homozygosity, some specific promoters is that their activity is strongly gave rise to sub-Mendelian ratios of viable offspring. influenced by steroid hormones. Furthermore, models Viable Brca1Tr/Tr mutants, encoding the Brca1-D11 employing the WAP-Cre and MMTV-Cre promoters splice variant, were found to be tumor-prone, although often require one or even multiple rounds of pregnancy only 12 out of 92 were mammary tumors and lactation for effective induction of mammary tumor (Ludwig et al., 2001a). A bias towards lymphoid and formation. To overcome these limitations, we created a sarcomatoid tumors became even more pronounced transgenic line expressing Cre under the control of the when these animals were crossed onto a p53À/À back- cytokeratin-14 (K14) promoter (Jonkers et al., 2001). ground; nevertheless, Brca1Tr/Tr;p53À/À mice developed Besides K14-Cre expression in salivary glands and skin, tumors significantly faster than the p53 single knock- stochastic Cre- activity was found both in outs. Similarly, Cressman et al. 1999 described three luminal epithelial cells and in myoepithelial cells of Brca1D223À763/D223À763;p53À/À animals that succumbed to virgin mammary glands, suggesting that K14-Cre is lymphomas. Finally, heterozygous p53 mutations intro- expressed in both cell types or in a common progenitor. duced in mice harboring two Brca1D11 alleles gave rise to The latter hypothesis is supported by the observa- mammary tumors in most of the animals produced, tion that K14 is expressed in LinÀCD29hiCD24 þ although also here lymphomas were observed (Xu et al., mammary progenitor cells (Shackleton et al., 2006) or 2001). For Brca2, all three models that enabled survival CD24medCD49fhigh mammary repopulating unit (MRU) of animals showed increased tumorigenesis in the cells (Stingl et al., 2006). The stochastic activity of absence of p53 mutations (Connor et al., 1997; Fried- K14-Cre results in Cre-mediated gene switching in only man et al., 1998; McAllister et al., 2002). However, the a small fraction of mammary epithelial cells. While this Brca2 models displayed a strong bias towards develop- trait is advantageous for sporadic tumor development, it ing thymic lymphomas. In conclusion, while these data precludes assessment of direct consequences of Brca clearly demonstrate tumor suppressor activities for loss-of-function. Brca1 and Brca2, the utility of these models to investigate properties of human breast cancer is limited Conditional Brca1 and Brca2 alleles because of the low incidence of mammary carcinomas. Three different conditional Brca1 alleles have been generated to date: Brca1F5-6 (Mak et al., 2000), Brca1Co (Xu et al., 1999a) and Brca1F5-13 (Liu et al., manuscript Conditional Brca1 and Brca2 mutants in preparation; see Figure 1). Cre-mediated deletion of exons 5–6 or exons 5–13 from the Brca1F5-6 or Brca1F5-13 Besides embryonic lethality and development of non- allele, respectively, induces a frameshift mutation that epithelial tumors, another important limitation of abrogates production of all three splice products conventional Brca1 and Brca2 homozygous mutants (full-length Brca1, Brca1-D11 and the putative Brca1- is that they cannot model development of sporadic IRIS). In contrast, deletion of exon 11 of the Brca1Co cancer, which arises amidst a genetically ‘normal’ allele results in a hypomorphic mutation that still allows background. To overcome these limitations, conditional for expression of Brca1-D11. For Brca2, four mutagenesis strategies have been developed, which rely different conditional alleles have been published to date on Cre recombinase that catalyzes specific genetic (Figure 2). Of these, the Brca2F9-10 and Brca2F3-4 alleles deletion of genomic regions flanked by loxP recombi- encode – upon Cre-mediated switching – truncated trans- nation sites (Jonkers and Berns, 2002). These recom- cripts that are susceptible to nonsense-mediated mRNA bination sites are inserted in non-coding sequences decay (Ludwig et al., 2001b; Cheung et al., 2002). Although to ensure that expression of the gene is not disrupted the Brca2F11 allele results in an in-frame deletion after prior to Cre-mediated recombination. Introduction Cre expression, early embryonic lethality of animals of Cre recombinase into these conditional mouse homozygous for the Brca2D11 allele suggests that also mutants – via intercrosses with Cre transgenic mice or this allele is a functional null (Jonkers et al., 2001). In

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5890 contrast, Cre-mediated deletion of the Brca2F27 allele closely resembled their human counterparts. Most did not result in embryonic lethality of homozygous mammary tumors of K14-Cre;Brca1F5-13/F5-13;p53F2-10/F2-10 mutants, despite the fact that the BRCA2 C-terminal females were highly aneuploid solid carcinomas with domain encoded by exon 27 contains a nuclear basal-like characteristics, including high proliferation localization signal, interacts with RAD51 in a CDK- index, poor differentiation, lack of ERa expression and dependent manner, and is required for maintaining continuous pushing margins. genomic stability after DNA damage (McAllister et al., 2002; Esashi et al., 2005). Mammarytumorigenesis in conditional Brca2 mutants To achieve mammary gland-specific recombination of a Mammarydevelopment in conditional Brca1/2 mutants Brca2F3-4 conditional allele, Ludwig et al. (2001b) used a To achieve mammary gland-specific inactivation of knock-in targeting stategy to place the Cre gene under Brca1, Xu et al. (1999a) crossed both MMTV-Cre and the transcriptional control of the endogenous mouse WAP-Cre transgenic lines with mice carrying one Wap gene promoter. Crossing of the resulting Wapcre/ þ conventional Brca111À allele and one conditional Brca1Co mice with Brca2ex11/F3-4 animals resulted in Wapcre/ þ ; allele. Female mice from both models showed abnormal Brca2ex11/F3-4 female mice, which, upon continuous development of mammary glands. Defects included mating to induce Cre expression, succumbed to non- incomplete ductal outgrowth, alveolar differentiation metastatic mammary carcinomas or adenosquamous and involution, suggesting that Brca1 function is carcinomas after a relatively long latency of B1.4 years. indispensable for normal mammary gland development. The tumors displayed aneuploidy and chromosomal Similar analyses for Brca2 showed reduced ductal side aberrations, were ErbB2/neu-negative and usually ERa- branching in mammary glands from Brca2D27/D27 female and -positive. Also MMTV-Cre;Brca2F9-10/F9-10 mice (McAllister et al., 2002). These data contrast animals developed mammary carcinomas after long to results obtained with MMTV-Cre;Brca2F9-10/F9-10 latency periods of B1.6 years (Cheung et al., 2004). This and with WAP-Cre;Brca2F9-10/F9-10 animals, in which latency was significantly reduced in a p53 heterozygous mammary gland development or involution appeared background. Although there was a difference in tumor to be unaffected (Cheung et al., 2004). However, no firm latency between MMTV-Cre;Brca2F9-10/F9-10;p53 þ /À mice conclusions can be drawn from the latter study, since and MMTV-Cre;Brca2F9-10/ þ ;p53 þ /À or p53 þ /À animals, Cheung et al. did not demonstrate the existence of the shift in tumor spectrum was the most striking effect Brca2-deficient mammary epithelial cells with both of mammary gland-specific Brca2 inactivation in p53 Brca2F9-10/F9-10 alleles recombined. heterozygous mice. Combined tissue-specific inactiva- tion of both Brca2 and p53, on the other hand, resulted in highly efficient mammary tumor formation in Mammarytumorigenesis in conditional Brca1 mutants K14-Cre;Brca2F11/F11;p53F2-10/F2-10 female mice (Jonkers With the aim of creating a mouse model for breast et al., 2001). A control group of K14-Cre;Brca2F11/ þ ; cancer, MMTV-Cre;Brca1Co/11À or WAP-Cre;Brca1Co/11À p53F2-10;F2-10 females showed a much longer median female mice were continuously mated to induce tumor latency (10 instead of 6 months), demonstrating sustained high-level expression of Cre. Some of the that Brca2 and p53 loss-of-function have a synergistic females indeed developed mammary tumors of diverse effect on mammary tumorigenesis. types, albeit with long latency (Xu et al., 1999a). This model was subsequently improved by introduction of a single p53 null allele, yielding MMTV-Cre;Brca1Co/Co; Conditional Brca1/2 mutation in other tissues p53 þ /À mice, which developed mammary tumors with Since cancer predisposition of human BRCA1/2-muta- reduced latency (Brodie et al., 2001). Follow-up studies tion carriers primarily concerns breast cancer, research showed that this model mimicked several aspects of efforts have primarily focused on modeling this cancer human BRCA1-associated . Tumors were type in Brca1/2 mutant mice. While the reason for this ERa-negative and displayed gross genomic instability as biased tumor spectrum is not understood, it is important shown by comparative genomic hybridization (CGH) to know whether the BRCA gene products exhibit tumor and spectral karyotyping (SKY) (Weaver et al., 2002). suppression functions in other tissues as well. Tumorigenesis in the MMTV-Cre;Brca1Co/Co;p53 þ /À T-cell-specific deletion of Brca1 in Lck-Cre;Brca1F5-6/F5-6 model appeared to be critically dependent on abrogation mice showed that thymocyte development, but not T- of p53 function, since most tumors showed loss of the cell receptor recombination, is dependent on Brca1 wildtype p53 allele (Brodie et al., 2001). This is similar to function. Concomitant p53 abrogation or overexpres- the human situation where aberrant p53 signaling sion of the apoptosis inhibitor Bcl-2 rescued thymocyte was more commonly found in BRCA1-associated breast survival and development (Mak et al., 2000). Similar cancers, compared to sporadic tumors (Crook et al., experiments using Lck-Cre;Brca2F9-10/F9-10 mice did not 1997). Cooperation between p53 and Brca1 in mammary show any changes in thymocyte cellularity or composi- tumorigenesis was also demonstrated in a K14-Cre; tion, although in vitro experiments showed increased Brca1F5-13/F5-13;p53F2-10/F2-10 mouse model with tissue- apoptosis and genomic instability in both Brca1- and specific inactivation of both Brca1 and p53 (Liu et al., Brca2-deficient thymocytes (Cheung et al., 2002). In manuscript in preparation). Female mice of this strain line with the lymphoma predisposition of hypo- developed, besides skin tumors, mammary tumors that morphic Brca2 mutants, a small acceleration of T-cell

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5891 lymphomagenesis was reported for Lck-Cre;Brca2F9-10/F9-10; BRCA deficiency relates to the recent appreciation of p53À/À mice, compared to Lck-Cre;p53À/À control animals. the potential role of tetraploid cells in tumorigenesis Brca1 and Brca2 also have tumor suppressor activity (Fujiwara et al., 2005). Tetraploid cells that were in skin epithelium. K5-Cre;Brca1Co/11À mice – in which obtained through induction of endoreduplication via a Cre expression from a bovine cytokeratin-5 promoter transient block in cytokinesis, were found to be induces recombination in, for example, the basal cell dependent on p53 dysfunction for survival. These cells layer of the epidermis, mammary myoepithelium and were genetically unstable and tumorigenic, properties oral cavity – develop squamous cell carcinomas (Berton that were not displayed by isogenic diploid cells. et al., 2003). Thus, Brca1 functions as a tumor Interestingly, it was recently shown that Brca2 deficient suppressor in other epithelial tissues besides mammary cells undergo abnormal cytokinesis (Daniels et al., gland. Interestingly, E2F1 overexpression in these 2004), and indeed knockdown of BRCA1 caused animals dramatically accelerated skin tumor develop- accumulation of multinucleated cells (Bae et al., 2005). ment, suggesting cooperation between the Rb-E2F1 It is thus conceivable that BRCA-deficiency may foster pathway and Brca1 in tumorigenesis. Similar coopera- the generation of endoreduplicated cells that require tion in skin tumorigenesis was found between Brca1 and dysfunctional p53 for survival. p53 (Liu et al., manuscript in preparation) and between Genetic interaction between BRCA1/2 and the Brca2 and p53 (Jonkers et al., 2001). The latter p53 pathway is supported by a large body of data. observation indicates that also Brca2 has tumor For example, p53 mutation is more often observed in suppressor activity in skin epithelium. BRCA-associated tumors than in sporadic cancers Tumor suppressor activity of Brca1 and Brca2 in yet (Crook et al., 1997), and BRCA1/2 tumors showed other tissues has not been convincingly documented to common changes at p53 codons that are not mutation date. However, broad tumor suppressor activity of hotspots (Smith et al., 1999; Greenblatt et al., 2001). Brca1 might be suggested by the fact that Brca1Tr/Tr mice As mentioned before, studies in mouse models developed, in addition to mammary tumors and have documented cooperation between loss of p53 and lymphomas, tumors in many other organs such as lung, inactivation of Brca1 or Brca2 in tumorigenesis, liver, uterus and colon (Ludwig et al., 2001a). From a although the exact nature of this cooperation remains clinical perspective, it would be especially relevant to unclear. However, p53 mutation is not sufficient for know whether ovarian-specific Brca inactivation would complete reversion of Brca-associated growth arrest, lead to tumors that resemble human BRCA-associated given the incomplete rescue of embryonic lethality and ovarian cancer. In conclusion, despite the tissue bias of proliferation defects of cultured embryonic fibroblasts human BRCA-associated cancers, Brca1 and Brca2 may (Shen et al., 1998). Taken together, these data suggest have tumor suppressor activity in multiple mouse that abrogation of the p53 pathway is necessary but not tissues, suggesting that the tumor spectrum seen in sufficient for survival of BRCA-deficient cells. human BRCA-mutation carriers is not due to cell-type specific tumor suppression. Other genetic interactions Besides p53, also other cell-cycle checkpoint and DNA damage response factors have been found to modulate phenotypes in Brca-deficient animals. Lee et al. (1999) Genetic interactions in development and tumorigenesis provided evidence that inactivation of the spindle assembly checkpoint may be involved in Brca2- BRCA1 and BRCA2 are unusual tumor suppressors in associated tumorigenesis. Following the observation that they are essential for normal cell survival, yet their that lymphomas arising from Brca2Tr/Tr mice contain inactivation seems pivotal for cancer development in mutations in the spindle checkpoint genes Bub1 and BRCA-mutation carriers. These paradoxical observa- Mad3L, it was shown that dominant-negative Bub1 tions suggest that BRCA-associated tumorigenesis rescues the proliferation defect of Brca2Tr/Tr embryonic might be fostered by (tissue-specific) environmental fibroblasts. However, direct cooperation between and/or genetic factors that somehow alleviate BRCA- mutated spindle checkpoint genes and BRCA2 inactiva- associated growth suppression. tion in tumorigenesis remains to be established. More clear are the genetic interactions between Brca1 Genetic interactions with the p53 pathway and Chk2, which have been documented in several It has long been hypothesized that genetic interaction Brca1 models. Chk2 deficiency was found to restore between BRCA1/2 and the p53 pathway results from the T-lymphocyte cellularity and development in Lck- DNA damage that accumulates in repair-deficient Cre;Brca1F5-6/F5-6;Chk2À/À mice and to promote mam- BRCA-mutated cells. DSBs in these cells would trigger mary tumor formation in WAP-Cre;Brca1F5-6/F5-6;Chk2À/À a p53-mediated that could be mice (McPherson et al., 2004). Furthermore, embryonic alleviated by, for example, mutation of p53 or its lethality of homozygous Brca1D11/D11 mutants was downstream target p21. This concept is supported by rescued in a Chk2-deficient or in an Atm-haploinsuffi- the observation that inactivation of p53 or p21 results in cient background (Cao et al., 2006). Together, these a prolonged survival of Brca-deficient embryos (Hakem data suggest that loss-of-function of either Atm or Chk2 et al., 1997; Ludwig et al., 1997). Another interesting can substitute for p53 inactivation in Brca1-associated hypothesis for how p53 mutations might cooperate with embryonic lethality and tumorigenesis, thus providing

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5892 strong evidence for the activation of the Atm–Chk2–p53 remains unclear. Cross-species comparison of gene DNA damage response pathway in Brca1-deficient cells. expression data from mammary tumors of the K14- Activation of this pathway by DSBs leads to p53- Cre;Brca1F5-13/F5-13;p53F2-10/F2-10 model revealed gene ex- induced cell cycle arrest, senescence and apoptosis pression signatures with features of basal-like breast (Kastan and Bartek, 2004). Chk2 activates p53 through cancers and strong resemblance to human BRCA1- phosphorylation, and may therefore not only function mutated breast cancers (Liu et al., manuscript in as an ‘amplifier’ of DNA damage response but also preparation). Analysis of DNA copy number alterations as a tumor suppressor. Indeed, heterozygous germline in mammary tumors from the MMTV-Cre;Brca1Co/D11, CHK2 mutations were found in families with non-p53 WAP-Cre;Brca1Co/D11 and WAP-Cre;Brca1Co/D11;p53 þ /À Li–Fraumeni syndrome (Bell et al., 1999). In addition, models by CGH and SKY revealed a pattern of the CHK2*1100delCvariant, encoding an unstable chromosomal gains and losses that resembled truncated protein lacking the kinase domain, was the pattern in human breast cancers, although a identified as a low- breast cancer suscept- cross-species comparison with data from human ibility gene in noncarriers of BRCA1 or BRCA2 BRCA1-associated tumors was not performed (Weaver mutations (Meijers-Heijboer et al., 2002). et al., 2002). GADD45a is a growth arrest- and DNA damage- With the most advanced models currently mimicking inducible gene, and a transcriptional target of BRCA1 several aspects of human BRCA-associated tumorigen- (Harkin et al., 1999). Since also Gadd45A-deficient esis, the time seems ripe to start using these mouse mouse embryonic fibroblasts exhibited models for addressing clinically relevant questions. amplification (Hollander et al., 1999), genetic inter- In fact, the first translational studies have already been actions between Brca1 and Gadd45 were studied in reported. Brca1D11/D11;Gadd45aÀ/À mice. Compound mutant em- bryos died at E9.5–10.5, exhibiting exencephaly and high rates of apoptosis, probably due to activation of Hormone dependencyof BRCA1-associated p53 as a result from increased genetic instability (Wang tumorigenesis et al., 2004b). The relevance of this interaction for Disruption of ubiquitously expressed BRCA genes leads BRCA1-associated tumor formation is still unclear. to a tumor spectrum that shows specificity towards steroid hormone-responsive target organs, such as the mammary glands, ovaries and prostate. Both BRCA1 Applications of current Brca1 and Brca2 mouse models and BRCA2 expression are, albeit indirectly, responsive to estrogen levels (Spillman and Bowcock, 1996), and Validation studies conversely, BRCA1 was found to inhibit ERa mediated The development of mice with mammary gland-specific signaling (Fan et al., 1999; Xu et al., 2005). Therefore, it deletion of Brca1 or Brca2 has not only improved was suggested that estrogen signaling might be impor- our understanding of BRCA-associated breast cancer in tant in BRCA-associated tumorigenesis, thus providing humans, but also provided tools that can be used to test a possible explanation for the observed tissue specificity. novel therapeutic intervention and tumor prevention In support of this, it has been shown that premenopau- strategies. To this end, it is important to validate the sal exerts an B50% reduction of the different mouse models with respect to the human risk of breast cancer in BRCA1 mutation carriers disease. Ideally, validation of the mouse models should (Rebbeck et al., 1999). Also, adjuvant antiestrogenic not only include comparative histopathology and tamoxifen resulted in a decreased risk of second cross-species comparison of profiles primary carcinomas and an improved overall survival and molecular genetic alterations, but also therapeutic of BRCA mutation carriers (Metcalfe et al., 2004; benchmarking by testing responses of mouse mammary Noruzinia et al., 2005). tumors to conventional chemotherapy and radiother- The mechanism by which oophorectomy exerts a apy. Although not necessarily all human aspects need to protective effect remains to be elucidated. Paradoxically, be recapitulated in one mouse model for it to be useful, BRCA1 associated tumors express ERa less often than careful assessment of all differences and similarities is sporadic tumors (Phillips, 2000). Since certain effects of important for correct interpretation of results obtained estrogen on normal mammary epithelial proliferation with the mouse model. and morphogenesis are mediated through paracrine Most Brca1 and Brca2 mouse mammary tumor mechanisms (Mallepell et al., 2006), it is conceivable models generated to date have been validated to only that similar paracrine signals from surrounding ERa- a limited extent (Table 1). In general, they show a positive cells might stimulate proliferation of ERa- relatively high incidence of mammary carcinomas, have negative BRCA1-deficient tumor cells. Alternatively, deregulated p53 pathways and show genetic instability tumors in which BRCA1 loss-of-function resulted in loss in the resulting tumor cells. Several mouse models, of negative control on estrogen signaling, may require especially the Brca2 mammary tumor models, developed less ERa expression (beneath the detection limit in mammary tumors with histopathological features that histochemical assays) for stimulation by estrogen. A were rather different from their human counterparts. third possibility is that ERa expression is expressed Whether this is reflective of a difference in cell-of-origin during initial stages of BRCA1-associated tumorigenesis or a more general difference between both species but is lost in the process of tumor progression.

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5893 Table 1 Human vs mouse comparison of BRCA-associated mammary tumors

+/- F2-10/F2-10 +/- 11 ;p53 3 ;p53 /∆ 11 /∆ ;p53 Co Co/Co 3/F5-1 +/- Co/ Co/Co Human BRCA1 F5-1 Property . 2001a) . 2001) 11 ;p53 breast cancers et al ∆ . 1999a) . , manuscript 11/ . 2001) et al (compared to Tr/Tr ∆ et al et al et al a sporadic tumors) Brca1(Ludwig Brca1 (Xu MMTV-Cre;Brca1WAP-Cre;Brca1(Xu MMTV-Cre;Brca1WAP-Cre;Brca1(Brodie K14-Cre;Brca1(Liu in preparation)

Histology Invasive ductal +/- ND +/- +/- + carcinoma, me- dullary carcinoma ER expression Less common +/- ND ND + + PR expression Less common +/- ND ND ND ND HER2/neu Less common + ND ND - + overexpression p53 mutations More common + - + + + Aneuploidy More common ND ND + + + Tumor incidence High - + - + +

+/-

0 ;p53 F2-10/F2-10

-4 1 ;p53 F9-10/F9/1 F9-10/F9/10 11/F3 1/F1 ex F1 Property Human BRCA2 . 2001b) . 2004) . 2004) . 2001) breast cancers et al et al et al et al (compared to a sporadic tumors) Wap-Cre;Brca2(Ludwig MMTV-Cre;Brca2(Cheung MMTV-Cre;Brca2(Cheung K14-Cre;Brca2(Jonkers

Histology Invasive ductal --ND+/- carinoma ER expression No difference +/- ND ND ND PR expression No difference +/- ND ND ND HER2/neu No difference + ND ND ND overexpression p53 mutations b No difference ++++ Aneuploidy More common + ND ND ND Tumor incidence High ++++

a b Chappuis et al. (2000); Phillips (2000) and Lakhani et al. (2002). It is unclear whether BRCA2 tumors show more p53 mutations, compared to sporadic tumors. However, since p53 mutations are found in 40-50% of BRCA2 tumors, we have scored p53 mutations in the mouse Brca2 models as being in concordance with the human condition. For each property, a '+' indicates similarity to the human situation, where as a '–' indicates lack of similarity. '+/–' symbols indicate heterogeneity of the factor involved, and 'ND' indicates no information has been reported.

To gain more insight in the role of estrogen signaling artifact, caused by decreased Cre activity. Hormone in BRCA-associated tumorigenesis, the MMTV-Cre; dependency of MMTV-Cre might also explain the Brca1Co/Co;p53 þ /À mouse model developed by the group unexpected results from tamoxifen treatment studies in of Deng, has been used to study the effects of the MMTV-Cre;Brca1Co/Co;p53 þ /À model, showing ac- interventions in this pathway (Bachelier et al., 2005). celerated development of mammary tumors in tamox- Concurrent with the human situation, there was a ifen-treated female mice (Jones et al., 2005). While the significant protective effect of oophorectomy on the experiments performed in this study suggest that loss of occurrence of mammary cancer in older animals. full-length Brca1 expression alters the agonist/antago- Although the mechanism underlying this effect remains nist activity of tamoxifen to the extent that the agonistic unclear, it was shown that mammary glands of function becomes more pronounced, firm conclusions oophorectomized mice underwent regression. Of note, can only be drawn when these results are confirmed in a oophorectomy studies in the MMTV-Cre;Brca1Co/Co; different model. p53 þ /À model are compromised by the fact that extent of Despite the technical limitations associated with MMTV-Cre mediated Brca1 mutation – and thus the the MMTV-Cre;Brca1Co/Co;p53 þ /À model, the studies of incidence and multiplicity of mammary tumors – may be Bachelier et al. and Jones et al. provide a proof- influenced by levels of steroid hormones. Hence, reduced of-concept for the utility of mouse Brca1/2 mammary mammary tumor formation in oophorectomized tumor models to investigate clinically relevant ques- MMTV-Cre;Brca1Co/Co;p53 þ /À females might be an tions. This type of research will no doubt help us to

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5894 address clinically relevant questions and provide both in development and tumorigenesis. Studies in knowledge that can subsequently be translated into conventional and conditional Brca1/2 mouse mutants clinical practice. have provided insight into the role of Brca1 and Brca2 in various cellular processes, and established genetic Targeting BRCA-deficient cells and tumors interactions with several other proteins, most notably components of the DNA damage response path- Another example that highlights the value of conditional way. Tumor suppressor activity of both BRCA proteins Brca1/2 mammary tumor models for preclinical studies was confirmed in several mouse models, not only in concerns the research on antitumoral and tumor the mammary gland but also in skin and lymphoid preventive activities of chemical inhibitors of poly- compartments. Nevertheless, the tumor spectrum of (ADP-ribose)-polymerase-1 (PARP1). PARP1 defi- the mouse models parallels the human tumor ciency results in increased Rad51 foci formation and sister chromatid exchanges, but does not affect DSB spectrum at least to some extent, since a clear bias towards mammary tumorigenesis is reported in condi- repair via HR (Schultz et al., 2003). Based on these tional Brca1/2 models employing MMTV-Cre and observations, it was hypothesized that inhibition of K14-Cre PARP1 activity in BRCA-deficient cells with defective , which express Cre in multiple tissues besides mammary gland. HR would result in cytotoxicity due to rapid accumu- Other features that are recapitulated by at least some lation of DNA damage. Since DNA lesions in of the mouse models include tumor histology and BRCA-proficient cells can be repaired by HR, PARP1 aneuploidy. inhibition may have a wide therapeutic window. Despite our increased understanding of the function Indeed, several groups showed that chemical inhibitors of BRCA1 and BRCA2, several important questions of PARP1 activity increased DNA damage and remain yet to be resolved. The intriguing observation caused specific killing of cells with impaired BRCA1 that BRCA inactivation induces growth arrest in normal or BRCA2 function (Bryant et al., 2005; Farmer et al., cells, yet promotes tumor formation in mutation 2005). In both studies, treatment with PARP1 inhi- bitors induced regression of Brca2-deficient tumors in carriers, strongly suggests the presence of secondary suppressor mutations that may overcome such an arrest transplantation models. during BRCA-associated tumorigenesis. Also stromal To further validate this approach to targeting BRCA- factors might foster in situ survival of BRCA-deficient deficient cells, a small intestine-specific inducible Cre mammary tumor cells. Cancer-associated fibroblasts line (Ah-Cre) was crossed to mice carrying conditional contribute to various stages of tumor growth (Kalluri Brca2F9-10 alleles as well as a reporter for Cre-mediated recombination, ROSA26R (Soriano, 1999; Hay et al., and Zeisberg, 2006), and also immune cells can et al 2005b). Subsequent treatment of mice with PARP1 support cancer development (de Visser ., 2006). inhibitors resulted in depletion of reporter-positive cells Clearly, mouse models are key to investigating these complex tumor–host interactions in the context of specifically in Ah-Cre;Brca2F9-10/F9-10;Rosa26R intestines BRCA-associated tumor formation. but not in Ah-Cre;Brca2F9-10/ þ ;Rosa26R intestines, suggesting specific depletion of Brca2-deficient cells Another unresolved aspect of BRCA-associated (Hay et al., 2005a). These findings pave the way for tumorigenesis concerns gender bias and tissue-specificity extending the application of PARP1 inhibitors from of the disease. This tissue specificity might be caused by therapeutic towards prophylactic settings. If loss of redundant pathways, specifically absent in mammary BRCA function is an early event in mammary tumor epithelium, that could mask BRCA-associated pheno- formation in human mutation carriers, such cells might types in other tissues. However, the embryonic lethality Brca1/2 be specifically depleted by PARP1 inhibition, even of homozygous mouse mutants argues against before turning malignant. the existence of pathways that are functionally redun- The potential use of PARP1 inhibitors for targeting dant with BRCA1 or BRCA2 in cell proliferation. It has BRCA-deficient or ‘BRCA-like’ tumors with defective therefore been proposed that the observed bias may be HR is exciting and, clearly, the current Brca1/2 explained by cell type-specific and/or environment- mammary tumor models are valuable tools for studying dependent survival of BRCA-deficient mammary and both therapeutic and prophylactic applications of ovarian epithelial cells (Elledge and Amon, 2002). Since PARP1 inhibitors in more detail. Also, evaluation of the spectrum of BRCA-related cancers is biased towards combination in these mouse models can organs that are targets of estrogen, it is conceivable that provide important information for the clinic, where survival factors in the form of hormones may have a the first clinical trials using PARP1 inhibitors have protective effect on BRCA-deficient cells. These and Brca1 already started. other possibilities may be effectively explored in and Brca2 mouse models. Future research is also expected to provide more insight into the genotype–phenotype correlations that Conclusions and future perspectives have emerged from the analysis of the various Brca1 and Brca2 mutants. It is conceivable that at least some of Since the discovery of BRCA1 and BRCA2, mouse these correlations may also be of relevance for models have proven of paramount importance for pathogenesis and clinical responses of BRCA-associated obtaining information on the function of these genes, tumors in human mutation carriers. Phenotypic analysis

Oncogene Mouse models of BRCA1/2 deficiency B Evers and J Jonkers 5895 of mice engineered to express one or more of the Existing as well as new mouse models for BRCA- observed Brca1 splice variants may shed light on the associated breast cancer – and perhaps other ‘BRCA- specific functions of the different Brca1 protein pro- like’ tumors with impaired HR – are expected to play an ducts. Similarly, mouse Brca1 and Brca2 mutants with increasing role in preclinical and translational research. specific changes may uncover functions of Robust validation of the utility of mouse models for specific protein domains or phosphorylation sites. One preclinical applications is important and should include such mutant, Brca1S971A, encoding a Brca1 protein with cross-species comparisons of tumor characteristics and an inactivated Chk2 phosphorylation site, has already responses to chemotherapeutics. Nevertheless, the first been described, showing predisposition to carcinogen- studies with targeted therapeutics in some of the existing induced tumorigenesis (Kim et al., 2004). Clinically more models have already been reported, thus sparking the relevant would be the generation of mouse mutants with hope that breast cancer patients will soon benefit from truncation mutations mimicking human BRCA1/2 germ- the collective research on mouse models of BRCA1 and line mutations. These models may be used to assess BRCA2 deficiency. tumor predisposition and clinical responses of known disease-associated mutations. Recently developed ‘huma- Acknowledgements nized’ mouse models carrying human BRCA1 BAC transgenes (Yang et al., 2003) or oligo targeting We thank Peter Bouwman, Rinske Drost, Xiaoling Liu and techniques that allow targeted insertion of subtle point Karin de Visser for critical reading the manuscript and mutations and small insertions into mouse embryonic providing helpful suggestions. BE is supported by a grant stem cells (Dekker et al., 2003, 2006) are particularly from the Netherlands Organization for Scientific Research suitable for such allelic series of Brca1/2 mutations. (NWO-VIDI 917.36.347).

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