Brca2 and Trp53 Deficiency Cooperate in the Progression of Mouse Prostate Tumourigenesis

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.


Introduction
Prostate cancer is the most common cancer in men in developed countries, with a rising incidence of the disease. However, the etiology of this malignancy is still unclear. Prostate cancer progresses through a pathologically defined series of steps involving increasing grades of PIN, invasive adenocarcinoma and metastatic cancer [1]. Androgens are crucial for normal prostate function, and act as pro-survival and proliferation factors in cancer cells. As such, prostate cancer is sensitive to androgen levels and androgen depletion therapy via chemical or surgical castration is an initial step in treatment, typically resulting in tumour regression. However, the cancer normally re-grows and develops as a castration-independent tumour.
Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility [2,3]. Approximately 10% of prostate cancers are thought to be hereditary, and this number increases with early on-set disease. In spite of this, little is known about the mechanisms of tumourigenesis of inherited prostate cancer. Prostate cancer frequently clusters in families that have breast cancer, indicating a genetic link between these two diseases [4][5][6]. Germline mutations in BRCA2 predispose to both breast and ovarian cancer making it a good candidate gene for prostate cancer etiology. There is an increased risk of prostate cancer in individuals carrying a mutation in BRCA2, particularly early-onset disease [7][8][9][10]. The Breast Cancer Linkage Consortium found a significant relative risk of 4.65 for prostate cancer in male carriers of a deleterious BRCA2 mutation that rose to 7.33 in men under 65 years of age [7]. Consistent with this, analysis of men with early-onset disease indicates that BRCA2 carriers account for between 0.8-2% of prostate cancer cases, compared with the prevalence of 0.1% BRCA2 mutations in the general population [11,12]. In addition, BRCA2 mutation carriers have been associated with aggressive prostate cancer [13][14][15][16].
BRCA2 is thought to act as a tumour suppressor, with tumours arising from BRCA2 mutations frequently demonstrating loss-ofheterozygosity with loss of the wild-type allele. BRCA2 plays an important role in the repair of DNA double-strand breaks (DSB) through homologous recombination (HR) [17]. When there is a second identical DNA copy (i.e. the sister chromatid after replication) HR is the primary method of repair and is a relatively error-free DNA repair pathway. After DNA damage, BRCA2 directly interacts with the recombinase RAD51, a process that is essential for HR-mediated repair of DSBs [18]. When HR is defective or no sister chromatid is available the error-prone methods of single-strand annealing and non-homologous end joining are used for DNA repair [19]. BRCA2-deficient cells form chromosomal aberrations spontaneously in culture and are more sensitive to certain DNA damaging agents [19][20][21]. Hence, loss of BRCA2 is thought to principally lead to tumour progression by the failure to repair DNA by HR, leading to genomic instability.
Mouse models have shown a direct in vivo tumour suppressor role for Brca2 in the mammary gland and have demonstrated a synergistic tumour suppressor activity with Trp53. However, Brca2 heterozygous animals do not show a predisposition to tumour formation and Brca2 null mice result in embryonic lethality [18,22,23]. To circumvent this prenatal lethality, the Cre-LoxP system has been used to conditionally delete Brca2 in a tissuespecific manner. Deletion of Brca2 from the mouse mammary epithelium either fails to produce mammary-gland tumours or results in mammary-gland tumour formation with long latency (1.4-1.6 years) [24][25][26]. Tumour latency was reduced in Brca2 mutant mice that were Trp53 heterozygous [24]. In addition, mice with conditional inactivation of Brca2 and Trp53 developed mammary tumours with high penetrance at 6 months [25].
To understand the role of Brca2 in prostate cancer we have used a prostate-specific Cre line and a conditional Brca2 allele to delete Brca2 in adult mouse prostate epithelia. We show that loss of Brca2 in the prostate results in focal hyperplasia and low-grade (LG) PIN. Mice with conditional deletion of Brca2 and Trp53 have a high incidence of high-grade (HG) PIN, which contain cells with elevated DNA damage. PIN lesions in Brca2;Trp53 homozygous mutant prostates persist and continue to proliferate after androgen depletion. This work confirms the role of Brca2 as a tumour suppressor in the prostate and provides a model to test potential therapeutics in Brca2-deficient prostate neoplasia.

Results
Deletion of Brca2 from prostate epithelia results in hyperplasia and low-grade PIN To investigate the role of Brca2 in the prostate we deleted Brca2 from the adult mouse prostate epithelia. To achieve this we mated mice carrying a Brca2 allele that has exon 11 flanked by loxP sites (Brca2 F/F ) to transgenic mice carrying Cre recombinase under the control of a prostate-specific composite rat probasin promoter, PBCre4 [25,27]. This Cre line has been used successfully to delete tumour suppressor genes and activate oncogenes to drive prostate neoplasia and tumour progression [28][29][30]. Deletion of this Brca2 conditional allele results in the loss of a Rad51-interacting domain, and consequently, homozygous germline deletion leads to embryonic lethality [25].
Cohorts of male control (Brca2 F/F ), Brca2 heterozygous (Brca2 F/+ ;PBCre4) and Brca2 mutant (Brca2 F/F ;PBCre4) animals were generated and analysed for tumour progression at 6 months, 10-14 months and 15-20 months of age. None of the Brca2 F/+ ; PBCre4 prostates had any observable morphological differences compared to control prostates at any time point analysed ( Figure 1 and Table 1). Focal hyperplasia that contained atypical cells was first observed in Brca2 F/F ;PBCre4 prostates at 10-14 months and was also present in these animals at 15-20 months (Table 1). In addition, at 15-20 months a significant number of Brca2 homozygous mutant prostates had focal LG PIN in their lumen compared to control animals (6/17 compared to 0/28; Z-test p = 0.0001) ( Figure 1A and Table 1). LG PIN lesions formed characteristic tufting and cribiform patterns ( Figure 1B). These focal lesions contained multiple atypical cells that had prominent nucleoli and hyperchromasia. Hyperplasia and LG PIN lesions were present in all four prostatic lobes of Brca2 mutants.

Author Summary
In Western countries, prostate cancer is the most common male cancer and the second biggest cause of cancerrelated deaths in men. Men with a familial history of either breast or ovarian cancer have an elevated predisposition to prostate cancer, suggesting there is a genetic element to this disease. Indeed, the inheritance of a mutated form of the breast cancer susceptibility gene BRCA2 has been linked to the development of prostate cancer, although the precise role that BRCA2 dysfunction plays in the development of prostate cancer is unclear. To address this, we have generated an animal model in which the mouse Brca2 gene is specifically deleted in the adult prostate. These mice develop precancerous prostate lesions, which progress in severity and incidence with the loss-of-function of an additional tumour suppressor, Trp53. Importantly, blocking male steroidal hormone production by castration leads to partial regression of the prostate lesions, however cells continue to proliferate after androgen withdrawal. This suggests human BRCA2 mutant prostate tumours, like the majority of prostate cancers, will respond to hormone therapy, but will relapse, as frequently occurs in this disease. In summary, our model suggests that BRCA2 acts as a tumour suppressor in the prostate and provides a preinvasive model to test novel therapeutics.
Combined deletion of Brca2 and Trp53 leads to frequent high-grade PIN The tumour suppressor TP53 is frequently mutated in BRCA2 cancers and studies in the mouse have shown a genetic interaction between Brca2 and Trp53 [24,31,32]. To test if Brca2 and Trp53 cooperate in the prostate we deleted both of these genes in the prostate epithelia using the PBCre4 transgene. Cohorts of male control (Brca2 F/F ;Trp53 F/F ), Brca2 homozygous and Trp53 heterozygous (Brca2 F/F ;Trp53 F/+ ;PBCre4) and Brca2 and Trp53 double homozygous (Brca2 F/F ;Trp53 F/F ;PBCre4) animals were generated and analysed for tumour progression.
In addition to hyperplasia and LG PIN observed in Brca2 mutants, deletion of Brca2 and Trp53 resulted in the formation of HG PIN lesions. At 10-14 months, Brca2 F/F ;Trp53 F/+ ;PBCre4 animals had focal LG PIN and hyperplasia ( Figure 2A and Table 1). By 15-20 months, LG PIN was still present and a significant number of animals had focal HG PIN compared to control animals (6/15 compared to 0/28; Z-test p = 0.0017). However, the frequency of HG PIN was significantly higher in Brca2 F/F ;Trp53 F/F ;PBCre4 animals compared to Brca2 F/F ;Trp53 F/+ ; PBCre4 animals at this age (27/32 compared to 6/15; Z-test p = 0.0058) ( Figure 2A and Table 1). In these animals, focal areas of hyperplasia consisting of atypical cells were present as early as 6 months. At 10-14 months, LG PIN was present and a significant number of Brca2 F/F ;Trp53 F/F ;PBCre4 animals had HG PIN compared to control animals (7/15 compared to 0/11; Z-test p = 0.0276). Hyperplasia and LG PIN lesions were similar to those found in Brca2 F/F ;PBCre4 mutants. Frequently multiple ducts of each lobe had HG PIN, which were present in proximal and distal regions of the prostate and consisted of many atypical cells filling the lumen. Atypical cells were unorganised with poor orientation, severe nuclear pleomorphism and abnormal nuclear to cytoplasm ratios ( Figure 2B). Mitotic figures, apoptotic bodies and areas of necrosis were also present within HG PIN lesions ( Figure 2B). In some cases, epithelial cells of the lumen protrude into the adjacent stroma and the smooth muscle surrounding the ducts was no longer continuous but was broken up ( Figure 2B). These areas contained atypical smooth muscle cells and desmoplasia in the surrounding stroma. HG PIN lesions were predominantly seen in the anterior prostate (AP) and dorsal prostate (DP) of Brca2;Trp53 homozygous mutant animals, with a small number observed in lateral and ventral lobes. Deletion of LoxP flanked Brca2 and Trp53 alleles by the PBCre4 transgene in the prostate was confirmed by PCR analysis on micro dissected tissue ( Figure S1).
Brca2;Trp53 HG PIN lesions display increased DNA damage and apoptosis As the predominant tumour suppressor function of BRCA2 is thought to be the repair of DNA DSBs, we assessed the level of prostates. An early response to DNA damage is the phosphorylation of histone H2AX (cH2AX) [33]. Areas of hyperplasia and PIN in Brca2 mutant and Brca2;Trp53 mutant prostates contained cells that were positive for cH2AX, which were not present in control prostates ( Figure 3A). While Brca2 F/F ;PBCre4 and Brca2 F/F ; Trp53 F/+ ;PBCre4 LG PIN lesions had individual or small groups of cH2AX positive cells, Brca2;Trp53 homozygous mutant prostates had large focal areas with many positive cells. These areas of cH2AX correlated with focal HG PIN lesions and were predominantly present in the AP and DP. Deletion of Brca2 frequently results in increased levels of cellular apoptosis, presumably as a result of increased constitutive DNA damage. To determine the level of apoptosis in prostate epithelia after deletion of Brca2 and Trp53 we used the TUNEL assay, which has been used to identify apoptosis in Brca2 null neural tissue [32].  Figure 3B). An anti-Caspase-3 antibody and histological analysis confirmed that TUNEL positive cells were apoptotic and not the result of labelling damaged DNA (data not shown).

Brca2;Trp53 mutant prostates have hallmarks of cancer
Ki-67 is a marker of proliferating cells and a prognostic indicator in prostate cancer [34]. Analysis of Ki-67 showed a low number of proliferating cells in control prostates that increased 6 fold in areas of hyperplasia and LG PIN in Brca2 F/F ;PBCre4 mutant prostates (2.2% Ki-67 positive cells vs 0.4% in control) ( Figure 4A). Levels of proliferation were 9 fold higher in Brca2 F/F ; Trp53 F/+ ;PBCre4 PIN lesions (3.5% Ki-67 positive cells vs 0.4% in control), and dramatically increased by 30 fold in Brca2 F/F ; Trp53 F/F ;PBCre4 HG PIN lesions (12% Ki-67 positive cells vs 0.4% in control) ( Figure 4A).
Levels of the androgen receptor (AR) expressed in the luminal epithelium usually increase in the nucleus during human prostate carcinoma progression [35]. Brca2 F/F ;PBCre4 and Brca2 F/F ;Trp53 F/+ ; PBCre4 mutant prostates had increased AR expression in the cytoplasm and nucleus of epithelial cells that correlated with regions of LG PIN ( Figure 4B). The level of AR increased significantly more in Brca2 F/F ;Trp53 F/F ;PBCre4 HG PIN lesions where it was found predominantly in the nucleus of luminal epithelial cells ( Figure 4B).
The human and mouse prostate comprise of basal, luminal and rare neuroendocrine epithelial cells. In addition, intermediate or transit amplifying (TA) cells are observed in human prostates [36]. Brca2;Trp53 homozygous mutant HG PIN lesions frequently contained large groups of p63-expressing cells, a marker of basal cells ( Figure 4C). Instead of their normal flat shape and position basal to the luminal cells, some p63-expressing cells were rounder and in a position near the lumen of the prostate. Sections fluorescently double labelled with p63 and the basal cell cytokeratin CK5 confirmed the presence of clusters of aberrant basal cells that protrude into the lumen ( Figure 4D). Brca2;Trp53 homozygous mutant HG PIN lesions were then double labelled with CK5 and CK8, a marker of differentiated luminal cells. Areas of neoplasia showed an increase in CK8-expressing luminal cells that were often adjacent to a population of expanded CK5expressing basal cells ( Figure 4D). These regions occasionally had cells that were labelled with both CK8 and CK5, similar to human TA cells that co-express basal and luminal markers, which are not seen in control mouse prostates ( Figure 4D) [36].

Brca2;Trp53 PIN lesions proliferate post-castration
Androgen ablation is the standard treatment for human prostate cancer. To assess the response of neoplasias formed after deletion of Brca2 and Trp53 to androgen ablation, we surgically castrated animals at 16 months when HG PIN lesions have already formed and analysed them 4 days post-castration. Castration of control animals resulted in normal prostate regression, with a reduction in lumen size ( Figure 5A). Brca2 F/F ;Trp53 F/F ;PBCre4 animals still contained focal areas of neoplasia with atypical cells following androgen-depletion (8/8 mutant animals) ( Figure 5A). However, we did not observe ducts with filled lumens as seen in areas of PIN in non-castrated mutant animals.
Following castration of control male mice, AR is expressed at low levels predominantly throughout the cytoplasm of luminal and Interestingly, Ki-67 staining showed an 18 fold increase in proliferating cells in areas of neoplasia in Brca2;Trp53 mutants that persisted after castration, compared to control castrated animals (5.5% Ki-67 positive cells vs 0.3% in castrated control) ( Figure 5D).

Discussion
Studies on human carriers of deleterious BRCA2 mutations have implicated this gene in prostate cancer etiology, but its function in this malignancy is unclear. We have undertaken a genetic analysis of Brca2 function in the adult mouse prostate to define its role in prostate cancer and to create an in vivo model of Brca2-dependent prostate disease progression. Our study has demonstrated that loss of Brca2 in the mouse prostate epithelium results in hyperplasia and LG PIN. These lesions have an increase in the number of cells with DNA damage and apoptotic cells, which could be the result of the impairment of DNA repair pathways. This demonstrates not only that Brca2 can play a role in the initiation of prostate neoplasia but also that other factors are required for prostate tumour progression.
Deletion of Brca2 and Trp53 in mouse prostate epithelia resulted in a shorter latency and increased frequency of prostate neoplasia compared to deletion of Brca2 alone ( Figure 6). Moreover, the severity of neoplasia increased in Brca2;Trp53 mutants, with the formation of hyperplasia and LG PIN at initial stages followed by a high incidence of multi-focal, proliferative HG PIN lesions with progressive cellular atypia ( Figure 6). HG PIN lesions that form after deletion of Brca2 and Trp53 contained many cells with DNA damage, indicating increased genomic instability [17]. Multi-focal lesions are a common feature of human prostate cancer and may be due to defects in the DNA damage response [37]. The formation of HG PIN lesions in Brca2;Trp53 mutant prostates may reflect the loss of key regulatory p53-dependent functions in response to DNA damage controlling cell-cycle checkpoints, apoptosis and senescence [38]. This demonstrates a co-operative tumour suppressor function of Brca2 and Trp53 in the prostate similar to the mammary gland [24][25][26].
A recent study investigating whether TP53 and BRCA2 are frequently mutated together in human prostate cancer found that TP53 overexpression could not distinguish BRCA2 carriers with prostate cancer from a control group of prostate cancer cases [39]. However, this study was limited by the small number of BRCA2 cases and inability to detect TP53 mutations that do not stabilize the protein, which are frequently detected in tumours with impaired homologous recombination [40].
Deletion of Brca2 in other tissues frequently leads to an increase in apoptosis, which is partially or fully rescued upon loss of Trp53 [31,32,41]. However, we see more apoptotic cells in Brca2;Trp53 mutant HG PIN lesions than in Brca2 mutant LG PIN lesions. The increase in apoptosis in HG lesions could be due to the rapid accumulation of additional mutations by proliferating cells, which causes catastrophic amounts of DNA damage. This suggests that while some Brca2-null cells may be rescued from apoptosis after loss of Trp53, other cells in Brca2;Trp53 deficient HG neoplastic lesions undergo p53-independent cell death. Several different DNA damage-induced p53-independent mechanisms of apoptosis have been reported in different cell types [42].
AR expression is increased in the nucleus of cells in Brca2;Trp53 HG PIN lesions suggesting they are androgen sensitive. Consistent with this, castration of Brca2 F/F ;Trp53 F/F ;PBCre4 animals led to regression of PIN and a reduction of cells within the lumen. This suggests that BRCA2-driven prostate cancer would initially respond to conventional androgen ablation. However, atypical cells persist in Brca2;Trp53 HG PIN lesions that continue to proliferate, indicating these lesions may be able to re-grow and become castration-resistant. Interestingly, some cells in Brca2;Trp53 mutant PIN lesions expressed AR at higher levels in the nucleus after castration, indicative of active AR signalling. Castration-resistant human prostate cancer growth commonly remains AR-dependent and is thought to occur through several mechanisms including AR amplification, AR mutation, changes in AR co-regulators and growth factor activation [43]. The presence of nuclear AR in castrated Brca2;Trp53 mutant prostates may indicate the regulation of proliferation by this factor after androgen depletion.
We   cells and luminal cell differentiation [44]. The increase in basal progenitor cell population could represent an expansion of cancerinitiating cells, indicating that HG PIN lesions in Brca2;Trp53 mutant prostates may originate from these cells. Several other murine models of prostate cancer that utilize the PBCre4 transgene display increased and aberrant p63 expression during early stages of cancer progression [29,30]. This transgene is expressed in both the basal and luminal cells of the mouse prostate [44]. In contrast, deletion of Pten using a PSA-driven Cre only expressed in luminal cells results in cancer without an expansion in p63 cells [45]. However, this model has slower kinetics than the Pten; PBCre4 model and tumours are initiated from cells in the luminal epithelial compartment. Frequently during human prostate tumour progression there is an increase in TA/progenitor cells, which has led to the proposal that these cells could be tumour-initiating cells [36,44,46]. Taken together, these data suggest that an increase in the basal progenitor cells could be a common early event in prostate neoplasia but may be dependent on the origin of the cancer-initiating cell.
Although we frequently observed HG PIN lesions in Brca2;Trp53 mutant prostates no invasive carcinoma formed. In contrast, deletion of Brca2 and Trp53 in the mouse mammary gland results in invasive carcinoma at 6 months, and consistent with this, human carriers of BRCA2 mutations have a high-risk of breast cancer. The lack of prostate carcinoma in our mutants may reflect the relatively low penetrance of prostate cancer in human BRCA2 mutation carriers and suggests there is only a subset of BRCA2 carriers that develop aggressive forms of the disease [13][14][15][16]. This subset may be dependent on additional genetic modifiers or environmental factors that influence the risk of individuals carrying a BRCA2 mutation forming prostate tumours [47]. It is possible that human carriers of deleterious BRCA2 mutations frequently form HG PIN lesions similar to our Brca2;Trp53 model but that never progress to carcinoma and therefore go undetected. Ongoing work into genetic modifiers of BRCA2 may identify which subgroups of patients with BRCA2 mutations are more at risk of developing aggressive forms of the disease.
Variations in genetic background can have a modifying effect on prostate tumour development in mice with tumour suppressor deletions [48]. Due to the complex nature of mouse breeding we were not able to investigate the effects of genetic background on the prostate lesions observed in Brca2;Trp53 mutant mice in this study. Although a change in genetic background may alter the frequency of tumour phenotype, we only observed PIN in mutant animals suggesting these lesions are an effect of Brca2 and Trp53 loss.
This murine study has demonstrated that deletion of the tumour suppressor Brca2 results in LG PIN, with the additional loss of a second tumour suppressor Trp53 leading to HG PIN. Other mouse models of tumour suppressor gene loss result in varying degrees of prostate tumour progression. Mice with prostatespecific homozygous Pten deletion progress to invasive carcinoma and metastasis [28]. The further loss of Trp53 in this model results in a shorter latency to invasive carcinoma [49]. Deletion of Nkx3.1, a gene involved in prostate epithelial cell differentiation, leads to mice that develop epithelial hyperplasia and dysplastic lesions that resemble human PIN, but do not progress to invasive carcinoma [50,51].
Our pre-invasive model could be used in the future to test the response to potential therapeutic agents and combination therapies. For example, a recent synthetic lethal approach using PARP inhibitors has been used successfully to specifically induce cytotoxicity in HR-deficient cells [52]. Promising phase I clinical data in BRCA2 carriers with a PARP inhibitor has shown antitumour activity, including resolution of bone metastases in one patient with prostate cancer [53]. These Brca2 mutant mice may provide a useful model to examine cellular responses, such as apoptosis, to combinations of therapies for optimisation of treatment. In addition, this study demonstrates that Brca2 acts as a tumour suppressor and can interact genetically with Trp53 deficiency in the prostate preventing DNA damage accumulation and neoplasia progression.

Ethics statement
Animals were handled in strict accordance with UK Home Office regulations.

Mouse prostate histology and statistical analysis
Histological phenotype of samples was assessed on haematoxylin and eosin stained sections. Serial sections were then stained for immunohistochemical analysis. Histological assessment was based on published guidelines and assisted by a pathologist [54,55]. PIN lesions noted as LG were equivalent to PIN I-II and those noted as HG were equivalent to PIN III-IV in Park et al [54]. The twosample Z-test was performed to test if there is a significant difference between groups of animals.

Quantification of proliferation and cell death
Ki-67 or TUNEL staining was performed by immunohistochemistry on sections and cells stained with nuclear brown DAB chromogen were counted as positive. Cells from at least 4 high power fields were counted per animal, which totalled more than 900 cells per animal. Five animals of each genotype were analysed. Randomly selected fields were counted for control analysis and sections corresponding to histologically identified areas of hyperplasia and PIN were counted for mutant animals. All values are significant with p,0.05 using Student t-test unless otherwise stated.