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Carcinogenesis Advance Access originally published online on March 15, 2007
Carcinogenesis 2007 28(7):1401-1407; doi:10.1093/carcin/bgm060
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Absence of full-length Brca1 sensitizes mice to oxidative stress and carcinogen-induced tumorigenesis in the esophagus and forestomach

Liu Cao, Xiaoling Xu, Longyue L. Cao, Rui-Hong Wang, Xavier Coumoul, Sang S. Kim1 and Chu-Xia Deng*

Genetics of Development and Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10/9N105, 10 Center Drive, Bethesda, MD 20892, USA
1 Present address: Division of Radiation and Nuclear Medicine, National Cancer Center, Goyang, Gyeonggi 411-769, South Korea

* To whom correspondence should be addressed. Tel: +301 402 7225; Fax: +301 480 1135; Email: chuxiad{at}bdg10.niddk.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Environmental and genetic factors are important both in affecting life span and neoplastic transformation. We have shown previously that mice, which are homozygous for full-length breast cancer-associated gene-1 (Brca1) deletion and heterozygous for a p53-null mutation (Brca1{Delta}11/{Delta}11p53+/–), display premature aging and high frequency of spontaneous lymphoma and mammary tumor formation. To investigate the role of Brca1 in regulation of organ homeostasis and susceptibility of Brca1 deficiency to environmental carcinogens, we examined biological function of Brca1 in maintaining organ homeostasis and carcinogen-induced tumorigenesis. Brca1{Delta}11/{Delta}11p53+/– mice showed altered gastrointestinal tract homeostasis, including hyperkeratosis in the esophagus and forestomach. At 6 months of age, most mutant mice displayed hyperplasia in their forestomach and esophagus, leading to dysplasia and carcinoma formation in older animals. Brca1 mutant mice exhibited increased expression of Redd1, elevated reactive oxygen species and are more sensitive to oxidative stress induced lethality. Upon methyl-N-amylnitrosamine (MNAN) treatment, 70% Brca1 mutant mice developed tumors within 4 months whereas only 14% control animals developed tumor at the same period of the time. Our further analysis revealed that the tumorigenesis is accompanied by the loss of p53 and increased expression of a number of oncogenes, including Cyclin D1, phosphorylated form of Akt, ß-catenin, Runx-2 and c-Myc. These results suggest that Brca1 is involved in renewable organ homeostasis, linking the environmental and genetic factors in carcinogenesis and aging, and providing new insights into genomic instability in organism maintenance and tumorigenesis.

Abbreviations: BRCA1, breast cancer-associated gene-1; DDR, DNA damage response; LOH, loss of heterozygosity; MEF, mouse embryonic fibroblast; ROS, reactive oxygen species; WT, wild type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Upper gastrointestinal (stomach and lower esophageal) cancers are common cancers with an increasing incidence in recent years (1,2). Prognosis of esophageal cancer is poor with an overall 5-year survival rate of <10% (3). Environmental factors and genetic alterations have been identified in these cancers, including loss of heterozygosity (LOH) in the region on chromosome 17q where the breast cancer-associated gene-1 (BRCA1) resides (4–6). BRCA1 is most well known for its tumor suppressor activity for breast and ovarian cancers (7–9), whereas its role in other types of cancers remains elusive (4–6,10,11).

Functions of BRCA1 have been extensively studied in multiple systems in the past decade. In mice, a number of different mutations have been introduced into Brca1 through gene targeting (reviewed in ref. 12). Depending on the nature of these mutations, Brca1 mutant mice die at varying development stages that are accompanied by pleiotropic effects, including growth retardation, apoptosis, defective DNA damage repair, centrosome amplification, loss of G2/M cell-cycle checkpoint, genetic instability and premature senescence (reviewed in refs 12–15). Studying mutant embryos carrying a targeted deletion of Brca1 full-length form (Brca1{Delta}11/{Delta}11), we showed that haploid loss of p53 in these mice could suppress embryonic lethality owing to the attenuation of apoptosis and relaxed G1/S cell-cycle checkpoint (16). The Brca1{Delta}11/{Delta}11p53+/– mice exhibit premature aging and develop tumors in mammary gland, ovary and thymus with LOH of p53 (16–19). The Brca1{Delta}11/{Delta}11p53+/– mice also display spermatogenesis failure due to impaired meiotic DNA damage repair, abnormal homologous chromosome pairing and crossover formation (20–22). Since all the organs/tissues in the Brca1{Delta}11/{Delta}11p53+/– do not contain the full length of Brca1, which is the major functional form of Brca1, it provides an excellent model for studying functions of Brca1 in development and tumorigenesis.

BRCA1 interacts with many proteins that function in multiple biological pathways (15,23). Recent studies indicated that proteins involved in DNA damage repair play an essential role in life span determination, as many mutant mice that carry targeted disruption of genes involved in DNA damage repair exhibit premature aging (24–26). Our data also indicated that Brca1{Delta}11/{Delta}11 mutation in mice results in genetic instability leading to ATM/CHK2/p53-mediated premature aging and tumorigenesis (18,19). This finding further extends the genomic instability as an initiating factor in aging. Consistent with this, human genetic disorders caused by mutations in genes that are essential for maintaining genetic integrity, including Bloom’s, Werner’s and Rothmund–Thomson syndromes, exhibited positive association between cancer predisposition and/or premature aging (27–30).

The observation that incidence of cancer increases with age in human and laboratory animals suggests that aging is an important factor for tumorigenesis (30,31). Abnormal organism homeostasis is known as an important pathogenic factor for age acceleration and cancer susceptibility. Stem/progenitor cells in somatic tissues control homeostasis, whereas self-renewal and differentiation of these cells are precisely regulated by genetic and environmental factors to avoid premature aging and neoplasm in renewable organs. Alteration of homeostasis and additional oxidative stress may contribute to progression of the carcinogenesis and aging processes (32,33). It is believed that DNA damage mediated by reactive oxygen species (ROS) contributes to the etiology of human cancer and aging, whereas augmented radical generation and/or impaired antioxidant enzyme may be important in affecting life span and neoplastic transformation.

To investigate the role of Brca1 in regulation of organ homeostasis and susceptibility of Brca1 deficiency to environmental stress-induced tumor formation, we studied the Brca1{Delta}11/{Delta}11p53+/– mice with a focus on organs, which contain highly renewable epithelium and are frequently targeted by environmental damage. Our data reveal an important tumor suppressor function of Brca1 in the development of esophagus and forestomach cancers and provide a link between the environmental and genetic factors in carcinogenesis and aging.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Mice and mouse embryonic fibroblast cells
Brca1{Delta}11/{Delta}11 embryos and Brca1{Delta}11/{Delta}11p53+/– mice were generated as described previously (16). Mouse embryonic fibroblast (MEF) cells were derived from E14.5 embryos generated from intercrosses of Brca1+/{Delta}11p53+/– mice. MEF cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum.

BrdU labeling of mice
Bromodeoxyuridine (BrdU) (100 mg/kg body wt) was injected intra-peritoneally into mice. The mice were killed 3 h after injection.

Histology and antibody staining
For histology, tissues were fixed in the 10% formalin, blocked in paraffin, sectioned, stained with hematoxylin and eosin and examined by light microscopy. K10, K14 and K18 were purchased from Covance (Berkeley, CA). Antibodies Run-X2 and Cyclin-D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), ß-catenin was purchased from BD Biosciences and P-Akt was purchased from Cell Signaling (Beverly, MA). c-Myc was purchased from UPSTATE Cell Signaling Solutions. Detection of primary antibodies was performed using the ZYMED HistomouseTM SP kit according to the manufacturer’s instructions.

Carcinogen treatment
N-amyl-N-methylnitrosamine (MNAN) was purchased from Toronto Research Chemicals (2 Brisbane Rd., North York, Ontario, Canada), and was dissolved in drinking water weekly to achieve the desired concentration. Black bottles filled with drinking water containing 10 p.p.m. of MNAN were fed to 2-months-old Brca1{Delta}11/{Delta}11p53+/– and Brca1+/{Delta}11p53+/– mice for 8 weeks. Mice were then maintained without further treatment for an additional 7 weeks and killed at experimental week 15–25.

Quantitation of cellular ROS
Intracellular levels of ROS were assayed using the fluorescent indicator 2',7'-dichloro dihydrofluorescein diacetate (CM-H2DCFDA, Molecular Probes (Carsbad, CA, USA)). Cells were stained with 1 mg/ml of the indicator in serum-free medium for 30 min, washed, trypsinized and then analyzed immediately. Analysis was carried out using the FACScan flow cytometer.

Paraquat experiments
The 3- to 4-month old Brca1+/{Delta}11p53+/– mice and Brca1{Delta}11/{Delta}11p53+/– mice were injected intra-peritoneally with 70 mg/kg paraquat in phosphate-buffered saline (n = 40).

Reverse transcription–polymerase chain reaction analysis
Total RNA was extracted from the esophagus of mutant and control mice. Reverse transcription reactions were carried out by using the first-strand cDNA synthesis kit (Roche, Cat. 1 483 188, Indianapolis, IN). One microgram of total RNA was used as template for each reaction. For polymerase chain reaction, the samples were heated to 94°C for 2 min, run through 22–31 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 10 min and then stopped at 4°C. Primers for Redd1 are F: 5'-GAGCCCTGCGGCCTTCGG-3' and R: 5'-GGAGTACAGTTTTTTCTT-3'. Primers for glyceraldehyde-3-phosphate dehydrogenase are F: 5'-ACAGCCGCATCTTCTTGTGC-3' and R: 5'-TTTGATGTTAGTGGGGTCTCGC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Brca1{Delta}11/{Delta}11p53+/– mice display altered forestomach and esophagus homeostasis
Aging is characterized by a gradual decline in organ functional reserves, reducing their ability to maintain homeostasis. To investigate whether Brca1 is involved in maintaining organ homeostasis, we performed histological analysis of young and aging mice, with emphasizes on organs containing highly renewable epithelium. Histological examination of 6-month-old Brca1 mutant mice revealed marked hyperkeratosis in the forestomach and esophagus (Figure 1B and D) compared with age-matched control mice (Figure 1A and C). BrdU-labeling assay detected remarkably increased proliferation in basal layers of forestomach and esophagus of mutant mice (Figure 1F and H) but not in controls (Figure 1E and G). To characterize the pathological changes, we stained epithelial cells using antibodies to keratin 14 (K14) and 10 (K10). Our analysis showed that K14 was weakly expressed in the basal epithelium layer of the control mice (Figure 1I and K) and K10 was not expressed (Figure 1M and O). In contrast, the K14-positive layer in the forestomach and esophagus in Brca1{Delta}11/{Delta}11p53+/– mice had undergone significant expansion (Figure 1J and L). A new layer of K10-positive cells also appeared in the mutant mice (Figure 1N and P). These data indicated that Brca1 deficiency resulted in marked hyperkeratosis and transdifferentiation of epithelium in the forestomach and esophagus. It is important to note that we have recently examined skin and gut epithelium of the Brca1{Delta}11/{Delta}11p53+/– mice and found that Brca1 deficiency resulted in epithelium hyperplasia of these organs (19). Thus, although Brca1 is involved in maintaining renewable organ homeostasis, it has different effects on different organs/tissues.


Figure 1
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  		Fig. 1. Loss of forestomach and esophagus homeostasis in Brca1 mutant mice. (A–D) Hematoxylin–eosin staining of the forestomach (A and B) and esophagus (C and D) of 6-month-old Brca1+/{Delta}11p53+/– (Co) and Brca1{Delta}11/{Delta}11p53+/– (Mt) mice. (E–H) BrdU-labeling of the forestomach (E and F) and esophagus (G and H) of 6-month-old control and mutant mice. (I–P) K14 (I–L) and K10 (M–P) staining of 8-month-old control and mutant mice forestomach (I, J, M and N) and esophagus (K, L, O and P). Over 20 mutant mice were analyzed and they all showed similar phenotypes. HE: hematoxylin–eosin; Co: control and Mt: mutant.

 
Spontaneous papilloma and carcinoma formation in forestomach and esophagus of Brca1{Delta}11/{Delta}11p53+/– mice
Hyperkeratosis and hyperplasia are rarely observed in normal mice. To investigate whether the ‘loss of homeostasis’ is age dependent, we monitored a cohort of mutant and control mice ranging from 1 to 12 months of age. As summarized in Figure 2 and supplementary Table I and II (available at Carcinogenesis Online), the esophagus and forestomach of the mutant mice were morphologically normal at young ages (Figure 2C and D) in comparison with those of control mice of all age-matched groups analyzed (Figure 2A and B and data not shown). Histological analysis also did not reveal an obvious difference between mutant and control mice (supplementary Figure 1A–D, available at Carcinogenesis Online). This observation suggests that the abnormalities observed in the mutant esophagus and forestomach are unlikely caused by an intrinsic defect associated with the absence of Brca1 full-length form. Notably, our analysis detected hyperplasia and metaplasia in 6-month-old mutant mice (supplementary Figure 1E and F, available at Carcinogenesis Online), dysplasia and carcinoma in situ in 8-month-old mutant mice (Figure 2E and F and supplementary Figure 1G and H, available at Carcinogenesis Online) and invasive carcinoma in 8- to 12-month old mutant mice (Figure 2G and H and supplementary Figure 1I and J, available at Carcinogenesis Online). No such histological abnormalities were detected in 12-month-old control mice (data not shown).


Figure 2
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  		Fig. 2. Pathological progression of esophagus and forestomach in Brca1{Delta}11/{Delta}11p53+/– mice. Photograph of 4- to 12-month old control and mutant mice esophagus (A, C, E and G) and forestomach (B, D, F and H). FS: forestomach; GS: glandular stomach; Co: control and Mt: mutant. Arrows point to esophagus and arrowheads point to abnormal areas of forestomach.

 
Involvement of p53 in esophagus and forestomach tumorigenesis in Brca1{Delta}11/{Delta}11p53+/– mice
Our previous study indicated that Brca1{Delta}11/{Delta}11p53+/– mice undergo premature aging due to p53 activation (18,19). Mutations of p53 are also frequently found in human esophagus cancers (34). To investigate whether p53 is involved in esophagus and forestomach abnormality and tumorigenesis in the Brca1{Delta}11/{Delta}11p53+/– mice, we examined p53 expression using immunohistochemical staining. Our data revealed that the total p53 protein levels in Brca1{Delta}11/{Delta}11p53+/– mice was higher than that of the control mice (Figure 3A and B), suggesting that the remaining wild-type (WT) allele of p53 in these mice was activated due to the absence of full-length Brca1. Thus, it is possible that Brca1 deficiency predisposes esophagus cancer formation whereas activation of p53 retards this process. To investigate this, we deleted the remaining copy of p53 allele and examined their esophagus and forestomach. Because all Brca1{Delta}11/{Delta}11p53–/– mice died of lymphoma after 4 months of age (17–19), we could only study Brca1{Delta}11/{Delta}11p53–/– mice that were 4 months of age and younger. Our study revealed that ~50% of Brca1{Delta}11/{Delta}11p53–/– mice exhibited hyperkeratosis and hyperplasia in forestomach and esophagus (supplementary Table I and II, available at Carcinogenesis Online), whereas these abnormalities were not detected in the Brca1{Delta}11/{Delta}11p53+/– mice at this stage of development. This observation suggests that p53 plays an important role in inhibiting Brca1-related tumorigenesis in the esophagus and forestomach. Consistent with this observation, our analysis by immunohistochemical staining revealed the absence of p53 expression in the esophagus and forestomach tumors of Brca1{Delta}11/{Delta}11p53+/– mice (Figure 3C).


Figure 3
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  		Fig. 3. Abnormal responses to pathological and physiological stress of Brca1{Delta}11/{Delta}11p53+/– mice. (A–C) Immunohistochemical staining of p53 in the esophagus of mutant (A) and control (B) mice and tumors (C). (D) Reverse transcription–polymerase chain reaction analysis of Redd1 gene expression in the esophagus of four control and four mutant mice. (E) Flow cytometric analysis of intracellular hydrogen peroxide using 2',7'-dichlorodihydrofluorescein diacetate assay content measured as the 2',7'-dichloro-fluorescein fluorescence intensity in passage 2 control and mutant MEFs (n = 3). (F) Percent of survivor mice (n = 40) after paraquat treatment. (G) Hematoxylin–eosin sections of livers of mice before and after paraquat treatment. Co: control and Mt: mutant.

 
Brca1{Delta}11/{Delta}11p53+/– mice were hypersensitivity to oxidative stress
Absence of Brca1 results in p53 activation that leads to premature aging of the Brca1{Delta}11/{Delta}11p53+/– mice (18,19). The increased levels of p53 in esophagus of Brca1{Delta}11/{Delta}11p53+/– mice prompted us to study p53 downstream genes that may play a role in oxidative stress and ROS production. It was shown previously that the activation of p53 results in the increased expression of Redd1, which may be involved in oxidative stress and ROS induction (35). Our reverse transcription–polymerase chain reaction analysis indicated that the mutant esophagus exhibited increased expression of Redd1 (Figure 3D). To determine whether inactivation of Brca1, indeed, induces ROS production, we compared ROS levels of mutant and WT MEFs by using the indicator dye, CM-H2DCFDA. In the mutant MEF cell, the ROS level is much higher than that in control cells (Figure 3E). Our data also indicated that the mutant MEFs were more sensitive to H2O2 damage-induced apoptosis than WT MEFs were (data not shown). This observation suggests that the absence of Brca1 triggers DNA damage, leading to p53 activation, which may partially contribute to the increased ROS in the mutant mice.

The mouse and fly mutants with enhanced resistance to oxidative stress exhibit increased life span (36,37). Therefore, we injected paraquat, an herbicide that induces formation of ROS into Brca1{Delta}11/{Delta}11p53+/– mice of 3–4 months of age, a time point prior to the onset of aging, to examine their response to oxidative stress. The mutant mice showed increased sensitivity to paraquat-induced severe lethality (Figure 3F) and organ damage (Figure 3G) than did the control mice. These results suggest that the premature aging of Brca1{Delta}11/{Delta}11p53+/– mice may be caused by accumulated ROS and sensitized response to oxidative stress.

Brca1{Delta}11/{Delta}11p53+/– mice exhibited increased susceptibility to MNAN carcinogenesis
Oxidative stress may accelerate aging and increase risk of tumor formation (38). To investigate whether inactivation of Brca1 increases susceptibility to carcinogen-induced carcinogenesis, we treated Brca1 mutant and control mice with methyl-N-amylnitrosamine (MNAN), a carcinogen that specifically induces esophageal tumors in mice and rats, probably due to the cytochrome P450 activation (39,40), leading to oxidative damage of protein and DNA. Remarkably, Brca1 mutant mice had significantly elevated tumor incidence compared with that of control mice (Table I and Figure 4A and E). Histological analysis revealed that the majority part of forestomach of control mice had no significant change although focal areas of hyperplasia were developed (Figure 4B–D). In contrast, the Brca1 mutant mice showed marked hyperkeratosis, hyperplasia and squamous cell carcinoma (Figure 4F and G). Consistently, immunohistochemical staining showed that K14 (a marker for keratinocytes) was strongly expressed (Figure 4I) whereas K18 (a marker for luminal epithelial cells) was negative (data not shown). Histological analysis also revealed duct-like and hair follicle-like structures in tumors, which were positive for K6 (Figure 4K). Indeed, K6 is constitutively expressed in the innermost layer of the outer root sheath of hair follicles and its expression is also induced in the interfollicular epidermis in response to stressful stimuli such as wounding.


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  		Table I. MNAN-induced tumor formation

 


Figure 4
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  		Fig. 4. Increased susceptibility of Brca1{Delta}11/{Delta}11p53+/– mice to MNAN carcinogenesis. Photograph of the forestomach and esophagus (arrows) of control mice (A) and mutant mice (E) after MNAN treatment. Hematoxylin–eosin staining of normal forestomach (B), hyperplasia and hyperkeratosis of control mice (C and D). Hematoxylin–eosin staining of forestomach hyperplasia and hyperkeratosis (F) and forestomach invasive squamous cell carcinoma (G) of mutant mice after MNAN treatment. (H–K) Immunohistochemical staining of forestomach of control mice (H and J) and mutant mice (I and K) using antibodies to K14 (H and I) and K6 (J and K).

 
Next, we studied molecular events associated with esophagus and forestomach cancers. As shown earlier, all tumors examined (n = 3) exhibited low levels of p53 (Figure 3C), which is consistent with our view that p53 plays an important role in the Brca1-associated tumorigenesis. Our data also revealed increased levels of several oncogenes that have been implicated in the human esophagus and/or stomach cancers, including Cyclin D1 (Figure 5A–C), phosphorylated form of Akt (Figure 5D–F), ß-catenin (Figure 5G–I) and Runx-2 (Figure 5J–L) and c-Myc (data not shown). These genetic alterations provide a better understanding of the etiology and pathogenesis of these cancers.


Figure 5
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  		Fig. 5. Marker analysis of tumors from MNAN-treated Brca1{Delta}11/{Delta}11p53+/– and Brca1+/{Delta}11p53+/– and untreated Brca1+/{Delta}11p53+/– mice. The genotypes of the mice and antibodies used were as indicated (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
Germ line mutations in BRCA1 have been detected in ~50% of familial breast cancer and 90% of familial breast–ovarian cancers (7–9). LOH in a region of chromosome 17q, where BRCA1 resides, was also found to increase risk of several other types of cancers, including prostate, liver, pancreas, stomach and esophagus cancers (4–6,10). However, the number of tumors analyzed in these studies were small and the increased risk might be attributed to some extent to misclassification of ovarian cancer (41) or misreported metastases from other sites (42). Studying the Swedish Family-Cancer Database that contains 944, 723 families involving >10 million people in at least three generation, Bermejo et al. (11) confirmed that BRCA1 mutation or BRCA2 mutation carriers had increased risk of ovarian, pancreatic, prostate or stomach cancers. In this study, we now demonstrate that the absence of full length of Brca1 results in the hyperkeratosis in the esophagus and forestomach, leading to the spontaneous esophagus and forestomach cancer formation in aging mice. The mutant mice also suffer increased susceptibility to MNAN-induced cancer formation. These data established an essential role of Brca1 in esophagus and forestomach cancer formation in mice.

However, a caution needs to be paid when interpreting this data because the frequency of spontaneous esophagus and forestomach cancer formation in the Brca1{Delta}11/{Delta}11p53+/– mice is much lower than that of mammary tumors (16). Moreover, due to a long latency of esophagus and forestomach cancers, which mainly are developed in the Brca1 mutant mice that are >1 year of age, they were usually undetectable or ignored when the animals were killed due to mammary tumors that primarily developed within 1 year of age. Nonetheless, the observation that esophagus and forestomach cancers develop at a much lower frequency and longer latency than those of mammary tumors in the same animals suggests that Brca1 may serve as a modifier for the formation of esophagus and forestomach cancers. Interestingly, a recent study revealed that the cofactor of BRCA1 (COBRA1) is over-expressed in 84% (60/70) of primary upper gastrointestinal adenocarcinomas (43). These observations suggest that BRCA1 may play a role in the upper gastrointestinal adenocarcinomas through its interacting proteins and/or downstream genes.

Of note, a previous investigation indicated that p53 mutant mice are sensitive to MNAN-induced esophagus cancer (44). Mounting evidence reveals a close relationship between BRCA1 and p53 in tumorigenesis. It has been shown that ~90% of human breast cancers derived from individuals with BRCA1 mutations carrying p53 mutations whereas p53 mutations are only found in less than half of sporadic breast cancers (45,46). Furthermore, in mouse model carrying mammary-specific disruption of Brca1, the deletion of one WT p53 allele (Brca1Co/CoMMTV-Crep53+/–) significantly accelerates the tumorigenesis (47,48). In this study, we have made the following observations. First, we showed that p53 protein levels are initially increased in the epithelium of the esophagus and forestomach and diminished in the tumors (Figure 3A–C). Second, our data indicated that deletion of the remaining WT p53 allele in the Brca1{Delta}11/{Delta}11p53+/– mice accelerates esophagus and forestomach hyperplasia (supplementary Table I and II, available at Carcinogenesis Online). Finally, like p53 mutant mice, our Brca1{Delta}11/{Delta}11p53+/– mice are also highly sensitive to esophagus tumors induced by MNAN (Table I). These data further strengthen the link between Brca1 and p53 in tumor suppression.

We have recently demonstrated that the genetic instability triggered by Brca1 deficiency is responsible for activation of ATM-Chk2-p53 signaling (19). It is conceivable that the activation of the ATM-Chk2-p53 signaling, so-called DNA damage response (DDR) (49,50), in turn, acts as a natural barrier that blocks cell proliferation and induces apoptosis in cellular level. At organism level, it may also cause abnormal organ homeostasis and premature aging with the gradual decline of the function of multiple organs. Thus, the activation of DDR serves as a double-edged sword that prevents neoplastic transformation at the expense of normal life span.

Why do Brca1 animals develop tumors while they are aging if the DDR is efficient enough in eliminating mutant cells? Indeed, many mouse mutants and human genetic disorders caused by mutations in genes that are essential for maintaining genetic integrity suffer both increased cancer predisposition and premature aging (30). These observations indicated that some mutant cells must have acquired the ability to break the cellular defense of DDR and develop further into full-grown tumors. Our finding that the tumor cells exhibited elevated levels of multiple oncogenes may provide an answer to this question. The genetic instability triggered by the absence of Brca1 full-length form may eventually cause genetic alterations, including LOH of p53 and activation of oncogenes that promote malignant transformation process.

In sum, we have studied a role of Brca1 in esophagus and forestomach cancer formation in our Brca1{Delta}11/{Delta}11p53+/– mouse model. Our data reveal a multi-step progression of tumorigenesis from normal epithelium to invasive carcinoma (supplementary Figure 2, available at Carcinogenesis Online). The Brca1 mutant mice exhibit abnormal organ homeostasis, increased ROS production and are more sensitive to oxidative stress due to p53 activation. Genetic instability as a result of Brca1 deficiency also increases susceptibility to spontaneous and carcinogen-induced tumor formation upon oncogenic stimulation. These data link the environmental and genetic factors together with carcinogenesis and aging. They also provide new insights into genomic injury in organism maintenance and tumorigenesis associated with Brca1 deficiency.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary Figures and Tables can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
We thank S.G.Brodie, R.Bachelier, W.Qiao and C.Li for their technical assistance and critical discussion of this work. This research was supported by the Intramural Research Program of the National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, USA.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received January 3, 2007; revised March 1, 2007; accepted March 2, 2007.


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