Indian Journal of Experimental Biology Vol. 39, May 2001 , pp. 391-400

Review Article

Implication of BRCAl gene in breast cancer

S K Kachhap* &. S N Ghosh

Cell Biology Division, Cancer Research Institute, Tata Memorial Centre, Parel, Mumbai-400012, India.

Breast cancer susceptibility gene (BRCAJ) is known to be responsible for hereditary breast and ovarian cancer. This gene is highly penetrant conferring a risk for 0.92 by the age of 70. Germline mutation in this gene leads to sus­ceptibility to breast and ovarian cancer, with a genotype phenotype correlation. Frequency of mutations of this gene in normal population of breast cancer is low suggesting that the effort of primary screening for BRCAJ gene should be restricted to only familial cases with a strong history of breast and ovarian cancer. Recent studies indicate that BRCA J is a tumor suppressor gene responsible for both normal development and carcinogenesis of the breast. Normal func­tion elucidated so far, reveal BRCAJ to be a multifunctional protein involved in DNA repair, cell cycle regulation and transcription. There is circumstantial evidence that gene interacts with p53, a protein involved in cell cycle control , DNA repair and apoptosis.

Cancer, the characteristic of uncontrolled growth is always accompanied by alterations in normal path­ways of differentiation and development. Repro­ductive endocrine factors such as menarche, meno­pause and age at first full term pregnancy which control breast development also influence breast cancer risk, supports the hypothesis that mammary gland development and mammary gland carcino­genesis are fundamentally related.

Epidemiological data indicate that incidence of breast cancer differs markedly in different coun­tries. It is high in North America and North Europe and low in Japan I. This indicates that breast cancers arise due to complex interaction between environ­mental factors and genetic factors. Among the envi­ronmental factors both endogenous and exogenous hormones and fat intake play a significant role. While among genetic factors susceptibility genes either common with less penetrant and rare with highly penetrant genes are found to be associated.

A variety of epidemiological and animal studies suggest that hormones play a critical role in both mammary gland development and mammary carci­nogenesis. An increased risk of breast cancer is

*Present address: John Hopkins Oncology Centre, John Hopkins School of Medicine, Bunting-Blanstein. Cancer Research Building, 1650 Orleans St.lRM132, Baltimore. MD 21231-1006, USA.

found among women with early onset of menarche and late menopause. It is reported that environ­mental estrogen or xenoestrogen have the ability to interfere with several physiological process that are normally estrogen regulated which could result in alteration in reproduction and susceptibility to breast cancer. Several investigators have reported that an excess risk of breast cancer is associated with oral contraceptive use in young women with family historl-s. Colditz6 has summarized the dif­ferent sources of data concerning the relationship of both endogenous and exogenous hormones to the etiology of breast cancer and concluded that evi­dence is now sufficient to infer that estrogen is a cause of breast cancer. However, a mechanism by which estrogen affects proliferation of certain target cells is not fully understood. Several evidences in­dicate that they can induce expression of cell cycle regulatory genes, that directly suggests a primary role of estrogen receptor (ER) in regulating cell cy­cle progression7

• Higher androgen levels have been reported in Caucasian women (high risk group) who showed familial clustering. Recent studies also in---­dicate that serum concentration of bioavailable es­tradiol and free testosterone8 and plasma prolactin9

are associated with future risk of breast cancer. Factor for increase of insulin concentration of insu­lin in plasma is also found to be associated with increase in risk of premenopausal but not post­menopausal breast cancer.

392 INDIAN J EXP BIOL. MAY 2001

Dietary intake of fats in particular (0-6 polyunsatu­rated fatty acids have been shown to increase breast tumor incidence in rodentslO,ll. On the contrary an intake of (0-3 polyunsaturated fatty acids have been shown to decrease tumor incidence l2

. Thus the type of fat intake can decide the outcome of the disease and a ratio of (0-6/(0-3 fatty acids might be a better indicator of cancer risk than the amount of fats 13 . Hilakivi -Clarke et al. 14 have reported that consumption of diet high in fat (primarily in the form of n-6 polyunsatu­rated fatty acid, linoleic acid) during pregnancy in­creased risk of developing carcinogen-induced mam­mary tumors, possibly by increasing the level of cir­culating estrogen.

Since 10-15% of breast cancer is familial, suscepti­bility to the disease is inherited. Lynch et al. 15 have estimated that among breast cancer patients about 15-20% patients have a positive history of breast cancer in an immediate family member, mother, sister or daughter. These cases are characterized by early onset and bilateral disease. Daughters, who have a mother with bilateral 16,17 or early onset of breast cancer have more risk of breast cancer than do daughters of mother with unilateral or late onset of breast cancer. Common environemntal exposure may also lead to familial clustering of the disease l7,18. This complicates the recognition of families in which clustering is due to common predisposing genes with low penetrance. Number of breast cancer cases caused by low pene­trance genes is however considered to be much higher than the number of hereditary cases caused by muta­tion in higher penetrance genes. A number of low and high penetrance genes have been discovered in breast cancer. Polymorphism in CYPIAI, NATl and CAG repeat in AR gene are among the low penetrance gene responsible for breast cancer, while pSJ, BRCAI, BRCA2, A TR and A TM are high penetrance gene that account for majority of hereditary breast cancerl9.

In 1992, Mary Claire King and her co-workers have identified a 8cM region in 17q12-q21 by linkage analysis in 23 breast cancer families using 11 poly­morphic marker20

. This region is linked to breast can­cer families that shows an early onset of the disease. The gene has been named as breast cancer suscepti­bility gene (BRCA 1) and found to be responsible for breast cancer in about half of all families with a dominant predisposition of the disease and between 80 and 90% of families in which multiple cases of both breast and ovarian cancer occurs. These muta­tions are highly penetrant, conferring a risk of about 90% of either breast or ovarian cancer by the age of

70 years21. Later, Miki et al. 22 have identified a strong candidate for BRCA 1 gene by positional cloning. Pre­disposing mutations have been identified in 5 of 8 kindreds in family linked to BRCA I. The gene has been found to be expressed in various tissues includ­ing breast and ovaries with maximum expression in thymus and testis. It encodes a predicted protein of 1863 amino acids. The protein contained a zinc finger domain in its amino terminal, indicating its role as a transcription factor. BRCAI is encoded by a gene which has 24 exons that spans a 81-kb genomic length.

Since in the breast connection between carcino­genesis and development is illustrated by the exis­tence of endocrine risk factors for breast cancer that are related to timing of normal development events such as menarche, menopause and age at full 151 term pregnancy, and if BRCAI , the susceptible gene for breast cancer is involved in breast cancer, then func­tion of this gene should alter during all these devel­opmental phases. This review illustrates hormonal regulation of BRCA 1 gene and its function at the time of development, growth and differentiation of mam­mary gland and how in the absence, mutations or down regulation of expression of this gene promotes breast carcinogenesis.

Germline mutations of BRCAI-The proportion of families attributable to BRCA 1 mutationls vary widely among different populations23. BRCAI gene does not seem to reveal a hotspot. Mutational studies in various populations differing ethnically and geographically have demonstrated BRCAJ mutations to be scattered throughout the length of the gene. However, majority of the mutations lie on exon 11 which is expected as this exon codes for 60% of the BRCA 1 protein. Using various techniques to identify BRCA 1 mutations which include the protein truncation test, single strand conformation polymorphism, heteroduplex analysis and direct sequencing, various researchers have iden­tified mutations throughout the length of the gene. Of all the mutations detected in the gene, about 90% re­sult in truncation. BRCA 1 mutation seems to be com­mon in Russian breast and/or ovarian cancer families (70%). Two alleles (5382insC and 4153de1A) are the most common in this population. Of these, 5382insC is also common among European population while 4153del is unique to the Russian. The second popula­tion that shows the highest BRCA 1 mutations are the Ashkenazi Jewish families of Israel. One of the most common alleles present in this population is 185de1AG, a founder mutation in majority of the

KACHHAP & GHOSH: IMPLICATION OF BRCA I GENE IN BREAST CANCER 393

Ashkenazi Jews. Almost every mutation analyzed in Italian breast cancer families seems to be unique and does not have a founder effece3

. Studying the propor­tion of 185delAG in various populations gives an in­sight about its origin and propagation along with the migration of population. The age of 185delAG muta­tion was estimated at 46 generations or 1000-1500 years24. However the Iraqi, Iranian and Ashkenazi Jewish families share the same haplotype indicating that mutation predates the separation of the commu­nities and is ~ 2000 years old. Many European muta­tions have been found in United States or Canada in­dicating the migration of this population to these ar­eas. BRCA I is estimated to be responsible for 20 to 25% of high risk families in Britain, France, Scandi­navia and Hungary and <20% of high risk families in Holland, Belgium, Germany and Norwal3

.

BRCAI mutations are by far rare in Japanese population with every mutation being unique to that country25. Further studies on Asian population should throw a light of the population specific mutations among this group. From a study comprising 33 breast and breast-ovarian familial cancer cases of Indian ori­gin, we have observed a frequency of 12% germ line mutations in Indian women. The mutations are unique to our population and have not been reported else­where26

.

Earlier studies on BRCAI mutation prevalence suggest that about half of breast cancer families are attributable to BRCAl mutations. However, recent analysis has revealed that actual number of BRCA 1 mutations in high risk families might be as low as 12.8 to 16% (Ref. 27, 28). Our findings are in accor­dance with other ethnic population. The reduced prevalence may be explained as early studies have involved families with large numbers of breast and ovarian cancer cases in comparison to more repre­sentative mixture of families with breast cancer alone and with both breast and ovarian cancer as reported in recent studies. The studies conclude that widespread screening of BRCAl is unwarranted and the efforts should be concentrated on high risk families with breast and breast ovarian cancer where BRCA I muta­tion is more prevalent.

Somatic mutations oj BRCA1~omatic mutations of BRCA I in sporadic breast tumors are yet to be de­tected. Although loss of heterozygosity (LOH) at BRCAl locus has been reported with varying fre­quency with the loss of one allele and the presence of mutation in the other allele has not been detected29

-31

,

few somatic alterations of BRCAI have been reported

in sporadic ovarian tumors (5/29) selected for LOH on 17q (Ref.32). One reason may be that in sporadic breast cancer BRCAI mutation could be present in a regulatory region other than the coding sequence. An­other possibility is that BRCAI expression is tempo­rally regulated by hormones and may function during puberty and this could possibly explain why germline mutation in the gene predisposes one for early onset breast cancer. Another reason could be that BRCAl may be regulated at the transcriptional level in spo­radic tumors. This hypothesis is supported by the findings of Thompson et al.33 who have reported lower levels of BRCAI mRNA in sporadic tumors in comparison to normal tissue.

Genotype and phenotype correlation and Jactors affecting BRCA1-Mutation analysis of BRCAI seems to indicate a phenotypic correlation. In a study of 60 families with a history of breast and/or ovarian cancer twenty-two different germ-line mutations have been detected34

. A significant correlation between location of mutation in the gene and the ratio of breast to ovarian cancer incidence within each family has been observed. Mutations at 3' end of the gene have been shown to be associated with a lower proportion of ovarian cancer. Their findings indicate that this region of the mutant BRCAl may have domain that might retain an active function in ovarian cell but not in the breast epithelium.

It has been shown that tumors from BRCA I mutant carriers are often histologically aggressive, steroid receptor negative, DNA aneuploidy, highly prolifera­tive as well as rp53 positive and ERBB2 negative by immunostaining3

S-39

• Eisinger et al. 36 have reported that there is an existence of two subgroups of BRCAI associated breast cancer families. The first group (78%) is composed of caf';:S with high proliferation rate and the second group (22%) is composed of cases with low proliferating breast cancer. To test for the existence of two types of BRCA I alleles, differently affecting breast tumor growth, Sobol et al.40 have analyzed the distribution of mitotic index, grade of tumor, and matching them with the location of germ­line mutation on breast cancer cases from 20 families. They have observed a prevalence of highly prolifera­tive tumors when the mutation occurs in two terminal domains (amino and carboxyl terminals) of BRCAI protein. This result therefore suggests that based on the site of germline mutation one might predict the development and behaviour of the tumor. Serry et a1.41

have identified an association between low levels of BRCAl expression and acquisition of distant metasta-

394 INDIAN J EXP SIOL, MAY 2001

sis in sporadic disease and suggested that a suppres­sion of BRCAI has a role to play in the progression of significant fraction of sporadic cancer and thus it can be used as a prognostic marker for the disease. How­ever, Marcus et aI.38 have reported that compared with other hereditary breast cancer (HBC), there is lower rate of recurrence in BRCAI related HBC group which seems to be more paradoxical because their study as well as other studies have shown that BRCAI related cancers are more likely to be aneuploid, estro­gen negative and highly proliferative, a feature asso­ciated with poor prognosis. According to these authors, these paradoxical phenomena can be ex­plained based on the sensitivity of these tumor cells and suggest that perhaps BRCAI linked tumors are more chemosensitive or radiosensitive and therefore have higher cured fraction as has been reported for other highly proliferative breast cancer42

• 43.

It is evident that despite of high penetrance of BRCAI, the development of tbe disease in carriers are not always expressed. Development is now known to be influenced by several other low risk genetic and environmental factors44

• Substantial variation exists in the age at which breast cancer is diagnosed in BRCA 1 mutation carriers. This suggests that genes other than BRCAI may modify BRCAI- associated age specific risk for breast cancer. Association studies using polymorphisms found in glutathione-S-transferase suggest that genetic modifiers of BRCAI penetrance do exist45

. Recently, Rebbeck et al.46 have studied the effect of a CAG repeat length polymorphism found in exon 1 of androgen receptor gene (AR-CAG) on age at diagnosis of breast cancer in known muta­tion carriers. The androgen receptor gene functions as a ligand dependent transcriptional activator in re­sponse to androgens and is thought to be involved in breast tumor growth and progression. The authors have compared AR-CAG repeat lengths in affected and unaffected women with germline BRCA 1 muta­tion. They have shown that carrier women who have at least one BRCAI allele with 28 or more CAG­repeats have an increased risk of cancer. The data suggest that women who carry larger repeat units are more prone to develop the disease than women who have shorter repeat lengths. We have compared free androgen levels in familial breast cancer individuals screened for BRCA I mutation with sporadic and con­trol individuals and found it to be higher in familial cancer cases and significantly higher in individuals carrying BRCA I mutations suggesting an increase in free androgen level and familial clustering26

• Besides,

other factors do exist that can lower BRCAI expres­sion in sporadic tumors. Several reports have indi­cated that unsaturated fatty acid like linoleic acid can regulate the expression of i 3 gene and H-ras onco­gene and estradiol that can modulate BRCA 1 gene expression thereby breast carcinogenesis. We have observed increased proliferation and anchoreage in­dependent growth with concomitant down regulation of BRCAI expression in MCF-7 cells treated with li­noleic acid and estradiol compared to untreated con­trols .Our results suggest that BRCAI expression is down regulated during breast carcinogenesis47

. Fur­thermore, it has been reported that such as hyper­methylation of BRCAI gene promoter could also be responsible for lower expression of BRCAI gene. Methylation is the main epigenetic modification in humans, and changes in patterns of methylation play an important role in the tumorigenesis. In particular, hypermethylation of normally unmethylated CpG is­lands located in the promoter regions of many tumor suppressor and DNA repair genes, such as pI6, pIS, Rb, VHL, E-cadherin, GTSTPI, and MLHI, is associ­ated with its loss of expression in cancer cell lines and primary tumours. Several reports suggest that aberrant methylation of BRCAI could occur in breast carci­noma48

-51

• Esteller et al 51 have reported that BRCAI promoter methylation shows an unusual distribution among histopathologic types of breast carcinoma. BRCAI promoter hypermethylation has been seen to be more frequent in medullary and mucinous sub­types of breast cancer.

Functions of BRCAI-8ince cloning of BRCAI gene, an attempt has been made to describe its possi­ble functions. Another aspect that is intriguing is that though BRCA I is ubiquitously expressed in various tissues with the maximum expression in thymus and testes22

, why is mutation in the gene does not have a pleiotropic effect? Why should it only predispose to breast, ovarian and prostrate cancer? Even though the expression is maximum in the testis and thymus none of the reported cases ever shows a thymic lymphoma or a testicular carcinoma. The answers to such ques­tions can only be elucidated by knowing the function of BRCAI and understanding various factors that gov­ern the expression of the gene.

Once the role of BRCAI as a tumor suppressor gene is established in familial tumors by LOH and mutation analysis and in sporadic tumors by tran­scriptional down regulation33 efforts have been fo­cussed on the discerning the function of BRCA I. Ear­lier studies using knock out mice reveal that homozy-

KACHHAP & GHOSH : IMPLICATION OF BRCAI GENE IN BREAST CANCER 395

gous mice lacldng BRCAl gene die within 10 and 13 days of embryonic development, suffering from a va­riety of neuro-epithelial defects52. Mice having het­erozygous BRCAl gene do not show any evidence of cancer52

, 53. This indicates that the gene is essential for early embryonic proliferation and development. Sub­sequently, BRACl has been shown to be widely ex­pressed in developing embryos and has a preference for replicating cells as it is associated with terminal differentiation of the ectoderm and mesoderm derived tissues54

.56

. Expression of BRCA 1 in testis is restricted to meiotic germ cell and in mammary tissue to alveo­lar and ductal epithelial cells. Since carriers of BRCAl mutant alleles exhibit an increased suscepti­bility to carcinoma of the breast, as well as ovary, colon and prostate, it is likely that these genes play an important role in regulation of growth and differen­tiation of epithelial cells, particularly those which are hormone responsive57

• BRCAl expression is enhanced considerably during pregnancy and lactation and de­clines after parturition54

• 55 indicating that expression is hormonally induced. Indeed steroid hormones like estrogen and progestrone as well as peptide hormones are shown to enhance BRCAl gene expression58

.60

.

BRCAl mRNA and proteins are highly expressed during late G 1- early S phase of the cell cycle6J

• 62. Cloning of BRCAl reveals a RING finger near the amino terminal (amino acids 1-112), the only se­quence homologous to a group of protein that binds DNA. RING fingers are zinc binding domains that show a region rich in cystines and histidine that meditate protein - protein or protein DNA interac­tions. Many proteins that form a macromolecular complex have such RING finger. They are also pres­ent on gene that facilitate ubiquitination, a common mechanism that results in degradation of proteins. Thus, BRCAl may function as one of the proteins that mediate ubiquitination and a loss of RING finger as a result of mutation may cause certain proteins to ac­cumulate thereby bringing about a disregulation of

l ·.{" . 63 pro heratlOn . Other clues to the function of BRCA 1 came from a

combination of different techniques like yeast two­hybrid system, immunoprecipitation and immunohis­tochemical localization that identified proteins physi­cally linked to BRCAl. The RING finger domain of BRCAl binds to the protein BARDl (BRCA] associ­ated RING domain) which is a de-ubiquitinating en­zyme. This association may probably regulate the ubiquitination pathway of BRCA] (Ref.64). At C ter­minal, BRCA] has a weakly conserved region BRCT

(BRCAl C-terminal) a motif that is shown to be pres­ent in pS3 binding protein like 53BPI and other pro­teins involved in DNA repair or metabolism65.

BRCAl immunostaining reveals discrete, nuclear foci during S phase of cell cycle. These foci have been shown to contain BARD 1 and RAD51.Eukaryotic RAD51 proteins are homologous of bacterial RecA gene and are reauired for recombi­nation during mitosis and meiosis . Rad 51 mediates double strand exchanges and brings about recombina­tion. BRCAl exon 11 encodes sequence(s) (residues 758-1064) that interact with Rad51. BRCAl-Rad may serve as complexes that particiRate in double strand break repair and recombination .67. BRCAl has also been found to co-localize with Rad50 complexes. Rad50 forms complexes with Mrell and p95-nibrin68

upon irradiation. It functions as homologous recombi­nation, non-homologous end joining, meiotic recom­bination, DNA damage response and telomere main­tenance68

• Formation of irradiation-induced foci posi­tive for Rad50, Mre11 , BRCA] is dramatically re­duced in HCC/1937 breast cancer cells carrying ho­mozygous mutation for BRCAl and is restored upon transfection with wild type BRCAl. These irradiation induced foci are discrete and different from Rad 50-BRCA] complexes, indicating that cells appear to have two distinct BRCA 1 foci. One fraction co­localizes with Rad50 and other co-localizes with Rad51 complexes. These two foci are mutually exclu­sive as cells with both Rad50 and Rad51 complexes are rarely seen. Thus, it appears that BRCA] may have different roles in two foci observed. Rad 50 complex participates in non-homologous end joining or ho­mologous recombination in DNA double strand breaks. In homologous recombination, it is postulated that Rad 50 complex brings about end joining and Rad 51 complex is involved in strand exchange dur­ing a subsequent step. BRCA] may be involved in coupling the two steps69.

Experiments based on response to various DNA damaging agents reveal distinct changes in BRCA] protein. Following treatment of MCF7 cells with hy­droxy urea and a low dose UV treatment BRCAl has been found to have relocated onto subnuclear sites of active DNA replication as evident by co-localization with PCNA, (a marker of replication). BRCA] under­goes specific phosphorylation at S phase which is dif­ferent from known phosphorylation that occurs during Gl-S transition70

• This phosphorylation seems to be dependent on mutation of Ataxia Telangiectasia (ATM) (Ref.71). Thus, BRCA] is engaged in double

396 INDIAN J EXP BIOL, MAY 2001

stranded break repair upon damage by various types of DNA damaging agents. Efficient DNA repair is important because unrepaired DNA leads to chromo­somal breaks, translocation and genomic instability. This is further supported by the fact that BRCAI defi­cient cell lines show an increased genetic instability. Xu et al.72 have used murine embryonic fibroblasts derived from mice with a targeted deletion of BRCAI exon 11 and have shown that the cells have a defec­ti ve G2-M checkpoint accompanied by chromosomal abnormalities, multiple functional centrosomes, une­qual chromosomal segregation, and abnormal nuclear division, leading to aneuploidy. Hsu and White73 have found that BRCA I is associated with gamma tubulin, a component of centrosome, during mitosis. The ob­servation that BRCAI co-localizes to centrosome sug­gests that it may act in a manner similar to other pro­teins that regulate cell cycle and co-localize with cen­trosome such as cyclin A, cyC\in B, cdc 2, and 14-3-3. It is also known that p53 associates with centrosomes and that p53 nullizygous mouse embryonic fibroblasts have a high frequency of amplified centrosome and abnormal mitosis. Retinoblastoma protein co-localizes with the centrosome, but retinoblastoma protein does not affect centrosome amplification. It is proposed that mutations in BRCA 1 might disregulate centro­some duplication and hence chromosome seggrega­tion leading to abnormal chromosomes73. The fact that BRCAI deficient cells and embryos both show a high level of abnormal chromosomes further strengthens the hypothesis that BRCAI gene has a major role in genomic integrity. Our results using clonal cell cul­ture of MCF-7 cell line, that has a low expression of BRCA 1, revealed that these cells have higher number of chromosomal breaks and dicentrics in comparison to Hela cells that have a normal level of BRCAI gene expression. Further, these cells are more sensitive to gamma rays in comparison to T -47D or Hela cells, suggesting a poor repair capacity in these cells. These results indicate that lower expression of BRCAI could also bring about genome wide chromosomal instabil­ity and such cells are more radiosensitive74.

Harkin et al.75 have reported the involvement of BRCAI in apoptosis. By using oligonucleotide arrays and functional assays, this group has shown expres­sion of BRCAI induced GADD45 and c-Jun N­terminal kinase/stress activated protein kinase (JNKlSAPK)-dependent apoptosis . Using a cell line engineered for inducible expression of BRCAl, they have shown an increase programmed cell death. This induction is a result of increase in BRCAI-Epression

through activation of JNKlSAPK, thereby leading to apoptosis. This observation let them to suggest that BRCAI has a role in induction of apoptosis.

There is also increasing amount of evidence that BRCAI may serve as a transcription factor and regu­lates gene expression76. BRCT repeats located at the C-terminus of BRCAI may serve as interacting do-

. . h 53 . 77 78 H . . f mams Wit p protem ' . owever, mteractlon 0

BRCAI with p53. is yet to be established. Neverthe­less, full length BRCA 1 when co-transfected with p53 is shown to induce transcription of p21 and MDM2 genes both of which are activated by p53. BRCAI in­teracts with both RNA helicase and with CtIP as a

f .. 1 I 79 80 M' 3' part 0 transcnptlOna comp ex ' . utatlOns at end of BRCAl, which are found in patients, reduce the ability of BRCAI to form a complex with RNA heli­caseA. The C-terminus of BRCAI (amino acids 1528-1863) when fuses in-frame with DNA binding domain of GAL4 protein induces transcription. Clinically, relevant mutations that truncate BRCAI C-terminus

, .. h .. 77 78 F 181 are mactlve III suc transcnptlOn assays ' . an et a

have shown that BRCAI protein suppresses estrogen dependent mammary epithelial proliferation by inhib­iting ER-alpha mediated transcriptional pathways re­lated to cell proliferation. ER-alpha is one of the two ligand-activated nuclear receptor type of transcription factors through which estrogen elicits its response. This study probably throws a light on functions of BRCAI in mammary epithelium, an estrogen respon­sive tissue 81. BRCA 1 has also been shown to be asso­ciated with the histone deacytylase complex82. His­tone deacytylases are a group of proteins that deacty­late histones thereby modulating their structure and hence the expression of the genes that they bind. BRCAI might repress transcription by recruiting his­tone deacytylase complex to specific promoters, where the complexes deacytylate certain core his­tones, modulating local structure of chromatin and makin~2 DNA less accessible to transcription en­zymes .

The question that arises that BRCAI is a caretaker or a gatekeeper. Cell proliferation is usually regulated by transcription factors for which these are classified as gatekeeper. Similarly, since BRCAI controls cell proliferation, it may be classified as gatekeeper. There are direct evidences to support the proliferation hy­pothesis, such as neutralization of BRCAI gene tran­script by antisense mRNA increases the proliferation rate of benign and malignant cells in culture. Moreo­ver, overexpression of BRCAI in ovarian cancer cells and MCF-7 cells inhibits their growth83. However, the

KACHHAP & GHOSH: IMPLICATION OF BRCAI GENE IN BREAST CANCER 397

repair function of BRCAJ indicates that it can be a caretaker as it maintains the integrity of the genome. Analyzing the sequence of events which occurs as a result of mutation or down regulation will throw some light about its function as a gatekeeper and/or care­taker.

As roles for BRCAJ and BRCA2 in DNA repair and cell cycle check points have been revealed, one para­dox of their biology might be closer to resolution, why BRCAJ and BRCA2 null mice die in early em­bryonic life, whereas, BRCAJ and BRCA2 null breast and ovarian cancer cells · develop tumours. Evidences from conditional knock-out mice suggest that loss of BRCA J in mammary cells leads t9 incomplete prolif­eration, apoptosis and tumor at low frequencies84

• In these mice the additional heterozygous mutations of p53 leads to many more mammary tumors, most of which loose the remaining p53 alleles. Genetic insta­bility caused by the loss of BRCAJ or BRCA2 could trigger mutation including mutations in checkpoint genes such as p53. Mutation in p53 would overcome incomplete proliferation to uncontrolled proliferation and basic growth. Rapid proliferation of breast epi­thelium during puberty and pregnancy would result in a loss of remaining allele in women inheriting a BRCAJ gene mutation. This may later lead to a loss of a gatekeeper like p53 leading to tumor formation. Thus, BRCAJ mutation is an earlier event in the de­velopment of the disease.

In conclusion, it appears that BRCAJ gene partici­pates in growth, development and differentiation of mammary gland abnormality manifesting as mutation seen mostly in familial breast and breast/ovarian can­cer and lower expression that would lead to develop­ment and progression of the disease even in sporadic cancer. Since several reports indicate that patients carrying BRCAJ germline mutation show increased incidence of p53 mutation in their tumors, it appears that probably both genes play in concert for onco­genesis. However, germline mutation of p53 is rare in breast or breast ovarian cancer families indicating that p53 mutation is a later somatic event occurring in the breast tissue and probably BRCAJ germ line mutation might render the cells more susceptible for p53 muta­tion by causing a genome wide instability. Since both caretaker and gatekeeper genes are affected, tumors of BRCAJ mutant carriers are more aggressive. How­ever, since BRCA J carrier tumors are more radiosen­sitive and chemosensitive, a timely treatment with these therapies would improve the life of the patient. Again since p53 mutation which is known to be a later

event in the breast tissue of BRCA J mutant carrier patient, it would be interesting to see whether p53 mutations can be studied from breast fluid cells pre­symptomatically in carrier individuals. Such findings would lead to an early diagnosis and treatment of the disease.

Acknowledgement This work was financially supported by CSIR, New

Delhi Grant No.37/884/95-EMR-II. The senior re­search fellowship awarded to Mr. Sushant K. Kach­hap by the Council of Scientific and Industrial Re­search (CSIR), India is gratefully acknowledged. We are grateful to Dr. M M Vaidya for critical reading and comments on the manuscript.

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