Section 2 BRCA Discovery

#### **Chapter 2**

## Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact

*Natalia B. Burachik, Ana Laura Ortiz and Edith C. Kordon*

#### **Abstract**

In the human genome, BRCA1 and BRCA2 (for **BR**east **CA**ncer 1 and 2) genes encode for proteins involved in several functions that are crucial for the maintenance of genome stability and integrity. They participate in DNA damage response and repair pathways and, therefore, act as tumor suppressor genes. Mutations in these genes, which are located in chromosomes 17q21 and 13q13 respectively, are responsible for a great fraction of inherited breast and ovarian cancers, as well as other pathologies, such as Fanconi Anemia. Approximately 30 years ago, a report from a group of the School of Public Health at the University of California about a hypothetical gene that led to predisposition to early-onset breast cancer in certain families changed the history of breast cancer research, diagnosis, and prevention. Nowadays, the accessibility of genetic testing and the availability of different approaches as wide coverage screenings, prophylactic mastectomies, and risk-lowering drugs benefits BRCA1 and BRCA2 mutation carriers enormously. This chapter summarizes the unique trajectory of BRCA research and its scientific and social implications.

 Introduction**

Breast cancer is the world's most prevalent cancer type. In 2020, 2.3 million women were diagnosed with breast cancer and 685.000 deaths were reported globally, ranking as the most common cause of cancer death among women [1]. From the 1930s to the 1970s breast cancer mortality rate remained quite constant, but since 1980, thanks to the implementation of early detection procedures, there has been notable progress in survival rates. Nowadays, some risk factors that lead to breast cancer are well identified and, in general, the population is better aware about them, as the information became more available in the era of digital communication. However, this progress has not been even among different regions in the world. Data suggest that the low income countries have diminished survival rates due to less access to information and early testing [2].

The high rates of breast cancer incidence and mortality in women of certain families led to a long search for the causes of inherent susceptibility to develop this illness. Around 1970, a global race to discover those factors started and reached the first milestone in 1994 that was the Science publication reporting BRCA1 sequencing, which was soon followed by the discovery and sequencing of a second gene, BRCA2. This chase indelibly marked the cancer and genetic research fields, but also labeled the initiation of the ongoing discussion about the role of private companies on gene discovery and patenting. Since then, medical progress based on gene and mutation discoveries have involved not only scientific, but also major ethical and commercial debates. Now, almost 30 years later, those issues are still controversial. In addition, and in spite of the huge advance of basic knowledge about those genes, several fundamental questions remain unanswered, such as why, in people carrying a mutated BRCA gene copy, hormone-sensitive tissues are the most prone to neoplastic transformation. In this chapter, we will focus on these topics, which are still under debate.

#### **2. Historical aspects about the discovery and diagnostic usage of BRCA1 and BRCA2 mutations**

In December of 1990, Mary Claire King's group published a research article reporting the analysis they performed in 23 extended families with a very high incidence of early-onset breast cancer. Analyzed cases were chosen meticulously according to their pathology records. Using different polymorphic markers and four statistical approaches, they associated those tumors with alterations in chromosome 17q21 [3]. Is important to underscore that only the position was then reported, and without the complete human genome sequenced, the gene that caused early-onset breast cancer was merely hypothetical. Then, the "race" to specifically establish the mutated gene responsible for hereditary breast cancer syndrome began.

A year later, Gilbert M. Lenoir and his colleagues confirmed King's team finding, but also associated that chromosomal location to a proportion of hereditary ovarian cancers [4] and Mary Claire King named this still hypothetical gene BReast CAncer 1 (BRCA1). In 1992, King's group reduced the previously located area to a 8-cM region that was very likely to include this gene. They pointed out that it would be a mistake to oversimplify and consider that mutated BRCA1 was involved in breast cancer initiation in the general population, since the analyzed cases corresponded specifically not only to families with high breast cancer incidence, but also to patients with earlyonset disease. In addition, they indicated that the data that linked the breast cancer syndrome to chromosome 17q was heterogenous, suggesting that it might be another gene involved in the development of familial breast cancer, and/or associated to a high frequency of sporadic cases in older-onset families. Importantly, they also reported that it was not possible to distinguish sporadic to familial breast cancer by clinical criteria. Therefore, the involvement of BRCA1 in the inherited risk of mammary tumor development was solely defined by the identification of mutations in breast cancer patients of susceptible families [5].

It was not until October 1994, that the predicted amino acid sequences of BRCA1 and some probable predisposing mutations were published by Mark Skolnick *et al* in *Science* [6]. Up to that time, King's laboratory as well as other groups were close but not able to sequence the whole gene, yet they were later responsible for identifying several different mutations in affected families. Importantly, a month earlier that same year (September 1994) the BRCA2 gene was identified in chromosome 13q

#### *Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact DOI: http://dx.doi.org/10.5772/intechopen.108648*

through a similar strategy to the previously used, but analyzing 15 families with multiple cases of early-onset breast cancer not linked to BRCA1. Interestingly, men bearing BRCA2 mutations showed higher breast cancer risk, but women had less chances to develop ovarian tumors than BRCA1 mutation carriers [7]. The following year, *Nature* published the BRCA2 predicted amino acid sequence as well as this gene mutations based on the Human Genome Project as well as data provided by the Sanger Center and Washington University [8].

Localization and sequencing of the BRCA genes did not provide any clue about the possible biological roles of the proteins encoded by them. They neither showed homologies to any other protein characterized up to that date, nor distinguishable functional domains. Therefore, it was not rapidily elucidated which were BRCA1 and BRCA2 biological roles and whether they overlapped totally or partially. Although the proteins are not similar in their primary sequences, they share several particular features. For example, both are surprisingly large and highly charged, and the corresponding genes have many exons, which suffer alternative splicing. Interestingly, exon 11, where the interactive regions with RAD51 are located, is particularly large and encodes about half a protein in both cases. Curiously, if not for the classic genetic approach to detect them, neither BRCA nor BRCA2 would have been selected as tumor suppressor candidates for to the information provided by their sequences.

The data that arose soon after their discovery were mostly related to the impact of different BRCA1 and BRCA2 mutations on cancer risk. It was determined that some subpopulations with a high tendency to develop breast cancer carried alternative genetic variants in their germline and that the prevalent mutations had been established as a consequence of a founder effect. This happens when a small group of people remains separated from the original population and, after several generations of interbreeding, rare mutations present in the first generation become more frequent. For example, Ashkenazi Jew families, whose ancestors lived in Central and Eastern Europe, are particularly affected by three well-known founder mutations; BRCA1-185delAG, 5382insC, and BRCA2-6174delT, with an overall rate of 2.6% (1/40), in contrast to the 0.2% (1/500) of these three BRCA1/2 mutation carriers in the general population [9]. Other founder mutations have been determined in various European populations, such as some Norwegian, Dutch, and Icelandic families [9, 10]. The possibility of having information about large numbers of people with the same mutation opened the door to analyze the penetrance of such variants, together with the importance of risk-modifying factors that could affect the outcome of the disease [9].

By the age of 70, women who carry a BRCA1 or a BRCA2 clinically relevant mutation, have a 50–65% or 50–55% chance, respectively, of developing breast cancer, while that probability goes down to 7% for women without any of those mutations. In the case of ovarian cancer, the risk is between 35 and 70% for women with a BRCA1 gene mutation, it is lower (10–30%) for those who bear a BRCA2 mutation, but less than 2% for women who do not have any of those variants [11]. For men, only BRCA2 mutation carriers have a significantly higher risk of developing breast or prostate cancer. However, all people who possess one BRCA1 or BRCA2 mutation have an increased risk of developing pancreatic cancer and Fanconi anemia. An increased susceptibility to melanoma has been observed only in the case of BRCA2 inherited gene mutations [12]. It is important to remember that BRCA mutations can also occur sporadically.

BRCA1/2 mutation carriers have between 40 to 80% more chances of getting a second primary contralateral breast tumor. However, it is remarkable that not all

women carrying a BRCA1 or BRCA2 mutation get breast cancer. Therefore, there are other risk factors involved in the occurrence of this illness, even for women who inherit the harmful mutations. Interestingly, BRCA1 and BRCA2 female carriers tend to develop different breast cancer subtypes. The first are more likely to have triplenegative tumors (*i.e.* Estrogen receptor-negative, Progesterone receptor-negative, and HER2-negative), which do not have specific clinically successful treatments, while the latter show a higher probability to develop estrogen receptor-positive (*i.e.* luminal) breast cancer, that usually receive endocrine therapy [11].

Even though most breast cancers do not involve a specific hereditary component, in particular cases it is recommended to take a genetic test in order to find out whether BRCA1 or BRCA2 is mutated in the germ line [11]. This analysis (patented on 1997 in the US) and owned by Myriad Genetics until 2013, is commonly recommended only under certain conditions, such as high incidence of breast or ovarian cancer in the family, belonging to certain ancestries (*i.e.* Ashkenazi Jew), etc. Nonetheless, until 2013, a critical aspect for which the test was not massively advised was the price, which was over US\$3000. Undeniably, a value not accessible to everyone. In addition, there were many other elements that the clinicians took into account before prescribing the BRCA1/2 genetic test, as the psychological impact of the result on the patient and their family as well as her/his predisposition to undergo prophylactic measures.

Almost from the beginning, it was clear that gene testing was not the magic answer to all clinical questions. In 1997, a review article clearly showed the problems associated with gene testing, which can be summarized into two main points intrinsically related: one related to technical and biological issues and the other associated to understanding and usefulness. Specifically, for the detection of BRCA 1/2 mutations, technology available in the late 90s was still slow, expensive and not too sensitive. In addition, there were variants still unknown and/or with no existing information about its biological association with cancer [13]. Genetic testing carries complex issues that even today are misunderstood by the general public and health professionals, so it is necessary to provide the required knowledge to both. Otherwise, gene testing may result useless, cause a waste of resources and be dangerously utilized.

The result of the gene test can be positive, negative, or indicate a variant of uncertain significance (VUS, when the harmfulness of the detected mutation is unknown). If a known variant is found, their relatives can be tested to determine whether they carry the same mutation, which is less expensive than sequencing the whole gene for each of them. In many cases, health insurance covers BRCA testing if the person meets the established criteria. With an accurate diagnosis and treatment at an early stage, women who have a BRCA1/2 inherited gene mutation present a similar survival rate than those who develop breast cancer without an inherent genetic component [10, 11]. In recent years, the recommendation of testing based only on familial high cancer incidence has been questioned. According to a report published in 2015, half the breast cancer patients with BRCA1/2 mutations did not meet family history criteria for testing, so they learned they were carriers after cancer had already developed. This was a breaking point in cancer prevention and, based on those and other similar results, some researchers and clinicians encourage population screenings that enable a much more complete and cost-effective identification of carriers [14].

In a study carried in 1999, among 200 women with breast and/or ovarian cancer, who were offered testing for BRCA1 and BRCA2 free of charge, a high proportion of them had overestimated their risk of having a mutation, and some of them faced difficulties with their health insurance if the outcome of their analysis resulted positive [15]. These facts reflect how the lack of general knowledge about genetics,

#### *Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact DOI: http://dx.doi.org/10.5772/intechopen.108648*

and particularly, cancer genetics may lead to unnecessary psychological stress, and the economical and social pressure suffered by who is (or suspects to be) a BRCA1/2 mutation carrier. A boom of testing occurred right after May 2013, when Angelina Jolie shared her breast cancer family history in the *New York Times* [16]. In an opinion article, the well-known actress and director, announced that upon learning that she carried a BRCA1 mutation, she decided to undergo double mastectomy to reduce her risk of dying from cancer. Later, she also had her ovaries and fallopian tubes removed. In that commentary, she encouraged women to take a genetic test if they believed they were highly susceptible to develop breast and/or ovarian cancer and, in that way, to take active action to prevent the onset and progression of these diseases. According to doctors and medical centers, this event significantly increased BRCA testing and public interest in this subject, a phenomenon baptized by the media as the "Angelina Jolie effect" [17].

There are different options for BRCA1/2 mutation carriers to decrease cancer risk, such as taking early detection tests and/or undergoing surgeries, like prophylactic mastectomy or oophorectomy (or both). When the first women diagnosed with those genetic variants had to make a decision, the data on the long term outcome of these procedures were very limited. Nevertheless, many social and clinical studies about their efficacy have taken place since then. In 1997, a special report showed that prophylactic mastectomy led to a higher increase in life expectancy than prophylactic oophorectomy, but there were much better benefits when both procedures were done [18, 19]. On the other hand, the advantages of those procedures tended to decrease with increasing age, being almost not significant at all when they were performed in women over 60 years old. That is why BRCA1/2 mutation testing is recommended for women under that age [19].

Some women may avoid BRCA testing for fear of the adverse effects of prevention treatments if they resulted positive. Breast and/or ovaries removal may affect them physically and emotionally as it may lead to fertility problems and issues with their body image. Oophorectomy causes early menopause, which can induce weight gain, an increased risk of cardiovascular diseases, osteoporosis and sexual discomfort. In addition, tamoxifen and raloxifene tratments have rare but severe side effects, such as uterine cancer, blood clots, and stroke [17]. Therefore, precise and early advice about pros and cons of these preventive approaches is required so candidates for these procedures can choose what is best for themselves [19].

Presently, prophylactic mastectomy can reduce the risk of breast cancer by over 90%, but for those who do not want to go through surgery, it is advisable to take a yearly screening with breast magnetic resonance imaging (MRI) (or mammography, which is less recommended because a harmful BRCA variant might be particularly sensitive to the DNA-damaging effects of radiation), as well as biannual pelvic ultrasonography and cancer antigen 125 (CA-125) testing. Noteworthy this protein has been identified in women with advanced ovarian cancer, but it has not been found in early stages of this illness, therefore screening based on its detection has not improved survival [10].

BRCA1/2 deficient cells may be sensible to classic chemotherapeutic drugs that arrest replication and DNA cross-linking agents, like those based on platinum components. Therefore, these drugs, may be recommended to mutation carriers [20]. Since normal BRCA1 protein participates in DNA repair mechanisms by homologous recombination, in the past few years, new drugs (like olaparib and talazoparib), which inhibit PARP1 enzyme and therefore block DNA reparation pathway by base excision repair, have been developed. These drugs leave a gap in the single strand

DNA that arrest the replication fork and convert the single strand break into a double strand break. This leads to cell death in cells whose repair mechanisms by homologous recombination are damaged because of BRCA1 function failure. Currently, there are four PARP inhibitors approved for clinical use, although it is essential to continue investigating possible new synthetic therapeutic and lethal targets, because PARP inhibitors have toxic effects on normal cells and some tumor cells may be resistant to these treatments [20, 21].

Shortly after BRCA1 sequencing was completed, Myriad Genetics, a biotechnology and molecular diagnostic company founded in 1991 in the US, requested patents over BRCA1 gene and over BRCA2 later on. They were granted in 1997 for the US, in 2000 for Canada and in 2001 for Europe, obtaining seven patents in total. In the country or countries where an invention has been patented, their owners control its making, using, and selling for a specific period, which, in the case of BRCAs, corresponded to twenty years. Laws of nature, physical phenomena and abstract ideas are not patentable. However, the BRCA applications were granted because the US Patent and Trademark Office (USPTO) argued that isolated human gene sequences were patentable because human labor was needed to extract and purify them. Noteworthy, a similar stance was taken by the European Union.

BRCA1 patent provided Myriad Genetics the rights over the diagnostic or therapeutic use of this gene, whatever technique was utilized for carry on the assays, as well as all mutations found in familial breast and ovarian cancers, and their usage for determining cancer predisposition. Similarly, BRCA2 most frequent allelic variants, identified mutations associated to disease and methods for determining nucleotide sequence variations were protected [22]. Therefore, Myriad had a wide span of patent rights that provided them the monopoly for BRCA testing [23]. The company required all laboratories to send the DNA samples to Myriad headquarters in Salt Lake City, Utah for testing. That involved an initial cost of 1600 US\$ and they could decide what research might be carried out on those genes, by whom and how much any resulting therapy or diagnostic test would cost [24]. This arrangement, which also included complete control over any further research on the diagnosis of certain breast tumors, was unprecedented in the field of genetic testing [22]. Furthermore, Myriad promoted the BRCA test to physicians and to the general US population on TV and print media, causing unnecessary anxiety about breast cancer risk [25]. In September 2007, the company released a questionable direct-to-consumer (DTC) marketing campaign offering genetic testing for 3100 US\$ without requiring personal medical advice for its solicitation. Noteworthy, the prices were increased while new technology actually made testing less expensive [26]. However, many laboratories performed diagnostic BRCA1/2 tests without observing patenting rights, putting themselves at risk of being sued.

With the aim of canceling Myriad patents, many plaintiffs coaligned across different countries. Europe was the first to invalidate the BRCA patents with different arguments, including the Art. 52 (4) of the European Patent Convention: "Methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body shall not be regarded as inventions …" and focusing on three points: Firstly, the lack of priority and absence of novelty, because gene sequences were already available in public databases when the third patent, which covered a specific set of mutations related to familial breast and ovarian cancer and their use in methods for determining predisposition for breast and/or ovarian cancer, was filed; secondly, the lack of inventiveness, for the same reasons indicated above, and thirdly because therapeutic uses of mutation sequences, in particular gene

#### *Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact DOI: http://dx.doi.org/10.5772/intechopen.108648*

therapy methods, were not sufficiently described for implementing them effectively. Also, by 2002, the Institut Curie demonstrated that the methods used by Myriad Genetics to detect mutations, failed to identify about 10-20% of all expected mutations [22, 23]. In addition, Dr. Mary Clair King's article in JAMA proving that Myriad's tests missed a significant number of mutations, discredit this company and its rights on BRCA1/2 in the UK and Canada. In the US, the news were mainly covered by Utah from the perspective of Myriad, who rebutted the claim [27].

In 2010, a US District Court stated that all BRCA patent claims were invalid and that isolated DNA is 'not patentable subject matter'. Then, in 2011, the US Court of Appeals for the Federal Circuit overturned this ruling (2 to 1 decision); but in 2013, the Supreme Court decided to re-examine the lower courts' decisions and to analyze the claims on gene patenting [28]. Finally, the Supreme Court agreed with the plaintiffs that isolated gene sequences were not patentable because they were not "markedly different" from gene sequences already existing in nature [29].

With the expiration and overturning of BRCA patents, there were no more limitations on the offering of commercial testing. Also, these legal changes occurred approximately at the same time as the widespread adoption of massively parallel sequencing (MPS) technology, which allowed less expensive testing panels featured in turn-around times (TAT) [30].

At this point, we would like to go back to the woman behind this ongoing story, Mary-Claire King, whose contributions for society exceed the discovery of the BRCA genes. Born in 1946 in the US, she attended college and got a degree in mathematics inspired by two of her high-school teachers. Then, she went to the University of Berkeley for graduate school, where she took a genetics course and decided to follow that path. It was the 60s and a lot of social and political situations were taking place all over the world, and the States were not the exception. King was involved in social justice causes like the civil rights movement, and subsequently the anti-war movement. For her Ph.D. Thesis, mentored by Allan Wilson, one of the firsts to approach evolution from a molecular perspective, she demonstrated that humans and chimpanzees share 99% of their coding sequences [31], which caused an evolution revolution at that time.

In the early 1970s, Dr. King moved to South America with her husband in an exchange program between the University of California and the University of Chile. She taught genetics, statistics, and evolution until 1973, when a military coup overthrew President Allende's government. They could not stay, so the couple decided to return to the US. Back then, postdoc positions did not quite exist in academia, but as she said in a conversation with Ushma S. Neill, "*there were many jobs available in cancer research, because President Nixon had just launched the war on cancer. One of those jobs was at UCSF with a lovely pediatric oncologist named Nicholas Petrakis who had become interested in breast cancer*" [32]. Petrakis soon became her post-doc mentor and she started studying an inherited genetic component in breast cancer.

She obtained that position thanks to affirmative action, a policy aimed to balance gender inequality, a fact she noted in more than one interview. Her involvement in social justice was not only about her feminist position, but also her scientific collaboration in the investigation of human rights abuse. In the early 1980s, Dr. Cavalli-Sforza from Stanford, started teaching Dr. King, molecular genetics. And by that time, the Committee on Scientific Freedom and Responsibility of the American Association of Advancement of Science (AAAS), contacted him as a consultant for a genetic issue. In 1977, the Grandmothers of Plaza de Mayo (the "Abuelas", as they are commonly known in Argentina) had organized themselves in Buenos Aires during

the last civil-military dictatorship, to demand the return of their grandchildren, who were born during the captivity of their missing ("desaparecidas") daughters. The "Abuelas" obtained good anecdotal evidence from some survivors and other witnesses about how those babies were given to different families. Then, they correctly proposed that it should be possible to establish the biological familial link by DNA comparison of the grandparents with their putative grandchildren, even if the parents were missing and presumed dead. To reach that goal, Dr. King and her colleagues created a genetic test that provided an "index of grandpaternity" ("índice de abuelitud"). Dr. King (who was fluent in Spanish) traveled to Buenos Aires to put the grandpaternity test into practice in June 1984, and that is how it began a 30-year collaboration with the organization of "Abuelas de Plaza de Mayo" [32–34]. Since then, and thank to that test, 130 grandchildren have been found by their biological families. Presently, Dr. King keeps on working at the University of Washington, trying to solve hereditary cases of breast cancer that cannot be explained by BRCA1 or BRCA2, but rather cryptic mutations that remain elusive [35].

#### **2.1 Understanding the biological role of the Breast Cancer genes**

The early reported BRCA1 and BRCA2 mutations coded for truncated proteins, and the loss of the wild-type allele (loss of heterozygosity, LOH) was detected in tumor samples from affected families [36]. The scientific community agreed on these genes being tumor suppressors, although the nucleotide sequences revealed nothing about their protein function. Starting in 1996, different groups around the world dedicated their efforts to decode the cellular localization and biological role of the BRCA proteins, because the first step to figure out how their deregulation could lead to disease was to understand their activities in physiological conditions.

Mouse models revealed that BRCA1 and BRCA2 were required for embryonic cellular proliferation, as different homozygous mutations resulted in embryonic lethality. The KO embryos showed no increased apoptosis, but cell proliferation was impaired and strong upregulation of the cell cycle regulator p21 was observed. In contrast, mouse with heterozygous mutations were phenotypically normal and fertile up to almost one year of age, although it was not ruled out the possibility of tumors may develop at more advanced age. The similarities displayed by embryos with homozygous null alleles for either BRCA1 or BRCA2 led to think that both genes acted in the same pathway during embryogenesis [37, 38].

BRCA1 and BRCA2 showed an incredibly similar pattern of expression through cell cycle when studied in normal and tumor-derived breast epithelial cells [39]. In synchronized cultured cells, gene expression reached a maximum in late-G1 and S-phase, suggesting a role for both genes in cell proliferation and cell cycle checkpoints. Soon, the first clue that BRCA1 and BRCA2 were actually involved in the DNA repair and homologous recombination came from the observation of co-localization and co-immunoprecipitation of BRCA1 with human Rad51 (hsRad51) in cultured cells [40]. However, it was then determined that it is BRCA2 the one that directly binds to hsRad51, while the interaction with BRCA1 would be indirect, requiring the participation of at least another protein [41]. hsRad51 is the human homolog of *E. coli* RecA and Rad51 in *Saccharomyces cerevisiae*, which are involved in DNA recombination and damage repair. In mouse and mammalian cells, homozygous knock-out of Rad51 displayed a very similar phenotype to BRCA1−/− and BRCA2 −/− mice, indicating the involvement of that protein in cell viability [42].

#### *Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact DOI: http://dx.doi.org/10.5772/intechopen.108648*

It has been shown that BRCA1 and BRCA2 colocalize in nuclear foci of somatic cells as a biochemical complex. In addition, these proteins also coincides with hRAD51 in DNA replication sites after exposure to damaging agents [43]. Furthermore, mouse cells with truncated *Brca2* gene have shown not only G1/S and G2 phase arrest, but also aberrant chromosomal number and structure [44]. Interestingly, cell cycle checkpoints and apoptosis mechanisms displayed no alterations [44]. These reports reinforced the idea that BRCA1 and BRCA2 share some common roles, acting coordinately to preserve chromosome stability. However, there must be functional differences between them that would account for the observed variation in cancer risk for BRCA1 and BRCA2 mutation carriers.

In 1996 and 1997 it was reported that both BRCA1 and BRCA2 contained domains that, when associated with the DNA-binding domain GAL4, induced transcriptional activation in yeast [45, 46]. Moreover, it has also been demonstrated that full-length BRCA1 was a component of the RNA Pol II holoenzyme [47]. Therefore, it has been proposed that the BRCAs may participate in transcription regulation. However, it has not been determined yet whether this activity would be independent from the DNA repair function. In addition, it also remains to be solved why mutation in genes coding proteins involved in very basic activities, such as DNA repair, required in every single cell type, induce high cancer risk only in very specific tissues. To date, there is still no right answer to this question. There are different theories about tissue-specific carcinogenesis caused mostly by BRCA1, but also BRCA2, in mutation carriers. The analysis and implications of this open question exceed the purpose of this chapter, but we briefly report here the leading hypothesis on this subject.

Based on certain BRCA deletion mutations found in cancer patients, it has been proposed that tissue specific carcinogenicity would be due to the loss of "breast-cancer cluster" regions (BCCRs) and "ovarian cancer cluster" regions (OCCRs) that are present in both *BRCA1* and *BRCA2* genes. Nevertheless, this proposition should be taken cautiously, because deletions in the proposed sequences would not only eliminate downstream exons coding for protein regions, but also might repress protein expression through nonsense-mediated mRNA decay. Therefore, those mutations, specifically those corresponding to the first few hundred nucleotides of the *BRCA1* or *BRCA2* coding sequences, would probably result into functionally "null" alleles, which should not cause tissue-specific effects [48]. Alternatively, it has been postulated that the higher cancer susceptibility of Estrogen-dependent organs to BRCA mutations would be due to the genotoxic effect that this hormone may exert on cells. Then, it has been suggested that hormone-responsive tissues might be particularly sensible to the failure of DNA damage reparation exerted by BRCA proteins [49]. On the other hand, another theory is based on the accumulation of R-loops, DNA–RNA hybrids, necessary for the differentiation of normal mammary luminal epithelial cells. In brief, BRCAs would be required for recruitment of some molecules involved in transcription (*i.e.* BRCA2 is necessary for PAF1 activity) and, in the absence of BRCA proteins chromatin disassembly may decrease. Then, transcription elongation would be obstructed causing accumulation of RNAPII in promoter-proximal pausing (PPP) sites generating R-loops which would lead to DNA breaks and consequently to genomic instability [50]. Growing evidence indicates the connection between R-loops and estrogen activity. These reports point out that genomic instability, increased in the scenario of BRCA1 or BRCA2 mutations, may be especially enhanced in estrogen-responsive tissues such as the breast [51]. This establishes another possible explanation for the high risk of BRCA mutation carriers to develop tumors in those organs.

### **3. Conclusions and final remarks**

As reported at the beginning of this chapter, breast cancer is a relevant issue due to its high incidence worldwide. Here, we summarized the very first steps that resulted in the discovery of BRCA1 and BRCA2 using basic genetic and statistics tools by many groups simultaneously. Then, the prognosis of having a mutation in these genes is explained and the strategies and treatments for cancer prevention in mutant carriers are indicated. Additionally, we report the legal history around the controversial patenting of these genes as well as a brief report about Mary Claire King a key scientist in BRCA discovery and in the recent history of our country. In the second part of this chapter, we review some BRCA1 and BRCA2 biological functions, particularly those relevant to the not completely answered question of why mutations in these genes cause high risk of developing tumors particularly in hormone-responsive tissues.

In summary, it can be said that the story behind the finding of BRCA1 and BRCA2 as well as its further scientific and clinical developments have not been linear at all. It have involved multiple actors as classical and molecular geneticists, clinicians, lawyers, entrepreneurs, ethical experts, pharmaceutical companies, and the judiciary systems of diverse countries. Undoubtly, is one of the best examples of how a scientific discovery may change human society for ever.

#### **Acknowledgements**

The authors would like to thank the Argentine National Council for Scientific and Technological Research (CONICET), the University of Buenos Aires (UBA) and the National Agency for Scientific and Technological Promotion (ANPCyT) for their support.

This chapter is in memoriam of Dr. Moisés Burachik, who has always emphasized the necessity of a fluid interaction between scientific research and human society, for inspiring several generations to study and do science in Argentina.

### **Author details**

Natalia B. Burachik1† , Ana Laura Ortiz2† and Edith C. Kordon2 \*

1 INGEBI-CONICET, FCEN, University of Buenos Aires, CABA, Argentina

2 IFIBYNE-UBA-CONICET, FCEN, University of Buenos Aires, CABA, Argentina

\*Address all correspondence to<EMAIL_ADDRESS>
† Equally contributed to this chapter.

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Discovery of BRCA Mutations: Historical Perspective of Its Scientific, Clinical and Social Impact DOI: http://dx.doi.org/10.5772/intechopen.108648*

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[19] Schrag D, Kuntz KM, Garber JE, Weeks JC. Decision analysis--effects of prophylactic mastectomy and oophorectomy on life expectancy among women with BRCA1 or BRCA2 mutations [published correction appears in N Engl J Med 1997 Aug 7; 337(6): 434]. The New England Journal of Medicine. 1997;**336**(20):1465-1471. DOI: 10.1056/ NEJM199705153362022

[20] Takaoka M, Miki Y. *BRCA1* gene: function and deficiency. International Journal of Clinical Oncology. 2018;**23**:36- 44. DOI: 10.1007/s10147-017-1182-2

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Section 3

## BRCA Structure and Function

**Chapter 3**

### BRCA Biological Functions

*Divya Bhargavi Pulukuri, Vijaya Babu Penke, Divya Jyothi Palati, Prudvi Raj Pulla, Shanker Kalakotla and Siddhartha Lolla*

#### **Abstract**

BRCA1 and BRCA2 genes encode proteins that have important roles in DNA repair and act as tumor suppressors. Though the sequence and structure of the proteins produced by BRCA1 and BRCA2 are different, they have similar biological activities. Both BRCA gene products are reported to interact with the RAD51 protein, which is essential for DNA repair through homologous recombination. BRCA gene mutations are associated with an increased risk of solid tumors. Their ubiquitously expressed protein products are involved in essential cellular functions. The defect caused by BRCA gene mutations might be leveraged to develop new targeted cancer treatments. This chapter outlines that BRCA1 and BRCA2 have unique roles in the pathways leading to DNA double-strand break repair and clinical findings show that BRCA genes play a crucial role in a variety of biological processes.

**Keywords:** BRCA1, BRCA2, RAD51 protein, DNA repair, cancer

#### **1. Introduction**

Mutations in the tumor suppressor genes BRCA1 (breast cancer susceptibility gene 1) and BRCA2 have an impact on various types of cancer (breast cancer susceptibility gene 2). More than 20 years ago, researchers first discovered an association between the BRCA1 and BRCA2 genes and the risk of developing ovarian and breast cancer [1]. Stomach, prostate, pancreatic, and colorectal cancers also have been linked to genetic mutations in the BRCA gene. There is some evidence that familial ovarian and breast cancers are linked to pancreatic, stomach, and prostate cancers via mutation, which accounts for 20% of these cancers [2].

While homologous recombination mends precise DNA damage, the BRCA1 and BRCA2 proteins are vital for the process [3]. This helps to ensure that the genome retains its complete and unaltered state. Depletion of BRCA capabilities consequences in genomic instability, which further drives the oncogenic conversion of non-tumorigenic cells into tumor-initiating cells, also referred to as cancer stem cells (CSCs), and further tumorigenesis. Several investigations over the last decade have shown that cancer cells within a single tumor varied significantly in terms of their potential to initiate tumors. A CSC population is capable of long-term self-renewal and differentiation into various tumor cell types and development. In addition to the prominent

genomic imbalance/instability that is connected to tumor tissues, CSCs also have a high ability to self-renew and clonogenic potential, which suggests that they may act as a catalyst for the growth of cancer [4]. consequently, intratumoral heterogeneity is contingent on the CSC's development, which is represented by the with-in quantity of newly forming tumor replicates [5].

In this chapter, we discuss the interaction between the BRCA gene and the RAD51 protein, which is important for DNA repair via homologous recombination, as well as the clinical significance and the central function of BRCA genes in a variety of biological functions.

#### **2. BRCA genes and encoded proteins**

The basic sequences of BRCA1 and BRCA2 are significantly diverse. BRCA1 (with 17q21, 17 chromosomes: 43,044,294 to 43,125,482base pairs) happens to be a 24-exon protein containing 1863 amino acids. It's made up of several domains for distinct purposes. It has the zinc-finger binding domain RING (Quite Fascinating New Gene) at the N-terminus, which is required for BRCA1 and BARD1 interaction and E3 ubiquitin ligase complex formation [6]. ABRAXAS, BRIP1/FACJ, CtIP, and 2 Phosphopeptide-binding BRCT (BRCA1 C-terminal) dominions mediate the association between BRCA1 and also its associated proteins (**Figure 1**). Exons 11–13, which encode the central region of BRCA1, are often mutated in patients with breast cancer. 2 Nuclear localization signals (NLS) along with 1 coiled-coil domain are required for the interaction of BRCA2 via PALB2 [7].

#### **Figure 1.**

*Schematic representation of BRCA1 and BRCA2 proteins. BRCA1 has 23 exons and 1863 amino acids, whereas BRCA2 has 27 exons and 3418 amino acids. BRCA1 contains a highly conserved zinc-binding RING (very intriguing new gene) finger domain near the N-terminus. Two BRCT (BRCA1 C-terminal) domains are located at the C-terminus. The core region of BRCA1 is made up of two NLS (nuclear localization signals) and one coiled-coil domain. BRCA2 has eight BRC repetitions of 20–30 amino acids. BRCA2 features a TAD (transcriptional activation domain) domain at its amino-terminus and two NLS and one TR2 domain at its carboxyl-terminus. The DNA-binding domain lies at the C-terminus and consists of a helical domain (H), three oligonucleotide-binding (OB) folds, and a tower domain (T). The domain names are displayed above. Braces are used to designate exons. Below are noted the locations of founder mutations.*

The BRCA1 gene has more than 1600 mutations, involving omissions, infusions, and single nucleotide mutations have been identified [8]. The majority of BRCA1 mutations have been detected in BRCT as well as RING dominions, in addition to exons from 11 to 13, that encode the NLS necessary for BRCA1 operations, then act as complex formation sites for additional BRCA1-interacting proteins, such as c-Myc, Rad50, Rad51, pRb, BRCA2 and PALB2 [9]. Ashkenazi Jews have a 5382insC frameshift alteration that appeared in Scandinavia and Russia, as well as 185delAG founder alterations in the RING and BRCT domains [10]. Mutations in BRCA1 exons 11–13 are linked with ovarian cancer and the breast to develop pancreatic, colon, rectal, and stomach cancer [11].

BRCA2 is an enormous protein that consists of around 3418 different amino acids and is located on chromosome 13 at position 13q12.3 (base pairs 32,315,479 to 32,399,671). Its genomic data accounts for about 84.2 kb and has 27 exons. The transcriptional activation domain (TAD) is present on the N-terminus of BRCA2. Exon 11 encodes the middle region with eight RAD51-binding BRC repeats [12]. BRCA2's carboxyl terminus features a DNA-binding domain made up of a tower domain (T), 3 oligonucleotide binding (OB) folds, as well as conserved helical dominion that makes it easier for BRCA2 to attach to double- and single-stranded DNA (ssDNA) (**Figure 1**). There are 2 NLS domains and 1 TR2 domain on the C terminus of BRCA2 [13]. It has about 1800 mutations. Frameshift replacements, erasures, and nonsense genetic mutations caused by these lesions result in untimely protein curtail or non-functional proteins [14]. Exon 11 of the BRCA2 gene encodes the most common germline frameshift mutation, 6174delT. BRCA2 mutations are connected to stomach, pancreatic, breast, ovarian, prostate, gall bladder, and bile duct cancer [15].

#### **3. Interaction of RAD51 and the BRCA proteins**

Amidst changes in order and structure, BRCA1 and BRCA2 share biological roles. BRCA1 and BRCA2 have comparable subcellular localization and expression patterns. Both are expressed during the cell cycle's S phase, suggesting DNA replication responsibilities [16]. When DNA is damaged, two subnuclear foci change their distribution. Both proteins are found in synaptonemal complexes of meiotic axial filaments. This expression and localization pattern is paralleled by RAD51, a human counterpart of the bacterial protein RecA, which *E. coli* requires to repair double-strand breaks (DSBs) through genetic recombination. It's been noted that RAD51 binds to both BRCA1 and BRCA2. In vitro, BRCA2-RAD51 interacts directly with recombinant protein fragments and the yeast two-hybrid system [17]. In yeast two-hybrid tests, murine BRCA2 binding to RAD51's first 98 residues was mediated by a C-terminal motif. Human BRCA2 homologous region 95% similar to murine sequence does not bind RAD51 [18].

Eight BRC repeats mediate RAD51's interaction with human BRCA2 [19]. With the exception of BRC5 and BRC6, each repetition can bind to RAD51 independently in two-hybrid experiments and in vitro when expressed as a GST fusion protein. BRC5 and BRC6 aren't capable. In two-hybrid experiments, BRC4 binds RAD51 four times more than BRC1. PCR mutagenesis identifies a 30-residue binding consensus in BRC1 and BRC4. Despite this core motif, BRC1 and BRC4 require different residues for RAD51 binding. This shows that BRC1 and BRC4 interact differentially with RAD51 [20].

BRCA1's interaction with RAD51 was first linked to a region covering residues 758–1064 [21]. Yet it is still unknown whether direct interaction between the two proteins is possible. Co-immunoprecipitated cell extracts show a low-stoichiometry interaction not confirmed by yeast two-hybrid or recombinant proteins. In meiotic and mitotic cells, BRCA1 and BRCA2 are co-localized. A region of BRCA1 (residues 1314–1863) that is unrelated to RAD51 binding is where these two proteins interact [17]. The connection may not be direct and involves 2–5% of each protein's cellular pool. Current biochemical purification and mass spectrometry efforts to characterize the BRCA1 protein complex have not detected RAD51 or BRCA2 [22].

Therefore, of the identified physical interactions between BRCA1, BRCA2, and RAD51, the BRCA2-RAD51 contact seems to be the most well-established. Although the functional significance of their observed interactions has not yet been determined, existing data suggest that BRCA1 interacts with BRCA2, RAD51, and BRCA2 in a multimolecular complex.

#### **4. Clinical relevance of BRCA genes**

The link between BRCA1/2 domain functions and tumor growth has been studied in animal models, but clinical data are required [23]. Most BRCA gene mutations (70–80%) induce protein dysfunction or absence. Certain clinically important mutations enhance the risk of hereditary cancers [24]. In addition, numerous studies have found a correlation between BRCA1/2 mutations and aggressiveness of tumors, and inadequate clinical results in cancer patients. In a recent investigation of 603 cases of sporadic pancreatic cancer in China, it was found that the germline missense variant rs1799966 (c.4837A > G[p.Ser1613 Gly]) in the BRCT dominion of the BRCA1 gene was related to lower overall patient life expectancies [25].

Contradictory results have been found in clinical studies investigating a potential link between BRCA1 and BRCA2 mutations and the prognosis of patients with breast cancer. In a recent prospective multihospital investigation of 2733 young breast cancer patients, 388 had BRCA1/2 mutations. Overall survival did not differ between people with and without BRCA mutations. According to the analysis of 558 triplenegative breast cancer (TNBC) patients, BRCA1/2 alterations/mutation transferors outlived non-carriers overall [26]. In the same time frame, 202 invasive breast cancer patients from Japan who underwent a retrospective study discovered that a loss of heterozygosity (LOH) at the BRCA1 gene is connected to notable shorter diseasefree endurance, remote metastasis-free survival, and overall endurance [27]. In yet another detailed overview, among 458 Chinese breast cancer patients with pathogenic germline BRCA2 mutations, lymph node metastases were more prevalent at diagnosis and had inferior results, such as disease-free life-span and distant relapse [28]. BRCA1 mutations enhance the prognosis for ovarian cancer, according to a meta-analysis of 33 scientific cases [29].

More prospective research on the impact of individual pathogenic mutations in tumor growth and patient responses to therapy is required to better understand how the prognosis of patients with ovarian and breast cancer is impacted by BRCA1/2 mutations. It may be possible to better target clinical treatment for BRCA-related malignancies by understanding the relationship between tumor aggressiveness and BRCA mutations.

### **5. Protective role of BRCA genes in the maintenance of genomic stability**

Through the control of homologous recombination, the BRCA1 and BRCA2 proteins are crucial for DNA double-strand break (DSB) repair (HR) (**Figure 2**) [30]. Using a homologous template, such as sister chromatids, HR is a DNA repair technique that achieves high precision. If sister chromatids are present, they can be effective in S and G2. Pre-synapsis, post-synapsis, and synapsis are processes in DNA repair [31]. Initial DSB ends are cut during the first stage by the Mre11-RAD50-Nbs1 (MRN) complex as well as C-terminal binding protein interacting protein (CtIP) with nuclease activity, leaving a 3′- single strand (ss) DNA tail that replication protein A (RPA) protects from destruction. Next, BRCA1 and BRCA2 regulate the invasion of RAD51-ssDNA filament into homologous duplex DNA. A D-loop (displacement loop) structure is created when the third DNA strand spans between two double-stranded DNA molecules. The resulting nucleoprotein filament searches for a similar DNA sequence on the sister chromatid and enters a duplex to produce a mutual molecule. In the final stage, during post-synapsis, RAD51 separates from dsDNA. DNA polymerases stretch the damaged DNA's 3′ end, followed by DNA ligation [32]. When DNA recombination results in Holliday junctions, the various mechanisms outlined elsewhere help resolve them, leading to an error-free repair [33].

Additionally, BRCA1 controls both HR-dependent DNA repair and non-homologous end joining (NHEJ) repair. NHEJ is a DNA repair process that ligates broken DNA ends without a template. HR takes longer but is error-free and accurate at rectification. Contrarily, DNA repair by NHEJ often causes mutations. It's the fastest DNA DSB repair process. Classical (C) NHEJ occurs most often in G0 and G1 but in every phase. The process is broken down into three steps: break recognition, endprocessing, and ligation. DNA-PK and NHEJ proteins are recruited when Ku70/Ku80 recognizes DSB ends. After that, DNA-PK recruits endonuclease Artemis to handle DSB ends. End ligation is promoted by the XRCC4 (X-ray repair cross-complementing protein 4)/Lig4 protein [34].

Unlike C-NHEJ, alternative (A)-NHEJ requires an MRN complex, CtIP, and poly (ADP-ribose) polymerase-1 (PARP-1) for protein recruitment and DNA lesion recognition. A-NHEJ is a backup repair route for C-NHEJ, but its mechanism is less wellknown [35]. According to recent studies, dephosphorylating 53BP1 allows BRCA1 to

#### **Figure 2.**

*The precise role of BRCA1 and BRCA2 in DNA DSB repair. The BRCA protein fixes DNA double-strand breaks, halted replication forks, and DNA cross-links. Protein kinases like ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutant (ATM) that activate the pathways are able to detect DNA damage. The RAD51 recombinase, which is responsible for mediating homology-directed (HR) repair and strand invasion is regulated by BRCA2 via the MRN complex (RAD50-MRE11-NBS1).*

switch from NHEJ to HR-dependent DNA repair (p53-binding protein 1) [36]. CDK phosphorylates CtIP at Ser327 when BRCA1, CtIP, and MRN activate HR [37]. PALB2/ FANCN recruits BRCA2 at DNA DSB sites during HR via BRCA1 [38].

Extensive research has also been conducted on the role of BRCA2 in DSB repair. BRCA2 plays a key function in HR by recruiting RAD51 to DSBs [39]. BRCA2 deletion causes tumorigenesis and genomic instability. This is partly because BRCA2 regulates RAD51's intracellular location and DNA-binding ability. The MRN complex recruits BRCA1 and CtIP to the DNA DSB site, which induces phosphorylation and ubiquitination. This complex promotes BRCA2 to DNA DSBs with Exo1 and DNA2-BLM (Bloom syndrome protein). BRCA2 recruits RAD51 to DNA damage sites, displacing RPA. BRCA2's BRC repeats and TR2 domain allow RAD51 to load onto ssDNA and search for a DNA template [36]. BRCA2 may work with RAD51 paralogs XRCC2 and XRCC3 to assemble RAD51 with ssDNA [38]. It regulates stalled DNA replication by binding to RAD51 BRC repeats. BRCA2 inhibits MRE11 to prevent chromosomal defects during replication stalling [40] 3′-repair exonuclease 2 (TREX-2) complexes recruit BRCA2 to handle R-loops, which form during transcription from hybrid DNA-RNA and ssDNA [41].

#### **6. Biological functions of the BRCA gene**

#### **6.1 Role of BRCA genes in the biological response to DNA damage and DNA double-strand break repair (DSB)**

BRCA1 and BRCA2 co-localize DNA damage responses were inevitable. Studies on breast-cancer-susceptibility-gene-mutated cells support this. DNA damage triggers cell cycle checkpoints and DNA repair mechanisms. Inactivating DNA checkpoints or repair enhances genotoxicity. Due to increased X-ray sensitivity, BRCA1 and BRCA2 take part in a significant role in the response to DNA damage in murine cells [42, 43]. DSB repair, the X-ray radiation-induced lesion, was found to be defective in BRCA1 or BRCA2-deficient cells. NHEJ and homologous recombination repair DSBs in mammalian cells. NHEJ ligates DNA without end-sequence homology. DSBs can be repaired by exchanging DNA from damaged templates and sister chromatids. In mammalian cells, its mechanism is unknown. Yeast recombination depends on Rad51p, Rad52p, Rad54p, Rad55p, Rad57p, Rad59p, and Mre11p/Xrs2p-Rad50. These yeast genes have similar mammalian homologs [44].

Existing research shows that BRCA2 might not be required for DSB repair through NHEJ. The V(D)J reconfiguration of T cell receptors or antibodies, known as an NHEJ reaction, can occur if the mouse Brca2 gene is shortened [42]. BRCA2-deficient cells can perform DNA-PK-dependent NHEJ in vitro [45]. BRCA2 is essential for DSB repair by homologous recombination, according to recent, convincing data. Truncated Brca2 cells develop tri-radial and quadri-radial chromosomes in culture [42]. Radial features indicate Bloom's syndrome and Fanconi's anemia. BRCA2 deficiency hinders DNA-damaged RAD51 nuclear foci and repair sites [45].

BRCA1 and BRCA2 play different roles in DSB recombination. BRCA2 appears to play a direct role in this mechanism. BRCA2 may influence RAD51's intracellular transport and function. BRCA2 may aid in the movement of RAD51 out of its synthesis site to its active position since RAD51, which lacks a nuclear localization signal, is poorly carried into the nucleus in cells with a defective form of the gene. Peptides BRC3, BRC4, or BRC7 suppress nucleoprotein filament formation in vitro. Gel filtration indicated

that binding RAD51 to BRC peptides prevents filament multimerization. These data suggest that BRCA2-RAD51 cannot promote homologous recombination in vivo. These in-vitro data show that BRCA2 is necessary for DSB repair in vivo. The BRCA2-Rad51 complex may exist in two states in vivo: an inactive state that precludes Rad51 from adhering to single-strand DNA as well as an active state where Rad51 generates nucleoprotein filaments that BRCA2 can transfer. A structural change in the BRCA2-Rad51 interaction that releases Rad51 from BRCA2 may be facilitated by increased phosphorylation. This in vitro biochemical prototypical may not apply to BRCA2's cellular action. Structural analysis of BRCA2-Rad51 may shed light on this [46].

BRCA1 is also important for homologous recombination DSB repair, but its mechanism is unclear (**Figures 2** and **3**). Due to the low stoichiometry of their interaction, it might not directly affect RAD51 function. BRCA1 binds to recombination proteins besides RAD51 [22]. RAD50, MRE11, and NBS1 co-localize and coimmunoprecipitate with BRCA1, but foci localization is unknown. Recent research shows BRCA1 regulates RAD50-MRE11-NBS1. MRE11's exonuclease may repair DSBs [47]. How this process aids DNA repair is unknown. The BRCA1-complex interaction indicates proximal roles.

#### **6.2 BRCA gene-dependent transcriptional regulation**

There is evidence from numerous studies indicating the transcriptional control of genes is regulated by both the BRCA1 and BRCA2 proteins [48]. The DNA plasmid

#### **Figure 3.**

*Functional features of BRCA proteins. The BRCA protein has many activities in diverse biological processes including DNA repair, transcription regulation, cell cycle progression, ubiquitination cycle, and chromatin structure regulation. Proteins that are interconnecting with RAD50 double-strand break repair protein (RAD50), Nijmegen breakage syndrome (NBS), meiotic recombination homolog a (MRE1), RAD51 double-strand break repair protein (RAD51), master regulator of cellular metabolism and proliferation (C-Myc), estrogen receptor (ER), tumor protein (P53), mothers against decapentaplegic homolog 3 (Smad3), histone deacetylases (HDAC), retinoblastoma protein (RB), BRCA1 associated Ring domain 1 (BARD1), ubiquitin-like, PHD, and RING finger domain 1 (UHRF1), checkpoint kinase 1 (CHK1), cyclin-dependent kinase inhibitor 1 (CIP1), mediator of DNA damage checkpoint 1 (MDC1) in numerous cellular pathways.*

that contained a BRCA1 C-terminal fragment (aa1528–1863) coupled to the yeast GAL4 (galactose–responsive transcription factor) DNA–binding dominion was used in the first investigations that revealed a role for BRCA1 in transcriptional regulation (GAL4-BRCA1). In both human and yeast cells, this recombinant protein triggered the transcription of genes. The prevalence of BRCA1 mutations in patients with breast and ovarian cancer dramatically decreased this transcriptional activity [49].

Despite the fact that the BRCA1 protein includes a DNA binding domain, recent research has shown that it is a co-regulator rather than a sequence-specific transcriptional component. Numerous transcriptional regulators, such as OCT-1, c-Myc, ER, p53, Smad3, and others, are controlled by BRCA1 [50]. For instance, BRCA1 interaction with ER controls VEGF transcription in breast cancer. It has been demonstrated that the C-terminal region of BRCA1 stimulates the p53 target gene MDM2 (Mouse Double Minute 2 Homolog) in breast cancer cells. It has been proven that BRCA1 and Smad3 work together to induce the Smad3-specific promoter. Various scientific investigations have linked BRCA2 to the control of transcription, for instance via forming a composite with p53 and Smad3 [51].

#### **6.3 Role of BRCA gene in cell cycle progression and regulation**

BRCA1 is discovered to be hyperphosphorylated in later G1 and S phases of the cell cycle and dephosphorylated in the M phase, indicating that it governs cell cycle progression, according to early research [52]. At DNA damage detectors, ATM, ATR, and Chk1 phosphorylate BRCA1 when there is DNA damage [53]. BRCA1 hinders G1/S by activating p21WAF1/CIP1. In addition to p21, the BRCA1-induced G1 arrest is dependent on Rb. BRCA1-induced G1/S arrest may be caused by the proteins ATM, ATR, BARD1, RB, p53, and p21 as well as their effectors. BRCA1-deficient cells show genomic instability, centrosome duplication, and DNA damage [54]. Also, BRCA1 acts with a DNA damage checkpoint mediator (MDC1). MDC1 recruits 53BP1, BRCA1, and MRN to DNA break spots to arrest S as well as G2/M cell cycle (**Figure 4**) [55].

The BRCA2 protein may be involved in the regulation of cell cycle progression, according to a large body of research. BRAF35/BRCA2 complexes on mitotic chromosomes phosphorylate histone H3 at Ser28 and Ser10 to aid in the condensing of mitotic chromosomes. This research verified that BRCA2/BRAF35 is crucial for cell cycle progression by microinjecting anti-BRAF35 or anti-BRCA2 antibodies into HeLa cell nuclei [56]. Recent research has shown that BRCA2-deficient cells are very susceptible to the anti-cancer medication S23906, which is attributable to both a deficiency in HR-dependent DNA repair and a malfunctioning S-phase checkpoint [57].

#### **6.4 BRCA facilitated chromatin remodeling and regulation of epigenetic gene expression**

Genome expression can be controlled by chromatin remodeling, which is regulated by BRCA1. RCA1 and its ubiquitin E3 ligase activities maintain gene silencing. Histone H2A is ubiquitinated to form heterochromatin. Tandemly repeated DNA sequences were produced when BRCA1 was deleted, a pathogenic BRCA1 mutant (T37R) was expressed, or BARD1 shRNA was used [58]. The chromatin remodeling complex SWItch/Sucrose Non-Fermentable (SWI/SNF) contains a BRG1 subunit, which BRCA1 can directly adhere to it. The dominant negative BRG1 mutant blocks p53-mediated BRCA1 transcription. BRCA1-containing chromatin remodeling complexes may lead to breast and ovarian cancer [59]. A novel BRCA1 cofactor (COBRA1) *BRCA Biological Functions DOI: http://dx.doi.org/10.5772/intechopen.107406*

#### **Figure 4.**

*The specific role of BRCA1 and BRCA2 in the regulation of the cell cycle. The proteins BRCA1 and BRCA2 and their associated proteins are responsible for cell cycle regulation. ATM and Chk1 phosphorylate BRCA1 at DNA damage detectors. BRCA1 impedes G1/S phase progression by activating TOPBP1 (topoisomerase 2- binding protein 1) and CTIP (C- terminal binding protein 1), induced G2 arrest is also dependent on RAD50 (RAD50 double-strand break repair protein), NBS1 (Nijmegen breakage syndrome 1 mutated gene), and MRE11 (meiotic recombination 11 homolog 1).*

binds directly with endogenous BRCA1 and it can activate chromatin decondensation and is recruited to chromosomes by BRCA1's BRCT1 domain [60].

Oncogenic microRNAs (miR) have been linked to BRCA1 by several studies. BRCA1-HDAC2 deacetylates H2A, H3, and miR-155. BRCA1 loss and HDAC inhibitors activate MiR-155 and diminish H2A and H3 acetylation, lowering oncomiR expression [61]. High PARP breast cancers upregulate miR-151-5p. This study implies PARP drugs could target miR-151-5p in BRCA1-mutant cancer patients. BRCA1 mutations silence the PEMT gene, which produces choline, a breast tumor nutrition. The PEMT promoter −132's hypermethylation of the DNA, which raises H3K9me and lowers H3K9ac, is the mechanism underlying this epigenetic repression [62]. BRCA1 regulates sirtuin 1, a NAD-dependent histone deacetylase in cancer. Imbalanced BRCA1 and SIRT1 activity can lead to cellular transformation and tumor growth, and BRCA1 regulates transcription-silencing chromatin modification PRC2. In order for H3K27 tri-methylation, heterochromatin formation, and PRC2 occupancy on chromatin to occur, BRCA1 interacts with the oncogenic lncRNA HOTAIR [63]. These results indicate that BRCA1 inhibits PRC2 to induce breast cancer.

#### **6.5 Degradation of the BRCA proteins via proteasomes and ubiquitination**

The cell cycle regulates the post-translational activity of the BRCA1 protein. Also, tumorigenesis ubiquitinates and degrades BRCA2, causing genomic variability and non-familial cancers. Numerous proteins influence the stability of BRCA1/BRCA2, such as the cysteine protease Cathepsin S (CTSS), which binds to the BRCT dominion of BRCA1 and encourages its proteolytic destruction, the E2 ubiquitin-conjugating enzyme E2T (UBE2T), and the E3 ubiquitin ligases Herc2 and F-box protein 44. (FBXO44). BARD1 stabilizes BRCA1 expression [64].

According to Kim *et al*., Fyn-related kinase (Frk)/Rak positively affects the stability of the BRCA1 protein by directly phosphorylating BRCA1 [61]. BRCA2 3'UTR interacts with miR-19a and miR-19b, lowering mRNA and protein levels. BRCA1/2 is regulated by protein and post-transcriptional control [65]. Chronic myeloid leukemia cells with BCR-ABL1 have less BRCA1. By attaching to 3'UTR ARE sites, the TIRA (TIA1 cytotoxic granule-associated RNA-binding protein-like1) protein prevents BRCA1 mRNA translation. HuR mRNA-binding protein improved mRNA stability and translation in the same research [66]. BRCA1 is silenced by UHRF1 (ubiquitinlike, PHD, and RING finger domain 1). By building up repressive histone marks on the promoter and restricting transcription factor binding, UHRF1 controls the transcription of BRCA1 [67].

#### **6.6 BRCA protein function in autophagy**

BRCA1 and BRCA2 are both necessary for quality control, the autophagy route for damaged mitochondria, and mitophagy. After being given oligomycin, antimycin A, or the PARP inhibitor AZD2281, mitophagy was reduced when BRCA1 and BRCA2 were knocked down. Under ER stress and serum fasting, siRNA-mediated BRCA1 knockdown induced pro-survival autophagy. BRCA1 activation triggers protective autophagy. Chemotherapeutic drugs enhanced pro-survival autophagy in BRCA1 mutated ovarian cells [68]. This study revealed that BRCA1 modulates chemotherapyinduced tumor cell death.

#### **6.7 Classical or novel BRCA1 cytoplasmic functions**

BRCA1 is widely recognized as a nucleoplasmic chromosomal caretaker. Several studies reveal BRCA1's cytoplasmic function. BRCA1 is recognized for regulating centrosomes. Centrosome amplification (CA) is common in human malignancies. CA can cause cancer and a poor prognosis [69]. BRCA1 ubiquitinates -tubulin in late S and G2/M via the BRCA1–BARD1–OLA1 complex. BRCA1 reduces early S-phase centrosome microtubule nucleation [70]. According to these results, loss of BRCA1 centrosome control may promote hypertrophy and aneuploidy in breast cancers. Cytoplasm BRCA1 is also implicated in apoptosis. BRCA1 promotes GADD45 independent apoptosis. BARD1 masks BRCA1's nuclear export signal and retains it in the nucleus. Nuclear export and cytoplasmic accumulation induce apoptosis with BRCA1 overexpression [71].

#### **7. The consequences of the BRCA gene**

When BRCA gene phenotypes are identified in malignancies, it may be possible to develop cytotoxic agent regimens that are targeted at the mechanisms that cause DNA-repair abnormalities in cancer cells. BRCA2 and other FA (Fanconi anemia) gene dysfunction cause cells to be extremely sensitive to cancer treatment that cause DNA crosslinks [72]. In contrast, Tumor cells with BRCA1 mutations are sensitive to DNA-crosslinking agents, but susceptible to mitotic-spindle poisons like taxanes [73]. Because taxanes are widely used in the treatment of breast and ovarian cancer, it's going to be essential for researchers to ascertain in clinical trials which of these tumors are resistant to taxanes. A study will randomize patients with metastatic familial-BRCA1/2 breast tumors among docetaxel and carboplatin treatment to test

whether in vitro investigations translate into better clinical efficacy [74]. If employed successfully, similar techniques might be utilized to treat tumors that have the BRCA gene.

### **8. Concluding remarks and perspectives**

The BRCA1 and BRCA2 genes encrypt proteins whose main task is to act as tumor suppressors and play a significant role in genome stability. These two genes contribute to DNA damage pathways, cell cycle, and apoptotic cascades in hereditary breast cancer. Despite changes in sequence and structure, BRCA1 and BRCA2 proteins have similar biological roles. Both BRCA gene products interact with RAD51, an important DNA-repair protein. Defective BRCA gene products impair their function as tumor suppressors, resulting in an elevated risk of cancer. An extensive study shows that BRCA1/2 gene mutations lead to the progress of breast, ovarian, and prostate cancers. BRCA-mutated malignancies are more susceptible to treatments that produce DNA DSBs, such as platinum-based drugs and PARP inhibitors. Reverting BRCA mutations that reinstate BRCA1/2 protein function is a solely clinical challenge that necessitates the careful analysis of restored gene frequencies during treatment. Notably, BRCA reversion mutations during antitumor therapy show that BRCA deficiency is crucial during oncogenesis. Combining other cancer-related therapies, CSC (cancer stem cells) therapy, and immunotherapy could resolve BRCA reversion resistance and enhance therapeutic effectiveness. Novel activities will result from the discovery of numerous additional BRCA protein binding proteins in the future. BRCA proteins' involvement in epithelial cell biology and transformation is as yet unclear.

### **Conflict of interest**

The authors declare no conflicts of interest.

### **Author details**

Divya Bhargavi Pulukuri1 , Vijaya Babu Penke2 , Divya Jyothi Palati1 , Prudvi Raj Pulla3 , Shanker Kalakotla1 \* and Siddhartha Lolla4

1 Department of Pharmacognosy, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, Nilgiris, Tamil Nadu, India

2 Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, Telangana, India

3 Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, Nilgiris, Tamil Nadu, India

4 Department of Pharmacology, GITAM School of Pharmacy, GITAM Deemed to be University, Hyderabad, Telangana, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 4**

## The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker for Cancer Treatment

*Yousef M. Hawsawi and Anwar Shams*

*"Our genomes carry the story of evolution, written in DNA, the language of molecular genetics, and the narrative is unmistakable." Kenneth R. Miller July 14, 1948*

#### **Abstract**

BRCA1-associated RING domain 1 (BARD1) constitutes a heterodimeric complex with BRAC1 that triggers several essential biological functions that regulate gene transcription and DNA double-stranded break repair mechanism. BARD1 gene was discovered in 1996 to interact with BRCA1 directly and encodes a 777-aa protein. Interestingly, the BARD1 has a dual role in breast cancer development and progression. It acts as a tumor suppressor and oncogene; therefore, it is included on panels of clinical genes as a prognostic marker. Structurally, BARD1 has homologous domains to BRCA1 that aid their heterodimer interaction to inhibit the progression of different cancers, including breast and ovarian cancers. In addition to the BRCA1-independent pathway, other pathways are involved in tumor suppression, such as the TP53 dependent apoptotic signaling pathway. However, there are abundant BARD1 isoforms that are different from full-length BARD1 due to nonsense and frameshift mutations and deletions associated with susceptibility to cancer, such as neuroblastoma, lung cancer, cervical cancer, and breast cancer. In the current chapter, we shed light on the spectrum of BARD1 full-length genes and isoform mutations and their associated risk with breast cancer. The chapter also highlights the role of BARD1 as an oncogene in breast cancer patients and its uses as a prognostic biomarker for cancer susceptibility testing and treatment

**Keywords:** BARD1, BRAC1/BARD1, BARD1 isoforms, BARD1 mutation, breast cancer, tumor suppressor, oncogene

#### **1. Introduction**

#### **1.1 A glance on The BRAC1/BARD1**

In recent decades, cell biology and molecular genetics have revolutionized our understanding of cancer in general and breast cancer in particular. In this book, we focused on the BRCA1 and BRCA2 mutations. BRCA1-associated RING domain 1 (BARD1) is the name of a protein that Wu et al. in 1996 found as a BRCA1 (BReast CAncer type 1) binding partner [1]. Here, we shifted our focus to the BARD1 as a potential prognostic biomarker for breast cancer.

Generally, the BRAC1/BARD1 constitutes a heterodimeric complex that mediates numerous fundamental biological functions, specifically in regulating gene transcription and DNA double-stranded break repair mechanism [2, 3]. Furthermore, BRCA1 and its partner BARD1 protein are essential in other cellular processes involving chromatin remodeling, telomere regulation, replication fork maintenance, cell cycle progression, apoptosis, and tumor inhibition [4]. BRAC1/BARD1 possesses an enzymatic activity through its E3 ubiquitin ligase capacity that assists in regulating the biological processes and controlling the activity and transcription of other protein complexes [2]. BRAC1/BARD1 acts as a nucleosome reader and writer to scan and correct the DNA breaks by following the homologous recombination pathway. The C-terminal domain of BARD1 serves as a reader/scanner player, while the N-terminal domain exhibits the writer/corrector capacity. Both domains interact with a nucleosome in a wrapping fashion, activating the Ub ligase function [2]. One study has identified a negative regulator of BARD1, DCAF8L2 (a DDB1-Cullin-associated factor (DCAF) associated with CRL4 E3 ligase). The interaction between BARD1 and DCAF8L2 resulted in the degradation and ubiquitination of BARD1 with subsequent disassembly and uncoupling of the BRCA1/BARD1 complex. Additionally, DCAF8L2 expression was upregulated in breast cancer cells suggesting an oncogenic function via disrupting the BRCA1/BARD1 complex stability [3]. The BARD1 gene plays two distinct roles in cancer progression, specifically breast cancer. Thus, several biological researchers have made important discoveries about the BARD1 gene's role in cancer evolution and its potential applications as a prognostic biomarker for breast tumors, or at the very least, to consider it a possible candidate for targeted breast cancer therapy [5].

#### **1.2 BARD1 structure, locations, and isoforms**

The human BARD1 gene has 11 exons that code for a 777 aa protein with a molecular weight of 87 kDa. BARD1 was discovered in 1996 and reported to interact with BRCA1 directly through their homologous N-terminal RING domains n chromosome 2 (2q34–35) [6]. Bard1 protein is structurally made up of a RING-finger domain at the N-terminal region, three repeating Ankyrin (ANK) domains in between, and two tandems of BRCA1 domains at the C-terminal area (BRCT) [6]. Interestingly, the BRCT repeats are essential for controlling how other partners' proteins interact with one another in a phosphorylation-based manner. These interactor proteins are crucial to mediate crucial cellular processes, including DNA damage checkpoints, DNA repair machinery, and cell cycle regulation [7, 8]. Notably, the RING-finger domain and BRCT repeats are essential for the BRCA1-BARD1 complex's ability to suppress cancer [9, 10]. The presence of several exomes in full-length BARD1 (FL-BARD1, resulted in different isoforms) **Figure 1** represents a scaled diagram depicting the comparison of protein structures of BARD1, BRCA1, and BRCA2

*The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker… DOI: http://dx.doi.org/10.5772/intechopen.107143*

#### **Figure 1.**

*A scaled diagram depicting the comparison of protein structures of BARD1, BRCA1, and BRCA2. The diagram illustrates the different protein structures of BARD1, BRCA1, and BRCA2. BRCT (dark red), RING (green), and ankyrin (dark blue) domains. The putative nuclear localization signals (NLS, light blue) and the nuclear export signal (NES, yellow) are shown. The location of BARD1's third NLS, at amino acid residue 321, is crucial for nuclear localization. With eight copies of a 70 amino acid motif known as the BRC repeats and a conserved transactivation domain (TD), BRCA2 is entirely unrelated to either BARD1 or BRCA1.*

There are many BARD1 isoforms with skipped exons and various molecular weights [11]. The isoforms are more frequently found in association with cancerous cells [12, 13]. For instance, isoform α has skipped exon 2. While the isoform β skipped exons 2 and 3, which causes the open reading frame (ORF) to a frameshift, resulting in the translation of shorter proteins (758 aa (85 kDa) and 680 aa (75 kDa), respectively. However, isoform γ is typically interrupted by the deletion of exon 4. Isoforms φ and δ skipped exons 2−6 and 3−6 to produce 326aa (37 kDa) and 307 aa (35 kDa) proteins, respectively. Isoform ε skipped exons 4−9, resulting in a protein with a molecular weight of 30 kDa (264 aa), while the skipping of exons 1, 10, and 11 leads to isoform η. Another splicing from exons 1 to 10 is worthwhile since it interrupts the ORF. As a result, additional alternative ORFs may host the translation's start codon,

#### **Table 1.** *Summarizes the different BARD1 isoforms.*

producing a short protein with 167 amino acids (19 kDa). Surprisingly, most of these isoforms had agonistic cancer susceptibility potential because they lack the RING finger and ankyrin repeats, which are essential for the full-length BARD1's tumor suppressor capabilities [14, 15]. **Table 1** summarizes the different BARD1 isoforms.

Since we highlighted some important aspects of the BARD1 structure, it is worth highlighting the function of the Bard1 protein.

#### **1.3 BARD1 as a tumor-suppressor gene and oncogene**

The Bard1 protein performs a tumor suppressor function in BRCA1-dependent and -independent pathways. Due to their homologous domains, the BRCA1/BARD1 heterodimer can be configured by the N-terminal RING-finger domains, altering the ubiquitin ligase's activity, which controls the cell cycle chromatin structure and hormone signaling pathways as well as DNA damage response pathways [16, 17]. BRCA1-BARD1 heterodimers are disrupted by mutations in cancer cells, which results in the degradation of both proteins [1].

Technology advancements have made it clear that numerous genes, including BRCA1/2 and BARD1, play a crucial role in hereditary and familial breast cancer and ovarian cancer [18, 19]. Research has identified BARD1's function in the BRCA1 dependent pathway as an anti-breast cancer agent [20]. Because it activates ubiquitination through E3 ubiquitin ligase activity and starts the degradation process for the damaged proteins, the BARD1-BRCA1 complex is essential to the DNA damage machinery [16]. The involvement of BARD1 and BRCA1 in a homology-directed repair (HDR) of chromosomal breaks that clarifies their presence alongside RAD51 in response to DNA damage was previously discussed by Westermark et al. [21–23]. Furthermore, through a particular interaction with the poly (ADP-ribose), the BARD1 BRCT domain promotes the early recruitment of the BRCA1/BARD1 heterodimer to DNA damage sites (PAR) [24]. Studies have also demonstrated that disruptive mutations in the phosphate-binding pocket of the BARD1 BRCT domain in mice (S563F and K607A) hinder the recruitment of the BRCA1/BARD1 heterodimer to the stalled replication fork (SRF), which ultimately causes chromosomal instability [25]. Such mutations do not affect recruitment to HDR [25], contrasting with the comparable modification in BRCA1 BRCT (S1598F) [10]. Additionally, BARD1 or BRACA1 mutations linked to the prevalence of breast cancer, such as alterations in the RING finger domain, interfered with the BRCA1/BARD1 heterodimer interaction [26, 27], missense mutations [28–30], and ANK sequences that are involved in the regulation of transcription [31]. Additionally, the heterodimer prevents inappropriate mRNA polyadenylation at DNA repair sites with cleavage stimulation factor subunit 1 (CSTF1) [32, 33]. Through the ubiquitination pathway, BRCA1/BARD1 also aids in the prevention of tumor growth [34] and BRCA1's subcellular location [35].

BARD1 also acts as a tumor suppressor in a BRCA1-independent manner by interacting with the repetitive regions of the BCL3 ankyrin domains and altering the transcription factor activities of NFKB in the TP53-dependent apoptotic signaling pathway [36, 37]. Furthermore, a decrease in Bard1 expression has been linked to cellular changes related to a premalignant phenotype [38]. BARD1 has a role in preserving genomic integrity, and BRCA1 null animals were also discovered to have this trait. Early embryonic death was caused by chromosomal instability and BARD1 damage or total deletion [39]. RNA polymerase II was shown to be ubiquitinated by BARD1, and its transcription of damaged DNA was inhibited [34], ubiquitination, beta, and other processes crucial to breast cancer growth [40]. Together, *The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker… DOI: http://dx.doi.org/10.5772/intechopen.107143*

these actions help FL-BARD1 to play a tumor suppressor role, in contrast to reports that BARD1 isoforms such as BARD1 work against this function and accelerate cancer development [41].

More recently, the Exome Sequencing Project and Exome Aggregation Consortium used 1915 patients to link the BARD1 gene and ovarian cancer [42], where the BARD1 gene has a mutation frequency of 0.2%. BARD1 is currently being studied to be included in panels of clinical gene testing for cancer susceptibility due to compelling data linking BARD1 mutations and breast/ovarian cancer susceptibility [43]. Since we mentioned the function of the BARD1 gene as a tumor suppressor, now we should turn to the other vital function of some isoforms of the BARD1 gene as an oncogene.

BARD1 has about 19 distinct expressed isoforms that have been identified so far [12, 44]; several of these isoforms, including BARD1β, BARD1κ, and BARD1π, have been implicated in the development of cancer by an oncogenic role [12, 13]. At the same time, it has been noted that the FL- BARD1, either on its own or in combination with BRCA1, has a tumor suppressor function [41]. However, BARD1β and BARD1δ were previously reported to have an antagonistic effect on full-length BARD1, resulting in oncogenicity and cancer susceptibility [12]. The majority of BARD1 isoforms possess BRCT domains but lack the RING finger domain necessary for the formation of BRCA1 heterodimers.  Additionally, it was revealed that the expression of BARD1 isoforms is significantly linked to a decline in the survival rate of patients with malignancies [12, 15].

BARD1 isoform anomalies result from protein translation from a different open reading frame (ORF). For instance, BARD1 can be translated as a noncontinuous ORF beginning with exon three and then exons 4 through 11. Additionally, it has been demonstrated that BARD1 isoforms inhibit the BRCA1-BARD1 ubiquitin ligase activity necessary to cause the death of cancer cells [12, 15, 40]. Furthermore, the expression of BARD1β has been associated with impaired homologous recombination (HR) and negatively impacted ubiquitin ligase activity in PARPi-sensitive colon cancer cells [45]. The epigenetic has a profound role in BARD1 gene expression and biological consequences. So, the question now is what is this role?

#### **1.4 The epigenetic effect on BARD1 gene expression and biological consequences**

Exons 6 to 11 (truncated isoforms) of the BARD1 gene were strongly expressed in Acute myeloid leukemia (AML) in vivo blasts compared to the BARD1-FL expression level. Lepore et al. demonstrated that HDACi (Vorinostat) treatment epigenetically controls the expression of BARD1 mRNA in AML cells, MCF-7 breast cancer cell line, and Kelly neuroblastoma cells. An increase in miR-19a and miR-19b levels were observed after vorinostat therapy, and when BARD1 3'UTR expression was targeted, this increased the apoptotic activity of malignant breast cells [46]. Following a similar pattern, estrogen also activated the estrogen response element (ERE) on BARD1's intron 9, which favorably controlled the protein expression of BARD1 [47].

While BARD1 9'L, a particular mutation of the BARD1 gene, was reported to compete with miRNAs (such as miR-101 and miR-203) on their binding sites of BARD1 3'UTR, it was also identified to act as competing for endogenous RNA (ceRNA) that negatively controls the expression of BARD1 mRNA [16]. The long non-coding RNAs (lncRNAs) display gene regulatory roles that modulate different biological mechanisms. GUARDIAN, a p53-responsive lncRNA, was determined to be involved efficiently in preserving genomic integrity and delivering protection against genotoxic stress. Additionally, GUARDIAN can facilitate the heterodimerization of BRCA1 with its partner interactor, BARD1, by acting as an RNA scaffold. Therefore, the breakdown of the BRCA1-BARD1 complex caused by GUARDIAN suppression increased the adverse effects of genotoxic stress, induced apoptosis, and caused genomic instability [48].

BRCA1 and BARD1 significantly influence the ATM/ATR pathway for DNA repair mechanisms important for a cell's decision to die. The BARD1 gene's epigenetic regulation was affected by histone modification of hESC. Splicing process regulation by H3K36 decreased BARD1 expression, suppressing the ATM/ATR signaling pathways that control hESC development [49]. Hepatocellular carcinoma (HCC) patients whose livers had developed cirrhosis were studied, and it was discovered that the BARD1 gene had much lower levels of methylation (13.3 percent) than in healthy controls. Additionally, it was proposed that BARD1 hypomethylation could be a biomarker for predicting aggressive illness in patients who do not have HBV [50]. Before we address the association of the BARD1 variant to breast cancer risk, we should have a breast cancer mutation in general.

#### **1.5 Breast cancer mutations**

The multiple risk factors that have been linked to the development of breast cancer include both genetic and environmental elements. To comprehend the pathogenesis and create a treatment plan, it is essential to identify the genetic and hereditary factors [51]. Numerous genes, including PALB2, ATM, BARD1, BRCA1, BRCA2, and CHEK2, have significantly maintained DNA fidelity and genomic integrity [52–54]. They are also essential in controlling the HR mechanics. As a result, it has been discovered that various mutations in genes are linked to an increased risk of developing many hereditary malignancies [55], including breast, prostate [56], ovarian, and pancreatic cancers [56–59]. Several vital polymorphisms in the domains of the BRCA1/ BARD1 protein-protein interaction (PPI) complex, including M18K, V11G, L22S, and T97R, were identified by a thorough mutational investigation. These mutations also impacted the stability of the BRCA1/BARD1 PPI complex [60]. The development of preventive and therapeutic strategies to thwart the advancement of breast cancer can therefore be facilitated by a thorough understanding and investigation of the interplay between the BRCA1 and BARD1 platforms [4].

A patient with a breast cancer diagnosis and a family history of the disease was seen as a significant factor in the hereditary predisposition to the condition. BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11 are rare but highly penetrant genes that account for about 30% of hereditary breast cancer cases. BRCA1 mutations were initially found in families with a similar pedigree in 1990. The BRCA2 gene variations were discovered four years later [61]. Hereditary Breast/Ovarian Cancer (HBOC) syndrome is caused by BRCA1 or BRCA2 mutations, yet some with this syndrome were negative for BRCA1 and BRCA2 mutations. BRCA1/2-mutant tumors have a basal, extremely aggressive character. Additionally, 2%–3% of breast cancer cases were found to have mutations in the uncommon but moderately penetrance genes, including; CHEK2, PALB2, ATM, RAD50, BRIP1, RAD51C, NBN, and MRE11. These genes engaged in DNA repair processes and interacted with BRCA1/2. A small number of SNPs, including the mutations for RAD51D, BARD1, RAD51C, ABRAXAS, NBN, and XRCC2BRIP, were linked to poor penetrance alleles and an increase in the risk of breast cancer in a polygenic manner. Clinical testing for mutations found in the high penetrance gene set was typically done

*The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker… DOI: http://dx.doi.org/10.5772/intechopen.107143*

on individuals with suspected genetic risk [62]. In addition, a genome-wide association study (GWAS) of breast cancer reported the discovery of 65 novel loci, including FES, MAP3K11, CLK2, GRK7, USP25, DFFA, PKP1, and ZKSCAN3, that are notably related with a high risk of breast cancer at P < 5 × 10−8 [63].

#### **1.6 The significance of BARD1 in genetic predisposition to breast cancer**

Many comprehensive sequencing studies have discovered several genetic variations among different clinical samples. Whereas the biological roles of BRCA1 have been exceptionally well documented, the functional machinery of BARD1 hasn't been fully understood. Two BARD1 cis mutations, P24S and R378S, were identified in a hereditary breast and ovarian malignancies report. BARD1 and BRCA1interaction m's affinity is decreased by the P24S mutation, whereas the R378S variant prevents the BRCA1/BARD2 complex from moving into the nucleus. The simultaneous presence of these two mutations contributed synergistically to tumor progress in vitro and in vivo models. Additionally, these two mutations mutually impair the DNA damage response, imposing genomic stability, although neither mutation alone can have a harmful effect [64]. Seven polymorphisms, including somatic missense mutations and germline modifications, were identified within the coding sequence of BARD1 in mutational research that included a variety of gynecological malignancies, ovarian, breast, and uterine tumors. These mutations caused the loss of the wild-type BARD1 allele, which led to the growth and spread of malignancies. A woman with breast and endometrial cancer presented simultaneously had the BARD1 mutation (Gln564His) [65]. Furthermore, the Gln564His mutation of BARD1 was reported to avoid p53-dependent apoptosis by reducing binding to the polyadenylation cleavage specification complex (CSTF-50) [33, 36].

Three non-synonymous variants in the BARD1 gene (Pro24Ser, Arg378Ser, and Val507Met) were assessed in a case-control analysis of 507 Chinese women with breast cancer and 539 matched controls. These SNPs demonstrated significant reductions in breast cancer risk and limited penetrance effects in the BARD1 gene on breast cancer propensity [66]. On the other hand, a large case-control study was carried out among the European (Polish and Belarusian) population to investigate the impact of the nonsense mutation c.1690C>T (p.Q564X). This nonsense variant was found to have a low/moderate increase in breast cancer risk (OR = 2.30, p = 0.04). The risk was further elevated in breast cancer forms that are more aggressive; TNBCs, bilateral breast cancers, early-onset cancer, and hereditary breast and ovarian cancers are a few examples [67]. According to the European study, the BARD1 mutation, one of the most prevalent non-BRCA1/2 mutations, was identified in 10901 TNBC cases and demonstrated a significant contribution to TNBC propensity with an incidence of 0.5−0.7%. Furthermore, Caucasian PVs American carriers of BARD1 gene pathological variants were at lower risk of TNBC (21%) than African American carriers of BARD1 gene mutations (39%) [41, 68].

To categorize the exon mutation of the BARD1 gene in 60 early-onset breast cancer patients and 240 healthy controls, direct sequencing and SNaPshot analysis were used. BARD1's rs28997575 site was found to have a deletion mutation, which increased the incidence of breast cancer by 3.4 times (P = 0.013) compared to the unaffected group. On the other hand, it was discovered that a different GC genotype missense mutation at the rs2229571 location of BRDA1 was associated with a 72.6 percent (P = 0.001) decreased risk of breast cancer. Remarkably, compared to the control group, the majority of variant carriers have a long family history of breast

cancer. Highlighting the significant contribution of breast cancer-positive family history to the elevated risk of breast cancer due to genetic predisposition, especially in BARD1 polymorphism carriers [69]. Likewise, several pathogenic variants (PVs) of the BARD1 gene were compiled in a sizable pooled analytical research of both breast cancer (48,000 cases) and ovarian cancer (20,800 cases). These BARD1 PVs had a moderate chance of developing breast cancer (odds ratio (OR) = 2.90, 95 percent confidence intervals [CIs]: 2.25–3.75, p 0.0001) but not ovarian cancer (OR = 1.36, CIs: 0.87 to 2.11, p = 0.1733). As a result, the BARD1 gene has been suggested as a diagnostic biomarker for evaluating breast cancer patients [70].

More recently, three BARD1 inherited missense mutations were found in the RING domain (Cys53Trp, Cys71Tyr, and Cys83Arg) in a family diagnosed with breast malignancy. However, according to the study, the mutant BARD1/BRCA1 complex was unable resulting nucleosomes and resulted in a loss of H2A ubiquitylation. Mutant BARD1 could heterodimerize with BRCA1 due to its mutations. These mutations also activate a defect in transcriptional repression of the BRCA1-regulated estrogen metabolism genes CYP1A1 and CYP3A4, which are usually controlled by the H2A ubiquitylation pathway [71]. 76 BARD1 cancer-associated missense and truncation variants were effectively identified in a whole-exome sequencing analysis on 10,000 cancer samples from 33 cancer types. Significantly, just two known benign mutations were found to be connected to HDR, whereas four pathogenic mutations are not linked to HDR. DNA damaging agents were more sensitive to BARD1 mutant cells [72].  With an incidence of 0.5–0.7%, BARD1 was statistically substantially related to a moderate to high risk of TNBC. A rare missense mutation of BARD1 gene c.403G>A or p.Asp135Asn was noticed in TNBC patients. This mutation was reported to increase the response of breast cancer cells to PARPi therapy [73]. While additional BARD1 isoforms are highly expressed in several types of cancer, their common pathogenic effect is owing to the expression of the oncogenic dominant-negative form and alternative splicing (**Table 2**) [15, 74].

BARD1 gene polymorphism was found in cases of neuroblastoma and breast cancer cases. The probability of developing neuroblastoma was strongly correlated with three BARD1 gene polymorphisms (rs7585356 GNA, rs6435862 TNG, and rs3768716 ANG). Using the TaqMan approach on 145 cases and 531 controls, only the rs7585356 GNA polymorphism demonstrated notable findings in relation to higher vulnerability to nephroblastoma (odds ratio (OR) = 1.78, 95 percent confidence interval (CI) = 1.01–3.12) with stage I + II clinically [76]. The impact of eleven BARD1 SNPs on NB development has been studied in a Chinese publication. Seven out of eleven BARD1 SNPs revealed an increased risk of high stage (III/IV) NB occurrence. One SNP in the 5'-UTR (rs17489363 G > A), two SNPs in exon (rs2229571 G > C and rs3738888 C > T), and four SNPs in intron (rs3768716 A > G, rs6435862 T > G, rs3768707 C > T, and rs17487792 C > T), were among the eleven BARD1 SNPs [77]. According to reports, the variant (rs17489363 G > A) in the BARD1 gene, which is tied to NB and linked to a decrease in BARD1-FL transcription, is the most common SNP in the gene [50].

Exon 5 is frequently skipped due to the mutation c.1361C>T, which interferes with ANK repeat domains, a critical component of the splicing factor SC35 that regulates apoptosis in the ovarian cancer cell line. NuTu-19 [78]. The NuTu-19 cell line was resistant to the induction of apoptosis. Still, after exogenous expression of the entire gene BARD1, it became susceptible to apoptosis, indicating that the absence of exon 5 results in abnormal isoforms that have lost their capacity to suppress tumors and affect the apoptosis pathway [79]. The BARD1 mutations c.1977A > G, p.Gln715Ter,

*The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker… DOI: http://dx.doi.org/10.5772/intechopen.107143*

#### **Table 2.**

*BARD1 variants in breast cancer predisposition.*

c.2148delCA, and p.Thr716fs\*12 have also been associated with other gynecological cancers, including fallopian tube, ovarian, and cervical cancers [79–81].

These findings from mounting data have collectively led to the conclusion that there is a context-dependent high/moderate risk of breast cancer associated with specific BARD1 SNPs. Therefore, additional practical and experimental studies are required to validate the aforementioned facts further.

#### **1.7 Correlation between The Cys557Ser BARD1 mutation and risk of breast cancer**

One meaningful known change to BARD1 is a missense mutation that causes the amino acid cysteine to be swapped out for the amino acid serine at position 557 (Cys557Ser) [75]. The 126 Finnish breast and ovarian cancer cases were used in a mutational analysis study to examine the possible impact of BARD1 alterations on tumor formation. Breast cancer cases were more likely to have the Cys557Ser missense mutation than healthy controls (7.4 vs. 1.4 percent, p = 0.001). To alter the transcriptional and apoptotic machinery, this variation is required. Intriguingly, the index cases were negative for BRCA1 and BRCA2 mutations highlighting that the occurrence of this mutation in familial predisposition to breast cancer is sufficient to cause the disease on its own [75]. In a study after this one, Stacey et al. and his colleague investigated the relationship between BARD1 Cys557Ser mutation and a familial group of breast cancer using a dataset of 1,090 Icelandic breast cancer patients with invasive type and 703 controls. Carriers of this variant are more likely than non-carriers to develop lobular and medullary breast carcinomas as single or multiple primary breast cancers. Additionally, this risk increased to 0.047 among individuals with the BARD1 Cys557Ser mutation and the BRCA2 999del5 mutation (OR 14 3.11, 95 percent CI 1.16–8.40, p 14 0.046) [82]. A case-control study of the Spanish and South American populations supported past investigations. Despite having a strong family history of breast cancer, the selected individuals have intact BRCA1/2 genes with no mutations. Examining the C-terminal of BARD1 Cys557Ser revealed a substantial increase in the risk of breast cancer (P = 0.04, OR = 3.4 [95 percent CI 1.2–10.2]). This likelihood was further elevated in patients with a family history of breast and ovarian cancer who were also found to have the BARD1 Cys557Ser joint mutation and the XRCC3 241Met variant (P = 0.02, OR = 5.01 [95 percent CI 1.36–18.5]) among patients with a family history of breast and ovarian cancer [83].

Contrarily, a study of Australian patients with a family history of breast cancer revealed that the frequency of the BARD1 Cys557Ser variant was not substantially

different from case-control cases (P0.3) and was not linked to an increased risk of breast cancer [84]. Similar to the Australian findings, numerous additional studies have been unable to establish a direct connection between the BADR1 variation and the development of breast cancer [85–87]. In a cohort of 5,546 BRCA1 and 2,865 BRCA2 mutation carriers, the function of the BARD1 Cys557Ser variation or BARD1 haplotypes as modifiers of BRCA1/2 linked with breast cancer risk was further evaluated. In both BRCA1 and BRCA2 mutation carriers, with a combined expected effect of 0.90 and 0.87, respectively, there was no evidence of either BARD1 mutation to indicate a significant connection with breast cancer risk [88]. Another team of researchers employed DHPLC analysis to identify nine BARD1 coding mutations, including two novel variants, in 210 breast cancer families of Australian descent (129 of which do not have BRCA1 or BRCA2 mutations) (Thr598Ile and Ile692Thr). Yet none of these mutations harbor a pathogenic impact based on their segregation, distribution, and frequency among the selected cases. In addition, non-pathogenic polymorphisms were found in the three variants (1139del21, G1756C, and A2285G) connected to breast cancer in other populations. Therefore, it was not advised in the Australian population to use BARD1 mutations or polymorphisms as a high penetrance susceptibility gene in the progression of familial breast cancer [87].

Collectively, studies linking this BARD1 Cys557Ser mutation to breast cancer incidence have been conducted in Iceland, Finland, Spain/South America, and Italy; however, other studies involving Yoruba, Chinese, Japanese, Australians, and African-Americans have shown different results [30, 65]. These contradictory results regarding the BARD1 Cys557Ser variant's relationship to familial breast cancer susceptibility raise the possibility that this mutation is restricted to a particular geographic substructure of the European population (as a result of regional migration) rather than being a de novo variant [82]. There are several reported BARD1 mutations, and it is worthwhile to highlight their impact on cancer predisposition risk.

#### **1.8 BARD1 gene as a potential target of new anticancer therapies including sensitivity to chemotherapy with a focus on breast cancer**

BARD1 has the potential to be a new target for the therapy of breast cancer, according to several research. According to Zhu Y et al. [14], tamoxifen-resistant breast cancer cells exhibit considerably greater BARD1 and BRCA1 expression levels, which confers resistance to treatment that causes DNA damage such as cisplatin and Adriamycin but not paclitaxel [89–92]. Watanabe et al. used bisulfite-pyrosequencing to study the aberrant DNA methylation status of the BARD1 gene in 30 TNBC core biopsy specimens from patients with pathologic complete response (noninvasive cancer) and noncomplete response after neoadjuvant chemotherapy (NACT). Even though BRCA1 gene hypermethylation is linked to the TNBC subtype and may affect chemosensitivity and progression under NACT, BARD1 gene hypermethylation only showed a low-to-moderate impact on these procedures [93]. Contrarily, the González-Rivera and his colleagues 2016 underline the low incidence and uncertain clinical implications of gene mutations other than BRCA1/2 (including BARD1) and the associated unfavorable outcomes for patients with breast cancer undergoing NACT [94]. Yet more recent research revealed that tamoxifen-resistant breast cancer cells had increased BRCA1 and its related protein BARD1, making them resistant to treatment that damages DNA [95]. Neoadjuvant chemotherapy now contributes significantly to breast cancer chemotherapy and is a transitional step to adjuvant regimens and other treatments [96]. It is crucial and beneficial to increase research into BARD1's role in

*The Fundamental Role of BARD1 Mutations and Their Applications as a Prognostic Biomarker… DOI: http://dx.doi.org/10.5772/intechopen.107143*

chemotherapy in women who are scheduled for NACT. Both ovarian and breast cancer patients with BRCA1 mutations who initially responded to platinum and PARPi therapy eventually developed resistance to both drugs [97]. In some populations, especially those with evidence of a higher occurrence of BARD1 gene mutations, it is fair to test BARD1 gene isoforms. Additionally, this method would need to be studied for its applicability to outcomes, survival rates, quality of life, influence on treatment choices, and cost-effectiveness for all patients with breast cancer.

#### **2. Concluding remarks and perspectives**