S

3418 aa

Figure 18.6 The BRCA1 and BRCA2 genes and proteins A: The genes as shown in the ensembl data base. B: The proteins and some of their interaction partners.

3418 aa

Figure 18.6 The BRCA1 and BRCA2 genes and proteins A: The genes as shown in the ensembl data base. B: The proteins and some of their interaction partners.

motif was also detected in several other proteins involved in DNA repair and/or cell cycle regulation. BRCA1 also has a transcriptional activation function, unlike BRCA2.

The BRCA2 gene is located at 13q12 and extends across «80 kb. Among 27 exons overall, exon 10 and 11 are - again - unusually large. The BRCA protein comprises 3418 amino acids. Nuclear localization signals are located C-terminally. Multiple protein interactions are supposed or documented. Most importantly, the central part of the molecule contains eight repeats of 'BRC' motifs, each consisting of «40 amino acids.

Germ-line mutations in both BRCA genes are spread out along almost the entire length of the genes. Most are small deletions or insertions resulting in frame-shift mutations. Nonsense mutations leading to truncated proteins are also prevalent. Splice-site mutations also occur as well as rare missense mutations. In each case, the mutations would be expected to inactivate the function of the proteins or - in some cases - perhaps to exhibit a dominant-negative phenotype.

In breast cancers arising in women with inherited mutations in a BRCA gene, the second allele is consistently inactivated as well, by deletion, recombination or point mutation. In this regard, therefore, BRCA1 and BRCA2 behave as classical tumor suppressor genes. However, mutations or deletions in these genes are almost never found in sporadic breast cancers. Instead, decreased levels of the proteins are often observed, which in some cases are associated with increased methylation of BRCA1 regulatory sequences. In this respect, therefore, the BRCAs do not follow the standard scheme for tumor suppressors (^5.4).

The BRCA1 and BRCA2 proteins are expressed in almost all tissues. They are important for DNA repair, prevention of chromosome breaks and checkpoint signaling. BRCA1 and BRCA2 have different, but related functions.

Lack of either protein causes an increase in chromosomal aberrations in proliferating cells, including deletions and translocations, but even fragmentation and formation of multiradial chromosomes. All these aberrations are found in breast cancers with BRCA mutations. They are primarily due to a deficit in homologous recombination repair and secondarily to a failure to activate appropriate cellular checkpoints. Homologous recombination repair (^3.3) is the preferred mechanism for dealing with DNA double strand breaks during the S and G2 phase of the cell cycle in mammalian cells, when homologous DNA strands from sister chromatids are available. In contrast, non-homologous end-joining predominates during G1 (^■3.3). This mechanism introduces a larger number of errors than homologous repair at the mended site, including small deletions and insertions. However, these errors do not seem to constitute the major problem in BRCA-deficient cells. Rather, non-homologous end-joining on its own appears insufficient to ensure chromosomal integrity.

The main function of BRCA2 in this regard appears to be control of RAD51 (Figure 18.7). BRCA2 binds and inhibits RAD51 through its BRC repeats, keeping it from binding to DNA as a multiprotein filament. During homologous recombination repair, double-strand breaks must first be processed. Then, RAD51 is released from BRCA2, likely as a result of BRCA2 being phosphorylated, and mediates recombination between the strands that are to be repaired and the intact homologous double helix. This is followed by extension of the single strands by a DNA polymerase, ligation and resolution of the recombinant Holliday junction structure (^3.3). At a later stage of this resolution, RAD51 is removed, apparently by reloading onto BRCA2. BRCA2 interacts with activated FANCD2 protein. This is activated via mono-ubiquitination by the FANC protein complex. It then moves to foci in the nucleus where BRCA2, RAD51 and the MRE11/RAD50/NBS1 (MRN) proteins reside and activates these (^3.3). Homozygous germ-line mutations in

Mre11 Rad50 Nbs1

Figure 18.7 Interaction between BRCA2 and RAD51 during DNA double-strand repair It is assumed that ATM activated by DNA double-strand breaks not only phosphorylates the MRN complex (left) that processes the open ends, but also BRCA2 (right) to release RAD51. RAD51 then mediates recombination between the processed single strand ends and homologous DNA. Rebinding of RAD51 by BRCA2 allows completion of the repair. Several further components are involved as shown in Figure 3.11.

Figure 18.7 Interaction between BRCA2 and RAD51 during DNA double-strand repair It is assumed that ATM activated by DNA double-strand breaks not only phosphorylates the MRN complex (left) that processes the open ends, but also BRCA2 (right) to release RAD51. RAD51 then mediates recombination between the processed single strand ends and homologous DNA. Rebinding of RAD51 by BRCA2 allows completion of the repair. Several further components are involved as shown in Figure 3.11.

BRCA2 cause a form of Fanconi anemia and, conversely, breast cancer incidence is enhanced in this recessive cancer syndrome (^3.4).

BRCA1 appears to have a still wider range of functions. In response to DNA double-strand breaks, it is phosphorylated by kinases sensing this damage, such as ATM, ATR or CHK2 (^-3.3). It may also be activated by other types of DNA damage, since it interacts e.g. with the MSH2 and MSH6 proteins involved in mismatch repair (^3.1), and perhaps also with the transcription-coupled nucleotide excision repair system (^3.2). Following its activation BRCA1 mediates selected transcriptional responses, such as induction of the GADD45 repair protein, and at the site of damage aids in chromatin remodeling. A crucial function is regulation of the MRN protein complex that resects the DNA ends at double-strand breaks (^■3.3). In a similar fashion to BRCA2 directing and limiting the function of RAD51, BRCA1 may control this complex and keep it from overdigesting. An important function of BRCA1 is exerted through its RING finger domain. This domain characterizes substrate recognition proteins of ubiquitin ligases. BRCA1 heterodimerizes with a protein named BARD1. Together they may support mono-ubiquitination of FANCD2 by the FANC protein complex (^-3.3), but certainly help to relocate the activated protein to the nuclear foci where BRCA2 resides. In this fashion, BRCA1 mediates repair of DNA crosslinks and stalled replication forks as well as actual strand breaks.

So, both BRCA1 and BRCA2 are involved in regulating or executing homologous recombination stimulated by the FANC proteins and perhaps also in

Figure 18.8 Interaction of BRCA1 andBRCA2 in the control of DNA repair See text for further explanations and cf. also Figures 3.9 and 3.11

regulating the MRN proteins. In a sense, BRCA2 acts 'downstream' of BRCA1 (Figure 18.8).

It is, of course, conspicuous that the functions of so many genes in which mutations convey an increased risk of breast cancer are closely related to DNA repair. On one hand, this has led to the conception of a sort of 'repairosome' that includes the BRCAs as crucial components, but also proteins actually recognizing and removing DNA damage as well as signaling components. The mutations predisposing to breast cancer would have the common effect of impeding the function of this dynamic 'organelle'. Its core structure may correspond to the nuclear foci in which BRCA2 resides.

On the other hand, the question arises why such mutations promote cancer of the breast in particular. In the case of BRCA mutations, the breast and the ovaries are the organs most susceptible to cancers by a large margin. A certain, but much lower increase of risk is observed for cancers of the pancreas, bile duct, stomach, colon, and prostate (in this approximate descending order). To date, the most likely explanation for this specificity seems that during the multiple cycles of growth and involution in the breast there is a high likelihood of incurring chromosomal aberrations unless the DNA damage surveillance and repair system works perfectly. This is plausible, but does not quite account for the increased risk of ovarian cancer as well and, even less, for the increased risk of male breast cancer in carriers of BRCA2 mutations. There are also continuing hints that some of the proteins in the 'repairosome' interact in a more direct fashion with the estrogen response. BRCA1 and TP53, among others, have been shown to interact with the estrogen receptor, mutually regulating each other's activity. So, a second (and perhaps additional) explanation might be that these proteins limit the pro-proliferative and anti-apoptotic action of the activated estrogen receptor in mammary epithelial tissue. Accordingly, BRCA1, in particular, does seem to exert a direct effect on the growth of breast and ovarian cells.

Women with germ-line mutations in BRCA1 and BRCA2 have a 40-80% estimated life-time risk ofbreast cancer compared to the «10% risk in the female population in Western industrialized countries at large. The risk of ovarian cancers is roughly 20-fold increased, as they are otherwise less prevalent. The precise increase in risk depends on the particular mutation, on genetic modifiers, and likely on environmental factors. The 4-8-fold increase in life-time breast cancer risk caused by BRCA mutations may seem moderate, but is exacerbated by the typical effects of an inherited tumor suppressor gene mutation. In familial cases, the disease appears earlier and the risk of a cancer in the contralateral organ (breast as well as ovary) is hugely increased over that in sporadic cases.

It is estimated that up to 10% of all breast cancers develop as a consequence of a mutation in a high-risk gene (Figure 18.9). Within this group, mutations in BRCA1 and BRCA2 may each underlie approximately one quarter of the cases. Mutations in all other genes listed in Table 18.1 may account for another «10%. This leaves >40% of familial cases unaccounted for by mutations in known genes. As during the initial search for the BRCA1&2 genes in the early 1990's, linkage studies are continuing in families with multiple cases of breast cancer, occuring at an younger than usual age and/or more often bilaterally. Today, such studies are greatly facilitated by the human genome sequence and technical advances allowing high-throughput analyses of genetic markers. It is therefore safe to conclude, even with research ongoing, that a "BRCA3" gene with the same impact as BRCA1 and BRCA2 will not be discovered. Instead, it is expected that mutations in several more individual genes conferring a very high risk will each be responsible in a small number of families. However, the majority of familial cases may result from mutations that increase the risk of breast cancer only moderately, but sufficiently so to cause an accumulation of cancers in some families that carry them (cf. 19.3). These same mutations will also be responsible for individual cases in other families that would be categorized as 'sporadic'.

Figure 18.9 Contribution of mutations in high risk genes to breast cancer risk Estimated proportions of cases contributed by mutations in the indicated genes. Note that the risk in 'sporadic' cases is also modulated by genetic polymorphisms.

This line of thought thus suggests a gradualism in cancer predisposition, at least for breast cancer (Figure 18.9). Mutations in certain 'high-risk' genes may confer such a pronounced increase in risk that they will regularly result in a clustering of cancers within families. They are highly penetrant and to a large degree independent of environmental modulators. Other mutations or polymorphisms in these same genes or mutations in 'low-risk' genes may emerge as a series of cancer cases in some families, but contribute to the development of many more cases categorized as sporadic. At the other end of the spectrum, certain polymorphisms in genes of DNA repair, cell protection, lipid or hormone metabolism etc. may confer only small increments in risk for breast cancer which may be strongly dependent on environmental modulators such as a high-fat diet or exposure to ionizing radiation. Since these polymorphisms are common, in contrast to actual mutations in high-risk genes, their impact on breast cancer may overall be more important. However, these effects are much more difficult to discern than the mutations in high-risk genes (cf.

Figure 18.9 Contribution of mutations in high risk genes to breast cancer risk Estimated proportions of cases contributed by mutations in the indicated genes. Note that the risk in 'sporadic' cases is also modulated by genetic polymorphisms.

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