Evaluation of: Konishi H, Mohseni M, Tamaki A et al. Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells. Proc. Natl Acad. Sci. USA 108(43), 17773–17778 (2011). In their study, Konishi et al. generate and study cell lines that carry one mutant and one normal copy of BRCA1. These heterozygous (noncancerous) cells are noteworthy in that they6 carry an increased number of copy number alterations and have diminished DNA repair capacity. These observations go beyond the classical two-hit hypothesis for inherited cancers, whereby the cell phenotype is normal until the second allele is lost through somatic mutation or epigenetic silencing. They propose that BRCA1 heterozygosity is associated with increased genomic instability, which accelerates the mutation rate of other critical genes, including the second copy of BRCA1. They conclude that BRCA1 is a haploinsufficient tumor suppressor gene, which when lost through inheritance, increases a cell's susceptibility to acquire further mutations.
Summary of methods & results
By generating two unique human cell lines, each with a single mutant copy of BRCA1 (BRCA1+/−), Konishi et al. were able to measure the consequences of decreasing the cellular dose of the BRCA1 protein. In contrast to previous studies, the BRCA1+/− cell lines were derived from noncancerous human breast epithelial cells. These unique cells were then employed in a battery of cellular and molecular assays designed to measure their capacity for DNA damage repair and their ability to maintain genomic stability. The authors demonstrated that heterozygous BRCA1 inactivation results in genomic instability. These cells also showed a higher degree of gene copy number loss and loss of heterozygosity as measured by single nucleotide polymorphism array analyses and FISH. It was concluded that BRCA1 haploinsufficiency accelerates hereditary breast carcinogenesis by facilitating additional genetic alterations.
Discussion
Genetic mutations can be of inherited or of somatic origin and can adversely affect the entire organism or an individual cell. Mutations are said to be recessive if the heterozygous mutant (one abnormal copy) displays a normal phenotype and the homozygous mutant (two abnormal copies) displays an abnormal phenotype. If the heterozygous mutant displays a mutant phenotype then a mutation is said to be dominant. The concept of tumor suppression was proposed by Knudson to explain the inheritance patterns of pediatric cancers. In his ‘two-hit hypothesis’, Knudson proposed that a tumor suppressor gene must be bi-allelically inactivated in order to initiate a fully fledged tumor [1]. This implied that the gene mutations in the cancer cells would act in a recessive manner.
Classically, the categories of dominant and recessive genes were dichotomous; recently, however, the demarcation has become blurred with the introduction of terms such as partial dominance and haploinsufficiency. Both terms describe a state whereby cells from heterozygote individuals exhibit features that are intermediates of the normal’ and ‘mutant’ phenotypes as a result of a reduced level of protein. Conventionally, we think of the breast–ovarian cancer syndrome as a dominant disease with generation-to-generation transmission. But at the cellular level this phenomenon appears to be recessive, because tumor cells are reported to almost always lose the wild-type BRCA1 allele. Consequently, several questions come to mind, including: do all breast and ovarian cancers in BRCA1 carriers exhibit loss of heterozygosity? And do cells with only one normal BRCA1 gene copy exhibit any abnormalities?
Women with a mutation in BRCA1 have a risk of developing breast cancer as high as 80% by the age of 70 [2]. In most of the associated breast tumors no functional BRCA1 protein is present. Notably, nullizygosity is incompatible with life in mouse models of Brca1 and it is not clear how cells can survive without any BRCA1 protein. In a recent study, Konishi et al. modeled allelic insufficiency of the cancer susceptibility gene BRCA1 [3]. Loss of BRCA1 in an otherwise normal cell leads to cell death owing to proliferation defects [4]; accordingly, Konishi et al. were unsuccessful in generating bi-allelic disruption of BRCA1 in cell lines [3]. Presumably, in the complete absence of BRCA1 a cell triggers a fail-safe mechanism to activate programmed cell death. Such behavior would place BRCA1 in a category previously termed obligate haploinsufficiency [5]. BRCA1 only partially conforms to this model because in the majority cases the other normal allele of BRCA1 succumbs to loss or mutation. This then raises two important questions: what happens in the precancerous cells prior to the disruption of the normal BRCA1 allele? And why is the normal allele of BRCA1 consistently a target for mutation? Konishi et al. propose some answers for the first question.
By generating two unique human cell lines, each with a single mutant copy of BRCA1 (BRCA1+/−), Konishi et al. were able to measure the consequences of decreasing the cellular dose of the BRCA1 protein. The BRCA1+/− cell lines in this study were derived from noncancerous human breast epithelial cells [3]. These cells were employed in a battery of cellular and molecular assays designed to measure their capacity for DNA damage repair and their ability to maintain genomic stability. They found that after double-strand breaks induced by γ-irradiation, the heterozygous cells had a reduced capacity for homologous recombination-mediated DNA damage repair and as a consequence, they were also sensitive to genotoxic stresses conferred by chemotherapy drugs such as doxorubicin and γ-irradiation, but not poly(ADP-ribose) polymerase inhibition.
The genetic alterations induced by exposure of BRCA1-deficient cells to γ-irradiation were analyzed through high-throughput methods and FISH and found to be moderately elevated [3]. Notably, changes at the genomic loci encompassing TP53 and MYC showed that there was an increased propensity to alter these genetic loci in BRCA1+/− patients. Together these findings suggest that normal cells from BRCA1+/− patients might be sensitive to genotoxic agents and that perhaps environmental mutagens may increase the inherent risk of cancer. These findings are reminiscent of observations made with patients afflicted with Li–Fraumeni syndrome. Malkin and colleagues report that individuals who carry a TP53 mutation also have an increased number of copy number variants [6]. Similar to BRCA1 mutations, TP53 mutations are associated with genomic instability that may act as the genetic foundation on which larger somatic chromosomal deletions and duplications build, leading to the development of cancer.
Future perspective
Konishi et al. suggest that an inherited mutation of a single allele of BRCA1 results in a decrease in the capacity of epithelial cells (in particular from the breast and ovaries) to repair DNA damage. On its own, this study offers little to help us understand why BRCA1 mutations affect cancers of specific organs and are so much more important in women than in men.
Importantly, the decreased capacity for DNA damage repair described in this study may explain the adverse effects of early chest x-rays on breast cancer risk in BRCA1 mutation carriers [7]. However, there is little evidence that radiotherapy of breast cancers or of adult exposure to the carcinogens in cigarettes modifies breast cancer risk in mutation carriers [8]. In fact, radiotherapy is associated with a marked reduction in the incidence of second primary cancer [9]. Perhaps the timing of exposure and intensity of radiation dose is critical [7,8].
We have recently proposed a stem cell-based model for breast cancer risk to explain the relative lack of evidence for carcinogens in breast cancer in the general public [10]. In this model the accumulation of mutations in precursor or progenitor cells is responsible for the generation of cancerous cells from mammary stem cells. Perhaps the accelerated rate of somatic mutations in the BRCA1 carriers is the underlying cause for the increase in cancer risk. An inherently high somatic mutation rate may explain the 80% increased lifetime risk of BRCA1+/− individuals to develop cancer as well as the long and variable latency period [2]. Perhaps reducing exposure to genotoxic stress will provide an opportunity for intervention, but it is most interesting to note that the risk factors for breast cancer observed to date in BRCA1 carriers are related to hormones and reproduction [11,12].
Executive summary
Konishi et al. have generated novel and unique BRCA1-heterozygous (BRCA1+/−) cell lines derived from normal human mammary cell lines.
BRCA1+1- cells have a reduced capacity for DNA damage repair, are genomically unstable and have a higher degree of genomic losses and loss of heterozygosity.
BRCA1+/− cell lines are sensitized to chemotherapy but not to poly(ADP-ribose) polymerase inhibition.
