IN THE SPOTLIGHT Tracking Evolution of BRCA1-Associated Breast Cancer

Author’s Affi liation: Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Corresponding Author: Jos Jonkers, Division of Molecular Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: 31-20-5122000; Fax: 31-20-5122050; E-mail: [email protected] doi: 10.1158/2159-8290.CD-12-0186 ©2012 American Association for Cancer Research. Women with heterozygous protein-disabling germline mutations in the BRCA1 gene are strongly predisposed to developing breast or ovarian cancer. BRCA1 is implicated in several cellular processes, most notably in the repair of DNA double-strand breaks (DSB) by homologous recombination (HR; ref. 1). Loss of BRCA1 function may therefore promote tumorigenesis by forcing cells to repair DSBs via error-prone mechanisms such as nonhomologous end-joining, resulting in increased genomic instability and accelerated acquisition of mutations in additional oncogenes and tumor suppressor genes that drive BRCA1-associated tumorigenesis. Examples of such collaborating cancer genes are TP53 and PTEN, which are frequently mutated in BRCA1-associated breast cancers (2, 3). The vast majority of BRCA1-associated tumors show loss of the wild-type BRCA1 allele through LOH (4). BRCA1 was therefore initially viewed as a classical tumor suppressor gene, that is, loss of the wild-type allele by a “second hit” mutation was considered to be the very fi rst tumor-initiating event in BRCA1 heterozygous cells. This notion was, however, confounded by the fact that normal cells do not tolerate acute loss of BRCA1. Genetic inactivation of BRCA1 in cultured cells induces a rapid proliferation arrest, and homozygous Brca1-mutant mice display early embryonic lethality (5). Together, these observations suggested that mutations in other genes should precede loss of the BRCA1 wild-type allele to render cells permissive to loss of this essential component of the HR machinery. TP53 was a plausible candidate given its frequent mutation in BRCA1-mutated tumors and its central role in the activation of cell-cycle checkpoints following DNA damage. Indeed, homozygous Brca1-mutant mice displayed prolonged embryonic survival and in some cases even postnatal viability when crossed onto a Trp53 knockout background (5). Moreover, analysis of fallopian tube epithelium from BRCA1-mutation carriers showed loss of the wildtype BRCA1 allele in tubal intraepithelial carcinomas (which are the precursors of high-grade serous ovarian cancer), but not in “p53 signature” foci of mutant p53–expressing cells present in the same tissue samples, arguing that TP53 mutation precedes loss of BRCA1 function in the evolution of BRCA1-associated ovarian cancer (6). In this issue of Cancer Discovery, Martins and colleagues (7) provide further evidence for the notion that BRCA1 inactivation may not be the fi rst event in BRCA1-associated tumorigenesis. Using a combination of histologic protein and DNA detection methods (immunohistochemistry, immunofl uorescence, FISH), they assessed at the single-cell level the expression status of PTEN and mutant p53 protein, as well as the mutational status of the BRCA1 wild-type allele in tissue sections from 55 BRCA1-associated breast cancers and 20 sporadic control cases. After counting the number of cells assigned to each of the 8 different states representing all possible combinations of 0, 1, 2, or 3 mutations, they determined the most probable tumor-initiating somatic mutation by identifying (within the 1-mutation class) the state with the largest number of cells (Fig. 1). They applied the same method to the 2-mutation and 3-mutation classes to determine the second and third somatic mutations, respectively. Using this approach, they found 2 main paths of tumor evolution within the BRCA1-associated breast cancer panel. Initial loss of PTEN followed by loss of p53 and/or BRCA1 was observed in the majority of BRCA1-associated tumors with a hormone receptorand HER2-negative (triple-negative) phenotype. In contrast, PTEN loss was never observed in hormone receptor–positive BRCA1-associated tumors, which showed early loss of p53 followed by loss of BRCA1. Strikingly, many BRCA1-associated tumors contained a substantial fraction of tumor cells that had retained the BRCA1 wild-type allele. This wild-type allele appeared to be functional, as nuclear BRCA1 foci were observed in tumors with retention of the wild-type BRCA1 allele, but not in cases with complete BRCA loss. The fi ndings of Martins and colleagues (7) suggest that loss of the BRCA1 wild-type allele may not only be a late event, but—at least in a proportion of cases—also a nonessential step in BRCA1-associated breast tumorigenesis, raising the intriguing possibility that in these cases tumorigenesis is promoted by BRCA1 haploinsuffi ciency rather than by BRCA1 loss. Although no defects have been observed in Brca1 heterozygous mutant mice, several studies have reported haploinsuffi cient phenotypes in BRCA1 heterozygous human cells. Impaired homology-mediated DNA repair and elevated