One BRCA BRCA delT and

One BRCA2 (BRCA2 6174 delT) and two BRCA1 (BRCA1 5382insC and BRCA1 185 delAG) founder mutations account for 90% of inherited BRCA1/2 mutations in patients of Ashkenazi Jewish descent (Petrucelli et al., 2010). Because 0.1% of the Ashkenazi population carries the BRCA1 5382insC mutation, we estimate that this mutation is present in approximately 10,000 individuals in the United States alone. A deeper understanding of how each of the Ashkenazi BRCA mutations leads to breast cancer is important because it will improve our understanding of phenotypic variabilities due to different mutations in the same gene. Although mice genetically engineered to have either Brca1 or Brca2 deficiency have been informative models for studies of breast cancer development (Drost et al., 2011; Drost and Jonkers, 2009; Evers and Jonkers, 2006; Shakya et al., 2011), differences in underlying biology exist between humans and mice. Thus, human models are necessary to complement animal models. It has been reported that mutation of a single BRCA1 allele leads to genomic instability in human cells, a phenomenon not observed in mice (Konishi et al., 2011). Generation of induced pluripotent stem SR 3576 Supplier (iPSCs) (Takahashi et al., 2007) from patients carrying BRCA1/2 mutations may provide a window into the cellular phenotypic differences driving their increased cancer risk. BRCA1/2 mutant iPSC lines may also prove useful for screening therapeutic compounds for the prevention and treatment of genetically predisposed cancer patients. To probe the cellular phenotypes of patients with BRCA1-associated cancers, we reprogrammed primary dermal fibroblasts from a family carrying the Ashkenazi BRCA1 5382insC mutation into 24 iPSCs. Here, we report on our characterization of these cell lines using in vitro and in vivo cellular differentiation assays as well as gene-expression and whole-genome sequencing (WGS) analyses.
Results
Discussion We report the generation of a series of iPSC lines from individuals with the BRCA1 5382insC mutation, which is a well-known predictor of high risk for breast and ovarian cancer (Futreal et al., 1994). Generation of iPSCs from individuals with BRCA1 mutations provides an opportunity to examine why cancer develops more frequently in these patients. In this study, it was easier to reprogram the mutant fibroblasts than the fibroblasts from relatives without the BRCA1 mutation. Determining whether this difference is due to the mutation itself or simply to line-to-line variations due to other characteristics (e.g., growth characteristics and handling) will require the generation of iPSCs from additional families and/or heterozygous mice with BRCA1 mutations. Our data contrast with those obtained by González et al. (2013) from mouse embryonic fibroblasts (MEFs), in which homozygous mutations of Brca1 led to reprogramming defects. Those authors did not include MEFs from heterozygous Brca1 mutant embryos, and the human BRCA1 mutations we used are different from the mouse Brca1 mutations used in their study. Finally, the parental fibroblasts in the mouse experiments were embryonic, whereas our fibroblasts were adult. The reprogramming experiments in mice with the BRCA1 5382insC mutation reported here, and TALEN-mediated targeting of the remaining human wild-type BRCA1 allele in the heterozygous iPSC or fibroblast lines generated in this study will begin to address these differences. We observed that mutant and wild-type BRCA1 alleles were expressed at equivalent levels in fibroblasts and their derivative iPSCs and teratomas. There was also a specific upregulation of PKC-theta in the BRCA1 5382insC iPSCs compared with the wild-type iPSCs. A functional relationship between BRCA1 and PKC-theta levels may involve the known interaction of BRCA1 with the HDAC2 complex (Chang et al., 2011) or the role of BRCA1 as a transcription factor (Haile and Parvin, 1999; Horwitz et al., 2006; Schlegel et al., 2000). A related finding in this report is that PKC-theta is overexpressed in some breast tumors. PKC-theta has been found to be a reliable marker of GISTs (Blay et al., 2004; Motegi et al., 2005; Ou et al., 2008), and prior reports have suggested that PKC activity, although not specifically PKC-theta activity, is upregulated in a subset of breast cancers (Gordge et al., 1996; O’Brian et al., 1989). We found PKC-theta to be upregulated in a fraction of breast tumors that are frequently hormone-receptor negative, a characteristic of BRCA1 mutant tumors. Because of the tumorigenic potential of PKC-theta, these results suggest that lower levels of PKC-theta in the presence of wild-type BRCA1 may be a mechanism for the tumor-suppressor function of BRCA1. In fact, Belguise et al. determined that PKC-theta is elevated in ER-negative human breast cancer cell lines (Belguise et al., 2012) and NF-kappaB-induced mouse breast tumors (Belguise and Sonenshein, 2007). They showed that PKC-theta inhibits ER expression. The MDA-436 BRCA1 mutant cell line, which expresses PKC-theta at levels higher than any of the other ER-negative breast cancer lines tested in their report, was the only BRCA1 mutant cell line they analyzed (Belguise et al., 2012). However, all of the ER-negative lines that were found to have high PKC-theta have been reported to be haploinsufficient for BRCA1 (Elstrodt et al., 2006). Upregulation of PKC-theta in our BRCA1 mutant iPSCs may be direct or indirect. If BRCA1 is directly upstream of PKC-theta, this would explain why BRCA1 mutant breast cancers are frequently ER negative (Hosey et al., 2007).