Functional Evaluation of BRCA2 Variants on Tumorigenesis

Division Of Basic Sciences - Nci

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Abstract

We have examined the biological role of several BRC repeats, individually as well as in combination using our mESC based functional assay. We expressed these variants in mESC and examined their ability to functionally complement the loss of endogenous Brca2. We examined the impact of these mutants on the viability of Brca2-null ES cells, IR induced RAD51 foci formation and sensitivity to PARP inhibitor, olaparib. We found BRCA2 containing BRC1-4, 5-8, 1-2, 2 only and 4 only to rescue Brca2-null ES cell lethality similar to the cells expressing WT BRCA2. Consistent with the previous studies, Brca2-null ESCs expressing BRCA2 with BRC5-8 were viable but were sensitive to olaparib. Furthermore, we found Brca2-null ESCs with BRC1-4 to be fully viable and not sensitive to olaparib. When we examined cells for their ability to form radiation induced RAD51 foci, we found 70% of the cells with WT BRCA2 had 5 or more RAD51 foci, BRC 4 and 2 resulted in 45-60% cells with RAD51 foci and only 15% of the cells expressing BRC1 had RAD51 foci. Interestingly, BRCA2 containing a single BRC, repeat 2 as well as repeat 4, were found to be functionally indistinguishable from WT BRCA2 based on the sensitivity of cells to olaparib. Cells expressing BRCA2 containing BRC1 had an intermediate phenotype. We have shown a single repeats, BRC2 and BRC4, to be sufficient for normal growth and development. Mice carrying a single BRC repeat are not predisposed to tumorigenesis. Stem cells (SCs) have a remarkable capacity to not only self-renew and differentiate to multiple lineages but also to self-assemble and self-organize into complex and functional tissues. Taking advantage of this physical property, ESCs and adult SCs can be channeled to form three-dimensional organ-like structures- organoids. While a majority of the organ system can be modelled using an ESC-derived organoid, generation of the mammary lineage has been unsuccessful so far. However, mammary-like organoids expressing luminal and basal markers that could be induced to produce milk protein have been generated from human induced pluripotent stem cells (iPSC). Early embryonic mammary gland develops via extensive epithelial-mesenchymal interactions between the surface epithelium and the mesenchyme. While most of the surface ectoderm thickens to become epidermis, a subset of ectodermal cells interacts with the underlying mesenchymal cells to form associated skin appendages like hair follicle, teeth and mammary gland. Skin appendages develop through a localized thickening in the surface ectoderm to form a placode which invaginates to form a bud like structure. We have used this knowledge of development of the mammary gland during embryogenesis to generate mammary organoids from mESCs and are now exploring the possibility of using organoids to examine the tumorigenic potential of BRCA2 variants.