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theses

Homologous recombination (HR) is the only error-free pathway for the repair of DNA double strand breaks (DSBs). BRCA1 and BRCA2, the two major breast cancer suppressor proteins, play essential roles in HR-mediated repair of DSBs by promoting the recruitment of RAD51, the recombination enzyme, to DNA damage sites for the initiation of HR. PALB2 (partner and localizer of BRCA2) plays a key role in this pathway by acting as a chromatin adaptor for BRCA2 and a linker between BRCA1 and BRCA2. Like BRCA1 and BRCA2, PALB2 is a tumor suppressor gene itself. Germline, heterozygous mutations in the gene increase the risk of breast, ovarian and pancreatic cancers. However, its mechanism is not fully understood. To investigate the in vivo role of the PALB2-BRCA1 interaction, we previously generated a Palb2 knockin mouse strain that contains a mutation that disrupts BRCA1 binding. This mouse model also allows us to bypass the embryonic lethality of the Palb2 KO mice. In this study, we hypothesized that the direct communication between the two proteins is critical for proper DNA damage repair and response in vivo and for suppression of tumorigenesis. Indeed, both immunohistochemistry (IHC) and immunofluorescence (IF) demonstrated that different tissues of the Palb2 mutant mice have higher levels of endogenous DSBs (gamma H2AX foci) and slower DSB repair kinetics after ionizing radiation (IR). Yet, mutant cells were more resistant to cell death. When aged under normal conditions, mutant mice showed increased tumor incidence in multiple tissues, particularly in the liver. Upon challenging of these mutant mice with carcinogen administration or gamma irradiation, they showed accelerated tumor development as compared with wild-type (wt) mice. When crossed with Trp53 mutant mice, the compound mutant mice showed greatly accelerated development of tumors typically associated with mutations in p53, i.e. thymic lymphoma, osteosarcoma and soft tissue sarcoma, etc. Whole exome sequencing (WES) of the tumors arising from Palb2m/m;Trp53+/- mice revealed loss of the wt allele of Trp53 in the majority of tumors, with at least some tumors showing focal deletions of the wt gene, suggesting that disruption of BRCA1-PALB2/BRCA2 axis promotes regional genomic deletions that may lead to loss of other tumor suppressors such as p53. These results underscore the importance of the BRCA1-PALB2/BRCA2 pathway for tumor suppression and suggest a potentially novel mechanism for BRCA/PALB2-mediated tumor suppression, which is by preventing Trp53/TP53 loss of heterozygosity (LOH), which would allow for tumor development. Finally, we also found constitutively elevated levels of reactive oxygen species (ROS) and activation of NF-kB, a redox sensitive transcription factor, in tissues and cells from the mutant mice. Given its established pro-survival function, NF-kB activation could explain why cells in the mutant mice are resistant to apoptosis upon irradiation despite having increased and prolonged DNA damage. This finding also suggests that the NF-kB pathway may be a potential target for treatment of PALB2 and BRCA1-associated cancers.

ETD_8035

Ph.D.

Includes bibliographical references

by Amar Hekmat Mahdi

doi:10.7282/T31G0Q4Q

ETD doctoral

The author owns the copyright to this work.