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Dissertation - Dongju Park- revised.pdf (3.32 MB)

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The role of PALB2 in BRCA1/2-mediated DNA repair and tumor suppression

Park, Dongju
http://rave.ohiolink.edu/etdc/view?acc_num=osu1502525517885784

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Germline mutations in the cancer susceptibility genes BRCA1 or BRCA2 confer an increased risk of developing breast, ovarian and pancreatic cancer. BRCA1 and BRCA2 are key molecules in DNA damage repair, specifically repair by homologous recombination (HR). Accumulating DNA damage, due to dysfunction of the BRCA genes, leads to genome instability and increased cancer risk. In 2006, a novel protein, PALB2 (partner and localizer of BRCA2) was identified as a DNA repair pathway component (1). PALB2 co-localizes with BRCA1 and BRCA2 at DNA damage sites and is thought to act as an adaptor protein that mediates the BRCA1-BRCA2 interaction (2). Indeed, prior studies showed that BRCA1 and PALB2 directly interact through their coiled-coil domain. The C-terminal WD40 domain of PALB2 binds to BRCA2 and the BRC repeats of BRCA2 recruit a central protein for HR, the Rad51 recombinase. Recently, mutations in the PALB2 gene were reported in the tumors of breast and pancreatic cancer patients. Interestingly, some of these mutations could inhibit its direct binding of PALB2 to BRCA1 or BRCA2 thus disrupting DNA repair. Furthermore, missense mutations in the coiled-coil domain of BRCA1 (M1400V, L1407P and M1411T) that mediates the interaction with PALB2 have been reported among familial breast cancer patients, which also could increase the risk of pancreatic cancer. Therefore, we hypothesized that physical and functional interaction between BRCA1 and BRCA2 through the linker protein PALB2 is required for BRCA1/2-mediated HR and tumor suppression. To test this hypothesis, we first determined whether PALB2 is a bonafide tumor-suppressor by deleting Palb2 in the pancreas specifically in genetically engineered KrasLSL-G12D/+; p53LSL-R270H/+; Pdx1-Cre mouse model (now referred to as KPC mice) and observing these animals for pancreatic tumors in comparison with Brca1flex/flex-KPC or Brca2 flex/flex-KPC pancreatic tumor mouse models. Homozygous deletion of Palb2 in the pancreas of KPC mice accelerated pancreatic tumor development compared to KPC mice, similarly to Brca1flex/flex-KPC and Brca2 flex/flex-KPC animals. However, the histopathology of tumors was different between the groups (KPC vs Palb2 flex/flex-KPC vs Brca1flex/flex-KPC vs Brca2flex/flex-KPC). Pancreatic tumor cell lines established from Palb2 flex/flex-KPC mice showed hypersensitivity to DNA damaging agents such as interstrand cross-linkers, Mitomycin C (MMC) and Cisplatin and poly ADP ribose polymerase inhibitor (PARP inhibitor) to an extent similar to that of cells from Brca1flex/flex-KPC or Brca2flex/flex-KPC tumors, compared to cells generated from KPC tumors. In addition, Palb2 flex/flex-KPC mice in vivo treated with MMC showed clearly prolonged survival. These results suggest that PALB2 plays a key role in BRCAs mediated HR and tumor suppression. Next, we determined if the BRCA1-PALB2 interaction per se is critical for BRCA1-mediated tumor suppression by generating Brca1 L1363P point mutant knock-in mice (L1407P in human sequence) that abolishes the binding of BRCA1 to PALB2. Mouse embryonic fibroblasts (MEFs) from homozygous Brca1 L1363P mutant animals exhibit hypersensitivity to DNA damaging agents (MMC, PARP-inhibitor and ionizing radiation (IR)), and following IR mutant cells fail to recruit the Rad51 recombinase to sites of DNA damage, implying a defect of DSBs repair by HR. Accumulation of unresolved DNA damage induces hyperactivation of p53 and its downstream target p21 in Brca1L1363P/L1363P primary MEFs resulting in impaired proliferation and premature senescence. While Brca1-/- null mice are early embryonic lethal, homozygous Brca1L1363P/L1363P mice are viable. However, mutant mice exhibit growth retardation to various extents. Some mutant animals were extremely small, developed aplastic anemia and died within a month. All other Brca1L1363P/L1363P animals developed T-cell acute lymphoblastic leukemia (T-ALL) with an average latency of 3 months. Interestingly, the majority of T-ALLs (53%) from Brca1L1363P/L1363P animals acquired activating Notch1 mutations such as discovered in patients with T-ALLs. The phenotypes observed in Brca1L1363P/L1363P cells and mice recapitulate clinical phenotypes seen in Fanconi Anemia (FA) patients. Therefore, this mouse model can provide insights for developing new therapies for FA. Furthermore, this results demonstrate the importance of the BRCA1-PALB2 interaction in vivo and suggests that the interaction is essential for BRCA1-mediated tumor suppression.
Thomas Ludwig (Advisor)
Kay Huebner (Committee Member)
Parvin Jeffrey (Committee Member)
Mark Parthun (Committee Member)
Cellular Biology; Molecular Biology
DNA repair and tumor suppression; cellular biology

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Citations

  • Park, D. (2017). The role of PALB2 in BRCA1/2-mediated DNA repair and tumor suppression [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1502525517885784

    APA Style (7th edition)

  • Park, Dongju. The role of PALB2 in BRCA1/2-mediated DNA repair and tumor suppression. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1502525517885784.

    MLA Style (8th edition)

  • Park, Dongju. "The role of PALB2 in BRCA1/2-mediated DNA repair and tumor suppression." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1502525517885784

    Chicago Manual of Style (17th edition)

osu1502525517885784
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This open access ETD is published by The Ohio State University and OhioLINK.