Asan, Alparslan
(2019)
Nitroalkene Repression of Homologous Recombination as a Treatment for Triple Negative Breast Cancer.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Triple-negative breast cancer (TNBC) is a heterogenous disease accounting for ~20% of all breast cancer (BC) cases. It is characterized by high genomic instability making it an aggressive BC subtype with higher rates of metastatic disease compared to other BC subtypes. In ~15% of TNBC, genomic instability is caused by loss of function mutations in BRCA1/BRCA2 genes leading to homologous recombination (HR) deficiency which increases sensitivity for PARP inhibitor (PARPi) therapy. Therefore, there has been a great interest in extending the utility of PARP inhibitors to patients who are wildtype for BRCA1/BRCA2. The nitro-fatty acid (NFA) 10-nitro-octadec-9-enoic acid (OA-NO2) was identified as an inhibitor of RAD51, an enzyme essential in HR. NFAs alkylate protein cysteines via Michael addition reaction and Cys319 in RAD51 is a specific target of OA-NO2. Thus, to mimic a BRCA mutant phenotype in wildtype BRCA TNBC cells, OA-NO2 was combined with a PARPi (olaparib or talazoparib) and other antineoplastic DNA-damaging therapies including doxorubicin, cisplatin and ɣ-irradiation (IR). Talazoparib combined with OA-NO2 displayed high levels of synergistic growth inhibition of MM231 TNBC cells in vitro and in vivo: mice treated with talazoparib plus OA-NO2 had significantly decreased tumor growth rates when compared to vehicle, talazoparib or OA-NO2 alone. Also, OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Additional analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies, enabling temporal resolution of recombination, revealed that OA-NO2 does not affect non-homologous end-joining (NHEJ). Rather, OA-NO2 inhibits post-resection DNA DSB repair pathways HR, single-strand annealing (SSA) and alternative end-joining (Alt-EJ). In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile–based combinational therapies for TNBC.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
12 June 2019 |
| Date Type: |
Publication |
| Defense Date: |
19 April 2019 |
| Approval Date: |
12 June 2019 |
| Submission Date: |
3 June 2019 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
121 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
DNA repair, TNBC, RAD51, nitroalkene |
| Date Deposited: |
12 Jun 2019 18:18 |
| Last Modified: |
12 Jun 2020 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/36887 |
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