Srinivasan Madhusudan - Targeting Replication protein A (RPA) for personalization of breast cancer therapy 2019 Pilot Grant
Lay summary
Background: Advanced breast cancer is a devastating disease. Not all patients respond to therapy. Chemotherapy and radiotherapy work by causing DNA damage in cancer cells. If we can increase DNA damage we can improve treatment.
Aims: We want to increase DNA damage in tumours by developing a new treatment aimed at blocking DNA repair.
Techniques: Using tissue culture, we will reduce the amount of a protein called RPA in breast cancer cells. We will then test if these cells respond better to chemotherapy and radiotherapy. We will also test if RPA blockade can also make breast cancer cells respond better to therapy.
Impact: Studies on RPA has not been done before in breast cancer. We will generate new information. Our study will help fine tune treatments in breast cancer
Scientific summary
Background: Modulating DNA repair has become an exciting possibility for personalized therapy; with potential for a real change in the way breast cancer patients are treated. We have generated a substantial body of clinical data to provide evidence that RPA, a critical DNA repair protein, is a valid and exciting novel target in breast cancer.
Aims:
1. Test RPA deficient breast cancer cells for sensitivity to chemotherapy (doxorubicin, cisplatin & 5-FU) and radiotherapy.
2. Test RPA deficient breast cancer cells for synthetic lethality interaction with PARP inhibitors (Olaparib and Talazoparib) and PARG inhibitor ((PDD00017273).
3. Test RPA inhibitors (TDRL-505 and NSC 111847) as synthetic lethality partners in BRCA1 deficient breast cancers. The ultimate purpose is to develop a novel targeted therapy for breast cancer.
Techniques and Methodology: We will generate transient RPA knock-downs (siRNAs) and RPA knockouts (CRISPR/Cas-9 methodology) in relevant breast cancer cell lines. We will conduct clonogenic survival assays for chemo-sensitization, radio-sensitization and synthetic lethality. For those cell lines where increased cell killing is observed functional studies will be completed to understand cellular response in control and RPA deficient cells. We will conduct DNA damage response signaling pathway analysis using phospho-western blotting, DNA damage and repair with time post treatment (H2AX, RAD51, 53BP1 foci), apoptosis (annexin V staining) and cell cycle progression (FACS).
Impact on breast cancer research: This will be the first comprehensive study of RPA in breast cancer. The data generated in this study will considerably improve our understanding of the role of RPA in breast cancer pathogenesis. Importantly, the ability to personalize breast cancer therapy based on RPA status has the potential to improve patient outcomes. RPA inhibitor could have clinical application as chemo/radio-sensitizer and as a synthetic lethality partner in DNA repair deficient breast cancers.