Abstract B36: Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC

Abstract

Abstract Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course, resistance to chemotherapy, and poor prognosis compared to other breast cancer subtypes. However, breast cancers arising in BRCA1 mutation carriers appear to be particularly sensitive to platinum-based chemotherapy agents. The tumor suppressor BRCA1 (breast cancer type 1 susceptibility protein) is part of a protein complex that repairs DNA damage by homologous recombination. Recent studies have shown that tumor cells expressing high levels of BRCA1 are resistant to both ionizing radiation (IR) and platinum-based chemotherapy agents, and that ablation of BRCA1 expression can restore sensitivity to these agents. We have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. However, established HSP90 inhibitors exhibit a high level of toxicity unrelated to HSP90. Recently, we observed that the natural compounds gedunin and celastrol modulate HSP90 activity without inducing side effects. We believe that combinatory use of these compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies. Study Design: The goal of these experiments is to demonstrate that combination treatment with the HSP90 inhibitors gedunin and celastrol will maximize BRCA1 degradation (via inhibition of HSP90), impair the DNA repair system, and allow carboplatin to effectively eradicate TNBC cells. We will confirm that this is a HSP90 mediated event that results in the impairment of DNA repair. Using in vivo models of breast cancer, we will demonstrate that natural compounds could sensitize BRCA1-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth. Results: Using Western blot analysis, we observed that the combination of gedunin and celastrol attenuates HSP90 activity, leading to a dose-dependent reduction in BRCA1 degradation in HCC1937BRCA1 TNBC cells. Additionally, we the combination facilitated carboplatin-mediated inhibition of proliferation in both BT-20 and HCC1937BRCA1 cells. Immunofluorescence staining for g-H2AX and RAD51 demonstrated that the two HSP90 inhibitors prevent BRCA1-mediated recruitment of DNA repair complexes following ionizing radiation-induced DNA damage. To test the effects of celastrol and gedunin in vivo, we generated mouse xenografts by performing fat pad injections of HCC1937BRCA1 cells. Mice were dosed I.P. every 5 days with celastrol (5 mg/kg), gedunin (5 mg/kg), and carboplatin (25 mg/kg) for 20 days. Treatment with the HSP90 inhibitors along with carboplatin resulted in significant reduction in both tumor size and weight. In contrast treatment with carboplatin alone resulted in tumors of size similar to vehicle-treated animals. Additionally, phospho-H3 and cleaved caspase 3 immunofluorescence staining indicated a corresponding decrease in cell proliferation and increased apoptosis in tumors from celastrol+gedunin+carboplatin -treated animals compared to either vehicle or carboplatin alone-treated. Future studies will focus on examining the effects of this combination treatment in TNBC xenografts created with patient specimens, and in defining the interaction between HSP90 and BRCA1. Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC. Citation Format: Kelli Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B36.