Abstract 4632: Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC

Abstract

Abstract Background: Breast cancer is the second leading cause of death for woman. Within breast cancer, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii). Methods: BT20, BT549, MDA-MB-157 and MDA-MB-231 cells (all TNBC cells), and immortalized human mammary epithelial cells (HMLE) were grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were incubated with IC50 concentrations of each compound for 24 h, after which they were allowed to grow and form colonies. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Celastrol and triptolide treatment suppressed proliferation and colony forming ability of all four TNBC cell lines, but not that of the immortalized HMLE cells. The compounds increased apoptotic cell death, based on increased Annexin V staining. Moreover, there was increased expression of the pro-apoptotic protein Bax but decreased expression of the anti-apoptotic protein, Bcl2 and BclXL. Immunoprecipitation-coupled western blots also showed that the compounds inhibit HSP90/CDC37 complex formation. Interestingly, the coupled immunoprecipitation-western blot analyses showed increased HSP90-BRCA1 interaction after treatment with the compounds. Coupled to this, western blot and immunostaining assays showed increased cytosolic levels and reduced nuclear levels of BRCA1 protein. Similar results were obtained in vivo with BT20 xenografts. In addition to decreased tumor size in response to treatment with celastrol or triptolide, the xenograft tissues showed an increase in cytoplasmic BRCA1 levels following treatment. In addition, there was increased in HSP90-BRCA1 complex formation in the treated xenograft tissues. Finally, knockdown of BRCA1 using specific silencer RNA resulted in partial inhibition in cell growth reduction after celastrol and triptolide treatment in both BT20 and MDA-MB-231 cells. Conclusion: Taken together, these data suggest that both celastrol and triptolide suppress TNBC cell growth, in part through increasing cytosolic HSP90/BRCA1 complex formation. Citation Format: Prabhu Ramamoorthy, Parthasarathy Rangarajan, Ossama Tawfik, Shrikant Anant, Roy A. Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4632.