Abstract PR05: Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells

Background: Breast cancer is the second leading cause of death for woman. Within breast cancer subtypes, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Tumors contain heterogeneous cell populations and it has numerically rare cancer stem cells with indefinite proliferative potential that is responsible for tumor invasiveness, heterogeneity, and therapy resistance. 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-231 and MDA-MB-157 cells (all TNBC cells) were obtained and 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 treated with IC50 concentration of each compound for 24h, and then allowed to grow and form colonies. Mammosphere assay was performed using 5000 cells/ml in ultra low attachment plates. Images were captured after 5 days. 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 the proliferation and colony formation ability of all four TNBC cell lines BT20, BT549, MDA-MB-231 and MDA-MB-157. Interestingly, the mammosphere assay (an assay used to evaluate the self-renewal capacity of the cancer stem cells) revealed that celastrol or triptolide significantly reduces the size and number of spheroids. Furthermore, expression of breast cancer stem cell markers ALDH1 and CD133 were significantly reduced in BT20 cells upon the treatment. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. Activation of the Notch receptor, a membrane spanning receptor involves the interaction with a ligand resulting in a series of proteolytic cleavage events culminating in the release of the Notch intracellular domain (NICD). This NICD translocates to the nucleus, and together with its interacting partner CSL/RBPJ binds to cognate element and activates the expression of downstream target genes such as Hes-1. In cells treated with either celastrol or triptolide, there was a significant reduction in NICD, and its downstream target Hes-1. Furthermore, there was a reduction in ALDH+ cells. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control vector transfected cells in reducing proliferation, colony formation or mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size of tumors in mice treated with celastrol or triptolide. In addition, western blot and immunohistochemistry analyses demonstrated a reduction in the number of ALDH+ and CD133+ cells. Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling. This abstract is also presented as Poster B31. Citation Format: Prabhu Ramamoorthy, Sydney Byrne, Satish Ramalingam, Parthasarathy Rangarajan, Dharmalingam Subramaniam, Scott Weir, Shrikant Anant, Roy Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR05.