Abstract 3682: Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human breast cancer cells.

Abstract

Abstract Withaferin A (WA), a constituent of the Ayurvedic medicine plant Withania somnifera, exhibits in vivo efficacy against human breast cancer xenografts in athymic mice as well as spontaneous mammary cancer development in MMTV-neu transgenic mice. However, the mechanism underlying anticancer effect of WA is not fully understood. The present study was undertaken to determine the role of Notch family of transcription factors in anticancer effects of WA using human breast cancer cells as a model. Notch activation is linked to mammary carcinogenesis as evidenced by the following observations: (a) overexpression of activated Notch1 and Notch3 in transgenic mice blocks mammary gland development but induces tumorigenesis of the breast; (b) high level expression of Notch1 and its ligands is associated with poor outcome in breast cancer; (c) expression of Notch ligand Jagged1 is associated with recurrence in lymph node-negative breast cancer; and (d) Notch1 is involved in migration and invasion of human breast cancer cells. The present study reveals differential effect of WA treatment on activation of Notch1 versus Notch2 in human breast cancer cells. Exposure of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 (an estrogen-responsive cell line with wild-type p53) human breast cancer cells to pharmacological concentrations of WA (2 and 4 μM) resulted in cleavage (suggesting activation) of Notch2, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved (active) Notch1. The WA-mediated activation of Notch2 was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was statistically significantly augmented by knockdown of Notch2 using siRNA and shRNA. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. Moreover, withanone and withanolide A were practically inactive against MDA-MB-231 cell migration. The results of the present study suggest that anti-metastatic effect of WA may be augmented in the presence of Notch2 inhibitors. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Joomin Lee, Anuradha Sehrawat, Shivendra V. Singh. Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3682. doi:10.1158/1538-7445.AM2013-3682