HER-2/neu protein is over-expressed in approximately 20-30% of breast cancers. Overexpression of this oncoprotein results in a more aggressive tumor phenotype and poor prognosis for patients. Current treatment strategies include passive administration of anti-HER-2/neu monoclonal antibody Trastuzumab as well as the small molecule inhibitor lapatinib. Both of these molecules function at the receptor level to block HER-2/neu signaling resulting in inhibition of cell growth and tumor regression in the majority of patients. However, a significant number of patients relapse or develop progressive disease due to acquired mechanisms of resistance to current therapy. Alternative approaches, that utilize multiple modes of action, are therefore necessary to combat this severe form of breast cancer. Withaferin A (WA) is a natural compound isolated from the Indian ayurvedic medicinal plant Withania Somnifera. Natural compounds are notorious for their multiple mechanisms of action, which make them ideal therapeutics for complex diseases such as cancer. Recent research has demonstrated that WA is effective in inducing cell death in several cancer cell lines by inhibiting molecular pathways such as Akt and MAPK proliferative cascades which are downstream of her-2/neu activation. Additionally, WA has also been shown to inhibit function of the molecular chaperone heat shock protein 90 (HSP90) in pancreatic carcinoma cells. Since HER-2/neu is a client protein of HSP90 and the chaperone functions to stabilize HER-2/neu protein expression on the cell surface, our goal was to determine whether WA would directly affect HER-2/neu expression. We initially established the cytotoxic activity of WA on HER-2/neu expressing human breast cancer cell lines such as SKBr3, MCF-7her-2neu and BT474 by MTT assay. We report that WA exhibits potent cytotoxic activity in these cells with IC50 concentrations of 1.6, 2.1 and 0.6 μM respectively after 48hr treatment. Comparison of WA sensitivity in a low HER-2/neu expressing cell line MCF-7 generated an IC50 of 5.1μM, indicating that cell lines that overexpress HER-2/neu may be more susceptible to the compound. Analysis of HER-2/neu expression both at the cell surface and intracellular localization were next performed in response to WA. WA treatment completely downregulated the expression of HER-2/neu protein from cell surface by 24hr and was both time and concentration dependent. Abrogation of intracellular protein expression also correlated to the downregulation from the cell surface. We determined that WA does not alter transcription of HER-2/neu, as there was no change in the gene expression with WA treatment compared to untreated cells. Further experiments are ongoing to address whether WA specifically targets signaling pathways that alter HER-2/neu signaling. Understanding the mechanisms by which WA induces down-regulation of HER-2/neu would serve as a means of developing WA as an alternative therapeutic strategy for HER-2/neu cancers not amenable to standard treatments.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A115.

Citation Format: Ananlise Smith, Sarah Poliquin, Darlah Lopez-Rodriguez, Samita Andreansky. Withaferin A downregulates her-2/neu protein expression in human breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A115.