Abstract 5606: Hedgehog pathway is a novel therapeutic target in tamoxifen resistant breast cancer that protects from autophagic cell death and is aberrantly activated by PI3K/AKT signaling
Authors: Majumder Sarmila , Lu Yuanzhi , Teng Kun-yu , Li Xiaobai , Shapiro Charles L. , Ramaswamy Bhuvaneswari 
Published: August 2012

Aims: Approximately 30-40% of patients with estrogen receptor positive (ER+) invasive breast cancer relapse despite targeted endocrine therapy with tamoxifen (TAM). Alternative cell survival mechanisms aberrantly activated over time supports tumor growth, upon blockade of estrogen dependent pathways by TAM. We have earlier identified Hedgehog (Hhg) signaling pathway as an alternative survival pathway activated in TAM resistant ER+ tumors (SABC Abst# 851859, 2011; S-9, 2010). We sought to determine if activation of Hhg pathway is critical for survival of the resistant cells, the mechanism underlying activation of this pathway, and determine if targeting such tumors with Hhg inhibitor would improve outcomes. Methods: SMO and GLI1 were depleted from TAM-resistant MCF7 (OHTR) and T47D cells using siRNA. These cells were tested in presence and absence of TAM for i) viability by MTT assay, ii) clonogenic survival, iii) apoptosis and autophagy by Western Blotting of respective markers. Pathway specific inhibitors were used to determine cross talk between Hhg pathway and different signaling pathways. Effect of GDC-0449, a small molecule inhibitor of SMO was tested on OHTR cell induced xenografts in athymic nude mice by monitoring tumor growth. Correlation of GLI1 expression in primary breast tumor tissues (assessed by IHC) and disease-free survival (DFS) was studied in women with ER+ breast cancer (N=230). Results: A. Depletion of SMO or GLI1 resulted in reduced cell proliferation, clonogenic survival and increased autophagy in TAM-resistant cells. We observed loss of p53 in SMO and GLI1 depleted cells as well as in Myc depleted cells. Expression of Myc, a Hhg target is reduced in SMO/GLI1 depleted cells, resulting in loss of p53. B. PI3K inhibitor Ly294002, inhibited GLI1 driven promoter activity, with simultaneous loss of SMO and GLI1 protein. Treatment with LiCl (GSK3B inhibitor) and/or MG132 (proteasome inhibitor) rescued these proteins from degradation. C. Proliferation of OHTR cells was significantly inhibited with GDC-0449 alone or TAM both in vitro and in vivo. D. Further, primary human ER+ breast tumors with high expression of GLI1 were associated with poor DFS in node-positive patients (p&lt;0.01). Conclusions: Our work has demonstrated for the first time that activated Hhg signaling is an alternate survival mechanism for TAM resistant breast cancer. A cross talk between Hhg pathway, MYC and p53 protects TAM-resistant cells from autophagy related cell death. PI3K/AKT signaling pathway plays a key role in activating this pathway in TAM-resistant cells. Furthermore, targeting these tumors with anti-Hhg therapy inhibited tumor growth and could be combined with PI3K inhibitor for additional benefit. Clinical trials using this combination in patients with advanced ER+ breast cancer are warranted and are under development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5606. doi:1538-7445.AM2012-5606