Abstract P5-03-11: Targeted disruption of transcriptional effector GLI2 attenuates breast tumor growth and metastasis
Authors: Beachy P , Cai S , Ma Y , Hatakeyama J , Zhao C , Stoffels M , Verhaegh W , van de Stolpe A , Pegram M 
Published: February 2018

Abstract
We reported that transcriptional regulation by the major Hedgehog (HH) pathway effector GLI2 in mammary gland stromal cells coordinates a hormone-responsive niche signaling program that directs epithelial stem cell activity during puberty [Zhao, et al., Science. 2017 Apr 21;356(6335)]. HH signaling is a key growth pathway in human carcinogenesis. The first targeted therapies aimed at G-protein coupled receptor Smoothened (SMO) resulted in rapid acquired resistance, underscoring the need for developing new HH pathway-targeting therapies downstream of SMO. In BC GLI-dependent transcription may involve a non-receptor-based mechanism of GLI2 activation involving cross-talk from other signaling pathways such as TGFß54, PI3K55,56, Wnt, or NF-κB. Accordingly, we investigated the role of HH pathway and its transcriptional effectors using the MMTV-PyMT BC mouse model. Development of mammary tumors arising proceeds through neoplastic lesions ranging from carcinoma in situ (10-12 weeks) to highly invasive ductal carcinoma with high incidence of pulmonary metastasis at 16-18 weeks. Epithelial ablation of GLI2 dramatically reduced tumor progression. Cre recombinase under control of the Ck14 promoter to genetically ablate GLI2 in basal cells of the mammary gland thus dramatically attenuated metastasis despite continued (albeit reduced) formation of primary tumors. In human BC, we applied Bayesian methods to gene expression data to identify metastatic BC with HH pathway activation. Kaplan-Meier analysis demonstrated significantly worse progression-free survival in patients with HH pathway activity (Log-rank p = 0.0013). Next, we analyzed 1294 BC samples, stratified according to HH activity, using the HH probability as a continuous score ranging between 0 and 1, univariate Cox regression analysis supports the hypothesis that HH activity in BC is a risk factor for relapse after surgery, HR = 2.45 (95% CI: 1.67 – 3.61, p = 2.72e-6). There is also a significant difference in survival between HH-actives and HH-inactives (Log-rank p = 3.91e-4), suggesting a pathogenic role of GLI activation in BC progression and metastasis. Arsenic trioxide (ATO), inhibits HH pathway activity by de-stabilizing the GLI transcriptional effectors of HH signaling, likely due to arsenic displacement of zinc within the DNA-binding zinc fingers of GLI proteins. ATO treatment of BC cell lines resulted in dose-dependent cell growth inhibition (Alamar Blue) and induction of apoptosis (PARP cleavage and Annexin V expression) at clinically achievable concentrations. In NSG mice with orthotopic transplant of 3e5 SU151 human BC PDX, with strongly positive GLI-active signature, daily IP injection of 10mg/kg ATO resulted in marked tumor growth inhibition in vivo (2.75-fold smaller tumor diameter vs. PBS vehicle controls, P= 0.006; 55d vs. 17d to reach 1.5 cm criterion for ATO vs. vehicle). We conclude the major HH pathway transcriptional effector GLI2 coordinates a transcriptional program with a central role in BC growth and metastasis in a significant subset of BC, and that systemic treatment with ATO (new oral formulation in commercial development), will reduce metastatic progression and improve clinical outcomes in patients whose tumors harbor a GLI-active transcript signature.
Citation Format: Beachy P, Cai S, Ma Y, Hatakeyama J, Zhao C, Stoffels M, Verhaegh W, van de Stolpe A, Pegram M. Targeted disruption of transcriptional effector GLI2 attenuates breast tumor growth and metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-11.