Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as ␤-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1 ؊/؊ macrophages have cholesterolladen rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1 ؉/؉ and Abca1 ؊/؊ macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1 ؉/؉ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1 ؊/؊ rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. Molecular & Cellular

We have generated a photoactivatable form of sonic hedgehog protein by modifying the N-terminal cysteine with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (Bzm). The Bzm modification on ShhN imparted a significant increase in activity as assessed in the C3H10T1/2 functional assay with potency comparable to that of the endogenous dual-lipidated form of ShhN (ShhNp). Reversed-phase HPLC analysis indicated that the increase in activity compared to unmodified ShhN may be due in part to the hydrophobic nature of the benzophenone group. In contrast to the fully processed ShhNp, Bzm-ShhN is monomeric as assessed by analytical SEC and does not require detergent to be soluble. Further, we demonstrated that the Bzm-ShhN was able to crosslink in vitro in the presence of a known binding partner, heparin. We suggest that Bzm-ShhN can serve as a relatively facile and preferred source of ShhNp for in vitro assays and as a probe to identify novel Hh protein interactions.

Introduction: The transcription factor, glioma-associated oncogene homolog 1 (GLI1) is a downstream marker for hedgehog (Hh) pathway activation. Recently, Hh-GLI has emerged as a central pathway target in several human cancers (breast, leukemia, lung etc.) with elevated GLI1 in breast cancer having been linked to poor survival outcomes. A number of GLI targeted inhibitors have been shown to inhibit Hh/GLI signaling with some having efficacy in tumor models. We have previously demonstrated that siRNA downregulation of GLI1 expression in inflammatory breast cancer reduced proliferation and migration[1]. Further, we previously demonstrated the feasibility of employing a high throughput (HT) approach to profile drugs in dose response in cell line panels [2]. In this study, we have taken a comprehensive approach to profile a panel of GLI antagonists (including GANTS, HPIs, ATO and JK184) in a range of breast cancer cell lines with varying GLI1 expression levels to identify those with effects on growth. Experiment procedure: to assess cell proliferation, we employed an automated quantitative high throughput (qHT) approach to profile compounds on three phenotypic subtypes of breast cancer cell line models: triple negative breast cancer (TNBC), inflammatory breast cancer, and Claudin low breast cancer cell lines. High-content imaging of nuclear count utilizing Hoechst staining was also used as an alternative measure of proliferation. Quantitative PCR and Western blotting was used to assess the effects of the inhibitors on the GLI1 expression. Results: A subset of GLI antagonists were identified as decreasing cell growth and viability in all the cell lines. Conclusion: Several agents showed efficacy in in vitro BC cancer models demonstrating that GLI inhibitors may be a valid therapeutic approach for targeting GLI-dependent BC cancers. [1] Z. Thomas, W. Gibson, J. Sexton, K. Aird, S. Ingram, A. Aldrich, H. Lyerly, G. Devi, K. Williams, Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104 (2011) 1575-1586. [2] K.P. Williams, J.L. Allensworth, S.M. Ingram, G.R. Smith, A.J. Aldrich, J. Z Sexton, G. R Devi, Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Lett. 337 (2013) 77-89. Funded in part by DOD/CDMRP IDEA W81XWH-13-1-0141 award (KPW); NIH U54CA156735 (KPW); Duke Cancer Institute - Cancer and Environment Initiative P3917733 sub-award (GRD); W81XWH-13-1-041 subcontract (GRD) and National Cancer Institute training grant T32CA009111 (SJS). Citation Format: Helen Oladapo, Jodie M. Fleming, Kezia Addo, Mike Tarpley, Ben Ehe, Shalonda Ingram, Scott Sauer, Gayathri Devi, Kevin P. Williams. Identification of GLI1 antagonists for breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2015-680