Inhibition of Hedgehog Signaling in Fibroblasts, Pancreatic, and Lung Tumor Cells by Oxy186, an Oxysterol Analogue with Drug-Like Properties

Abstract

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has motivated the clinical development of Smoothened (Smo) antagonists, such as vismodegib and sonidegib. However, Smo antagonists have failed to benefit patients suffering from Hh pathway-dependent solid tumors, such as pancreatic, colorectal, or ovarian cancer. Hh-dependent cancers are often driven by activating mutations that occur downstream of Smo and directly activate the transcription factors known as glioma-associated oncogenes (Gli1-3). Hence, the direct targeting of Gli could be a more effective strategy for achieving disease modification compared to Smo antagonism. In this study, we report on the biological and pharmacological evaluation of Oxy186, a semisynthetic oxysterol analogue, as a novel inhibitor of Hh signaling acting downstream of Smo, with encouraging drug-like properties. Oxy186 exhibits strong inhibition of ligand-induced Hh signaling in NIH3T3-E1 fibroblasts, as well as in constitutively activated Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells. Oxy186 also inhibits Gli1 transcriptional activity in NIH3T3-E1 cells expressing exogenous Gli1 and Gli-dependent reporter constructs. Furthermore, Oxy186 suppresses Hh signaling in PANC-1 cells, a human pancreatic ductal adenocarcinoma (PDAC) tumor cell line, as well as PANC-1 cell proliferation in vitro, and in human lung cancer cell lines, A549 and H2039.

Inhibition of Non-Small Cell Lung Cancer Cells by Oxy210, an Oxysterol-Derivative that Antagonizes TGFβ and Hedgehog Signaling

“…Having had identified the Hh pathway inhibitory effects of Oxy16 and Oxy186 [22,23], we examined the effects of Oxy210 on Hh signaling in NIH3T3-E1 mouse fibroblastic cells treated with CAPAN-1 conditioned medium (CM) that we previously reported to contain Hh ligands [22] and found that Oxy210 robustly inhibited the mRNA expression of ligand-induced Hh target genes Gli1 and Ptch1 (Figure 4a). Oxy210 exhibited strong inhibitory effects comparable to established Gli1 inhibitors, such as HPI-1 (10 μM) and Gant61 (20 μM), with an IC 50 for inhibition of Gli1 expression of 1.83 ± 0.21 μM (Figure 4b).…”

“…NIH3T3-E1 fibroblasts were obtained from ATCC (Manassas, VA, USA) and cultured as previously described [25,26]. The human lung cancer cell lines A549 and H2030 were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and antibiotics as previously described [23]. CAPAN-1 human pancreatic adenocarcinoma cell line was purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and antibiotics as previously described [23].…”

“…The human lung cancer cell lines A549 and H2030 were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and antibiotics as previously described [23]. CAPAN-1 human pancreatic adenocarcinoma cell line was purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and antibiotics as previously described [23]. Sufu-/- mouse embryonic fibroblasts were a gift from Dr. Matthew Scott of Stanford University.…”

“…NIH3T3-E1 cells cultured in 24 well-plates at 90% confluence were transiently transfected with 0.1 μg of Smad response-element reporter (SBE-Luciferase) plasmid (Qiagen), or with 0.1 μg of Gli response-element reporter (pGL3b-8xGli-Luciferase) plasmid, and 10 ng of pSV40-Renilla-Luciferase plasmid (Promega, Madison, WI, USA) with or without co-transfection with 10ng of Gli1 overexpression vector, pSRα-Gli1 using a Lipofectamine LTX Plus transfection reagent, as previously described [23]. Sufu-/- cells cultured in 24 well-plates at 90% confluence were transiently transfected with 0.1μg of pGL3b-8xGli-Luciferase plasmid, and 10 ng of pSV40-Renilla-Luciferase plasmid.…”

“…We previously reported on the ability of Oxy16 (20α,22( R )-dihydroxycholesterol, Figure 1), a naturally occurring oxysterol, to block Hh signaling downstream of the Smoothened (Smo) receptor, a plasma membrane-associated signal transducer of Hh signaling that mediates ligand activated Hh signaling [22]. In subsequent studies, through analysis of structure activity relationships (SAR) between analogues of Oxy16, we were able to identify more potent semisynthetic oxysterol derivatives, such as Oxy186, that inhibit Hh signaling and the proliferation of NSCLC and pancreatic tumor cells in vitro [23]. More recently, we have identified structural analogues of Oxy186 that in addition to their Hh pathway inhibitory properties also block TGFβ signaling.…”