Published OnlineFirst October 1, 2019; DOI: 10.1158/1535-7163.MCT-19-0233 MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS Molecular Inhibitor of QSOX1 Suppresses Tumor Growth In Vivo Amber L. Fifield1, Paul D. Hanavan2, Douglas O. Faigel3, Eduard Sergienko4, Andrey Bobkov4, Nathalie Meurice5, Joachim L. Petit5, Alysia Polito5, Thomas R. Caulfield6,7,8,9,10, Erik P. Castle11, John A. Copland12, Debabrata Mukhopadhyay13, Krishnendu Pal13, Shamit K. Dutta13, Huijun Luo14, Thai H. Ho14, and Douglas F. Lake1 ABSTRACT ◥ Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme over- inhibitors, known as “SBI-183,” suppresses tumor cell growth in a expressed by many different tumor types. QSOX1 catalyzes the Matrigel-based spheroid assay and inhibits invasion in a mod- formation of disulfide bonds in proteins. Because short hairpin ified Boyden chamber, but does not affect viability of nonma- knockdowns (KD) of QSOX1 have been shown to suppress lignant cells. Oral administration of SBI-183 inhibits tumor tumor growth and invasion in vitro and in vivo, we hypothesized growth in 2 independent human xenograft mouse models of that chemical compounds inhibiting QSOX1 enzymatic activity renal cell carcinoma. We conclude that SBI-183 warrants further would also suppress tumor growth, invasion, and metastasis. exploration as a useful tool for understanding QSOX1 biology High throughput screening using a QSOX1-based enzymatic and as a potential novel anticancer agent in tumors that over- assay revealed multiple potential QSOX1 inhibitors. One of the express QSOX1. Introduction largest cohort (N ¼ 126), to our knowledge, of matched patient primary and renal cell carcinoma (RCC) metastases, we identified the Cancer is a leading cause of death worldwide and distant metastases upregulation of ECM-related genes in metastases relative to primary are the major cause of patient mortality. Initially the primary tumor RCC tumors. Because these ECM genes are upregulated in metastases, grows in its microenvironment which consists of tumor cells and they may play an important role in the metastatic cascade across nonmalignant stroma, each secreting extracellular matrix (ECM; multiple solid tumors (5). ref. 1). The constituents of tumor ECM are a critical factor for cancer Because every protein in the ECM contains disulfide bonds, we invasion and metastasis (1) and many changes occur in the tumor hypothesized that QSOX1, an enzyme that generates disulfide bonds in microenvironment (TME) prior to physical migration of metastatic substrate proteins, is important for tumor cell growth, adherence, and cells away from the primary tumor. These changes include upregula- invasion. Overexpression of QSOX1 in cancer was discovered after a tion of matrix metalloproteinases (MMP; ref. 2), aberrant integrin C-terminal peptide was detected by mass spectrometry of plasma from signaling (3), and a loss of adherens junctions (3, 4). Each step of the patients with pancreatic cancer (6). Subsequently, QSOX1 overexpres- metastatic process is mediated by various ECM constituents (1). In the sion has been reported in many other cancers (7–12). Inhibition of QSOX1 activity with a mAb revealed that QSOX1 is active extracel- lularly in stromal fibroblasts and is required for proper incorporation 1School of Life Sciences, Arizona State University, Tempe, Arizona. 2RenBio, Inc., of laminin and fibronectin into the ECM (13, 14). Further, a small New York, New York. 3Division of Gastroenterology and Hepatology, molecule inhibitor of QSOX1, ebselen, reduced proliferation, and Department of Internal Medicine, Mayo Clinic, Phoenix, Arizona. 4Assay Devel- invasion of pancreatic and renal cancer cell lines in vitro, and reduced opment, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, tumor growth in vivo (15). California. 5Hematology/Oncology, Mayo Clinic, Scottsdale, Arizona. 6 7 Herein we demonstrate a novel small molecule derived from a high Department of Neuroscience, Mayo Clinic, Jacksonville, Florida. Mayo Grad- uate School, Neurobiology of Disease, Mayo Clinic, Jacksonville, Florida. throughput screen of 50,000 compounds, 3-methoxy-N-[4-(1-pyr- 8Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. 9Health rolidinyl)phenyl]benzamide (“SBI-183”), inhibits the enzymatic activ- Sciences Research, Division of Biomedical Statistics & Informatics, Mayo Clinic, ity of QSOX1, thereby suppressing the proliferative and invasive Jacksonville, Florida. 10Center for Individualized Medicine, Mayo Clinic, phenotype of 2 renal cancer cell lines (786-O and RCJ-41T2), a triple 11 Jacksonville, Florida. Department of Urology, Mayo Clinic, Phoenix, Arizona. negative breast cancer (TNBC) cell line (MDA-MB-231), a lung 12 13 Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. Department adenocarcinoma cell line (A549), and a pancreatic ductal adenocar- of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, Florida. 14Division of Hematology/Oncology, Mayo Clinic, Phoenix, Arizona. cinoma (MIA PaCa2). Furthermore, we did not observe any com- pound-related toxicity of normal adherent fibroblasts or nonadherent Note: Supplementary data for this article are available at Molecular Cancer peripheral blood mononuclear cells (PBMC) supporting a role for Therapeutics Online (http://mct.aacrjournals.org/). QSOX1 in tumor-derived ECM. Corresponding Authors: Thai H. Ho, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, AZ 85054. Phone: 480-301-8335; Fax: 480-301-4675; E-mail: [email protected]; and Douglas F. Lake, School of Life Sciences, Arizona Materials and Methods State University, Tempe, AZ. E-mail: [email protected] Compounds Mol Cancer Ther 2019;XX:XX–XX SBI-183 (molecular weight 296.3723 g/mol) was purchased from doi: 10.1158/1535-7163.MCT-19-0233 ChemBridge Corp. Compounds were dissolved in tissue culture-grade Ó2019 American Association for Cancer Research. DMSO (Sigma-Aldrich) and kept at À80 C as 100 mmol/L stock AACRJournals.org | OF1 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 1, 2019; DOI: 10.1158/1535-7163.MCT-19-0233 Fifield et al. solutions. See Supplementary Fig. S1 for the chemical structure of SBI- 150 mmol/L NaCl pH 8.0 overnight at 4oC. The labeling ratio À À 183. was estimated using e ¼ 250,000 M 1cm 1 at 655 nm for DyLight À À 650 and e ¼ 93110 M 1cm 1 at 280 nm for QSOX1, and found Cell culture to be 1.1. RCC line 786-O was purchased from the ATCC and maintained in Microscale thermophoresis (MST) experiments were performed in RPMI1640 (Corning) containing 10% FBS (Atlanta Biologicals), 1% a Monolith NT.115 (Nanotemper). Sixteen serial dilutions of SBI-183 Penicillin-Streptomycin (Pen-Strep; Corning), and 1% Glutamax (from 250 to 0.0076 mmol/L) with 50 nmol/L Dylight 650-labeled (Gibco). A recently derived sarcomatoid RCC line from Mayo Clinic, QSOX1 in 1x PBS, pH 7.4, 5% DMSO, and 0.05% Tween 20 were RCJ-41T2 (16), was maintained in DMEM in 10% FBS, 1% Pen-Strep, loaded into standard MST capillaries and scanned at MST power of and 1% Glutamax. The TNBC adenocarcinoma cell line MDA-MB- 20% at 23 C. To obtain Kd, MST data were fitted using MO Affinity 231 (ATCC), lung adenocarcinoma cell line A549 (ATCC), and the Analysis software (Nanotemper). pancreatic ductal adenocarcinoma cell line MIA PaCa2 (ATCC) were also maintained 10% DMEM. MDA-MB-231-Luc (Cell Biolabs) was Small molecule docking maintained in 10% RPMI1640 without Pen-Strep. De-identified fibro- Docking for SBI-183 was performed using Glide (v.5.6) within the blasts derived from a 28-year-old Caucasian male with no overt disease Schrodinger€ software suite (Schrodinger,€ LLC; ref. 18). Our modeling were a kind gift from Dr. Clifford Folmes. PBMCs were obtained under techniques have been described (19–25). Briefly, we started with an IRB-approved protocol (#06010000548) from Arizona State Uni- conformation searches of the ligand via the method of Polak-Ribiere versity. The identity of all cell lines was confirmed by STR analysis. conjugate gradient (PRCG) energy minimization with the optimized Each cell line also tested negative for mycoplasma and mouse patho- potentials for liquid simulations (OPLS) 2005 force field (26) for 5,000 gens throughout the study, and were maintained at 37 Cin5%CO2. steps (or until the energy difference between subsequent structure was All cell lines were used immediately upon thawing throughout the less than 0.001 kJ/mol-Å; ref. 18). Our docking methodology has been study. described (19, 25, 27), and the scoring function utilized described elsewhere (28). Briefly, molecular refracting molecules were removed Stable lentiviral QSOX1 KD generation from the human QSOX1 crystal structure (PDB Codes: 3Q6O; ref. 29). Short hairpin (sh) lentiviral particles were purchased from Gene- Schrodinger's€ SiteFinder module focused the grid on the active site Copoeia containing either sh742 RNA as described (7) (catalog no. region for QSOX1 (Fig. 1C). Using this grid, initial placement for SBI- LPP-CS-HSH273J-LVRU6GP-100) or a shScramble (shScr) control 183 was docked using the Glide algorithm within the Schrodinger€ suite (catalog no. LPP- CSHCTR001-LVRU6GP-025). 786-O cells were as a virtual screening workflow (VSW). The docking proceeded from seeded at 2.5 Â 104 cells/well in a 6-well plate in complete RPMI1640. lower precision through SP docking and Glide extra precision (XP; Adherent cells were transduced in triplicate with lentiviral particles Glide, v.5.6; Schrodinger,€ LLC;