Sulindac Sulfide Research Articles

Abstract 5243: Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in the APC +/min-FCCC mouse model

Abstract Sulindac can reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. Here we characterize the antineoplastic properties of a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity. ADT-061 potently and selectively inhibited colon tumor cell growth in vitro with IC50 values of 0.3-0.5 μM by inducing apoptosis without affecting the growth of normal colonocytes at concentrations 100x higher. By comparison, sulindac sulfide (SS) inhibited colon tumor cell growth with IC50 values of 40-60 μM, and had only modest tumor cell selectivity. While SS non-selectively inhibits multiple cyclic GMP phosphodiesteraes (PDE) isozymes, previous studies have suggested an important role for PDE10 in colon cancer (Li et al, Oncogene, 2014; Lee et al, Oncotarget, 2015). At concentrations comparable to its IC50 values, ADT-061 selectively inhibits PDE10 to induce an increase in intracellular cGMP levels and activate protein kinase G (PKG). ADT-061 also suppressed nuclear levels of β-catenin and induced cell cycle arrest and apoptosis, within the same concentration range that inhibits tumor cell growth. With attractive drug-like properties, the chemopreventive activity ADT-061was assessed using the APC+/min-FCCC mouse model of spontaneous colorectal tumorigenesis. Prior to treatment, colonoscopy exams were performed on all APC+/min-FCCC mice (males, 6-7 weeks of age) to ensure the absence of colorectal adenomas. At 7-8 weeks of age, mice were assigned (19 per group) to receive either unsupplemented chow (control) or chow supplemented with ADT-061 (1500ppm) for 14 weeks. No discernible toxicity was observed during the experimental period. Double-blinded pathological analysis of fixed colon tissues revealed that ADT-061 not only reduced the multiplicity of colonic adenomas by 50% (Mean ± SEM, control vs. ADT-061, 3.95 ± 0.81 vs. 1.95 ± 0.58, P < 0.05), but also decreased the incidence of colonic adenomas by 36.7% (control vs. ADT-061, 94.7% vs. 57.9%, respectively). ADT-061 treatment reduced the multiplicity of polypoid adenomas (36%), flat adenomas (100%, P < 0.05), and microadenomas (69%). The formation of colonic adenocarcinomas found in 15.8% of untreated mice was completely inhibited in mice treated with ADT-061. These data indicate that ADT-061 inhibits colon tumorigenesis when given to APC+/min-FCCC mice prior to the detection of gross colonic tumors. These observations support further evaluation of ADT-061 and analogs for colorectal cancer chemoprevention. Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Harry Cooper, Margie Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in the APC+/min-FCCC mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2017-5243

Abstract 1359: How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC

Abstract Introduction: We reported that Wnt Pathway (WP) upregulation, one of the salient genetic features of metastatic TNBC, controls the acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes of TNBC cells (Dey et al., 2013). RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. Aim: Here we tested the role of the mechanism of involvement of RAC1 in the regulation of WP-mediated ID-MA phenotypes in TNBC cells and examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Method: Oncoprints showed that the major alterations of RAC1 gene in TNBC were constituted by the amplification/gain of the gene. The collective percentage of alteration(s) in RAC1 in ER+ve BC was 35% as compared to 57% in ER-ve BC (brca/tcga/pub2015). Using pharmacological agents (sulindac sulfide), genetic tools (siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed migration and various motility parameters (actin dynamics, filopodia and lamellipodia by confocal microscopy) in the context of fibronectin-directed RAC1 and Cdc42 activation. Result: Breaking down by molecular subtypes of BC, RAC1 gene alterations are observed in 20% in PAM50 Luminal A, 35% in PAM50 Luminal B, 37% in PAM50 HER2 enriched and 40% in PAM50 Basal-like. For the samples set of TCGA, Cell 2015, RAC1 gene was altered in 42% total while it was altered 37% in PAM50 Luminal A, 57% in PAM50 Luminal B, 55% in PAM50 HER2 enriched and 60% in PAM50 basal-like. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) altered actin dynamics and (d) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report (De et al., 2016) that WP upregulation stimulates ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in an RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Citation Format: Nandini Dey, Jennifer H. Carlson, Casey Williams, Tyler Jepperson, Pradip De, Brian Leyland-Jones. How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1359. doi:10.1158/1538-7445.AM2017-1359

Inhibition of cyclooxygenase-2-mediated matriptase activation contributes to the suppression of prostate cancer cell motility and metastasis.

Chronic inflammation plays an important role in cancer development and progression. Cyclooxygenases-2 (COX-2) is a key enzyme in generating prostaglandins causing inflammation, is often found to be overexpressed in prostate cancer (PCa) and is correlated with PCa cell invasion and metastasis. We aim to investigate the molecular mechanism of how COX-2 promotes PCa cell invasion and metastasis and to evaluate the effect of COX-2 inhibitors in a selected model of PCa progression. Our results showed that the expression of COX-2 and Interleukin 1β (IL-1β) was upregulated in highly invasive PCa cells and was correlated with the activated levels of membrane-anchored serine protease matriptase. The expression levels of COX-2 were increased and were correlated with matriptase levels in PCa specimens. Moreover, results showed that COX-2 overexpression or a COX-2 product Prostaglandin E2 (PGE2) caused an increase in matriptase activation and PCa cell invasion, whereas COX-2 silencing antagonized matriptase activation and cell invasion. In addition, the inhibition of COX-2-mediated matriptase activation by Celebrex and sulindac sulfide suppressed the androgen-independent and COX2-overexpressing PCa PC-3 cell invasion, tumor growth and lung metastasis in an orthotopic xenograft model. Our results indicate that COX-2/matriptase signaling contributes to the invasion, tumor growth and metastasis of COX-2-overexpressing and androgen-independent PCa cells.

Marmoset Flavin-Containing Monooxygenase 3 in the Liver Is a Major Benzydamine and Sulindac Sulfide Oxygenase.

Common marmosets (Callithrix jacchus) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non-cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1-FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%-95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N-oxygenation and sulindac sulfide S-oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets.

Abstract A30: Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model

Abstract Sulindac has been reported to reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. We report here a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity, yet potently and selectively inhibits colon tumor cells with IC50 values of 0.3-0.5 μM with no significant effect on the growth of normal colonocytes. By comparison, sulindac sulfide inhibits colon tumor cell growth with IC50 values of 40-60 μM, and has only modest tumor cell selectivity. ADT-061 inhibits phosphodiesterase 10A (PDE10), which increases intracellular cGMP levels, thus activates protein kinase G, suppresses nuclear levels of β-catenin, and induces cell cycle arrest and apoptosis, all within the same concentration range that inhibits tumor cell growth. ADT-061was evaluated for chemopreventive activity in the APC+/min-FCCC mouse model of colon cancer, which harbors colorectal adenomas that express PDE10. Colonoscopy exams were initially performed on all mice to ensure the absence of colorectal adenomas prior to treatment. At 7-8 weeks of age, male mice were treated with ADT-061 (1000ppm and 1500ppm) in the diet for 14 weeks. ADT-061 reduced the incidence of colorectal adenomas from 95.8% in the control group to 73.9% in the 1000ppm group (p=0.048), and 59.1% (p=0.004) in the 1500ppm group. Control untreated mice had an average of 2.98 colorectal adenomas per mouse, while mice treated with 1000ppm and 1500ppm ADT-061 had an average of 2.04 (p=0.102) and 1.91 (p=0.039) adenomas per mouse, respectively. These observations suggest that ADT-061, or analogs thereof, merit further drug development efforts for colorectal cancer chemoprevention. Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Margie L. Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A30.

Bioelectrochemical profiling of two common polymorphic variants of human FMO3 in presence of graphene oxide

Genetic variation of phase I drug metabolising enzymes has been shown to greatly influence inter-individual reaction to pharmacological treatments. Among these enzymes, human flavin-containing monooxygenase 3 (hFMO3) plays a crucial role and understanding its pharmacogenetics is fundamental for the prediction of individual drug response and the efficacy of therapy. In this work the altered drug metabolism of two common polymorphic variants of hFMO3 (E158K and E308G) are studied by using an electrochemical platform modified with graphene oxide (GO). Electrochemistry was used to characterise the properties of these two engineered and purified hFMO3 variants followed by electrocatalysis experiments in the presence of three different hFMO3 substrates benzydamine, tamoxifen and sulindac sulfide. HPLC quantification of the electrochemically produced metabolites showed that E158K mutation leads to an impairment of N-oxygenation activity while E308G mutation enhances the same activity.Results demonstrate that electrocatalysis on GO modified glassy carbon electrodes provides a fast and reliable method for measuring kinetic parameters of hFMO3 polymorphic variants. This method can be considered suitable for deciphering metabolic implications of polymorphisms that might lead to adjustment of drug dosages depending on the individual’s genetic makeup, a step closer to the development of personalised medicine.

Effects of FMO3 Polymorphisms on Pharmacokinetics of Sulindac in Chinese Healthy Male Volunteers.

Sulindac is a nonsteroidal anti-inflammatory drug, which is clinically used for the ailments of various inflammations. This study investigated the allele frequencies of FMO3 E158K and E308G and evaluated the influences of these two genetic polymorphisms on the pharmacokinetics of sulindac and its metabolites in Chinese healthy male volunteers. Eight FMO3 wild-type (FMO3 HHDD) subjects and seven FMO3 homozygotes E158K and E308G mutant (FMO3 hhdd) subjects were recruited from 247 healthy male volunteers genotyped by PCR-RFLP method. The plasma concentrations of sulindac, sulindac sulfide, and sulindac sulfone were determined by UPLC, while the pharmacokinetic parameters of the two different FMO3 genotypes were compared with each other. The frequencies of FMO3 E158K and E308G were 20.3% and 20.1%, respectively, which were in line with Hardy-Weinberg equilibrium (D′ = 0.977, r2 = 0.944). The mean values of Cmax, AUC0–24, and AUC0–∞ of sulindac were significantly higher in FMO3 hhdd group than those of FMO3 HHDD group (P < 0.05), while the pharmacokinetic parameters except Tmax of sulindac sulfide and sulindac sulfone showed no statistical difference between the two groups. The two FMO3 mutants were in close linkage disequilibrium and might play an important role in the pharmacokinetics of sulindac in Chinese healthy male volunteers.

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.

Abstract B31: Sulindac inhibition of tumor cell transformation

Abstract Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA's promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered two tumor suppressor miRNAs, let-7b and let-7g, which were previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b and let-7g could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b and let-7g were knocked down by CRISPR/cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. These results suggest that upregulation of let-7b and let-7g is the key step for SS to inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-kappaB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-kappaB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Zhipin Liang, Xiangling Feng, Hong Chang, Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac inhibition of tumor cell transformation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B31.

Abstract B34: Inhibition of breast cancer cell metastasis with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFbeta/miR-21 signaling

Abstract Compelling efficacy on intervention of tumor occurrence and progression by nonsteroidal anti-inflammatory drugs (NSAIDs) has been intensively reported in numerous preclinical and clinical studies. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for human cancer chemoprevention. A novel derivative of the NSAID, sulindac sulfide (SS), referred as sulindac sulfide amide (SSA) was recently developed, which lacks COX inhibitory activity, yet shows greater suppressive effect than SS on growth of variable cancer cells. In this study, we focus on the inhibitory activity of SSA on breast tumor cell motility, which has not been studied previously. Our results showed that SSA could inhibit breast tumor cell invasion and migration at sub-cytotoxic concentrations; whereas we previously reported that SS has similar activity on breast and colon tumor cells but at a concentration (50 µM) over 10 times higher than SSA (4 µM). We found that miR-21, a well-documented oncogenic miRNA for promoting tumor cell metastasis, could be downregulated by SSA and involved in mediation of anti-invasive activity of SSA in breast tumor cells. When studying the mechanisms responsible for the regulation of miR-21 by SSA, we demonstrated that TGFbeta signaling suppressed by SSA could ultimately result in downregulation of miR-21 through the transcriptional control. Reduction of Smad2 and Smad3 phosphorylation was the direct response to SSA treatment in metastatic breast tumor MDA-MB-231 cells. By using ChIP assay, we demonstrated that phosphorylated Smad2/3 could bind to the promoter of miR-21 gene. Therefore, in this project, we demonstrate that a non-COX inhibitory derivative of sulindac can inhibit breast tumor metastasis through a new mechanism involving the TGFbeta/miR-21 signaling axis. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Bin Yi, Hong Chang, Xiangling Feng, Ruixia Ma, Gary A Piazza, Yaguang Xi. Inhibition of breast cancer cell metastasis with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFbeta/miR-21 signaling. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B34.

Human flavin-containing monooxygenase 3: Structural mapping of gene polymorphisms and insights into molecular basis of drug binding

Human hepatic flavin-containing monooxygenase 3 (hFMO3) catalyses the monooxygenation of carbon-bound reactive heteroatoms and plays an important role in the metabolism of drugs and xenobiotics. Although numerous hFMO3 allelic variants have been identified in patients and their biochemical properties well-characterised in vitro, the molecular mechanisms underlying loss-of-function mutations have still not been elucidated due to lack of detailed structural information of hFMO3. Therefore, in this work a 3D structural model of hFMO3 was generated by homology modeling, evaluated by a variety of different bioinformatics tools, refined by molecular dynamics simulations and further assessed based on in vitro biochemical data. The molecular dynamics simulation results highlighted 4 flexible regions of the protein with some of them overlapping the data from trypsin digest. This was followed by structural mapping of 12 critical polymorphic variants and molecular docking experiments with five different known substrates/drugs of hFMO3 namely, benzydamine, sulindac sulfide, tozasertib, methimazole and trimethylamine. Localisation of these mutations on the hFMO3 model provided a structural explanation for their observed biological effects and docked models of hFMO3-drug complexes gave insights into their binding mechanism demonstrating that nitrogen- and sulfur-containing substrates interact with the isoalloxazine ring through Pi-Cation interaction and Pi-Sulfur interactions, respectively. Finally, the data presented give insights into the drug binding mechanism of hFMO3 which could be valuable not only for screening of new chemical entities but more significantly for designing of novel inhibitors of this important Phase I drug metabolising enzyme.

Abstract 4605: Metastasis-associated tumor cell phenotypes in TNBC is regulated by Wnt-beta-catenin pathway signals

Abstract Introduction: Acquisitions of metastasis-associated phenotypes (MA) by Triple Negative Breast Cancer (TNBC) cells are imparted by genetic alterations which cause deregulation of different signaling pathways associated to those phenotypes. We have recently reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the salient genetic features of TNBC and demonstrated that the WP signaling in TNBC is associated with poor prognosis and metastasis (Dey et al., 2013). We have also identified that the functional upregulation of secreted-MMP7, a transcriptional target of WP in TNBC is associated to the functional loss/absence of PTEN gene (Dey et al., 2013), the most common first event associated with basal-like subtype (Martins et al., 2012). Aim: In order to understand the relevance of WP pathway in the biology of metastasizing TNBC tumor cells, here we have undertaken a study in which the involvement of WP is tested in the context of MA phenotypes including (1) integrin-directed migration, (2) matrigel-invasion and (3) clonogenic growth and proliferation in 15 TNBC cell lines. Methods: CHIR9902, Wnt-C59, XAV939, sulindac sulfide, beta-catenin SiRNA as well as physiological stimulation of the WP (LWnt3A CM) were used for the study. Podia-parameters were studied using confocal microscopy for architecture of filamentous actin while matrigel invasion was studied by MMP7 specific caesin-zymography. Results: The collective% of alterations in CTNNB1, APC and DVL1 genes among total breast invasive carcinomas (TCGA 2012) were 17% in contrast to 46% breast invasive carcinomas, PAM50 Basal. A similar trend was observed among subtypes of BC from brca/tcga/pub2015 (cBioPortal). Wnt-C59, XAV939, sulindac sulfide and beta-catenin SiRNA (1) inhibited fibronectin-directed migration (2) decreased maximum relative distance from the origin and average velocity of the movement by real-time video microscopy, (3) altered cytoskeletal organization of the filamentous-actin and decreased the podia parameters, (4) decreased matrigel-invasion, and (6) inhibited cell proliferation and 3D colony formation. Mechanistically the treatment with sulindac sulfide and beta-catenin SiRNA decreased cellular levels of beta-catenin, active beta-catenin and MMP7 in the same TNBC cell lines wherein fibronectin-directed migration, invasion, and colony formation were found inhibited. LWnt3ACM stimulated cell proliferation, clonogenicity, migration and invasion was perturbed by WP modulators, sulindac sulfide and PI3K pathway inhibitor in brain-metastasis specific TNBC cells. Conclusion: Here we present the first conclusive evidence to demonstrate that the WP activation is functionally responsible for MA phenotypes in TNBC. Our data explains the why different components of the WP is upregulated in TNBC and how WP activation is associated with poor prognosis and metastasis in this subtype. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian R. Leyland-Jones. Metastasis-associated tumor cell phenotypes in TNBC is regulated by Wnt-beta-catenin pathway signals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4605.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Abstract B67: Matriptase is involved in COX-2 signaling-induced prostate cancer cell invasion

Abstract Chronic inflammation plays an important role in the early development and progression of cancer. Cyclooxygenases-2 (COX-2) is a key rate-limiting enzyme which regulates of inflammation-producing prostaglandins. Several studies have shown that COX-2 is found with overexpression in human cancers (colon, breast, lung, prostate cancers, etc), and implicated in cancer cell invasion. Moreover, high COX-2 expression has been correlated with prostate cancer metastasis. However, the molecular mechanism that COX-2 promotes prostate cancer cell invasion is still unclear. In this study, we established a PC-3 cell invasion progression model (parental and M2I2 PC-3 cells) and found out that several inflammation-associated proteins, COX-2, p-JNK and IL-1β were up-regulated in M2I2 PC-3 cells. Therefore, we purposed that if COX-2 signaling plays a role in prostate cancer cell invasion and the involvement of matriptase in COX-2 signaling-induced cancer cell invasion. The results showed that COX-2 inhibitors (celebrex and sulindac sulfide) could suppress prostate cancer PC3 cell invasion and inhibited matriptase expression and activation. A COX-2 product PGE2 could induce matriptase activation and prostate cancer cell invasion. COX-2 overexpression also could up-regulate matriptase and enhance the cancer cell invasion, while COX-2 silencing antagonized both matriptase activation and cell invasion. Thus, our results indicate that COX-2 signaling can promote prostate cancer cell invasion, at least in part via up-regulating matriptase. Citation Format: Chun-Jung Ko, Ying-Chieh Lu, Pee-Fang Lai, Pei-Wen Hsiao, Ming-Shyue Lee. Matriptase is involved in COX-2 signaling-induced prostate cancer cell invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B67.

Sulindac Sulfide Induces the Formation of Large Oligomeric Aggregates of the Alzheimer's Disease Amyloid-β Peptide Which Exhibit Reduced Neurotoxicity.

Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide induced Aβ aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aβ peptide in the aggregate.

Inhibition of breast cancer cell motility with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFβ/miR-21 signaling.

Compelling efficacy on intervention of tumorigenesis by nonsteroidal anti-inflammatory drugs (NSAIDs) has been documented intensively. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for human cancer chemoprevention. A novel derivative of the NSAID sulindac sulfide (SS), referred as sulindac sulfide amide (SSA), was recently developed, which lacks COX inhibitory activity, yet shows greater suppressive effect than SS on growth of various cancer cells. In this study, we focus on the inhibitory activity of SSA on breast tumor cell motility, which has not been studied previously. Our results show that SSA treatment at non-cytotoxic concentrations can specifically reduce breast tumor cell motility without influencing tumor cell growth, and the mechanism of action involves the suppression of TGFβ signaling by directly blocking Smad2/3 phosphorylation. Moreover, miR-21, a well-documented oncogenic miRNA for promoting tumor cell metastasis, was also found to be involved in inhibitory activity of SSA in breast tumor cell motility through the modulation of TGFβ pathway. In conclusion, we demonstrate that a non-COX inhibitory derivative of sulindac can inhibit breast tumor metastasis by a mechanism involving the TGFβ/miR-21 signaling axis.

Sulindac sulfide inhibits colon cancer cell growth and downregulates specificity protein transcription factors

BackgroundSpecificity protein (Sp) transcription factors play pivotal roles in maintaining the phenotypes of many cancers. We hypothesized that the antineoplastic effects of sulindac and its metabolites were due, in part, to targeting downregulation of Sp transcription factors.MethodsThe functional effects of sulindac, sulindac sulfone and sulindac sulfide on colon cancer cell proliferation were determined by cell counting. Effects of these compounds on expression of Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes were determined by western blot analysis of whole cell lysates and in transient transfection assays using GC-rich constructs.ResultsSulindac and its metabolites inhibited RKO and SW480 colon cancer cell growth and the order of growth inhibitory potency was sulindac sulfide > > sulindac sulfone > sulindac. Treatment of SW480 and RKO cells with sulindac sulfide downregulated expression of Sp1, Sp3 and Sp4 proteins. Sulindac sulfide also decreased expression of several Sp-regulated genes that are critical for cancer cell survival, proliferation and angiogenesis and these include survivin, bcl-2, epidermal growth factor receptor (EGFR), cyclin D1, p65 subunit of NFκB and vascular endothelial growth factor (VEGF). Sulindac sulfide also induced reactive oxygen species (ROS) and decreased the level of microRNA-27a in colon cancer cells, which resulted in the upregulation of the Sp-repressor ZBTB10 and this resulted in downregulation of Sp proteins.ConclusionsThe results suggest that the cancer chemotherapeutic effects of sulindac in colon cancer cells are due, in part, to its metabolite sulindac sulfide which downregulates Sp transcription factors and Sp-regulated pro-oncogenic gene products.

Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion.

ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp(23)-Lys(28) salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly(33), a residue involved in Met(35) oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.

Abstract 1966: Role of Wnt-Beta-Catenin signals in the control of vascular mimicry in TNBC

Abstract Background: TNBC or basal-like subtype of breast cancer confers poor clinical outcome mostly due to its inherent aggressive nature and its high frequency of metastasis. Malignancy in TNBC is associatedwith micro-vascular proliferations. Vascular Mimicry (VM) of solid tumor is an endothelium-independent matrix-embedded, blood-perfused non-angiogenic micro-circulatory phenomenon. VM of tumor cells refer to the characteristic plasticity of aggressive cancer cells forming de novo vascular networks which perfuse rapidly growing tumors, transporting fluid from leaky vessels and/or connect with the constitutional endothelial-lined vasculature. In a study comparing the VM in three subsets of BC, the presence of VM was more profoundly observed in TNBC than in luminal and HER2+ subtypes. Hypothesis: Recently we have identified a differential activation of Wnt-b-Catenin pathway (WP) in TNBC (Dey et al., PLOSONE; 2014) and demonstrated that Wnt signaling in TNBC is association with metastasis (Dey et al., BMC Cancer 2013). Since TNBC is an aggressive form of BC and VM is associated with the aggressiveness / poor outcome in many solid tumors including BC, we hypothesized that the upregulation of WP in TNBC may have a functional relationship with VM. Methods: Four strategies were used to study the role of WP in VM. They are (1) Wnt-receptor interaction by using WntC59, (2) Wnt cytosolic signaling by using XAV939, (3) Wnt nuclear signaling by using sulindac sulfide and beta-catenin SiRNA and (4) Wnt-regulating pathway by using pan-PI3K inhibitor (LY294002 and SF1126). The signaling of VM was characterized by bevacizumab (HUVEC cell cord formation used as positive control). Results: Using pharmacological and genetic tools, we demonstrated that perturbation of WP (1) at its different nodes and (2) via its upstream regulatory pathway (PI3K) can regulate VM in TNBC cells. Data showed that (1) sulindac sulfide, (2) XAV939 and WntC59 and (3) LY294002 or SF1126 (which decreased pGSK3beta) caused an abrogation of VM in TNBC cell lines while bevacizumab failed to affect the vessel formation. Considering the involvement of VM in mediating the aggressive/metastatic nature of TNBC, we finally tested the effect of downregulation of WP on VM in brain metastasis specific EGFP-tagged MDA-MB231BR cells using a real time recording device (Incucyte) to demonstrate that VM in these cells is regulated by WP. Significance: To our knowledge, this is the first report to demonstrate a direct functional role of WP in the regulation of VM in TNBC. Citation Format: Brian Leyland-Jones, Jennifer H. Carlson, Pradip De, Nandini Dey. Role of Wnt-Beta-Catenin signals in the control of vascular mimicry in TNBC. [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 1966. doi:10.1158/1538-7445.AM2015-1966