Repurposing the selective estrogen receptor modulator bazedoxifene to suppress gastrointestinal cancer growth
2019; Springer Nature; Volume: 11; Issue: 4 Linguagem: Inglês
10.15252/emmm.201809539
ISSN1757-4684
AutoresPathum Thilakasiri, Jennifer Huynh, Ashleigh R. Poh, Chin Wee Tan, Tracy L. Nero, Kelly Tran, Adam C. Parslow, Shoukat Afshar‐Sterle, David Baloyan, Natalie J. Hannan, Michael Büchert, Andrew M. Scott, Michael D. W. Griffin, Frédéric Hollande, Michael W. Parker, Tracy L. Putoczki, Matthias Ernst, Ashwini L. Chand,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoResearch Article18 March 2019Open Access Source DataTransparent process Repurposing the selective estrogen receptor modulator bazedoxifene to suppress gastrointestinal cancer growth Pathum Thilakasiri Pathum Thilakasiri Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Jennifer Huynh Jennifer Huynh Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Ashleigh R Poh Ashleigh R Poh Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Chin Wee Tan Chin Wee Tan The Walter and Eliza Hall Institute, Melbourne, Vic., Australia Search for more papers by this author Tracy L Nero Tracy L Nero ACRF Rational Drug Discovery Centre, St Vincent's Institute, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Kelly Tran Kelly Tran Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Adam C Parslow Adam C Parslow Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Department of Molecular Imaging and Therapy, Austin Health, Melbourne, Vic., Australia Search for more papers by this author Shoukat Afshar-Sterle Shoukat Afshar-Sterle Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author David Baloyan David Baloyan Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Natalie J Hannan Natalie J Hannan Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael Buchert Michael Buchert Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Andrew Mark Scott Andrew Mark Scott Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Department of Molecular Imaging and Therapy, Austin Health, Melbourne, Vic., Australia Department of Medicine, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael DW Griffin Michael DW Griffin Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Frederic Hollande Frederic Hollande Department of Clinical Pathology, University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael W Parker Michael W Parker ACRF Rational Drug Discovery Centre, St Vincent's Institute, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Tracy L Putoczki Tracy L Putoczki The Walter and Eliza Hall Institute, Melbourne, Vic., Australia Search for more papers by this author Matthias Ernst Corresponding Author Matthias Ernst [email protected] orcid.org/0000-0002-6399-1177 Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Ashwini L Chand Corresponding Author Ashwini L Chand [email protected] orcid.org/0000-0002-1245-729X Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Pathum Thilakasiri Pathum Thilakasiri Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Jennifer Huynh Jennifer Huynh Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Ashleigh R Poh Ashleigh R Poh Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Chin Wee Tan Chin Wee Tan The Walter and Eliza Hall Institute, Melbourne, Vic., Australia Search for more papers by this author Tracy L Nero Tracy L Nero ACRF Rational Drug Discovery Centre, St Vincent's Institute, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Kelly Tran Kelly Tran Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Adam C Parslow Adam C Parslow Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Department of Molecular Imaging and Therapy, Austin Health, Melbourne, Vic., Australia Search for more papers by this author Shoukat Afshar-Sterle Shoukat Afshar-Sterle Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author David Baloyan David Baloyan Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Natalie J Hannan Natalie J Hannan Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael Buchert Michael Buchert Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Andrew Mark Scott Andrew Mark Scott Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Department of Molecular Imaging and Therapy, Austin Health, Melbourne, Vic., Australia Department of Medicine, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael DW Griffin Michael DW Griffin Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Frederic Hollande Frederic Hollande Department of Clinical Pathology, University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Michael W Parker Michael W Parker ACRF Rational Drug Discovery Centre, St Vincent's Institute, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Tracy L Putoczki Tracy L Putoczki The Walter and Eliza Hall Institute, Melbourne, Vic., Australia Search for more papers by this author Matthias Ernst Corresponding Author Matthias Ernst [email protected] orcid.org/0000-0002-6399-1177 Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Ashwini L Chand Corresponding Author Ashwini L Chand [email protected] orcid.org/0000-0002-1245-729X Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia Search for more papers by this author Author Information Pathum Thilakasiri1, Jennifer Huynh1, Ashleigh R Poh1, Chin Wee Tan2, Tracy L Nero3,4, Kelly Tran1, Adam C Parslow1,5, Shoukat Afshar-Sterle1, David Baloyan1, Natalie J Hannan6, Michael Buchert1, Andrew Mark Scott1,5,7, Michael DW Griffin4, Frederic Hollande8, Michael W Parker3,4, Tracy L Putoczki2, Matthias Ernst *,1,‡ and Ashwini L Chand *,1,‡ 1Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, Vic., Australia 2The Walter and Eliza Hall Institute, Melbourne, Vic., Australia 3ACRF Rational Drug Discovery Centre, St Vincent's Institute, Melbourne, Vic., Australia 4Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, Vic., Australia 5Department of Molecular Imaging and Therapy, Austin Health, Melbourne, Vic., Australia 6Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Vic., Australia 7Department of Medicine, University of Melbourne, Melbourne, Vic., Australia 8Department of Clinical Pathology, University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, University of Melbourne, Melbourne, Vic., Australia ‡These authors contributed equally to this work *Corresponding author. Tel: +61 3 9496 9775; E-mail: [email protected] *Corresponding author. Tel: +61 3 9496 9373; E-mail: [email protected] EMBO Mol Med (2019)11:e9539https://doi.org/10.15252/emmm.201809539 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Excessive signaling through gp130, the shared receptor for the interleukin (IL)6 family of cytokines, is a common hallmark in solid malignancies and promotes their progression. Here, we established the in vivo utility of bazedoxifene, a steroid analog clinically approved for the treatment of osteoporosis, to suppress gp130-dependent tumor growth of the gastrointestinal epithelium. Bazedoxifene administration reduced gastric tumor burden in gp130Y757F mice, where tumors arise exclusively through excessive gp130/STAT3 signaling in response to the IL6 family cytokine IL11. Likewise, in mouse models of sporadic colon and intestinal cancers, which arise from oncogenic mutations in the tumor suppressor gene Apc and the associated β-catenin/canonical WNT pathway, bazedoxifene treatment reduces tumor burden. Consistent with the proposed orthogonal tumor-promoting activity of IL11-dependent gp130/STAT3 signaling, tumors of bazedoxifene-treated Apc-mutant mice retain excessive nuclear accumulation of β-catenin and aberrant WNT pathway activation. Likewise, bazedoxifene treatment of human colon cancer cells harboring mutant APC did not reduce aberrant canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof of concept to support the repurposing of bazedoxifene for the treatment of gastrointestinal cancers in which IL11 plays a tumor-promoting role. Synopsis Inhibition of gp130-receptor/STAT3 activity confers anti-tumor effects in mouse models of gastrointestinal cancers. This effect is recapitulated in mice treated with bazedoxifene, an FDA-approved drug for osteoporosis treatment, supporting its repurposing as treatment in gastrointestinal cancers. First proof-of-concept demonstration that bazedoxifene, an FDA-approved drug for postmenopausal osteoporosis, inhibits the growth of gastric and colon cancers using three independent mouse models. Mechanistically, this arises from the capacity of bazedoxifene to systemically inhibit gp130/STAT3 signalling as demonstrated in the gp130Y757F mouse model of intestinal-type gastric cancer. This data provides a strong rationale to support future clinical efforts for repurposing bazedoxifene as an inhibitor of gp130/STAT3 signaling. Introduction The contribution of inflammatory cytokines to the progression and treatment resistance of solid cancers is now widely accepted, even for malignancies that occur in the absence of overt inflammation (Putoczki et al, 2013). Among the cytokines most prominently involved in this process are those of the interleukin (IL)6/11 family, characterized by the shared use of the transmembrane receptor β-subunit gp130 (Ernst & Putoczki, 2012; Putoczki et al, 2013). We have recently identified a hitherto unrecognized tumor-promoting role for IL11 in mouse models of gastrointestinal cancers that arise in the mucosal epithelium of the stomach, small intestine, or colon (Ernst et al, 2008; Putoczki et al, 2013). Strikingly, genetic restriction of IL11 signaling by either ablation of the IL11Rα co-receptor subunit, or therapeutic administration of the antagonistic "IL11-Mutein" variant, limits the growth of tumors driven by excessive signaling of the canonical WNT pathway resulting from oncogenic β-catenin/CTBNN1 or APC mutations that underpin 80% of human colon cancer (Putoczki et al, 2013). Meanwhile, others have proposed a role for IL11 signaling to enable metastatic spread and to retain tumor heterogeneity that underpins an aggressive phenotype (Kang et al, 2003, 2005; Marusyk et al, 2014). We therefore proposed that tumors exploit the IL6/11 family cytokines, which provide a rheostat function to link an effective, proliferative wound-healing response to the ensuing inflammatory response of the intestinal epithelium (Ernst et al, 2014). We surmised that the activity of these cytokines becomes rate limiting for the growth of tumors and can be exploited as therapeutic vulnerability. Due to their solubility and cellular expression, cytokines and their receptors are preferred targets for the development of antibody-based therapeutics and have become mainstream targets for the treatment of inflammation and other chronic diseases. Accordingly, antibodies directed against IL6, or its cognate ligand-specific IL6Rα co-receptor subunit, are either already in the clinic, or in advanced clinical trials for the treatment of autoimmune conditions and some hematological malignancies. Surprisingly, no such clinically approved antibody reagents have been developed to target IL11 signaling either at the level of the ligand or the IL11Rα receptor subunit. Previous in silico modeling has revealed that the selective estrogen receptor modulators (SERM) raloxifene and bazedoxifene could interfere with the protein–protein interactions between IL6 and gp130 (Li et al, 2014). These third-generation SERMs are approved in Europe and the United States for the prevention and treatment of postmenopausal osteoporosis without conferring agonistic effects on endometrial, ovarian, and breast tissues (Silverman et al, 2008; Pinkerton et al, 2012; Flannery et al, 2016). Here, we provide the first in vivo evidence that bazedoxifene treatment of mice, which harbor epithelial tumors in the glandular stomach, the small intestine or the colon, with drug doses corresponding to treatment regimens for osteoporosis patients, suppresses tumor growth irrespective of the gender of the host. Akin to our observations with the IL11Rα receptor antagonist IL11-Mutein (Putoczki et al, 2013), we find that bazedoxifene restricts the growth of intestinal tumors by suppressing IL11-mediated signaling rather than by interfering with excessive canonical WNT signaling that arises from bi-allelic inactivation of the Apc tumor suppressor gene. Collectively our observations suggest that bazedoxifene could be readily repurposed for the treatment of gastric and colon cancers and that bazedoxifene serves as a tool compound for further chemical refinements to increase specificity and affinity of future small molecule IL11 signaling antagonists. Results Bazedoxifene blocks IL11 signaling Bazedoxifene is thought to inhibit IL6 signaling by interfering with the formation of the signaling-competent hexameric receptor complex. Bazedoxifene prevents the aggregation of two trimeric receptor complexes, comprised of an IL6 ligand, an IL6Rα co-receptor subunit, and one gp130 subunit, and resulted in suppressed activation of STAT3 (Li et al, 2014). Given the proposed similar hexameric 2:2:2 nature of the IL11:IL11Rα:gp130 complex (Veverka et al, 2012; Putoczki et al, 2014), we investigated whether bazedoxifene could also inhibit IL11-mediated, gp130-dependent STAT3 signaling. We co-expressed human IL11Rα alongside the STAT3-responsive pAPRE-luciferase (luc) reporter construct in HEK293 cells. Treatment with IL11 induced a 15-fold increase in APRE-luc reporter activity, which was antagonized in a dose-dependent manner by bazedoxifene (Fig 1A and Appendix Fig S1A). To ensure this was not a generic effect conferred by antagonistic-acting estrogen analogs, we also tested these cells with tamoxifen (Appendix Fig S1A). Tamoxifen failed to suppress IL11, suggesting a selective effect of bazedoxifene in the inhibition of IL11:IL11Rα:gp130 signaling. Figure 1. Bazedoxifene suppresses IL11-mediated STAT3 signaling activity A. Effect of bazedoxifene (BZA) on IL11-induced and STAT3-dependent pAPRE-firefly luciferase reporter activity in HEK293T cells expressing human IL11Rα. Cells were co-transfected with a non-responsive Renilla luciferase plasmid. Results are expressed as relative luciferase units (RLU), that is, firefly luciferase activity normalized against Renilla luciferase activity in each individual culture. B. Effect of BZA treatment on proliferation of IL11 stimulated BAF/03 murine B-cell lines, as determined by MTS-assay. IL6 stimulation was used as a positive control. Cells were engineered to express human either IL6Rα or IL11Rα, respectively. C. Effects of BZA treatment, as determined by MTS-assay, on parental BAF/03 cells stimulated with IL3, of LIF receptor (LIFR)-expressing cells stimulated with LIF, or of cells expressing the constitutive active L-gp130 construct. Data information: Data are mean ± SEM, n = 3 individual cultures, *P < 0.05, **P < 0.01, ***P < 0.001, 2-way repeated-measures ANOVA, Tukey's multiple comparison test. Download figure Download PowerPoint To extend our findings to a more biologically complex response, we exploited the IL3-dependency of the murine BAF/03 pro-B-cell line for their survival and proliferation in vitro (Hilton et al, 1994; Nandurkar et al, 1996). For this, we installed expression constructs encoding gp130 alongside either IL6Rα, IL11Rα, or LIFR in BAF/03 cells. This enabled the subsequent propagation of the corresponding BAF/03 clones with IL6, IL11, or LIF, respectively, in the absence of IL3 (Fig 1B and C). We confirmed that bazedoxifene treatment antagonized IL11-mediated cell proliferation in a concentration-dependent manner (Fig 1B). Corroborating the selective effect that we observed with bazedoxifene on STAT3 transcriptional activity in HEK293T cells, we also found that bazedoxifene but not tamoxifen suppressed IL11-mediated proliferation of BAF/03 cells expressing human IL11Rα (Appendix Fig S1B). Our observations are consistent with the proposed inhibitory mechanism of bazedoxifene on the hexameric gp130 signaling complex, as bazedoxifene also inhibited IL6-dependent BAF/03 proliferation (Fig 1B). By contrast, bazedoxifene treatment not only failed to antagonize IL3-dependent parental BAF/03 cell proliferation, but also that of the LIFR-expressing clones stimulated with human LIF (Fig 1C) and consistent with LIF forming trimeric LIF:LIFR:gp130 complexes (Gearing et al, 1991; Hammacher et al, 1998). We also excluded that bazedoxifene interfered with gp130 signaling in the absence of ligand or of receptor α-subunits. For this, we exploited a synthetic form of gp130 in which a leucine zipper region of c-jun substitutes for the native extracellular receptor domain and confers ligand-independent homodimerization of the resulting chimeric L-gp130 proteins (Stuhlmann-Laeisz et al, 2006). Thus, L-gp130-dependent BAF/03 cell proliferation should be refractory to bazedoxifene inhibition, which we confirmed experimentally (Fig 1C). We surmise from this collective functional data that bazedoxifene disrupts IL11 signaling akin to its proposed action on the signaling-competent, hexameric IL6 receptor complex. In silico modeling of bazedoxifene bound to gp130 Site III residues It was been previously predicted that bazedoxifene competes with binding of IL6 in the trimeric IL6:IL6Rα:gp130 complex. It is the interaction between IL6 of one trimer and the gp130 co-receptor of the second trimeric unit that is inhibited thereby preventing the formation of the signaling-competent hexameric complex (Fig 2A and B; Li et al, 2014; Wu et al, 2016). Specifically, structural analysis of the IL6:IL6Rα:gp130 hexameric complex (PDB ID: 1P9M) revealed that IL6 binds to gp130 via residues leucine-57, glutamic acid-59, asparagine-60, leucine-62, tryptophan-157, and leucine-156 in Site III. A 30 Å resolution cryo-electron map of the IL11:IL11Rα:gp130 hexameric complex suggested that its subunits are organized in a similar arrangement to that of the hexameric IL6:IL6Rα:gp130 complex (Neddermann et al, 1996; Matadeen et al, 2007; Putoczki et al, 2014). Site-directed mutagenesis studies indicated that IL11 interacts with gp130 at Site III via tryptophan-168 and IL6 interacts via tryptophan-157 as amino acids that form part of helix D of these ligands (Barton et al, 1999). We therefore aligned helix D from the crystal structure of IL11 (PDB ID: 4MHL; Putoczki et al, 2014) with that of IL6 to model how IL11 is likely to interact with gp130 at Site III (Fig 2C). When aligned in this manner, tryptophan-157 in IL6 and tryptophan-168 in IL11 indeed interact with the same gp130 residues (Fig 2D). Figure 2. Structure of the gp130/IL6 hexameric signaling complex and the corresponding gp130/IL11 complex A. Crystal structure of the extracellular domains of the IL6 hexameric signaling complex (PDB ID: 1P9M; Boulanger et al, 2003), depicted in cartoon format. The signaling complex consists of two molecules each of IL6 (cream colored), IL6Rα (magenta), and gp130 (one colored gray and the second yellow). B. 90° rotation about the x-axis from the view shown in (A). IL6 binds with its specific IL6Rα subunit via interaction Site I and with gp130 via Site II and Site III. Bazedoxifene has been proposed to interact with Site III, thereby disrupting the interaction of IL6 and IL11 with gp130 (Li et al, 2014; Wu et al, 2016). C. Proposed binding of IL11 to gp130 via Site III residues. The structure of IL11 (PDB ID: 4MHL, orange ribbon structure; Putoczki et al, 2014) was superimposed on IL6 in the hexameric receptor complex. D. Close-up view of the Site III interaction sites between IL6 (cream ribbon, amino acid residues in black) or IL11 (orange ribbon, amino acid residues in orange) with gp130 (gray ribbon, amino acid residues in blue). The side chains of some of the residues at the Site III interface are shown as sticks. Download figure Download PowerPoint We next used our own unbiased in silico modeling approach and identified two possible binding modes of bazedoxifene with gp130 (Appendix Fig S2A–E). In the first one, the indole ring and azepanyl ring of bazedoxifene mimic the interactions between gp130 and the tryptophan-157 and leucine-57 residues in IL6 (Appendix Fig S2C and D). The indole ring hydroxyl substituent of bazedoxifene is able to interact through hydrogen bonds specifically with glutamine-78 in gp130, and the indole and phenol rings both form π-π interactions with tyrosine-94. Meanwhile, the leucine-3 residue in gp130 engages through hydrophobic interactions with the indole ring and phenol substituent, as well as with the pendant phenyl ring of bazedoxifene. Finally, the bazedoxifene azepanyl ring extends in a largely hydrophobic region of gp130 comprising cysteine-6, cysteine-32, phenylalanine-36, isoleucine-83, and glutamine-91. In an alternative second binding model, the phenol ring and azepanyl ring of bazedoxifene mimic the interactions between gp130 and tryptophan-157 and leucine-57 in IL6 (Appendix Fig S2E and F). In this binding mode, the bazedoxifene indole ring can form π-π interactions with tyrosine-94 in gp130. Meanwhile, the phenol substituent of bazedoxifene can form a hydrogen bond with asparagine-92 in gp130, and the indole ring hydroxyl substituent could form hydrogen bonds with either the hydroxyl group of tyrosine-94 or the side chain of glutamic acid-12. The leucine-3 residue in gp130 would then form hydrophobic interactions with the pendant phenyl substituent of bazedoxifene. Importantly, in either of the putative interaction models, bazedoxifene makes multiple interactions with Site III residues of gp130 consistent with the proposed models by Li et al (2014) and Wu et al (2016). Bazedoxifene inhibits IL11-dependent STAT3 activation and growth of patient-derived colon cancer organoids Given our previous observations that IL11 signaling confers potent activation of STAT3 in gastrointestinal cancer cells (Putoczki et al, 2013), we next examined the effects of bazedoxifene on cytokine-stimulated human gastric and colon cancer cells. We observed that bazedoxifene treatment suppressed IL11-mediated induction of the transcriptionally active, phosphorylated STAT3 (pSTAT3) isoform in a dose-dependent manner (Fig 3A). Reduced pSTAT3 levels were also observed in the estrogen receptor (ER)-negative breast cancer cell line, MDA-MB-231, confirming the ER independence of the bazedoxifene effect on IL11/gp130 signaling (Fig 3A). To ascertain the biological importance of this observation in a pathologically relevant setting, we assessed the effects of bazedoxifene on IL11 signaling in patient-derived samples of colon cancer cells grown as primary cultures. We consistently observed that bazedoxifene treatment decreased pSTAT3 levels in a dose-dependent manner (Fig 3B). These tumor epithelial cells showed higher expression of IL11 mRNA transcript when compared to that of IL6 (Fig 3C). Furthermore, mRNA transcripts for IL6R, IL11R, and SOCS3 were readily detectable, with IL6ST transcripts, encoding gp130 as being the most abundant (Fig 3C). We then exploited patient-derived human colon cancer organoids grown in 3D cultures in the presence of IL11. We observed significantly smaller organoids in the presence of 10 μM bazedoxifene (Fig 3D and E). Collectively, our in vitro data in factor-dependent BAF/03 cells, human colon and gastric cancer cell lines, patient-derived colon cancer cells, and organoids provide compelling evidence that bazedoxifene effectively antagonizes IL11-elicited STAT3 signaling and associated cell proliferation in vitro. Figure 3. Bazedoxifene suppresses IL11-mediated STAT3 signaling activity in human cancer cell lines and patient-derived colon cancer primary cultures and organoids A. Effects of bazedoxifene (BZA) on IL11-stimulated STAT3 activation (pSTAT3) in gastric (MKN1), colon (LIM2405), and breast (MDA-MB-231) cell lines. B. Effects of bazedoxifene (BZA) on IL11-stimulated STAT3 activation (pSTAT3) in primary cell cultures of isolated colon cancer epithelial cells of three individual colorectal cancer (CRC) patients. C. mRNA expression levels of IL6, IL11, IL6R, IL11R, IL6ST encoding gp130 and SOC3, a STAT3-regulated gene in epithelial cells derived from six colorectal cancer patients. D. Effects of BZA treatment on IL11-dependent growth of human colon cancer organoids. Organoids were cultured for 6 days in the presence of IL11 (50 ng/ml) and then for a further 7 days in IL11 plus the indicated concentration of BZA. Representative bright-field microscopy images of organoid cultures at 7 days after BZA treatment. Scale bar = 300 μm. E. Relative changes in organoid size after 7 day treatment with vehicle control or BZA as described in (D). Data are mean ± SEM, n = 3 individual cultures, one-way ANOVA, Dunnett's comparison test. Source data are available online for this figure. Source Data for Figure 3 [emmm201809539-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint Therapeutic bazedoxifene treatment impairs IL11-dependent gastric tumor growth in vivo In order to extend our in vitro findings to an in vivo cancer setting, we utilized the gp130Y757F mouse model of intestinal-type gastric cancer. In this model, gastric adenomas spontaneously and reproducibly develop with 100% penetrance in 4-week-old mice with an absolute genetic dependence on bi-allelic expression of the il11rα and Stat3 genes, but completely independent of IL6 signaling (Jenkins et al, 2005; Ernst et al, 2008). Importantly, we had previously shown that therapeutic treatment of tumor-bearing, 10- to 13-week-old gp130Y757F mice with either the antagonistic IL11-Mutein peptide (Putoczki et al, 2013), JAK1/2 kinase inhibitors (Stuart et al, 2014), STAT3 antisense oligonucleotides (Ernst et al, 2008), or inducible short hairpin STAT3-RNA (Alorro et al, 2017), reduces tumor burden associated with reduced cell proliferation and increased apoptosis. Thus, we treated 13-week-old gp130Y757F mice with established gastric tumors for 7 weeks with bazedoxifene at a dose of 3 mg/kg i.p. five times per week (Fig 4A). This dosing was previously used to document inhibitory effects on estrogen-sensitive mouse tissue (Sakr et al, 2014; Flannery et al, 2016). We consistently detected significantly smaller and fewer tumors in the bazedoxifene-treated cohorts compared with the vehicle-treated cohorts (Figs 4B–D and EV1A). Importantly, we observed reduced tumor burden in both, bazedoxifene-treated gp130Y757F male and female m
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