Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor B–Specific Kernel Nicholas Underhill-Day and John K

Research Article

Oncostatin M (OSM) Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor B–Specific Kernel Nicholas Underhill-Day and John K. Heath

Cancer Research UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

Abstract OSM is a multifunctional, 28-kDa glycoprotein that was originally cloned based on its ability to inhibit the growth of The interleukin-6 cytokine oncostatin M (OSM) induces potent melanoma cells (2, 3). OSM is produced by activated T lymphocytes growth-inhibitory and morphogenic responses in several and monocytes (2, 4) and is a member of the glycoprotein 130 different tumor cell types, highlighting the importance of (gp130) cytokine family, which exert their actions via a shared OSM signaling mechanisms as targets for therapeutic inter- signal-transducing receptor, gp130 (5). Members of this family vention.The specific molecular pathways involved are not well include interleukin (IL)-6, human herpesvirus 8 IL-6, IL-11, understood, as OSM can signal through two separate hetero- leukemia inhibitory factor (LIF), ciliary neurotrophic factor, dimeric receptor complexes, glycoprotein 130 (gp130)/leuke- cardiotrophin-like cytokine, cardiotrophin-1, and neuropoietin mia inhibitory factor receptor (LIFR) A and gp130/OSM (6, 7). Different members of the gp130 family signal either by the receptor B (OSMRB).In this investigation, we used a LIFR formation of homodimeric gp130 complexes (facilitated in some antagonist to help resolve signaling responses and identify cases by nonsignaling coreceptors) or via heterodimeric receptor patterns of gene expression elicited by the different receptor complexes containing gp130 and a second signaling receptor. complexes.OSM-induced biological effects on breast tumor– Human OSM is unusual in that it can form two types of derived cell lines were specifically mediated through the heterodimeric signaling complexes. The type I OSM receptor gp130/OSMRB complex.Each cytokine tested exhibited dif- complex consists of the gp130 receptor and the LIF receptor a ferential signaling capability and manifested both shared and (gp130/LIFRa; ref. 8). This configuration is also used by LIF. As a unique patterns of gene activation, emphasizing compositional consequence, human OSM and LIF can exhibit similarities in differences in activator protein-1 transcription factor activity biological activity. The type II OSM receptor complex comprises and expression.In particular, OSM strongly activated the c-Jun gp130 and the OSM receptor h (gp130/OSMRh), and this is NH -terminal kinase (JNK) serine/threonine kinase and 2 activated by OSM only (9). The type II receptor complex may downstream components, including activating transcription activate signaling pathways distinct from the OSM type I complex factor (ATF)/cyclic AMP-responsive element binding protein (10, 11), thus conferring OSM-specific signaling functions. family member, ATF3.JNK/stress-activated protein kinase There is a particular interest in dissecting the specific signaling kinase inhibition abrogated cell morphogenesis induced by functions of human OSM, as it has been identified as a potent OSM, indicating an important role for this pathway in OSM suppressor of tumor cell multiplication in a variety of contexts. specificity.These findings identify a core signaling/transcrip- Targets for human OSM-mediated growth inhibition include B tional mechanism specific to the OSMR in breast tumor cells. melanoma (3, 4), glioblastoma (12), lung carcinomas (13), ovarian (Cancer Res 2006; 66(22): 10891-901) carcinomas (14), and breast tumors (15, 16). In addition, human OSM induces differentiation of several tumor cell types (12, 17). Introduction These two effects highlight the potential application of human Receptor-mediated signal transduction pathways are a major OSM as a therapeutic agent. However, a key outstanding issue is target for anticancer drug development (1). In principle, suppres- the role played by the different OSM receptor complexes in sion of tumor cell growth can be achieved by inhibiting pathways mediating these biological outcomes. that activate cell multiplication or by activating pathways that The generic features of gp130 cytokine signaling are well defined inhibit cell multiplication. Molecular definition of the pathways (6). The formation of a high-affinity transmembrane signaling that negatively regulate cell multiplication in tumor cells would complex results in activation of the receptor-associated Janus- therefore open up new prospects for the manipulation of tumor cell activated kinase (JAK) kinase family, leading to phosphorylation of growth. The characterization of negative growth-regulatory signal gp130, recruitment of the adaptor protein Shc, and activation of transduction pathways has been greatly aided by the identification the mitogen-activated protein kinase (MAPK) cascade. The second of ligands that suppress tumor cell growth. Here, we analyze target of receptor-activated JAK kinases is the signal transducers the molecular mechanisms of signal transduction mediated by the and activators of transcription (STAT) family of transcription cytostatic cytokine oncostatin M (OSM), which result in the factors, which, on JAK-mediated phosphorylation, migrate to the suppression of tumor cell proliferation. nucleus and activate the transcription of downstream gene targets. However, this generic pathway cannot readily account for the distinctive properties of OSM in suppressing tumor cell growth, Note: Supplementary data for this article are available at Cancer Research Online suggesting that either dynamic features of gp130 cytokine signaling (http://cancerres.aacrjournals.org/). Requests for reprints: Nicholas Underhill-Day, Cancer Research UK Growth dictate cell responses or additional receptor-activated growth- Factor Group, School of Biosciences, University of Birmingham, Edgbaston, inhibitory pathways remain to be discovered. Birmingham B15 2TT, United Kingdom. Phone: 44-121-414-7529; Fax: 44-121-414- In this study, we exploit the properties of a specific type I receptor 5925; E-mail: [email protected]. I2006 American Association for Cancer Research. antagonist LIF-05 (18, 19) to identify the receptor complexes doi:10.1158/0008-5472.CAN-06-1766 involved in mediating the growth-inhibitory functions of human www.aacrjournals.org 10891 Cancer Res 2006; 66: (22). November 15, 2006

OSM on a panel of breast tumor cell lines. We find that growth extracellular signal-regulated kinase 1/2 (ERK1/2; Thr202/Tyr204), JNK/SAPK 183 185 261 271 275 inhibition and morphologic transformation of these cells is an (Thr /Tyr ), MAPK kinase kinase (MKK) 4 (Thr ), MKK7 (Ser /Thr ), 71 63 exclusive feature of the type II receptor complex. Using microarray activating transcription factor (ATF) 2 (Thr ), c-Jun (Ser ), anti-STAT1, approaches, we identify both shared and specific molecular STAT3, c-Jun, c-Myc, IFN regulatory factor 1 [IRF1; New England Biolabs (UK) Ltd., United Kingdom], anti-ATF3, JunB, ETS-2 (Santa Cruz Biotech- signatures of type I and type II OSM signaling. Further analysis of nology, Santa Cruz, CA), anti-phosphorylated STAT5A/B (Tyr694/Tyr699; the specific type II cytostatic signaling pathway reveals that the type Millipore, Billerica, MA), and anti-a-tubulin (Sigma). Anti-KLF10/TIEG II complex is a potent activator of the JNK/stress-activated protein antibody was a kind gift of Malayannan Subramaniam (Mayo Clinic College kinase (SAPK) pathway, which leads to activation of a specific set of of Medicine, Rochester, MN). Membranes were then probed with secondary target genes that have been implicated in mediating cytostasis in anti-rabbit or anti-mouse horseradish peroxidase–conjugated antibodies other cell systems. Thus, we conclude that human OSM suppresses (Amersham Biosciences, United Kingdom), and blots were developed using breast tumor cell proliferation and induces morphologic transfor- SuperSignal West Pico Enhanced Chemiluminescence (Pierce). mation, in part, by activating a specific stress/senescence program cDNA microarray hybridization and scanning. T47D breast tumor cells F of gene expression mediated by the JNK/SAPK pathway. were stimulated with IL-6 (100 ng/mL) or OSM (100 ng/mL) 30-minute preincubation with LIF-05 (500 ng/mL) for 30/60 minutes or with LIF (100 ng/mL) or LIF-05 (500 ng/mL) for 60 minutes. MCF-7 and MDA-MB-231 Materials and Methods breast tumor cells were stimulated with OSM (100 ng/mL) for 60 minutes. Cells and reagents. T47D, MCF-7, and MDA-MB-231 human breast Control cDNA was hybridized against cDNA from OSM/LIF-05-, OSM-, IL-6-, cancer cells were obtained from the American Type Culture Collection LIF-, or LIF-05-treated cells. Total RNA was isolated by Trizol extraction (Manassas, VA). Cells were cultured in DMEM (Life Technologies Ltd., according to the manufacturer’s instructions (Life Technologies), and equal United Kingdom) supplemented with 10% FCS, 1 mmol/L glutamine, and amounts (20 Ag) of RNA were used as starting material for cDNA synthesis. 1 mmol/L streptomycin/1 mmol/L penicillin. Chemical inhibitors SU6656, This was done overnight at 42jC using SuperScript II RNase H Reverse RG13022, SU5614, PD153035, PD98059, SP600125, LY294002, and AG490 Transcriptase (Invitrogen). Random nonamers (3 mg/mL), oligo(dT) (1.0 Ag/ were purchased from Merck Biosciences Ltd., United Kingdom and used in AL), and Cy3-dCTP or Cy5-dCTP were obtained from Amersham Biosciences, a final concentration of DMSO of <0.1%. United Kingdom. The RNA template was removed by alkaline hydrolysis, and Cytokines. The human OSM recombinant expression plasmid was unincorporated nucleotides were removed with QIAquick PCR purification prepared by K.R. Hudson (University of Birmingham, United Kingdom). The spin columns as per manufacturer’s instructions (Qiagen Ltd., United expression vector pGEX-3C-OSM was modified from an original clone as Kingdom). Equal amounts (130 pmol) of Cy3- and Cy5-labeled probe from described previously (20). The design of pGEX-2T-hLIF and pGEX-2T-hLIF05 control and stimulated samples were mixed together in the presence of plasmids (LIF wild-type and LIF antagonist) has also been described human Cot-1 DNA (Life Technologies) and concentrated using YM30 filter previously (18, 21). OSM, LIF, and LIF-05 were expressed as glutathione (Millipore). Prewarmed (37jC) hybridization buffer (Invitrogen), formamide S-transferase fusion proteins in Escherichia coli strain JM109. Expression, (BDH Chemicals, United Kingdom), and pd-dA40-60 (Amersham Biosciences, purification, and cleavage of proteins were carried out as described (21, 22). United Kingdom) were mixed with the probe. Genome-wide microarray chips Protein concentrations were determined using the Coomassie blue protein were spotted at the Cancer Research UK DNA microarray facility (Institute of assay (Perbio Science UK Ltd., United Kingdom). Human recombinant IL-6 Cancer Research, Sutton, Surrey, United Kingdom) and carried human DNA was purchased from Calbiochem. representing 6,528 pairs of duplicate DNA spots.1 The mixed probe was Cell growth/morphology. Cells were seeded in 24-well plates (growth/ heated at 92jC for 3 minutes and cooled to room temperature for 10 minutes inhibitor studies) or onto coverslips in six-well Costar (Cambridge, MA) before application to the array. This was covered by a LifterSlip (Erie cluster plates (morphology studies) at an initial density of between 0.5 Â 104 Scientific Co., Portsmouth, NH). Hybridization was done overnight at 42jCin and 1 Â 104 cells/mL. Fresh medium and cytokines/inhibitor were added 24 a hybridization chamber (Fisher Scientific Co., United Kingdom). The array hours after initial seeding and replenished daily. After 7 days of cytokine/ was washed five times (2 minutes per wash) in prewarmed (42jC) 2Â SSC inhibitor exposure, cells were washed with PBS and trypsinized and viable cell buffer (Sigma) containing 0.2% SDS and once each (2 minutes) in 1Â SSC and numbers were counted by trypan blue exclusion. For morphology studies, 0.5Â SSC buffer, respectively. The array was dried quickly with nitrogen gas, coverslips supporting T47D cells were transferred to chamber slides contain- and signals were detected with an Axon slide scanner 4000B and GenePix Pro ing normal medium and cell morphology of live cells was assessed by light 3 software (Molecular Devices, United Kingdom) at a resolution of 10 Amat microscopy. MDA-MB-231 cells, which are less visible in live culture, were 100% laser power and photomultiplier tube voltage of 550 to 650. Data were fixed in 4% paraformaldehyde for 20 minutes, permeabilized in TBS contain- analyzed with GeneSpring data software package (Agilent Technologies, ing 0.1% Triton X-100, and stained with May-Grunwald-Giemsa (Sigma- United Kingdom). Briefly, each data set was transformed into log base 2 Aldrich, United Kingdom) for viewing by light microscopy. Images for both expression ratios, normalized using ‘‘Lowess’’ intensity-dependent normal- cell types were captured using OpenLab (Improvision Ltd., United Kingdom). ization, and subjected to minimal expression restriction (intensity minimum Western blot. Following serum starvation for 24 hours, T47D cells were of 400 for control/raw data). For each pathway, two hybridizations were done stimulated with increasing concentrations of cytokine for varying times at (dye swap) to give mean values of n = 2. Only genes overexpressed or under- 37jC. Cells were washed in PBS and solubilized in either Triton X-100 lysis expressed (2-fold cutoff) in both hybridizations were deemed to be signif- buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L icant. All GenePix ‘gpr’ data files and associated image files have been EGTA, 50 mmol/L NaF, 1% Triton X-100) or radioimmunoprecipitation assay deposited at the Gene Expression Omnibus repository2 [expression platform (RIPA) buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, accession number GPL996 (23); sample accession numbers GSM102927-928, 50 mmol/L NaF, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) plus GSM104792-793, GSM104751-770, GSM104788-781; series entry GSE4661].

1 mmol/L sodium orthovanadate (Na3VO4) and one complete protease inhibitor tablet (Roche Diagnostics GmbH, United Kingdom) per 10 mL lysis Results buffer. Even amounts of whole-cell lysate were loaded onto 4% to 20% Tris- glycine gels (Invitrogen Life Technologies Ltd., United Kingdom) and The LIF antagonist LIF-05 discriminates between type I and electrophoresed at 100 V for 1 hour. For Western blotting, proteins were type II OSM actions on T47D and MDA-MB-231 breast tumor transferred onto polyvinylidene difluoride (PVDF; Millipore, Billerica, MA) cells. Effects of gp130 cytokines on breast tumor cell proliferation using a standard protocol. Membranes were blocked overnight in TBS buffer [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20] plus 5% bovine serum albumin and subjected to immunodetection using specific antibodies: 1 http://www.crcdmf.icr.ac.uk. anti-phosphorylated STAT1 (Tyr701), STAT3 (Tyr705), p38 (Thr180/Tyr182), 2 http://www.ncbi.nlm.nih.gov/geo/.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. OSMRb-Specific Cytostasis of Breast Tumor Cells have been reported previously (15, 24). Reverse transcription-PCR T47D and MDA-MB-231 cell growth (Fig. 1A), indicating that analysis confirmed expression of gp130, LIFRa, OSMRh, and IL-6 gp130/LIFRa heterodimer formation does not mediate sustained receptor (IL-6R) mRNA by T47D, MCF-7, and MDA-MB-231 cells growth suppression. These findings confirm the significant (data not shown). Here, we assessed the antiproliferative effect of cytostatic functions of human OSM on human breast tumor cells. OSM on estrogen receptor–positive T47D and estrogen receptor– The specific LIFR antagonist, LIF-05, has previously been shown negative MDA-MB-231 breast tumor cells. We examined breast to block ligand-stimulated tyrosine phosphorylation of both gp130 tumor cell growth by measuring cell numbers after 7 days of and the LIFR and suppress biological responses mediated by the exposure to OSM, LIF, and IL-6 (Fig. 1A). Cytokine concentrations gp130/LIFRa heterodimeric complex (19). LIF-05, therefore, of between 10 and 1,000 ng/mL OSM and IL-6 exhibited potent, represents a useful tool to discriminate between responses dose-dependent growth inhibition of the T47D and MDA-MB-231 mediated by the type I and type II OSM receptor complexes. cell lines. OSM was more potent than IL-6 in mediating growth T47D and MDA-MB-231 cells were incubated for 7 days in the inhibition in these experiments. In contrast, only the highest presence of OSM (200 ng/mL) or OSM plus increasing concen- nonphysiologic concentration (1,000 ng/mL) of LIF suppressed trations of LIF-05 (10, 100, and 1,000 ng/mL). OSM, on its own,

Figure 1. OSM mediates growth inhibition and altered cell morphology of T47D and MDA-MB-231breast tumor cells via the gp130/OSMRh complex. Breast tumor cells were plated at between 0.5 Â 104 and 1.0 Â 104 cells/mL and cultured for 7 days in the presence of increasing concentrations (1.0-1,000 ng/mL) of OSM, LIF, or IL-6 (A)in the presence of 200 ng/mL OSM and increasing concentrations (10-1,000 ng/mL) of the LIFR antagonist LIF-05 (B)orin the presence of 100 ng/mL OSM, LIF, IL-6, or OSM (100 ng/mL) plus LIF-05 (1,000 ng/mL; C, a-e). Fresh medium and cytokine were replaced daily. For cell growth measurements (A and B), viable cells were counted and cell number is expressed as a percentage of the corresponding untreated control. Columns, mean of triplicate experiments; bars, SE. To view cell morphology (C), images were captured at Â40 and are representative of cell morphology for each condition (minimum of three experiments).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research inhibited proliferation of T47D and MDA-MB-231 cells by 41% and OSM stimulation resulted in JNK/SAPK phosphorylation (data not 30%, respectively (Fig. 1B). Addition of LIF-05 up to 1,000 ng/mL shown). did not block OSM growth inhibition, confirming that this effect is Cells were then stimulated with OSM or LIF in the presence of mediated through the type II gp130/OSMRh complex and not the LIF-05 to identify signals propagated by the type II gp130/OSMRh type I gp130/LIFRa complex. In fact, LIF-05 enhances the cytostatic complex. As the concentration of LIF-05 increased (10-1,000 activity of OSM, suggesting that type I and type II receptor ng/mL), LIF-induced STAT3 phosphorylation decreased but was signaling may exhibit antagonistic functions. unaffected in OSM-stimulated cells (Fig. 2B). Thus, human OSM In the course of these experiments, we noted, as previously has the potential to mediate STAT3 phosphorylation through both reported by others (15, 25), a striking effect of OSM on breast gp130/LIFRa and gp130/OSMRh complex formation. LIF-05 had tumor cell morphology (Fig. 1C). T47D cells, in the absence of any no effect on OSM-mediated STAT1 (Fig. 2B) or STAT5 phosphor- added cytokine, are typically epithelioid in shape growing tightly ylation (data not shown). This reveals that OSM activates both in flat, monolayer colonies (Fig. 1C, a, top). MDA-MB-231 cells STATs primarily via the gp130/OSMRh heterocomplex because LIF grow as a monolayer of dissociated epithelial-like cells due to loss cannot stimulate effective phosphorylation of STAT1 or STAT5. of E-cadherin expression and intercellular adhesion (Fig. 1C, a, LIF-05 also had no effect on OSM-induced JNK/SAPK phosphor- bottom). Exposure to OSM dramatically altered morphology, with ylation, again defining a specific requirement for the gp130/OSMRh T47D cells becoming spherical or fusiform in appearance, heterocomplex in this pathway. Conversely, stimulation with OSM exhibiting decreased intercellular contact and forming aggregated or LIF in the presence of LIF-05 decreased the level of ERK1/2 colonies (Fig. 1C, b, top). MDA-MB-231 cells became elongated, phosphorylation (Fig. 2B), suggesting that gp130/LIFRa dimeriza- exhibiting a more fibroblast-like phenotype and developed long tion significantly contributes to OSM-induced ERK1/2 activation. pseudopodia (Fig. 1C, b, bottom). OSM also altered MCF-7 cell OSM, LIF, and IL-6 activation of STAT and MAPK pathways by each morphology (data not shown). These cells became very heteroge- of the representative receptor complexes are summarized (Fig. 2C). neous in size and shape, developed numerous cytoplasmic The type II gp130/OSMRh complex specifically activates signaling processes, and also exhibited decreased intercellular contact. via the JNK/SAPK and STAT1/STAT5 pathways, whereas both type T47D cells exposed to LIF (Fig. 1C, c, top) or IL-6 (Fig. 1C, d, top) I and type II complexes strongly activate the canonical STAT3 and remained epithelioid in shape and still grew in flat monolayer MAPK/ERK1/2 signaling pathways. colonies, but there were a higher number of cells showing rounded OSM and IL-6 regulate ‘public’ and ‘private’ subsets of early- or fusiform morphologies than occurred in control colonies. Cell response genes in T47D breast tumor cells. The previous borders also appeared more defined, indicating a small effect on experiments show that human OSM activates different signaling cell-cell adhesion. Similarly, LIF and IL-6 had a less dramatic effect pathways depending on the identity of the receptor complex used. than OSM on MDA-MB-231 cell morphology, which remained These signals result in the activation of gene expression, which predominantly epithelial-like (Fig. 1C, c and d, bottom), although leads to the biological response. In this case, we were interested to cells developed higher numbers of small, cytoplasmic extensions define those genes that are specifically regulated by the gp130/ compared with untreated control cells. This suggests that LIF and OSMRh type II complex as mediators of the cytostatic and IL-6 have a small effect on breast tumor cell morphology but OSM morphologic effects of human OSM on breast cancer cells. We seems the prominent cytokine of this family to stimulate gross characterized the repertoire of genes regulated by signaling structural changes in both cell lines. Incubation with OSM in the through different OSM receptor complexes using a custom presence of LIF-05 did not block OSM-induced changes in cell genome-wide cDNA microarray containing f6,500 genes selected morphology of either T47D or MDA-MB-231 cells (Fig. 1C, e, top for their roles in cell proliferation, checkpoint control, and and bottom). LIF-05 alone did not stimulate any change in cell survival.1 shape or structure compared with untreated controls (data not T47D breast tumor cells were either preincubated with LIF-05 for shown). Thus, the characteristic changes OSM induces in breast 30 minutes before stimulation with OSM or stimulated with OSM, tumor cell morphology are also mediated via the type II gp130/ IL-6, LIF, or LIF-05 alone (Fig. 3A, pathways A to E). Sets of genes OSMRh receptor complex. up-regulated as a result of stimulation of the gp130/OSMRh OSM, LIF, and IL-6 exhibit differential STAT and MAPK heterocomplex (pathway A) or arising via the combined activities activation. Activation of both the JAK/STAT and MAPK pathways of the gp130/OSMRh and gp130/LIFRa receptor complexes by gp130 cytokines has been well defined as a mechanism of signal (pathway B) were compared (Fig. 3B and C). After 30 and 60 transduction (6). In this study, we used our panel of cytokines to minutes, 10 and 25 genes, respectively, were significantly (>2-fold) determine the repertoire of STATs and MAPKs activated by each up-regulated in the presence or absence of the LIFR antagonist LIF- receptor complex in T47D cells. We studied STAT1, STAT3, and 05, indicating OSMRh-specific gene induction. LIF-05 stimulation STAT5 only, as other STATs are not implicated in gp130 family (pathway E) had no effect on gene expression. LIF stimulation cytokine signaling in breast tumor cells. OSM, LIF, and IL-6 (pathway D) also had no effect on gene expression. This mirrors the stimulated phosphorylation of STAT3 (Fig. 2A). In contrast, only lower signaling capability of LIF and lack of growth suppression OSM and IL-6 were able to induce appreciable STAT1 and STAT5 mediated by 100 ng/mL of this cytokine. Genes up-regulated as a phosphorylation. This suggests that gp130/LIFRa heterodimeriza- result of IL-6 activation of the gp130/gp130 homodimer (pathway tion does not initiate efficient activation of STAT1 or STAT5. In C) were compared with the OSM-stimulated gene set of pathway B addition, we tested for activation of MAPKs, ERK1/2, p38, and JNK/ (Fig. 3B and C). Collectively, these define a set of 12 ‘shared’ OSM- SAPK. OSM, LIF, and IL-6 activated p42/44 ERK1/2 with LIF being and IL-6-regulated genes (7 and 11 common genes up-regulated more potent than OSM or IL-6 (Fig. 2A). None could induce p38 after 30 and 60 minutes, respectively) and a set of 15 ‘specific’ phosphorylation (data not shown). OSM and IL-6 mediated p46 gp130/OSMRh-regulated genes and 5 ‘specific’ gp130/gp130- JNK/SAPK phosphorylation, but the former cytokine was 10-fold regulated genes (Fig. 3B and C). Of the shared genes up-regulated, more potent (Fig. 2A). In the MCF-7 breast tumor cell line, only five were early-response transcription factors (junb, atf3, klf10/tieg,

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Figure 2. OSM, LIF, and IL-6 activate differential signaling of STAT and MAPK pathways in T47D breast tumor cells. T47D cells were starved of serum for 24 hours and stimulated with increasing concentrations (1.0-1,000 ng/mL) of OSM, LIF, or IL-6 for 15 minutes (A) or preincubated with increasing concentrations (10-1,000 ng/mL) of LIF-05 for 30 minutes before stimulation with 10 ng/mL OSM or LIF (B). Even amounts of Triton X-100–extracted whole-cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, blocked, and probed with anti- phosphorylated STAT1(Phospho-STAT1 ; Tyr701), STAT3 (Phospho-STAT3; Tyr705), STAT5A/B (Phospho-STAT5; Tyr694/ Tyr699), ERK1/2 (Phospho-Erk1/2; Thr202/Tyr204), or JNK/SAPK (Phospho- Jnk/SAPK; Thr183/Tyr185) antibody. Membranes were stripped and reprobed with anti-a-tubulin, STAT1, STAT3, ERK1/2, or JNK/SAPK antibody to confirm equal protein levels in all lanes. C, results of pathways activated by each of the representative heterodimeric and homodimeric receptor complexes are summarized.

irf1,andfra2). OSM specifically up-regulated a further four response to OSM and IL-6 signaling. These results (Fig. 4B) transcription factors (c-fos, c-myc, c-jun, and ets-2). showed that, at the protein level, ATF3 and JunB exhibited robust To identify an OSM-induced ‘gene signature’ common to induction in response to OSM signaling, c-Jun and c-Myc were multiple breast tumor–derived cell lines, microarray experiments induced at lower levels, whereas IRF1, ETS-2, and KLF10/TIEG were done on MCF-7 and MDA-MB-231 cells in addition to the expression were unaffected. In summary, fold changes for ‘shared’ T47D cell line (Fig. 4A). OSM mediated up-regulation of six genes genes (Fig. 3B) and protein levels (Fig. 4B) were generally higher (klf10/tieg, c-jun, junb, irf1, rgs16, and efna1) in all three cell lines for OSM (especially JunB and ATF3) compared with IL-6 and and a further 15 genes (c-fos, egr1, atf3, c-myc, ets-2, socs3, fosl2/fra2, potentially reflect the greater potency of biological action cdk5r1, ccl2, ccl7, ccl11, ctgf, icam1, ier3, and pggt1b) in at least two exhibited by OSM. of the three cell lines. Long-term regulation of gene expression by OSMRB. Distilling this data, we arrive at a common core set of OSM/ We extended the microarray study to longer time points IL-6-regulated transcription factors (junb, atf3, klf10/tieg, and irf1) (3 hours and 1, 3, and 6 days) to coincide with the onset and and a specific ‘OSM-only’ set of transcription factors (c-jun, c-myc, establishment of stasis with the objective of discerning the and ets-2) whose expression may be tightly associated with the signature of gene expression associated with the cytostatic cytostatic and morphologic responses. We next validated these response to activation of gp130/OSMRh. The results are detailed genes by examining the induction of protein expression in in Supplementary Table S1.2 Prominent in this data set is the www.aacrjournals.org 10895 Cancer Res 2006; 66: (22). November 15, 2006

Figure 3. OSM and IL-6 induce shared and specific gene expression in T47D breast tumor cells. T47D cells were stimulated with OSM + LIF-05 30-minute preincubation (100 and 500 ng/mL, respectively), OSM (100 ng/mL), IL-6 (100 ng/mL), LIF (100 ng/mL), or LIF-05 (500 ng/mL) for 30 and/or 60 minutes. RNA was isolated, cDNA synthesis was carried out, and equal amounts of purified Cy3- or Cy5-labeled cDNA were hybridized against Cy3- or Cy5-labeled control cDNA (unstimulated cells). Microarray chips carried 6,528 pairs of duplicate human DNA spots. A, pathways A to D, hybridizations done representing OSM, IL-6, and LIF heterodimeric and homodimeric receptor utilization are displayed as pathways; pathwayE, aberrant gene expression mediated by stimulation with LIF-05 alone was also assessed. B, descriptions of all genes up-regulated after 30 and 60 minutes as a result of OSM F LIF-05 and IL-6 stimulation. Values are the mean fold change from two hybridizations (n =2; dye swap), and error values represent the SE. C, gene sets (up-regulated genes only) representing pathways A (gp130/OSMRh), B (gp130/OSMRh and gp130/LIFRa), and C (gp130/gp130) were assessed for similarity and are expressed in Venn diagram format.

up-regulation of genes associated with inhibition of cell multi- consistent with the proposal that OSM-induced arrest of breast plication and growth arrest, such as RBP1 (26) and GADD45 (27), tumor cell multiplication and associated morphologic differences and down-regulation of genes associated with DNA synthesis resemble the induction of a partial terminal differentiation (histones) and G2-M cell cycle progression, such as CDC20 (28) program of pleiotropic changes (17). and CENPF/mitosin (29). OSM specifically activates the JNK/SAPK pathway in T47D These findings are consistent with an OSM-dependent growth breast tumor cells. As OSM seemed to be a potent activator of arrest mechanism that has multiple axes acting at both the G1-S JNK/SAPK serine/threonine kinase, we assessed activation of and G2-M checkpoints. In addition to genes implicated in cell cycle downstream effectors ATF2 and c-Jun. JNK/SAPK was originally control, this data set also displays significant changes in genes associated with stress-induced and inflammatory responses but involved in a wide variety of metabolic processes and immune has known roles in other biological outcomes, such as growth regulatory functions (Supplementary Table S1). These results are and differentiation. JNK/SAPK is activated by stress signals (UV,

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. OSMRb-Specific Cytostasis of Breast Tumor Cells g-radiation, and osmotic shock) as well as inflammatory kinase to be upstream of JNK/SAPK. OSM stimulation for 2 hours cytokines (tumor necrosis factor-a, IL-1h, and IFN-g) becoming also caused a dose-dependent up-regulation of ATF3 (Fig. 5C), a phosphorylated on threonine and tyrosine residues in response to known target of c-Jun and ATF2 (31). upstream MAPK kinases MKK4 and MKK7 (30). Once activated, We used a specific ATP-competitive inhibitor of JNK/SAPK JNK/SAPK translocates to the nucleus where it can phosphorylate serine/threonine kinase, SP600125, to confirm that phosphoryla- members of the activator protein-1 family of transcription factors tion of c-Jun and ATF2 was mediated by OSM-induced JNK/SAPK (c-Jun, JunB, JunD, and ATF2). We used antibodies to phosphory- activity. At a concentration of 10 Amol/L SP600125, phosphoryla- lated MKK4, MKK7, c-Jun, and ATF2 to investigate the ability of tion of both transcription factors was blocked on stimulation with OSM and IL-6 to activate JNK/SAPK pathway components. OSM OSM (Fig. 5D), confirming a requirement for JNK/SAPK kinase stimulation resulted in phosphorylation of c-Jun and ATF2 in a activity. Phosphorylation of JNK/SAPK itself was also partially dose-dependent manner, whereas IL-6 activated ATF2 and, to a blocked. These results show that OSM, acting through the gp130/ much lesser extent, c-Jun (Fig. 5A). LIF stimulation did not OSMRh, is a potent activator of the JNK/SAPK pathway. JNK/SAPK phosphorylate JNK/SAPK or downstream components. In contrast inhibition also reduced OSM-mediated ATF3 and c-Jun expression to MKK7 (data not shown), T47D cells displayed a low basal level of (Fig. 5E). However, there was minimal effect on c-Myc expression MKK4 phosphorylation. This increased after OSM stimulation with and no effect on JunB expression. This suggests that gp130/OSMRh maximum levels at 10 to 15 minutes coinciding with optimum JNK/ signaling via the JNK/SAPK pathway results in regulation of at least SAPK phosphorylation (Fig. 5B), implicating this threonine/tyrosine two key early-response genes, ATF3 and c-Jun.

Figure 4. OSM stimulates gene up-regulation of a small subset of early-response transcription factors in multiple breast tumor cell lines. T47D, MCF-7, and MDA-MB-231cells were stimulated with OSM (100 ng/mL) for 60 minutes. RNA was isolated, cDNA synthesis was done, and equal amounts of purified Cy3- or Cy5-labeled cDNA were hybridized against Cy3- or Cy5-labeled control cDNA (unstimulated cells). A, fold change of genes up-regulated in at least two of the three cell lines and are also expressed in Venn diagram format. Values are the mean fold change from two hybridizations (n = 2; dye swap), and error values represent the SE. T47D cells were stimulated with 100 ng/mL OSM or IL-6 for up to 6 hours. Even amounts of RIPA-extracted whole-cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, blocked, and then probed with antibodies against a small subset of transcription factors, including anti-ATF3, c-Jun, JunB, c-Myc, IRF1, ETS-2, and KLF10/TIEG. B, membranes were stripped and reprobed with anti-a-tubulin antibody to confirm equal protein loading in all lanes.

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Figure 5. Inhibition of JNK/SAPK abrogates OSM-induced activation/expression of JNK/SAPK pathway components. T47D cells were starved of serum for 24 hours and stimulated with increasing concentrations (0.01-100 ng/mL) of OSM or IL-6 for 30 minutes (A), 10 ng/mL of OSM from 5 to 60 minutes (B), and increasing concentrations (0.1-100 ng/mL) of OSM for 2 hours (C) or preincubated with increasing concentrations (0.1-50 Amol/L) of JNK/SAPK serine/threonine kinase inhibitor, SP600125, for 30 minutes before stimulation with 10 ng/mL OSM for 30 (D) or 60 minutes (E). Even amounts of Triton X-100–extracted or RIPA-extracted whole-cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, blocked, and then probed with anti- phosphorylated JNK/SAPK (Thr183/Tyr185), ATF2 (Phospho-Atf2; Thr71), c-Jun (Phospho-c-Jun; Ser63), MKK7 (Ser271/Thr275), MKK4 (Phospho-Mkk4; Thr261), or anti-ATF3, c-Jun, c-Myc, or JunB antibody. Membranes were stripped and reprobed with anti-a-tubulin antibody to confirm equal protein loading in all lanes.

Inhibition of JNK/SAPK abrogates OSM-induced morpho- However, none of the tyrosine kinase inhibitors blocked the logic changes of T47D breast tumor cells. We assessed the effect cytostatic effects of OSM on T47D cells (Fig. 6A) or MCF-7 cells of JNK/SAPK inhibition on OSM-induced growth inhibition and (data not shown). This indicates that these effectors and associated morphologic changes of T47D cells (Fig. 6A and B). Ligand downstream targets, such as ERK1/2 and STATs, cannot, on their stimulation of IL-6R oligomerization and subsequent phosphory- own, mediate OSM-induced cytostatic activity, which may require lation of tyrosine residues in the receptor cytoplasmic regions have combinatorial input from multiple signaling pathways. the potential to attract several effector molecules, which link these For morphology studies, cells were grown for 7 days in the receptors to downstream signaling pathway. We assessed the effect presence or absence of OSM (50 ng/mL) and SP600125 (100 nmol/L, of a panel of chemical inhibitors of tyrosine kinase activity on 10 Amol/L). In normal growth medium, cells displayed flat, OSM-mediated growth inhibition. T47D cells, exposed to OSM, monolayer, epithelial-like cell morphology (Fig. 6B, a), whereas were incubated for 7 days in the presence or absence of specific OSM (50 ng/mL) induced the characteristic alterations in morphol- kinase inhibitors. Viable cell numbers were counted by trypan blue ogy (Fig. 6B, b) as described above. Cells exposed to OSM in the exclusion. Effects of tyrosine kinase inhibition are shown for T47D presence of 100 nmol/L SP600125 underwent morphologic changes, cells (Fig. 6A). Inhibition of platelet-derived growth factor receptor/ but these did not seem as pronounced as with OSM alone (Fig. 6B, c). vascular endothelial growth factor receptor (PDGFR/VEGFR; In the presence of a higher concentration of SP600125 (10 Amol/L), SU5614), JNK/SAPK (SP600125), phosphatidylinositol 3-kinase at which phosphorylation of c-Jun and ATF2 is blocked, T47D cells (PI3K; LY294002), and the JAK family of kinases (AG490) partially retained their epithelial-like shape (Fig. 6B, d). Exposure to inhibited cell growth in the absence of OSM, indicating a SP600125 alone (100 nmol/L or 10 Amol/L) had little effect requirement for these molecules in effective growth and survival. (Fig. 6B, e and f ). These findings suggest that JNK/SAPK pathway

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. OSMRb-Specific Cytostasis of Breast Tumor Cells activation is integral in OSM-induced changes on T47D breast Two conclusions emerge from these experiments. The defining tumor cell morphology. However, because SP600125 alone did not morphologic features stemming from gp130/OSMRh complex abrogate OSM growth inhibition, this indicates that integration of formation involve the JNK/SAPK signaling pathway. Second, the additional signaling pathways (ERK1/2 or STATs) at the level of gene gp130 cytokine family as a whole should be considered as a single transcription is a necessary requirement for OSM cytostasis. multifunctional entity whose signaling outputs are qualitatively and quantitatively dictated by the repertoire of receptors expressed by the responding cell type. These considerations highlight the use Discussion of receptor-specific agonists and antagonists in eliciting receptor- The aim of this study was to delineate the molecular pathways specific cell responses in both experimental (19) and therapeutic involved in the cellular responses of breast tumor cells to the settings (22, 32). cytostatic cytokine OSM. Studies using the receptor-specific A second OSMRh-dependent cytokine, IL-31, has recently been antagonist LIF-05 revealed that the characteristic functions of reported (33), which signals via heterodimerization with the gp130- OSM are executed by signaling through the gp130/OSMRh complex related receptor GPL (34, 35). Characterization of the signaling and not by the gp130/LIFRa complex, which has a broader ligand pathways elicited by the OSMRh/GPL heterodimer has revealed specificity (5). Using this platform, we revealed that the gp130/ activation of the STAT1, STAT3, and STAT5 pathways but not the OSMRh and gp130/LIFRa dimers exhibit shared and distinct ERK1/2 or JNK/SAPK pathways, suggesting that OSMRh signaling signaling pathways. The widely used STAT3 and ERK1/2 systems may be further influenced by the identity of the coreceptor are common to both receptors, whereas a defining feature of participating in the activated signaling complex. signaling via the gp130/OSMRh complex activates the JNK/SAPK The execution of OSM-dependent biological responses is pathway. We also noted that gp130/OSMRh formation shares dictated by the transcriptional response to gp130/OSMRh activa- features with gp130 homodimer signaling, but not gp130/LIFRa tion. Here, this is defined by microarray methods and validated at signaling, in the activation of STAT5 and STAT1. the level of protein expression for selected targets. In agreement

Figure 6. Inhibition of JNK/SAPK abrogates OSM-induced morphologic changes of T47D breast tumor cells. T47D cells were plated at between 0.5 Â 104 and 1.0 Â 104 cells/mL and cultured for 7 days in the presence of specific chemical kinase inhibitor F OSM at 50 ng/mL (A) or plated in the presence or absence of 50 ng/mL of OSM F a 30-minute preincubation with JNK/SAPK serine/threonine kinase inhibitor SP600125 (at 100 nmol/L or 10 Amol/L; B, a-f). Fresh medium and cytokine/kinase inhibitor were replaced daily. For cell growth measurements (A), viable cells were counted and cell number is expressed as a percentage of the corresponding untreated control. Columns, mean of triplicate experiments; bars, SE. Kinase inhibitor concentrations were as follows: SRC family inhibitor SU6656, 10 Amol/L; epidermal growth factor receptor (EGFR) inhibitor RG13022, 10 Amol/L; PDGFR/VEGFR inhibitor SU5614, 20 Amol/L; EGFR/ErbB2 inhibitor PD153035, 2.5 nmol; MAPK/ERK kinase (MEK) inhibitor PD98059, 20 Amol/L; JNK/SAPK inhibitor SP600125, 10 Amol/L; PI3K inhibitor LY294002, 14 Amol/L; and JAK family inhibitor AG490, 20 Amol/L. To view cell morphology (B), images were captured at Â40 and are representative of cell morphology for each condition (minimum of three experiments).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research with similar studies (36, 37), a time-dependent profile of such as JNK/SAPK. This implies that, as described above, seeking a transcriptional remodeling is observed where rapid induction, single target gene for mediation of OSMRh-mediated cytostasis and subsequent disappearance, of an ‘‘early-response’’ set of genes may be a false errand if the response depends on the integrated is later followed by the induction and suppression of a broader set totality of transcriptional changes. The early gene transcriptional of genes in a process that extends in our study for 6 days. By responses described here are, however, entirely consistent with the comparing the early-response profile in several OSM-responsive ability of signaling via OSMRh to elicit robust and prolonged breast tumor cell lines and using a variety of stimuli, including activation of the JNK/SAPK pathway, which leads to activation of a targeted activation of OSMRh, we define a ‘kernel’ transcriptional small kernel of early gene responses that, in turn, are modified by profile of early responses to activation of the gp130/OSMRh coinduction of response modifiers in a cell type–dependent context heterodimer common to the breast tumor cell lines under leading to morphologic changes and cytostasis. investigation. Further analysis of the kernel by validation of protein The long-term transcriptional responses to OSMRh activation expression revealed four OSMRh-dependent early genes whose are more diverse and numerous than early changes, and at time expression was common to multiple breast tumor cell lines: atf3, points when the cytostatic effect of OSM signaling is firmly c-jun, junb, and c-myc. established, we observe pleiotropic changes in the gene expression Members of the Jun family of transcription factors are well profile, including alterations in key components of G1 and G2 documented to respond to activation of both the ERK1/2 and JNK/ checkpoint controls, immune response regulators, and diverse SAPK pathways (38, 39) and are defined as having growth- metabolic enzymes. These findings support the proposals of Grant promoting and growth-suppressing functions, respectively, with et al. (17, 45) that the effects of OSM on breast cancer cells involve JunB having the property of suppressing the growth-promoting significant long-term changes in cell phenotype, which resemble a properties of c-Jun (40). ATF3 induction is a well-documented differentiation program. To conclude, these results identify a core transcriptional response to a wide variety of genotoxic and signaling/transcriptional mechanism specific to human OSM, nongenotoxic stress stimuli [including cytokine-mediated activa- which feed into the downstream biological effects induced on tion of the JNK/SAPK pathway (36)], which mediates cytostatic breast tumor cells. In addition, these findings provide the first responses in tumor cell lines in part through stabilization of the OSMRh-specific gene expression profile, at both early and late time p53 response at the protein level (41) and possibly via activation or points, which provide a framework from which to study OSM suppression of downstream ATF3 gene targets (42). However, in functions in other pathologic and nonpathologic contexts. other biological contexts, ATF3 has been reported to exert a multiplication enhancing effect (43). Hai et al. (44) have argued that the divergent cell responses to ATF3 activation reflect the context Acknowledgments in which ATF3 functions are executed. In this respect, the results Received 5/15/2006; revised 8/14/2006; accepted 9/14/2006. presented here support this notion in that transcriptional Grant support: Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page responses to a ‘pure’ OSMRh signal differ quantitatively in cells charges. This article must therefore be hereby marked advertisement in accordance that undergo a similar biological response (cytostasis). Recall that with 18 U.S.C. Section 1734 solely to indicate this fact. h We thank Celine Jones and Anthony Jones for skilled technical support and advice, signaling via OSMR involves activation of shared or core Ian Giddings (Institute of Cancer Research) for supplying the microarrays, and Suzie pathways, such as STAT3 and ERK1/2, alongside private pathways, Grant and Glenn Begley for discussions and sharing of data.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Oncostatin M (OSM) Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor −β Specific Kernel

Nicholas Underhill-Day and John K. Heath

Cancer Res 2006;66:10891-10901.

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