Oncostatin M Modulates the Mesenchymal–Epithelial Transition of Lung Adenocarcinoma Cells by a Mesenchymal Stem Cell-Mediated Paracrine Effect

Published OnlineFirst November 8, 2012; DOI: 10.1158/0008-5472.CAN-12-1568

Cancer Tumor and Stem Cell Biology Research

Mong-Lien Wang1, Chih-Ming Pan1, Shih-Hwa Chiou2,5, Wen-Hsin Chen1, Hsiang-Yi Chang1, Oscar Kuang-Sheng Lee4,6, Han-Sui Hsu4,7, and Cheng-Wen Wu1,3,4,8

Abstract Mesenchymal stem cells (MSC) are strongly associated with tumor progression and have been used as novel cell-based agents to deliver anticancer drugs to tumors. However, controversies about the direct involvement of MSCs in tumor progression suggest that MSCs mediate tumor progression in a cancer type-dependent manner. In this report, we analyzed the functional interactions between human MSCs and lung adenocarcinoma (LAC) cells to determine the therapeutic potential of MSCs in lung cancer. We showed that MSCs effectively inhibited the migration, invasion, and cell-cycle progression of several LAC cell lines. MSCs also enhanced the mesenchymal– epithelial transition (MET) pathway, as evidenced by the reduction of several epithelial–mesenchymal transition- related markers in LAC cells cocultured with MSCs or in MSC-conditioned medium (MSC-CM). By cytokine array analysis, we determined that Oncostatin M (OSM), a differentiation-promoting cytokine, was elevated in the MSC- CM derived from primary MSC cultures. Furthermore, OSM treatment had the same effects as MSC-CM on LAC, whereas neutralizing antibodies to OSM reversed them. Notably, short hairpin RNAs against STAT1, an important downstream target of OSM, hindered the OSM-dependent induction of MET. In vivo xenograft tumor studies indicated that OSM inhibited tumor formation and metastasis of LAC cells, whereas neutralizing OSM in the MSC-CM hampered its inhibitory effects. In conclusion, this study showed that OSM is a paracrine mediator of MSC-dependent inhibition of tumorigenicity and activation of MET in LAC cells. These effects of OSM may serve as a basis for the development of new drugs and therapeutic interventions targeting cancer cells. Cancer Res; 72(22); 6051–64. 2012 AACR.

Introduction ical cord matrix stem cells have been shown to inhibit murine Lung cancer remains one of the leading causes of cancer- lung adenocarcinoma (LAC) growth (2). However, further work related mortality worldwide despite continuous efforts to ﬁnd in human lung cancer models is still needed to clarify the effective treatments (1). Lung adenocarcinoma (LAC) is the therapeutic potential of this approach. Mesenchymal stem most common histologic type of lung cancer. The highly cells (MSC) have recently gained much attention for their invasive and metastatic phenotypes of LAC are the major potential applications in tissue engineering and disease ther- reasons for treatment failure and the poor prognosis associ- apy. Their tropism for sites of tissue damage and the tumor ated with this disease. Stem cell-based therapy is an emerging microenvironment has led to a great deal of interest in the strategy to treat various diseases, including cancer. Rat umbil- functions of MSC in tumors and their ability to serve as delivery vehicles for therapeutic agents (3–5). For example, MSC-based delivery of the death receptor ligand TRAIL leads to the Authors' Afﬁliations: 1Institute of Biochemistry and Molecular Biology, inhibition of tumor growth and the elimination of cancer 2Institute of Pharmacology, 3Institute of Microbiology and Immunology, metastasis in xenograft lung tumor models (6, 7) as well as 4 5 Institute of Clinical Medicine, National Yang Ming University; Department a reduction in the number of putative lung cancer stem cells of Medical Research and Education, 6Department of Orthopaedics and Traumatology, 7Department of Surgery, Taipei Veterans General Hospital; (8). However, the effects of MSCs themselves on cancer pro- and 8Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan gression, as well as the crosstalk mechanisms between MSCs Note: Supplementary data for this article are available at Cancer Research and cancer cells, remain controversial and unclear. Online (http://cancerres.aacrjournals.org/). The epithelial–mesenchymal transition (EMT) is a critical M.-L. Wang and C.-M. Pan contributed equally to this work. process during embryonic development that is reengaged in adults during wound healing, tissue regeneration, and cancer Corresponding Author: Cheng-Wen Wu, Institute of Biochemistry and Molecular Biology, National Yang Ming University, No. 155, Sec. 2, Li-Nong progression (9). EMT is a key step in the induction of tumor St., Peitou, Taipei 112, Taiwan. Phone: 886-228267919; Fax: 886- metastasis and is associated with a poor clinical outcome in 228236518; E-mail: [email protected] cancer patients (10). An aberrant upregulation of EMT tran- doi: 10.1158/0008-5472.CAN-12-1568 scription factors is associated with poor overall and metasta- 2012 American Association for Cancer Research. sis-free survival in patients with non–small cell lung cancer

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(11, 12). Targeting the EMT pathway or inducing the mesen- Collection of CM chymal–epithelial transition (MET), the inverse of EMT, has MSCs were seeded at a density of either 5 105 (low) or been reported to suppress lung cancer progression and metas- 2 106 (high) cells/100-mm plate in complete RPMI-1640 or tasis (13–15) and may therefore represent a potential treat- MesenPRO RS (Invitrogen) medium supplemented with pre- ment strategy for LAC patients. Notably, the composition of the selected FBS, P/S, and L-glutamine. CM was collected after microenvironment, including oxygen tension, growth factors, 24 hours of incubation with MSCs, filtered with a 0.2-mm and cytokines, was recently suggested to modulate the MET pore size Millex filter unit (Millipore),andthenstoredat and EMT process in cancer cells (16–19). MSCs are an impor- 80C. Control medium was produced simultaneously after tant source of many chemokines and cytokines that affect the a 24-hour incubation without MSCs. tissue repair process (20). MSC-secreted cytokines have also been implicated in the MSC-mediated regulation of tumor Animals and tumor cell transplantation cells. However, whether MSCs mediate LAC tumor progression Luciferase-expressing CL1-5 (CL1-5-luc) cells were har- through a paracrine mechanism and whether MSC-derived vested, washed, resuspended in PBS, and mixed with an equal cytokines are involved in LAC tumorigenesis and MET remain volume of Matrigel (BD Biosciences). CL1-5-luc cells, MSCs, or open questions. a mixed population of CL1-5-luc cells and MSCs (in a total Oncostatin M (OSM) is a differentiation-promoting cyto- volume of 100 mL) were injected subcutaneously into the right kine that has been reported to induce hepatocyte differen- dorsolateral side of the flank region of 8-week-old male BALB/c tiation of human embryonic and putative liver cancer stem nude mice (BioLasco Taiwan Co.). The tumors were measured cells (21, 22). Recently, OSMwasshowntopromoteMSC with an IVIS Lumina II system (Caliper Life Science). differentiation into osteoblasts (23). OSM is also reported to have antiproliferation effects against several types of cancers Migration and invasion assay and is thus considered a potential therapeutic target (21, 24– A FluoroBlok 24-Multiwell Insert System with an 8-mm pore 28). However, the role of OSM in tumor progression and size polyethylene terephthalate membrane (BD Falcon) was metastasis has yet to be evaluated. In this study, we aimed to used to test cell mobility. Each well was filled with 700 mL investigate the role of MSCs and their mediators in the medium, and cell suspensions were seeded into the insert regulation of lung cancer progression. We first showed that chamber at a density of 2.5 104 cells in 300 mL medium. After both MSCs and MSC-conditioned medium (MSC-CM) inhib- 24 hours, the medium was removed, and the chamber was ited in vivo tumor growth and the in vitro proliferation, washed with 1 PBS and fixed in 100% methanol overnight. migration, and invasion of LAC cells in a dose-dependent The reverse side of the membrane facing the lower chamber manner. Moreover, both MSCs and MSC-CM promoted the was stained with propidium iodide (Sigma-Aldrich) for 30 MET process in LAC cells, as evidenced by elevated pro-MET minutes, and the migratory cells were then visualized under markers, inhibited pro-EMT regulators, and suppressed an inverted microscope. Cell number was quantitated using Nanog stemness factor. We then analyzed the cytokine ImageJ software. For the invasion assay, the membrane was composition of the MSC-CM and identified OSM as an coated with Matrigel (BD Biosciences) diluted with an equal important mediator of the MSC-dependent inhibition of volume of serum-free medium and incubated for at least 1 hour LAC tumorigenicity. Treatment with recombinant OSM at 37C before the cells were seeded. repressed LAC proliferation, migration, invasion, and promoted MET as well as epithelial-like phenotypic trans- Proliferation assay formation. Conversely, neutralizing OSM in the MSC-CM Cells were seeded into 96-well cell culture plates at a density 3 hindered these effects on LAC cells. Most importantly, a of 1 10 cells/well in 100 mL media and allowed to adhere xenograft tumor model revealed that OSM reduced the overnight. The media was aspirated and replaced with MSC- tumor size, suppressed the incidence of metastasis, and CM or MesenPRO RS medium with or without recombinant enhanced MET markers. In conclusion, this study identified OSM (Peprotech) as described. A WST-1 (Roche Applied OSM as a crucial paracrine mediator of the MSC-dependent Science) assay was carried out according to the manufacturer's inhibition of LAC tumorigenicity, in part by enhancing the protocol to assess the changes in relative cell density every MET pathway. 24 hours. Statistical analysis Materials and Methods The results are reported as the mean SD. Statistical t P Cancer cell lines and culture conditions analyses were carried out using Student test. A value < fi A549 LAC cell line, H1299 large cell carcinoma cell line, 0.05, as denoted with " "in gures, was considered statisti- fi A431 epidermal carcinoma cell line, and breast adenocar- cally signi cant. cinoma cell lines (including MCF-7 and MDA-MB-231) were obtained from the American Type Culture Collection before Results 2007 and tested positive for human origin. The CL1-5 LAC Bone marrow-derived MSCs suppressed LAC tumor cell line was established previously (29). All cell lines were growth in vivo maintained in RPMI-1640 medium (Sigma-Aldrich) supple- Bone marrow-derived MSCs are known to specifically mented with 10% FBS. migrate to and engraft at tumor sites (30, 31). Although several

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Oncostatin M Enhances Mesenchymal-Epithelial Transition

reports in the past decade have investigated the physical and 2 cell lines revealed that treated cells were partially sequestered functional interactions between MSCs and different types of in G1 phase: MSC-CM increased the percentage of CL1-5 and cancer cells (e.g., breast, liver, and colon), their relationships A549 cells in G1 phase by 15% and 8%, respectively, compared remain controversial. Moreover, the roles of MSCs in lung with cells treated with control medium (Fig. 2B). This cancer progression and development are still unclear and need decreased rate of cell proliferation and restrained cell-cycle to be further investigated. To examine the effects of MSCs on progression could explain the inhibition of tumor growth lung cancer cell tumorigenicity, we first conducted xenotrans- observed in the animal model. plantation experiments in immunocompromised mice. Prima- ry bone marrow-derived MSCs were isolated from clinical MSC-CM inhibited migration, invasion, and EMT in LAC specimens taken from 3 healthy donors and analyzed by flow cells cytometry to confirm the expression of MSC markers and the Migration and invasion are critical properties of the cancer absence of macrophage, endothelia, and hematopoietic cell cells that initiate metastasis. To clarify the involvement of markers (1 representative MSCs shown in Supplementary MSCs in LAC migration, we carried out a wound-healing assay Fig. 1). The MSCs were then mixed with luciferase-labeled with both LAC cell lines. As shown in Fig. 2C, the numbers of CL1-5 human LAC cells at a ratio of 1:5 (CþM (low)) or 1:10 migrating cells were decreased by MSC-CM in a dose-depen- (CþM (high)) before subcutaneous injection into the right dent manner compared with those treated with control medi- flanks of male mice. Injections of either MSCs or CL1-5 cells um. This effect was not due to a difference in proliferation rate, alone were conducted simultaneously (Fig. 1A, top; n ¼ 4), and as we observed an approximately 3- to 5-fold difference in the tumor volumes were monitored for 7 weeks. As expected, the number of migrating cells between control- and high CM- CL1-5-induced xenograft tumors significantly increased in treated cells at 12 hours (Fig. 2C), whereas cell proliferation was size. However, when mixed with MSCs, regardless of the ratio, not affected within the first 24 hours (Fig. 2A). the growth of the CL1-5 xenograft tumors were dramatically An indirect coculture assay was carried out to further assess inhibited (Fig. 1A). At day 49, the volume of the CL1-5/MSC cell mobility (Fig. 3A, left). Reduced migration of CL1-5 cells tumors was approximately one tenth that of the CL1-5 tumors was observed when the bottom chamber was coated with (Fig. 1A, bottom right). These data indicated an in vivo inhib- MSCs or filled with MSC-CM compared with noncoated or itory effect of MSCs on LAC tumor growth. control medium-filled controls, respectively (Fig. 3A, middle The inhibitory effect of MSCs on LAC cell-derived tumors and right). The same experiment was also conducted with could occur through direct cell–cell contacts or in a paracrine other 4 primary MSCs isolated from bone marrow or endo- fashion without direct cell contact. To distinguish between metrium of 4 individuals (Supplementary Information). Each these possibilities, we first subcutaneously transplanted lucif- of the MSCs showed inhibitory effect on CL1-5 migration, erase-labeled CL1-5 cells in the right flank of immunocom- indicating that the MSC-mediated inhibition of LAC cell promised mice. Two weeks after transplantation, when the migration is rather a general phenomenon than a unique case tumor mass was visible using an IVIS Lumina Imaging system, for a specific strain of MSC (Supplementary Fig. 2). Moreover, control or CM that had been incubated for 24 hours without or this phenomenon was not unique to CL1-5 cells; the migratory with MSCs, respectively, was injected at the tumor locus. The abilities of 2 other lung cancer cell lines (A549 and H1299) were injections were conducted on days 14, 21, 28, and 35, and the also suppressed when cultured in MSC-CM (Fig. 3B, left). Apart tumor volumes were assessed on day 55 by measuring changes from migration, invasion is another important property of in the mean total photon flux using the IVIS system. The CL1-5 malignant tumor cells. Using a Matrigel-coated Transwell tumors in the CM-treated mice were significantly smaller than culture chamber, we showed that the invasion of both CL1- those of the mice treated with control medium (Fig. 1B; n ¼ 3). 5 and A549 cells was inhibited by MSC-CM (Fig. 3B, middle). These data suggested that the inhibitory effect of MSCs on LAC Whether MSCs promote or inhibit cancer progression tumor growth occurs through a paracrine pathway and may remains controversial. Karnoub and colleagues reported that not require direct cell–cell contact. MSCs within tumor stroma promote breast cancer metastasis (32), and similar phenomena have also been observed in colon MSC-CM inhibited the proliferation and cell-cycle cancer (33). On the other hand, accumulating reports show progression of LAC cells that MSCs inhibit the tumorigenesis of several types of cancers, The MSC-mediated inhibition of tumor growth in the animal such as hepatomas and lymphomas (34–37). To further vali- model indicated that MSCs may suppress cell proliferation or date our results, we carried out a migration assay using CL1-5 cell-cycle progression in LAC cells. We therefore carried out cells, the A431 epidermal carcinoma cell line, and the MCF-7 WST-1 proliferation assays for 2 LAC cell lines (CL1-5 and and MDA-MB-231 breast cancer cell lines, which have been A549) incubated in control medium or 2 concentrations of reported to be stimulated, not inhibited, by MSCs in tumor- MSC-CM. No significant differences in total cell number were igenicity assays (32, 38). As shown in Fig. 3B, only CL1-5 cells observed between groups on the first day. However, after day 2, were inhibited by MSC-CM; migration of the other cell lines CL1-5 and A549 cell numbers were reduced by MSC-CM in a was slightly increased by this treatment. It should be noted that dose-dependent manner compared with the cells cultured in the ratio of MSCs to cancer cells used in this experiment was control medium (Fig. 2A, left). By day 4, treatment with high much less than that used in previous reports (32), which likely CM reduced the cell numbers by more than 50% compared explains the decreased stimulatory effect of MSC-CM on the with control cells (Fig. 2A, right). Flow cytometry analysis of the migration of breast and epidermal carcinoma cell lines

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Figure 1. Bone marrow-derived MSCs inhibited LAC tumor growth in an animal model. A, immunocompromised mice were subjected to subcutaneous transplantation of 8 106 MSCs, 4 106 luciferase-labeled CL1-5 cells (CL1-5-luc), or mixed MSC: CL1-5 at the ratio of 1:5 (CþM- high, with 8 105 MSC) or 1:10 (CþM-low, with 4 105 MSC). The mean total photon ﬂux, representing the tumor volume, was monitored weekly using an IVIS Lumina Live Imager. Representative tumor photographs of IVIS images taken on day 49 are shown (top). The tumor growth curves and the chart of tumor volumes on day 49 are presented (bottom, n ¼ 4). B, Immunocompromised mice were subcutaneously transplanted with 4 106 CL1-5-luc cells and allowed to form tumors. Once the tumor locus could be visualized, 100 mL of control or MSC-derived conditioned medium (CM; supernatant from 2 106 cultured MSCs) was injected at the tumor locus every 7 days until day 55. The mean total photon ﬂux was assessed on day 55 and presented as fold change in the graph (n ¼ 3).

compared with previous reports. However, even under this low with RPMI, the standard culture medium for CL1-5 cells, for the ratio of MSCs:cancer cells, CL1-5 cell migration was still indicated durations (Fig. 3C, top left). Cells were subcultured severely repressed (Fig. 3B, right). when they reached 90% conﬂuence and subjected to the Next, we investigated the reversibility of the inhibitory effect migration assay for the last 24 hours of RPMI incubation. We on migration. CL1-5 cells were cultured in control medium or found that a 24-hour rest in RPMI medium had little effect on MSC-CM for 24 hours, after which the medium was replaced cell mobility in the MSC-CM-treated cells. However, 72 hours of

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Figure 2. MSCs inhibited lung adenocarcinoma cell proliferation, cell-cycle progression, and wound-healing capacity. A, CL1-5 and A549 cells were subjected to a WST-1 proliferation assay in the presence of high density CM (supernatant from 2 106 MSCs), low density CM (supernatant from 5 105 MSCs), or control medium (medium incubated overnight without MSCs). The proliferation curves are shown on the left, and the fold changes in cell number on days 1 and 4 are shown on the right. B, CL1-5 and A549 cells were incubated in high density CM, low-density CM, or control medium for 24 hours and then subjected to ﬂow cytometry cell-cycle analysis. The percentages of cells in each stage of the cell cycle are presented. C, CL1-5 and A549 cells were subjected to a wound-healing assay in the presence of control medium or CM. The numbers of cells within the gap at 0, 6, and 12 hours were calculated and shown in the graphs in the right panels.

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Figure 3. MSC-CM inhibited cell migration, invasion, and induced the MET. A, diagram showing the indirect coculture of MSCs and LAC cells for the Transwell migration and invasion assays (left). CL1-5 cells were subjected to a 24-hour Transwell migration assay with or without either MSCs or MSC-CM. The migrating cells that moved through the 8-mm pores to the other side of the membrane were counted under a microscope (middle) and presented as a percentage relative to controls (right). B, another LAC cell line, A549, and the H1299 large cell carcinoma cell line, were also subjected to the migration assay with or without MSC-CM as indicated (left). CL1-5 and A549 cells were subjected to a Matrigel-coated Transwell migration assay with MSC-CM or control medium, and the results are presented as the percentage of migrating cells relative to the control (middle). Two breast cancer cell lines, MDA-MB-231 and MCF-7, an epidermal cancer cell line, A431, and the CL1-5 cell line were subjected to the migration assay in the presence or absence of MSC-CM (right). C, CL1-5 cells were cultured in either MSC-CM or control medium for 24 hours and then switched to ordinary RPMI maintenance medium for the indicated durations. Cells were subcultured when they reached 90% confluence. For the last 24 hours of RPMI incubation, cells were seeded in the Transwell and subjected to the migration assay. The procedure is summarized in the top left diagram. Blue arrow, control medium; red arrow, CM; black arrow, RPMI medium. The migrating cells were quantified and presented as the percentage of the control value in each set (bottom left). Micrographs of the migratory cells are presented (top right). D, CL1-5 cells with 24-hour treatment of CM or control medium were subjected to immunofluorescent staining and confocal microscopic observation.

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Oncostatin M Enhances Mesenchymal-Epithelial Transition

Figure 4. MSC-CM inhibited the migratory ability of Nanog-, Snail-, and Slug- overexpressing LAC cells. A, CL1-5 cells were subjected to Western blotting analysis (right) after 24 hours of incubation in CM or control medium. Micrographs show the morphologic changes induced by CM treatment (left). B, Western blot analysis of Nanog, Snail, and Slug expression in A549 and CL1-5 stable cell lines. C, CL1-5 stable cell lines were subjected to the Transwell migration assay in the presence of MSC-CM or control medium. The results are presented as the percentage of migratory cells treated with MSC-CM relative to the number of migratory cells treated with control medium (left). CL1-5-Vec, CL1-5-Nanog, A549-Vec, and A549-Nanog were subjected to a wound-healing mobility assay for 24 hours in the presence of MSC-CM or control medium (right). D, the aforementioned CL1-5 stable cell lines were treated with CM or control medium for 24 hours, and Western blottings were carried out to analyze protein expression.

rest in RPMI medium resulted in a slight recovery of cell proposed as a mechanism to inhibit cancer progression mobility (10%); after 7 days of rest, the recovery was even (13–15, 39). Upon incubation in MSC-CM, the mesenchymal- more apparent (Fig. 3C, bottom left and top right). Moreover, like CL1-5 cells adopted a more epithelial-like morphology (Fig. by immunoﬂuorescence analysis and confocal microscopy, we 4A, left). Western blot analysis also revealed increased cyto- observed elevated expression of E-cadherin, a pro-MET mark- keratin-18 (CK-18) and E-cadherin expression, and decreased er, and reduced levels of vimentin, a pro-EMT marker, in cells vimentin, Snail, and Slug expression in MSC-CM-treated LAC treated with MSC-CM (Fig. 3D). These data indicate that the cells compared with control cells (Fig. 4A, right). However, the MSC-mediated inhibition of LAC migration may be caused by level of another EMT-related transcription factor, Twist, was certain molecule(s) released by MSCs into the medium and not altered (data not shown). These data suggested that MSC- that removing the molecule(s) by replacing the medium can CM treatment of LAC cells promotes a MET process that may reverse the inhibition of cell mobility. involve Snail and Slug but not Twist. Interestingly, although the stemness factor Oct4 was not affected, Nanog expression was MSC-CM promoted MET in LAC cells suppressed by MSC-CM, indicating the involvement of Nanog The EMT is strongly linked to cell mobility and cancer stemness signaling in the crosstalk between MSCs and LAC metastasis. Repressing EMT or enhancing MET has been cells. We previously reported that coexpression of Nanog and

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Figure 5. OSM contributed to the MSC-mediated inhibition of LAC migration and promoted the MET. A, MSC-CM and control medium were subjected to a cytokine array analysis using a commercial membrane-bound array kit (left). The expression levels of the OSM receptor (OSM-R) subunits (LIFR and OSMR-b), the IL-6 receptor (IL6-R) subunit (IL6R), and their common receptor subunit (gp130) were analyzed by quantitative real-time PCR. The data are presented as relative log2-fold change relative to the 18S RNA expression level (middle). CL1-5 cells were incubated in CM or control medium for 24 hours and then subjected to quantitative real-time PCR analysis to determine the OSM-R subunits expression levels. The relative mRNA levels of each subunit in CM-treated cells are presented relative to the level measured in control medium-treated cells (right). B, CL1-5 and A549 cells were subjected to the Transwell migration and invasion assays after a 24-hour treatment with or without OSM (5 or 20 ng/mL). The percentages of migratory or invasive cells in OSM-treated cultures compared with untreated controls are shown in the graphs (top). The CM was pretreated with control IgG or various amounts of anti-OSM (aOSM; 0.5 and 2 mg/mL for A549, 2 and 5 mg/mL for CL1-5) antibodies for 2 hours before it was applied to CL1-5 and A549 cells. Migration and invasion activities were measured 24 hours after incubation, and the results are presented as percentages relative to control medium-treated cells (bottom). C, CL1-5 treated with control medium, CM, OSM, or OSM-neutralized CM were subjected to immunoﬂuorescent staining of E-cadherin, vimentin, and nucleus, and observed by confocal microscope.

Oct4 can induce EMT and promote lung cancer metastasis CL1-5/Nanog, CL1-5/Snail, and CL1-5/Slug) using a lentiviral (40). It is possible that Nanog plays a key role in the MSC- transfection system; empty vector-transfected control cell mediated regulation of lung cancer cell mobility. We therefore lines (A549/Vec and CL1-5/Vec) were also generated. The generated A549 and CL1-5 stable cell lines overexpressing expression of Nanog, Slug, and Snail was conﬁrmed by Western Nanog, Snail, or Slug (A549/Nanog, A549/Snail, A549/Slug, blotting (Fig. 4B). Notably, overexpression of Nanog enhanced

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Figure 5. (Continued) D, the morphology of CL1-5 cells after 24 hours of treatment with or without OSM is shown (left). CL1-5 and A549 cells were treated with or without recombinant OSM (20 ng/mL) for the indicated durations and then subjected to Western blot analysis to determine the expression levels of EMT- related proteins (middle). CL1-5 cells with stable knockdown of STAT1 were treated with or without OSM (20 ng/mL) for 24 hours, followed by Western blot analysis of the indicated proteins (right).

the protein levels of Snail and Slug in A549 cells, whereas compositional differences between control medium and MSC- neither Snail nor Slug overexpression affected Nanog levels, CM. Within the array, the anticancer cytokine OSM presented a suggesting that Nanog may be an upstream regulator of Snail more than 3-fold difference (Fig. 5A, left). An ELISA assay also and Slug expression in some LAC cells (Fig. 4B). The stable cell detected secreted OSM in the CM derived from 3 MSCs lines were treated with MSC-CM or control medium before a compared with control medium (Supplementary Fig. 3A). migration assay. Compared with the vector control, overex- Moreover, the OSM mRNA was detected in MSCs by quanti- pression of Nanog increased the number of migrating cells in tative real-time PCR and visualized in agarose gel (Supple- MSC-CM-treated cultures by more than 2-fold (Fig. 4C, left; mentary Fig. 3B and C). OSM is a multifunctional cytokine that from 11.7% to 27.1%), whereas Snail- and Slug-overexpressing inhibits the growth of many types of tumor cells (25, 28, 41, 42). lines presented a 50% increase in migration (from 11.7% to Quantitative real-time PCR analysis of OSM receptor expres- 18.0% and 17.4%, respectively). However, compared with con- sion in LAC cells indicated that the level of OSM-specific trol medium-treated cells, exposure to MSC-CM still induced a receptor subunits (LIFR and OSMR-b) was higher than that pronounced reduction in migratory cell number, making the of IL6 (IL6R), a cytokine reported to mediate MSC-dependent slight rescue effect induced by Nanog, Snail, and Slug over- regulation of cancer cells (Fig. 5A, middle). Treating LAC cells expression scarcely noticeable (Fig. 4C, left). These data sug- with CM further enhanced the expression of the 2 OSM gested that enhancing Nanog, Snail, or Slug levels is not receptor subunits, OSMR-b and LIFR (Fig. 5A, right), which sufficient to render cells resistant to the MSC-CM-induced may magnify cellular sensitivity to OSM. inhibition of mobility. Similar results were observed in the To investigate the effect of OSM on LAC cells, we first carried wound-healing assay. Though Nanog-overexpressing cell lines out a WST-1 cell proliferation assay on CL1-5 and A549 cells showed better mobility than control cell lines, a 24-hour treated with different concentrations of recombinant OSM. In treatment with MSC-CM still dramatically reduced their line with our previous findings, OSM treatment inhibited cell migratory capacity and abrogated the significant difference proliferation: 3 days of 20 ng/mL OSM treatment reduced CL1- between control and Nanog-expressing cells (Fig. 4C, right). 5 and A549 cell numbers by 40% and 25%, respectively, com- Western blot analysis of the CL1-5 stable cell lines showed that pared with untreated controls (Supplementary Fig. 4). the ectopically expressed Nanog and Slug were suppressed by Although OSM has been reported to inhibit the growth of MSC-CM, whereas E-cadherin expression was elevated (Fig. many types of tumor cells (25, 28, 41, 42), its role in metastasis is 4D). Together, these data indicated that MSC-CM promotes less well understood. We determined that addition of OSM to the MET process in LAC cells by suppressing EMT markers and the culture medium significantly inhibited cell migration and the Nanog stemness factor. invasion of both LAC cell lines (Fig. 5B, top), as well as H2170 and H520 squamous carcinoma cell lines (Supplementary Fig. Oncostatin M mediated the MSC-dependent inhibition of 5), in a dose-dependent manner. The use of a specific antibody cell proliferation and migration against OSM (aOSM, R&D Systems) to neutralize the OSM The inhibitory effects of MSC-CM on LAC cells suggested the secreted into the MSC-CM blocked the inhibitory effect of involvement of a paracrine mechanism mediated by molecules MSC-CM and increased the migration and invasion of both secreted from MSCs. We used a cytokine array to determine the LAC cell lines compared with control anti-IgG-treated MSC-

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CM (Fig. 5B, bottom). Immunofluorescent staining of the CL1-5 CMþaOSM abrogated the inhibitory effect. Histochemical cells treated with CM, OSM or OSM-neutralized CM staining of the lung sections from CL1-5-injected mice showed (CMþaOSM) indicated that OSM, like CM, elevated E-cadherin that CM or OSM treatment reduced the spread of tumors in and suppressed vimentin expression, whereas antibody against lung tissue (Fig. 6C, left). Quantitative real-time PCR analysis of OSM blocked these effects of MSC-CM (Fig. 5C). OSM also the tumor tissues from the A549-injected mice showed that CM affected cell morphology: 24 hours of OSM treatment induced and OSM elevated E-cadherin and suppressed Slug mRNA an epithelial-like phenotype in CL1-5 cells (Fig. 5D, left). levels, which were reversed by CM-aOSM (Supplementary Fig. Western blot analysis of OSM-treated LAC cells showed that 7D). Taken together, these data showed that OSM mediated the OSM inhibited the expression of Nanog, Snail, and Slug but CM-dependent elevation of MET and suppression of tumor elevated E-cadherin after 24 hours of treatment, suggesting an growth and metastasis in vivo. enhanced MET process in both LAC cell lines (Fig. 5D, middle). STAT1 is an important downstream target of OSM-dependent pathways. Activation of STAT1 has been reported to be critical Discussion for the efficacy of anti-metastatic immunotherapies (43). The Tumor progression with metastasis is one of the major anticancer effect of OSM against chondrosarcoma is thought causes of mortality in lung cancer patients and therefore to work through the JAK3/STAT1 pathway (27). The activation represents an important clinical challenge. EMT has been of STAT1 upon OSM treatment was confirmed in the LAC cells considered a key mechanism responsible for the metastatic (Supplementary Fig. 6A). We then specifically knocked down progression of lung cancer (44). Enhanced EMT characteristics STAT1 or STAT3, another downstream target of OSM, using are associated with poor overall and metastasis-free survival in lentiviral expressed short hairpin RNAs. After puromycin patients with non–small cell lung cancer (11). Targeting the selection for 2 weeks, the knocked down pool of cells were EMT pathway or enhancing MET has been proposed as a treated with or without OSM (Fig. 5D, right; Supplementary Fig. promising therapeutic method to address cancer metastasis 6C). Western blotting indicated that without OSM treatment, and improve patient survival (45). Slug and Snail, EMT-related knockdown of STAT1 had little effect on Nanog or Slug protein transcription factors, have been reported to increase the levels. However, with OSM treatment, knockdown of STAT1, metastatic risk of lung cancer (11, 12, 15). In addition, stemness but not STAT3, resulted in elevated levels of Nanog, Snail, and factors like Nanog and Oct4 are also involved in EMT regula- Slug compared with a scramble control (SC). These data tion and are correlated with a poor clinical outcome for LAC indicated that the OSM-dependent inhibition of Nanog and patients (40). In this report, we showed that BM-MSCs isolated elevation of MET signaling may be mediated by a STAT1- from 3 different donors inhibited the migration, invasion, dependent pathway. proliferation, and cell-cycle progression of LAC cells through a paracrine mechanism. The EMT regulators Nanog, Snail, and OSM reduced LAC tumor growth and metastasis in vivo Slug were all inhibited and the MET markers CK-18 and E- To investigate the anticancer effect and therapeutic poten- cadherin were elevated by MSC-CM, resulting in elevated MET tial of OSM in vivo, we pre-treated luciferase-labeled CL1-5 cells process in LAC cells. The inhibitory effect on cell migration was with control medium, CM, OSM, or OSM-preneutralized CM also observed in endometrium-derived MSCs (Supplementary (CMþaOSM) and transplanted the cells into immunocompro- Fig. 2), suggesting that this effect on LAC cells may share in mised mice. As shown in Fig. 6A, tumors appeared 2 weeks after different types of MSCs. In an animal model, both MSCs and transplantation in mice transplanted with control medium- MSC-CM suppressed LAC tumor growth and metastasis; the treated cells, whereas the luciferase signals in mice trans- treatment of MSC-CM enhanced MET and suppressed prolif- planted with CM- or OSM-treated cells were barely detectable eration in the xenograft tumor tissues. Our data suggested that or significantly lower. Interestingly, preneutralizing OSM in the molecules secreted from MSCs may have a therapeutic poten- CM with a specific antibody reduced the inhibitory effect of the tial for lung cancer treatment. CM and resulted in an elevated luciferase tumor signal com- OSM is a regulator of stem cell pluripotency and modulates pared with CM-treated mice (Fig. 6A). The same result was also the differentiation of specific lineages. OSM induces osteogen- observed in mice transplanted with A549 cells (Fig. 6B, left; esis in MSCs (23) and is involved in the differentiation of Supplementary Fig. 7A). Immunofluorescent staining of the hepatocytes from human ESCs (22, 46, 47). OSM has also been tumor sections indicated that CM reduced the population of reported to inhibit the proliferation of several tumor cell lines, proliferating cells (Supplementary Fig. 7B). Treatments of CM including melanoma, glioblastoma, and breast cancer cells or OSM increased E-cadherin (Fig. 6B, right) but suppressed (21, 24–28). Yamashita and colleagues further showed that vimentin (Supplementary Fig. 7C) expression level in the OSM renders liver cancer stem cells sensitive to chemotherapy subcutaneous tumors. To evaluate the anti-metastatic effect by inducing hepatocyte differentiation (21). However, the effect of OSM, we conducted a tail vein injection of pretreated CL1-5 of OSM on cancer metastasis is less understood. Lacreuseette and A549 cells in immunocompromised mice. Two weeks after and colleagues reported that some metastatic melanoma cells transplantation, control medium, CM, OSM, or CMþaOSM was lost the expression of OSMR-b (48), implying a role for OSM in injected into the mice 4 times at 7-day intervals. The tumor cancer metastasis. In the present study, we identified OSM as a nodules in the lungs were counted and shown in Fig. 6C. The mediator of the MSC-dependent inhibition of LAC tumorige- data clearly showed that CM and OSM reduced the number and nicity. Interestingly, MSC-CM treatment increased the mRNA volume of metastatic tumor nodules, whereas exposure to expression of OSMR-b, which could be a mechanism to

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Oncostatin M Enhances Mesenchymal-Epithelial Transition

Figure 6. OSM suppressed xenograft tumor growth and metastasis in an animal model. A, immunocompromised mice were subcutaneously transplanted with CL1-5-Luc cells pew-treated with control medium, CM, OSM (20 ng/mL), or OSM-neutralized CM (CMþaOSM; CM preincubated with 2 mg/mL aOSM for 1 hour). On day 5 and day 10, the same treatments were applied through peritumoral injections. Tumor volumes were monitored with an IVIS Lumina Live Imager, and the graph shows the mean SD of luciferase signal intensity on day 14 (n ¼ 3). B, a similar experiment as described in A was conducted with A549 cells. The tumor volumes were measured by a caliper on day 58 (left, n ¼ 3). Tumor sections were stained with anti-E-cadherin and Hoechst33258, and observed under confocal microscope (right). C, immunocompromised mice were transplanted through tail veins with CL1-5 or A549 cells pretreated as indicated. Two weeks after transplantation, control medium (100 mL), CM (100 mL), OSM (20 ng in 100 mL of control medium), or OSM-neutralized CM (CMþaOSM; 100 mL of CM preincubated with 2 mg/mL aOSM for 1 hour) was injected into the mice 4 times at 7-day intervals through intraperitoneal injection. Mice were sacriﬁced 3 months after transplantation. Tumor formation and histochemical staining of the CL1-5 tumor sections in lung were photographed (left). The numbers of metastatic nodules in the lung was counted (top right), and the tumor volume in lung was calculated from the histographs using the formula (length width2)/2 (bottom right).

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Wang et al.

magnify the OSM pathway. The increased OSMR-b might be actions with tumor cells, the large range of cytokines and possibly due to the OSM in MSC-CM, as Blanchard and growth factors they produce, and the receptors expression colleagues reported that OSM triggers the synthesis of levels of the tumor cells might also explain the inconsistent OSMR-b (49), though we did not rule out the involvements of effects of MSCs on tumorigenicity. Bergfeld and colleagues other soluble factors in regulating OSMR-b expression. We have stated that MSCs can be the friends or foes of cancer cells, showed that OSM inhibited LAC cell proliferation and reduced depending on their origin, degree of differentiation, and the xenograft tumor size in animal model. Moreover, OSM sup- type of tumor cells with which they interact (50). Our inves- pressed cell mobility and tended to induce MET in LAC cells; tigation of the interactions between MSCs and LAC cells not mice transplanted with OSM-incubated LAC cells and treated only revealed inhibitory effects of MSCs on lung cancer growth with OSM showed reduced metastatic tumor nodules in the and metastasis but also provided insight into how this cross- lungs, along with increased pro-MET parkers and suppressed talk is mediated. We proposed a model in which MSC-derived pro-EMT markers in tumor tissues. More importantly, pre- OSM signals in a paracrine manner to LAC cells, resulting in neutralizing OSM in MSC-CM reduced the effects of MSC-CM increased MET, and thereby suppress tumorigenicity. Further on LAC cells. It should be noted that there may be other studies are needed to evaluate the prospect of the extracellular cytokines also contributing to the MSC-CM-inhibited tumor cytokine-MET signaling pathway as a target for therapeutic progression, as neutralizing OSM in MSC-CM did not development. completely block this inhibition. Given that there is a gap in OSM concentrations between high-CM and the recombinant Disclosure of Potential Conflicts of Interest OSM we used, we suspected a synergistic cooperation of No potential conflicts of interest were disclosed. cytokines in MSC-CM, presuming that natural and recombinant OSM have equal efficacy. Potential candidates are cur- Authors' Contributions rently under investigation. The downstream effector of OSM, Conception and design: M.-L. Wang, S.-H. Chiou STAT1, has been linked to anti-metastasis signaling in mela- Development of methodology: M.-L. Wang, C.-M. Pan, S.-H. Chiou, O.K.-S. Lee Acquisition of data (provided animals, acquired and managed patients, noma cells (43). We showed that knockdown of STAT1, but not provided facilities, etc.): M.-L. Wang, C.-M. Pan, W.-H. Chen STAT3 (Supplementary Fig. 6C), hampered the OSM-mediated Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.-L. Wang, C.-M. Pan, W.-H. Chen, O.K.-S. Lee inhibition of Nanog and Slug, suggesting a role for STAT1 in the Writing, review, and/or revision of the manuscript: M.-L. Wang, C.-M. Pan, OSM-regulated induction of MET involved in the metastasis of S.-H. Chiou, C.-W. Wu LAC cells. We also showed that knockdown of STAT1 resulted Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): M.-L. Wang, C.-M. Pan, H.-Y. Chang, H.- in increased cell mobility in LAC cells (Supplementary Fig. 6B). S. Hsu Further studies are needed to dissect the signaling pathways in Study supervision: C.-W. Wu LAC cells that are activated or inactivated in response to extracellular OSM stimulation. Acknowledgments The relationship between MSCs and tumor cells is still We thank Dr. Oscar Kuang-Sheng Lee and Dr. Shih-Hwa Chiou for providing bone marrow- and endometrium-derived MSCs, respectively. We acknowledge controversial (50). For example, BM-MSCs have been reported the Taiwan Mouse Clinic funded by the National Research Program for Bio- to promote colon cancer growth by enhancing angiogenesis pharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan for the and inhibiting tumor cell apoptosis (33). Direct coculture of animal core facility; the National RNAi Core Facility in Academia Sinica (NSC 97- 3112-B-001-016) for lentiviral shRNA clones. BM-MSCs with breast cancer cells suggested that BM-MSCs stimulate the EMT in breast cancer cells and enhance cancer metastasis (32). On the contrary, studies of liver cancer indi- Grant Support This research was supported by the Institute of Biomedical Sciences, Aca- cated that human MSCs inhibited the cell-cycle progression, demia Sinica, the National Yang-Ming University, the Department of Health tumorigenesis, and metastasis of hepatocellular carcinoma (DOH101-TD-C-111-007), and the National Science Council (NSC100-2321-B- b 010-020; NSC100-2325-B-010-011; NSC100-2321-B-010-021), Executive Yuan, (34, 35), possibly through downregulated TGF- or Wnt sig- Taiwan, R.O.C. naling (34, 35). These discordant studies suggest a cancer type- The costs of publication of this article were defrayed in part by the payment of dependent mechanism for MSC-mediated pro- or antitumor page charges. This article must therefore be hereby marked advertisement in effects. MSCs are the source of many chemokines and cyto- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. kines that can be anti- or pro-tumorigenic and contribute to a Received April 25, 2012; revised August 16, 2012; accepted August 27, 2012; state of immunotolerance (50). The complexity of their inter- published OnlineFirst November 8, 2012.

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6064 Cancer Res; 72(22) November 15, 2012 Cancer Research

Oncostatin M Modulates the Mesenchymal−Epithelial Transition of Lung Adenocarcinoma Cells by a Mesenchymal Stem Cell-Mediated Paracrine Effect

Mong-Lien Wang, Chih-Ming Pan, Shih-Hwa Chiou, et al.

Cancer Res 2012;72:6051-6064. Published OnlineFirst November 8, 2012.

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