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Potent EMT and CSC Phenotypes Are InducedBy Oncostatin-M in Pancreatic CancerJacob M. Smigiel1, Neetha Parameswaran1, and Mark W. Jackson1,2

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is referred to as asilent killer due to the lack of clear symptoms, a lack of earlydetection methods, and a high frequency of metastasis at diag-nosis. In addition, pancreatic cancer is remarkably resistant tochemotherapy, and clinical treatment options remain limited.The tumor microenvironment (TME) and associated factors areimportant determinants of metastatic capacity and drug resis-tance.Here, oncostatinM(OSM), an IL6 cytokine familymember,was identified as an important driver of mesenchymal and cancerstem cell (CSC) phenotypes. Furthermore, the generation of cellsthat harbor mesenchymal/CSC properties following OSM expo-sure resulted in enhanced tumorigenicity, increased metastasis,and resistance to gemcitabine. OSM induced the expressionof ZEB1, Snail (SNAI1), and OSM receptor (OSMR), engaginga positive feedback loop to potentiate the mesenchymal/CSCprogram. Suppression of JAK1/2 by ruxolitinib prevented

STAT3-mediated transcription ofZEB1, SNAI1 andOSMR, as wellas the emergence of a mesenchymal/CSC phenotype. Likewise,ZEB1 silencing, by shRNA-mediated knockdown, in OSM-drivenmesenchymal/CSC reverted the phenotype back to an epithelial/non-CSC state. Importantly, the generation of cells with mesen-chymal/CSC properties was unique to OSM, and not observedfollowing IL6 exposure, implicating OSMR and downstreameffector signaling as a distinct target in PDAC. Overall, these datademonstrate the capacity of OSM to regulate an epithelial–mes-enchymal transition (EMT)/CSC plasticity program that pro-motes tumorigenic properties.

Implications: Therapeutic targeting the OSM/OSMR axis withinthe TME may prevent or reverse the aggressive mesenchymal andCSC phenotypes associated with poor outcomes in patients withPDAC. Mol Cancer Res; 15(4); 478–88. �2017 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) has been referred to

as a silent killer due to the lack of clear symptoms, and the lack ofearly detection methods. In the great majority of cases, patientspresent with advanced disease that has already metastasized. Inaddition, pancreatic cancer cells are remarkably resistant to che-motherapy, and additional treatment options remain limited. Asa result, the 5-year survival rate is approximately 5% (1), withnearly asmany patients dying each year of pancreatic cancer as arediagnosed. The problem lies, in part, due to the early cellulardissemination of PDAC cells to distant organs, which can precedeprimary tumor formation (2). This highly invasive characteristicof PDAC (3) is largely associated with the gain of mesenchymalproperties by the ductal epithelial cells, indicative of an epithe-lial–mesenchymal transition (EMT; ref. 2). Expression of key

transcription factors capable of driving EMT, such as ZEB1, Snail(SNAI1), and TWIST1/2, initiate the repression of tight cell–cellprotein interactions (claudin, occludin, ZO1, and E-Cadherin),ultimately releasing cells to invade neighboring tissue (4–6).These invasive and motile properties allow the tumor cells toescape a primary site of disease, systemically disseminate, andseed metastases at secondary sites. Concomitant with thistransition to an invasive, mesenchymal phenotype is the acqui-sition of cancer stem cells (CSC) properties responsible forenhanced tumorigenicity and acquired resistance to therapeuticdrugs (7–11).

Emerging evidence suggests a pivotal role for the tumor micro-environment (TME) in promoting epithelial–mesenchymalplasticity (E–M plasticity) and the acquisition of CSC properties(12–15). The TME is composed of secreted factors emanatingfrom infiltrating immune components, fibroblasts, and otherstromal cells. Notably, severe immune infiltration and stromalfibrosis (desmoplasia) is observed in early and late stages of PDAC(16, 17), and enhances tumor growth/survival (18). Identifyingkey mediators within the TME that engage mesenchymal/CSCplasticity will be important for developing effective therapeutictargets for the treatment of PDAC.

Oncostatin M (OSM), an IL6 family cytokine, is elevated inthe serum of PDAC patients relative to healthy controls (19).Moreover, tumor-associated macrophages (at both primary andmetastatic tumors) exhibit increased secretion of OSM in murinePDACmodels (20). OSM signals through a gp130/OSM receptor(OSMR) complex, which activates the JAK/STAT3 pathway.STAT3 promotes gene expression both as a transcription factoras well as by epigenetic mechanisms, and is critical in inducing

1Department of Pathology, Case Western Reserve University, Cleveland, Ohio.2Case Comprehensive Cancer Center, Case Western Reserve University, Cleve-land, Ohio.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

J.M. Smigiel and N. Parameswaran contributed equally to this article.

Corresponding Author: Mark W. Jackson, Department of Pathology, CaseWestern Reserve University School of Medicine, 2103 Cornell Road, WolsteinResearch Building 3-503, Cleveland, OH 44106. Phone: 216-368-1276; Fax: 216-368-8919; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0337

�2017 American Association for Cancer Research.

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inflammation and cancer. Furthermore, constitutive activation ofoncogenic STAT3 signaling promotes tumorigenesis and is asso-ciated with poor outcomes in PDAC (21, 22). Importantly, OSMcan more potently activate STAT3 relative to other IL6 familymembers (23, 24). However, very little is known about thebiological effects of OSM-induced STAT3 in PDAC. Here, weexamine the ability of OSM to promote the emergence of mes-enchymal/CSC properties in PDAC. We demonstrate that OSM iscapable of inducing a potent EMT/CSC program that enhancestumorigenic potential, cell motility, invasiveness, and metastasis.Importantly, OSM conferred resistance to gemcitabine, a currentfirst-line therapy for PDAC. We propose that targeting OSMwithin the TME or OSM signaling within the tumor is likely toreduce the aggressive, metastatic and tumorigenic propertiesassociated with PDAC, thereby improving patient response totherapy and extending patient survival.

Materials and MethodsCell culture

All PDAC cell lines (HPAC, Panc 04.03, Panc 08.13, Panc05.04) were obtained from ATCC and grown in a humidifiedatmosphere containing 5% CO2 at 37�C. PDAC cell line HPACand human fibroblasts were grown in DMEM/F12 (#10-092-CV;Corning) with 10% FCS (#S11150; Atlanta Biologicals), 0.005mg/mL of human transferrin (#T2252; Sigma Aldrich), 10 ng/mLof human epidermal growth factor (#01-107; Millipore), 0.002mg/mL of human insulin (#I9278; Sigma Aldrich), and 40 ng/mLof hydrocortisone (#H4001; SigmaAldrich). Panc 04.03 andPanc08.13 cells were grown in RPMI1640 (#SH30027.01; Hyclone)with 15% FBS (#S11150; Atlanta Biologicals). Panc 05.04 cellswere grown inRPMI1640 (#SH30027.02;Hyclone)with15%FBS(#S11150; Atlanta Biologicals) and 0.2 U/mL of human Insulin(#I9278; Sigma Aldrich). Treatments were done either with10 ng/mL human recombinant Oncostatin M (OSM; #OSM01-13; DAPCEL), 10 ng/mL IL6, or 10 mmol/L ruxolitinib (#R-6688;LC Laboratories). All short-term treatments were performed asdenoted in the figure legend; all long-term treatments were givenat each medium change unless denoted otherwise (�48 hours).Human fibroblast cultures were established from the digestionmedium filtrate of primary reduction mammoplasty tissue asdescribed previously (25). The fibroblast-containing filtrate wasplated into DMEM (#10-013-CV; Corning) with 10% FBS(#S11150; Atlanta Biologicals) and immortalized using pBabe-Puro-hTERT.

Microscopy, Western blot analysis, and quantitative real-timeRT-PCR

Bright-field images were captured at 40� and 200� on aNikonEclipse TE2000-S usingMetaMorph (MolecularDevices).Westernblots were conducted as described previously (12). Primary anti-bodies used were actin (#MS-1295-P; Thermo Scientific), E-cad-herin (#sc-27191; Santa Cruz Biotechnology, #3195; Cell Signal-ing Technology), Snail (#3879; Cell Signaling Technology), phos-phorylated STAT3Y705 (#9145; Cell Signaling Technology), STAT3(#9139; Cell Signaling Technology), and ZEB1 (#3396; CellSignaling Technology). Secondary antibodies used were HRP-linked anti-mouse (#7076; Cell Signaling Technology) andHRP-linked anti-rabbit (#7074; Cell Signaling Technology). Forquantitative real-time RT-PCR (qPCR), total RNA was isolated asdescribed previously (12). RNA (1 mg) was reverse transcribed by

iScript cDNA Synthesis Kit (#170-8891; Bio-Rad). Gene expres-sion was identified using iQ SYBR Green Supermix (#170-8880;Bio-Rad) and a CFX96 thermocycler (Bio-Rad). All sampleswere normalized to GAPDH expression, and error bars represent� SEM for a representative experiment performed in triplicate. Atwo-tailed unpaired Student t test was performed to determinesignificance (�, P � 0.05; ��, P � 0.01; ��, P � 0.001; ��, P �0.0001). Primer sequences were as follows: OSMR forward:50-TCCCAATACCACAAGCACAG-30; OSMR reverse: 50-GCAAG-TTCCTGAGAGTATCCTG-30; SNAI1 forward: 50-GGAAGCCTA-ACTACAGCGAG-30; SNAI1 reverse: 50-CAGAGTCCCAGATGAG-CATTG-30; ZEB1 forward: 50-ACCCTTGAAAGTGATCCAGC-30;ZEB1 reverse: 50-CATTCCATTTTCTGTCTTCCGC-30; GAPDHforward: 50-TGCACCACCAACTGCTTAGC-30; GAPDH reverse:50-GGCATGGACTGTGGTCATGAG-30; CDH1 forward: 50-CCCAA-TACATCTCCCTTCACAG-30; CDH1 reverse: 50-CCACCTCTAAG-GCCATCTTTG-30.

Flow cytometry, migration, and growth assaysFor flow cytometry and FACS, HPAC cells (�1–2 � 106) were

stained with anti-human CD44 APC (clone BJ18; #338806;BioLegend), anti-human CD24 PE (clone ML5; #311106; BioLe-gend), and anti-human CD133 VioBright FITC (clone AC133;#130-105-225; Miltenyi Biotec). Cells were analyzed using a BDLSRII and FACSDiva software version 6.2. Migration assays wereperformed using the live cell IncuCyte ZOOM imaging system(Essen BioScience). Briefly, HPAC cells (1,000 cells/well) weresuspended in DMEM/F-12 containing 0.5% FBS and were seededonto 96-well ClearView-Chemotaxis plates with 8-mm pores.Wherever indicated, plates were coated with 50 mg/mL Matrigel,5 mg/mL collagen, or 5 mg/mL fibronectin, prior to the seeding ofthe cells. The plates were incubated and imaged over the indicatedtime points. Cells migrating to the bottom chamber across thepores were imaged and quantified. Similarly, growth assays wereperformed using 96-well tissue culture dishes seeded with HPAC-VEC, HPAC-OSM, parental HPAC, or OSM-treated HPAC (day 7)cells (2,000 cells/well). Each well was imaged at regular intervalsto determine confluence over time.

Mouse xenograftsAthymic NCr (nude) and NSG mice were bred and main-

tained at the Athymic Animal and Xenograft Core facility at theCase Western Reserve University (Cleveland, OH). For tumor-igenicity assays, 1� 106 of HPAC-VEC or HPAC-OSM cells wereresuspended in 50 mL of 1:1 mix of Matrigel:media mix. Ortho-topic pancreatic injections were performed on 8- to 12-week-oldmale or female, nude or NSG mice anesthetized using isoflur-ane. The mouse was laid on its right side and after appropriatesterilization of the surgical site; a small incision was madeslightly medial to the splenic silhouette. The pancreas wasexternalized and 50 mL of cells were slowly injected close tothe tail of the pancreas. The pancreas was then internalized andabdominal wall and incision site closed with sterile sutures.Tail-vein injections were performed by slightly warming the tailunder IR lamp and injecting 100 mL of cell suspension contain-ing 1� 106 tumor cells. The in vivo, coinoculation studies (usinghuman fibroblasts and HPAC cells) were performed as follows.HPAC cells were cocultured at a 1:3 ratio with FB-VEC or FB-OSM for 7 days. GFP positivity (from the GFP-expressing HPACcells) was used to assess the ratio of HPAC:FB in both groupsand the ratio was normalized to 4:1 prior to orthotopic injection

OSM Drives EMT and Stemness in PDAC

www.aacrjournals.org Mol Cancer Res; 15(4) April 2017 479




of 1.2 � 106 cells into the pancreas of nude mice. Limitingdilution assays were performed by injecting the indicated num-ber of tumor cells in 100-mL Matrigel-media mix into the flanksof the mice. For all experiments, tumor growth was monitoredby bioluminescence imaging after injecting luciferin 10 minutesprior to imaging. Tumor volume was also measured manuallyusing calipers and tumor weight assessed following euthanasiaand tumor retrieval. All animals were used in compliance withthe guidelines approved by the Case Western Reserve UniversityInstitutional Animal Care and Use Committee.

Viral constructs and virus productionLentiviruses were packaged and used to infect target cells as

described previously (26). PLK0.1 vectors containing shRNAstargeting ZEB1 (TRC Version: TRCN0000017565; Clone Name:NM_030751.2-572s1c1 and TRC Version: TRCN0000017566;Clone Name: NM_030751.2-70s1c1) were purchased fromSigma Aldrich. PLK0.1 vector containing shRNA-targeting GFPhas been described previously (26). The SORE6 cancer stemcell reporter construct was a kind gift from Dr. Lalage Wakefieldat Center for Cancer Research, NCI, Bethesda, MD. (27).Lentiviral pLenti-CMV-GFP was obtained from Addgene (plas-mid #17447). pBABE-puro-hTERT was purchased fromAddgene (plasmid #1771). pLenti-Neo-VEC was generatedusing gateway cloning and recombining the multiple cloningsite of PCR8 topo vector (Invitrogen; catalog no. 46-0899) intothe lentiviral vector pLenti-CMV-Dest (Addgene; plasmid#17451). pLenti-Neo-OSM as generated by subcloning OSMcDNA (OriGene; catalog # SC-121421) into gateway entryvector pLenti-ENTR4 (Addgene; plasmid# 17424) and recom-bined into lentiviral destination vector (Addgene; plasmid#17451). pFLUG-GFP-LUC (Firefly) fusion construct was a kindgift from Dr. Huiping Liu at Northwestern University, FeinbergSchool of Medicine, Chicago, IL. (28).

ResultsElevated OSM and OSMR induce EMT in PDAC cells

Utilizing publicly available gene expression datasets, we inter-rogated the levels of OSM and OSMR in invasive pancreaticadenocarcinomas. PDAC tissue exhibited a significant increasein OSM and OSMR gene expression relative to normal pancreas(Fig. 1A), which correlates with an elevated STAT3 gene expres-sion signature (Fig. 1B). Additional datasets also demonstratedelevated OSM and OSMR expression in PDAC tissue (Supple-mentary Fig. S1A–S1D; refs. 29–32). Moreover, studies docu-menting the presence of OSM within primary and metastaticlesions in murine models, and elevated levels of OSM in theserumofPDACpatientswho respondpoorly to treatment providefurther rationale for examining OSM in the biology of PDAC(19, 20). The elevated level of OSM, OSMR, and STAT3 targetgenes support our further investigation into the biological role ofOSM in PDAC. For this, we treated four PDAC cell lines withrecombinant humanOSM, and observed an emergent mesenchy-mal morphology consistent with an epithelial–mesenchymaltransition (EMT), concomitant with STAT3 activation (Fig. 1Cand D). The mesenchymal morphology was accompanied by therepression of epithelial junctional protein, E-cadherin (CDH1;Fig. 1E), and induction of ZEB1 (Fig. 1E), an EMT transcriptionfactor capable of repressing E-cadherin expression. Further anal-ysis of OSM-induced EMT confirmed the time-dependent induc-tion of ZEB1 and Snail (SNAI1; another key EMT transcriptionfactor), which correlated with the upregulation of CD44, a cellsurface marker used to enrich for CSCs from various cancer types(Fig. 2A–C). Moreover, exposure to OSM increases expression ofthe OSM receptor (OSMR), creating a positive feedback loopcapable of enhancing the impact of TME OSM (Fig. 2A). Con-versely, the repression of E-cadherin correlated with the loss ofCD24 expression, indicating that the emergentmesenchymal cells

Figure 1.

Elevated OSM and OSMR in PDAC drive EMT. A, Oncomine data of whole tumor tissue versus normal pancreas tissue examining OSM and OSMR mRNAlevels in PDAC and normal pancreas (60). B, Expression of a STAT3 gene expression signature (generated by filtering pancreatic cancer vs. normalanalysis; then applying filter "genes differentially expressed in melanoma cells in response to STAT3 expression") in PDAC and normal pancreas(Supplementary Fig. S9). C–E, A panel of PDAC cells lines were treated with recombinant OSM for one week and analyzed for morphologic changesusing bright-field microscopy (D; images shown at 200�). STAT3 phosphorylation (Tyr 705), total STAT3, and Actin were assessed using Western blotanalysis (D), and E-cadherin (CDH1), and ZEB1 were assessed using qRT-PCR (E).

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have acquired a CD24LOW/CD44HI phenotype, a well-describedCSCphenotype acrossmultiple tumor types, including pancreaticcancer (Fig. 2C; refs. 10, 28, 33).

In addition, exposure of HPAC cells to recombinant OSMincreased activity of a GFP-reporter construct containing OCT4/SOX2 response elements (SORE6), which has been previouslydescribed to identify cancer stem cell populations (ref. 27; Fig.

2D). To confirm the connection between aCD24LOW/CD44HI cellsurface profile and a mesenchymal phenotype, the small fractionof CD24LOW/CD44HI cells present in parental HPAC populationwere separated by FACS (Supplementary Fig. S2). Indeed, theCD24LOW/CD44HI population expressed elevated levels of SNAI1andZEB1, as well as reduced levels ofCDH1when comparedwiththe CD24HI/CD44LOW population (Fig. 2E). To further confirm

Figure 2.

OSM-induces mesenchymal/CSC plasticity. A–C, HPAC cells were exposed to recombinant OSM for 4, 8, 16 hours (H) and 1, 3, 5, 7, 10, 14 days (D). qRT-PCRanalysis was used to quantify expression of the indicated genes. (A). Western blot analysis was used to assess the indicated proteins (B). CD24 and CD44 surfaceexpression was assessed by flow cytometry (C). D, HPAC cells were infected with lentiviral particles encoding a CSC-specific SORE6 GFP-reporter construct(HPAC-SORE6), and left untreated or treated with recombinant OSM for 8 days. Flow cytometry was performed for CD24/CD44 surface expression, as wellas GFP intensity (numbers represent the percentage of cells that were GFP positive) (Supplementary Fig. S10). E–G, HPAC cells were sorted using FACS for CD24HI/CD44LOW and CD24LOW/CD44HI populations. qRT-PCR analysis was used to quantify the expression of the indicated genes (E). CD24HI/CD44LOW cells were treatedwithOSM for 3, 7, and 18 days andCD24 andCD44 expressionwas assessed by flow cytometry (F). CD24HI/CD44LOW cellswere sorted fromnative HPACpopulationsand cultured in basal HPAC medium for 7 days (far left); CD24LOW/CD44HI sorted cells were cultured for 3 and 7 days in the presence or absence of OSM(adjacent three panels) and CD24 and CD44 expression was assessed by flow cytometry (G). H, HPAC cells were treated with recombinant OSM for 7 days andsubsequently were monitored for growth over a period of 4 days. The graph shows percent confluence of cells at the indicated times. I and J, HPAC cells weretreated for 7 days with recombinant OSM or IL6.Western blot analysis (I) and flow cytometry (J) were performed after 3 and 7 days of OSM treatment.






thatOSM converts cells into a CD24LOW/CD44HI state rather thanselecting for a preexisting CD44HI population, sorted CD24HI/CD44LOW cells were exposed to OSM for various times. Again,OSM induced the acquisition of CD44 and the loss of CD24(Fig. 2F), while untreated CD24HI/CD44LOW cells retained theirepithelial, CD24HI/CD44LOW state, indicating the epithelial/non-CSC state is quite stable (Supplementary Fig. S3). Conversely, theCD24LOW/CD44HI-sorted population spontaneously generateddifferentiated CD24HI cells (Fig. 2G). The ability to producedifferentiated progeny is a property of CSCs. Notably, the de novogeneration of a differentiated CD24HI population was preventedby adding OSM to the medium following the sort for CD44HI

cells, indicating that OSM can suppress the differentiation of theCD44HI population. Finally, OSM generated the emergence of aCD44HI/CD133HI population (Supplementary Fig. S4), whichpossesses significant tumor-initiating capabilities and metastaticpotential within PDAC (11, 34). Importantly, analysis of cellconfluency using the Incucyte Zoom imaging systemor analysis ofcell number determined that OSM does not alter proliferation atthe doses used in our experiments (Fig. 2H; Supplementary Fig.S5B), further indicating that OSM is not selecting for preexistingCD24LOW/CD44HI cells. In contrast to OSM, IL6 was unable to

convert parental HPAC into CD24LOW/CD44HI, likely due to theweaker induction of STAT3Y705 phosphorylation and ZEB1expression (Fig. 2I and J). This further implies that OSM may bea more potent driver of aggressive phenotypes in PDAC in certaincontexts. While we do not observe comparable phenotypicresponse to IL6 in our studies, we note that IL6 has also beendemonstrated to play a significant role in PDAC initiation andprogression in transgenic murine mouse models (22, 35).

OSM induces motility, gemcitabine resistance, and increasedtumorigenic potential

Our observation that OSM induces key EMT transcriptionfactors, generates cells with a CD24LOW/CD44HI phenotype, andactivates a CSC-reporter prompted us to assess whether OSMexposure enhanced biological characteristics consistent withmesenchymal/CSC. For this, OSM was exogenously expressed inHPAC cells by lentiviral transduction (HPAC-OSM) so as to enrichfor a stable CD24LOW/CD44HI population. An empty lentiviruswas used as a control (HPAC-VEC). One week postinfection,HPAC-OSM cells exhibited increased STAT3Y705 phosphorylation,increased ZEB1 expression, a loss of E-cadherin expression, and aconversion from CD44LOW to CD44HI (Fig. 3A). As observed

Figure 3.

OSM induces properties of CSC and increases cell motility. HPAC cells (expressing GFP-LUC fusion protein) were infected with lentiviruses encoding OSM(HPAC-OSM) or control vector (HPAC-VEC). A, One week postinfection, Western analysis and flow cytometry was performed, as indicated. B, Migration ofHPAC-VEC and HPAC-OSM cells was assessed using a live cell IncuCyte Zoom imaging system. Graph shows number of migrated cells (purple colored cells in theadjacent images), error bars represent � SEM for a representative experiment performed in triplicate. A two tailed paired t test was performed to determinesignificance; � , P � 0.01. C, HPAC-VEC and HPAC-OSM cells were cultured in the presence or absence of gemcitabine (30 nmol/L) and cell number wasmonitored using the IncuCyte Zoom live cell imaging system. Graph shows percent confluence of cells at the indicated times, error bars represent � SEM for arepresentative experiment performed in triplicate; a two-way ANOVA was performed in order to determine significance; �� , P � 0.001. D, HPAC-VEC andHPAC-OSM populations were injected subcutaneously at limiting dilutions of 100,000, 10,000, and 1,000 cells/injection. Images of resected primary tumors andquantification of primary tumor volume are presented. P values were calculated using a two-tailed Student t test; �� , P � 0.01; ��, P � 0.01.

Smigiel et al.





with recombinant OSM, exogenous OSM expression did notimpact the proliferation of HPAC cells (Supplementary Fig.S5). However, HPAC-OSM cells were significantly more migra-tory onmultiple matrices (including fibronectin andMatrigel, aswell as uncoated wells, but not collagen; Fig. 3B; SupplementaryFig. S6A–S6C).

Next, we assessed the ability of OSM to induce therapeuticresistance and tumorigenic potential, key characteristics ofmesenchymal/CSCs (36–38). HPAC-OSM and HPAC-VEC cellswere treated with gemcitabine; a first-line therapy for PDAC,and cell growth was monitored over time. HPAC-OSM cellswere significantly less sensitive to gemcitabine (Fig. 3C), whencompared with HPAC-VEC cells, suggesting that elevatedOSM in the TME may promote gemcitabine resistance. Finally,limiting dilutions of HPAC-VEC and HPAC-OSM cells weresubcutaneously injected into the flank of NSG mice (Sup-plementary Fig. S7). Importantly, HPAC-OSM cells formedsignificantly larger tumors (�5–10 times larger) at each of thecell numbers tested, implicating the OSM-induced CD44HI

population as highly tumorigenic (Fig. 3D). The existence ofthe small number of CD44HI cells in the HPAC-VEC population(ranging from �2%–12%) is likely contributing to the smalltumors generated from this population. Taken together, ourdata support a role for OSM in generating PDAC cells withmesenchymal/CSC properties that are highly migratory, tumor-igenic, and resistant to gemcitabine.

OSM increases tumor burden and metastases in vivoWe next assessed whether the increased motility and tumori-

genicity induced by OSM impacted tumor invasiveness andmetastatic spread using orthotopic injection of HPAC-VEC andHPAC-OSM. OSM expression conferred a significant increase inprimary tumor burden as well as increased intraperitoneal met-astatic spread (Fig. 4A), a common feature in patients with PDAC.Furthermore, HPAC-OSM demonstrated a greater capacity tocolonize the lung following tail-vein injection, when comparedwith HPAC-VEC (Fig. 4B). To better represent a tumor microen-vironment with high levels of OSM, we cocultured GFP-labeledHPAC cells (HPAC-GFP) with control human fibroblasts(FB-VEC), or fibroblasts expressing OSM (FB-OSM). HPAC cellscocultured with FB-OSM showed a nearly 3-fold increase in theCD24LOW/CD44HI population (Fig. 4C). Moreover, orthotopiccoinjection of HPAC and FB-OSM into the pancreas of miceyielded greater tumor burden andmetastatic dissemination whencompared with HPAC/FB-VEC coinjection (Fig. 4D and E).HPAC/FB-OSMcoinjections spreadmoreoften to every site exceptthe body wall, resulting in more rapid lethality (Fig. 4E). Thissuggests that a stroma high in OSM will impart a more invasiveand aggressive phenotype within the tumor by eliciting a mes-enchymal/CSC program.

OSM-inducedmesenchymal/CSCplasticity requires JAK/STAT3signaling

OSM induces activation of the heterodimeric receptorgp130/OSMR, which activates JAK1/2–mediated STAT3 phos-phorylation, dimerization, and subsequent STAT3-mediatedtranscription (of ZEB1, SNAI1, OSMR, as shown in Fig. 2A).To assess whether the suppression of JAK/STAT3 activationprevents OSM-induced mesenchymal/CSC plasticity, HPACcells were treated with recombinant OSM in the presence orabsence of a JAK1/2 inhibitor, ruxolitinib. Indeed, ruxolitinib

inhibited STAT3Y705 phosphorylation and suppressed ZEB1and OSMR expression induced by a 4-hour treatment withOSM (Fig. 5A). Furthermore, upon longer term treatment,ruxolitinib prevented CD44 acquisition, transcription of OSMRand key EMT transcription factors ZEB1 and Snail, and E-Cadherin (CDH1) repression (Fig. 5B–D). Finally, ruxolitinibinhibited OSM-induced cell motility (Fig. 5E). Taken together,the invasive and stem-like properties induced by OSM requireJAK-activated STAT3, thus opening up avenues of therapytargeting the intracellular signaling required for OSM-inducedmesenchymal/CSC plasticity in PDAC.

STAT3-activated ZEB1 drives OSM-induced mesenchymal/CSCplasticity

As ZEB1 has been shown to be amaster regulator of EMT, and adirect target of OSM-activated STAT3, we hypothesized that ZEB1was a key component of OSM-induced mesenchymal/CSC plas-ticity. To test this hypothesis, HPAC-OSM, which have a 95.9%CD24LOW/CD44HI profile, were infected with lentiviruses encod-ing short hairpin RNAs targeting ZEB1 (or shGFP as a control),and knockdown of ZEB1 was confirmed by Western blot analysis(Fig. 6A). Importantly, knockdown of ZEB1 did not alterSTAT3Y705 phosphorylation (Fig. 6A) or downstream transcrip-tional target OSMR (Fig. 6B). ZEB1 knockdown increasedE-cadherin expression (CDH1; Fig. 6B), though to a lesser extentin the population with less ZEB1 knockdown (shZEB1-566). Theincrease in CDH1 correlated with a more epithelial morphologycompared with shGFP control (Fig. 6C). Most importantly, thesuppression of ZEB1 in HPAC-OSM cells led to a marked reduc-tion in the number CD44HI cells at both 1 and 3 weeks followinginfectionwith theZEB1 shRNAs (from�96% in shGFPcontrols to42.2% for shZEB1-565 and 36.4% for shZEB1-566). These datasupport our hypothesis that ZEB1 is a crucial JAK/STAT3–activat-ed gene required for OSM-induced mesenchymal/CSC plasticityin PDAC cells.

DiscussionPatients diagnosed with pancreatic cancer currently have dis-

mal survival rates, with 75% of patients succumbing to theirdisease within one year of diagnosis. The observation that trans-formed pancreatic epithelial cells can disseminate to distantorgans even before a frankmalignancy is detectable at the primarysite underscores the difficulty in managing patients with aggres-sive PDAC (2). Our understanding of how OSM, (39) present inthe PDAC TME, impacts mesenchymal/CSC plasticity and givesrise to metastatic and therapy-resistant PDAC growth is currentlylimited and would provide important insights into how PDACmay be targeted.

The CSC hypothesis places cells with self-renewal capacity andthe ability to differentiate at the top of the tumor cell hierarchy.Importantly, CSCsharboring amesenchymal phenotype display adecreased sensitivity to chemo- and radiotherapy, resulting in anenrichment of CSCs following treatment. Evidence is emergingthat suggests that non-CSCs can be induced into a transientCSC-like, drug-tolerant state when exposed to chemotherapies.Our studies support such a model, whereby OSM exposure in-duces the conversion of epithelial/non-CSC into mesenchymal/CSCs (Fig. 7).Mechanistically, we demonstrate that gp130/OSMRactivation results in JAK/STAT3–mediated ZEB1 transcription,and ZEB1-dependent expression of CSC marker CD44.






Consequently, a TME high in OSM creates more aggressive PDACcells with greater tumorigenicity, increased invasive and meta-static potential, and resistance to gemcitabine, classified here as aCD24LOW/CD44HI population. However, early PDAC stem cellswere identified as CD24HI/CD44HI, yet we did not observe aCD24HI/CD44HI population following OSM exposure (10).Rather, CD24HI cells typically reduced CD24 expression first, andthen gained expression of CD44. This finding underscores theheterogeneity of CSC populations and the difficulties in identi-fying/targeting cellular plasticity, and points to the importance ofusing functional assays when defining PDAC stem biology. More-over, OSMR is induced by OSM ligand, suggesting that a positivefeedback loop is engaged to promote greater OSMR/JAK/STAT3activation andmaintain the inducedmesenchymal/CSC cell state.This OSM/OSMR–positive feedback loop provides a number of

potential therapeutic targets, which, if disrupted, may prevent theemergence of aggressive mesenchymal/CSCs in response OSM.In line with our observations, a recent study also demonstratedthat OSM induces EMT in CFPAC1 pancreatic cancer cells, inaddition to lung cancer cell lines. OSM induced mesenchymalproperties and differential 3D growth and colony architecturethat was regulated through a JAK-dependent mechanism. Ourstudies build upon these observations by demonstrating that themesenchymal/CSC phenotype induced by OSM also promoteswidespread metastasis and gemcitabine resistance (40).

Elevated levels of OSM in the tumor microenvironment (TME)have been associated with highly aggressive metastatic cancers,increased risk of tumor recurrence, and a poor prognosis in avariety of cancers (24, 41–44). OSM in the TME may originatefrom a host of immune cells as well as cancer-associated

Figure 4.

OSM increases tumor burden and metastases in vivo. A, HPAC-VEC and HPAC-OSM cells were injected orthotopically into the pancreas of athymic, nude mice.Tumor weight and representative bioluminescence (BLI) images at the end of the experiment are shown (23 days postinjection). A two-tailed Student t testwas used to determine significance; �� , P � 0.01. B, HPAC-VEC and HPAC-OSM cells were injected via the tail vein in athymic, nude mice. BLI images showing lungcolonization at 14 days postinjection are shown. C–E, HPAC-GFP were cocultured with control fibroblasts (FB-VEC) or fibroblasts expressing OSM (FB-OSM).Followingoneweekof coculture, CD24andCD44 surface expression onHPAC-GFPwas assessedviaflowcytometry bygating on theGFPþ cells (C). HPAC-GFPwereorthotopically coimplanted with FB-OSM or FB-VEC into nude mice (D). BLI images at 21 days postinjection and quantification of primary tumor weightat the time animals were sacrificed. A two-tailed Student t test was used to determine significance; ��, P � 0.01. Representative BLI images, site and frequencyof metastasis, and Kaplan–Meier survival curve of mice that received HPAC GFP and FB-VEC or HPAC GFP and FB-OSM cells (E).

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fibroblasts or adipose tissue (45–47). There ismounting evidencethat the OSM in the TME contributes to tumor progression inmany ways. First, macrophages secreting OSM are localized at the

advancing, infiltrative margins of carcinomas, which may impli-cateOSM in tumor invasion (48). Second, highly aggressive basal-like and triplenegativebreast cancer subtypes express higher levels

Figure 5.

OSM-induced mesenchymal/CSC plasticity requires activated STAT3. A, HPAC cells were treated with recombinant OSM, ruxolitinib (RUX), or a combinationof both for four hours. Western blot (top) and qRT-PCR (bottom) were performed as indicated. B–D, HPAC cells were treated with recombinant OSM,ruxolitinib, or a combination of both for 8 days. Flow cytometry (B), qRT-PCR (C), and Western blot analysis (D) were performed as indicated. The percentageof CD24LOW/CD44HI cells is indicated by the number below the flow cytometry scan in B. E, Migration of HPAC cells treated as indicated was assessed usinga live cell IncuCyte Zoom imaging system. The graph (left) and pictures (right) show the number of migrated cells at the indicated times; error bars represent� SEMfor a representative experiment performed in triplicate; a two-way ANOVA was performed to determine significance; �� , P � 0.001.

Figure 6.

ZEB1 is crucial to OSM-inducedmesenchymal/CSC plasticity.CD24LOW/CD44HI HPAC-OSM cellswere infected with lentivirusesencoding two short hairpin RNAstargeting ZEB1 (shZEB1-765 andshZEB1-766) or a control (shGFP).Following selection, Western blotanalysis (A), qRT-PCR (B), bright-fieldmicroscopy (C), and flow cytometry (D)were performed as indicated.






of OSMR which is associated with adverse clinical outcomes andincreased expression of the CSCmarker CD44 (47). Finally, DNA-damaging chemotherapy induces additional OSM secretion fromperitoneal and bone marrow–derived macrophages, potentiallyexacerbating the aggressive properties associated with mesenchy-mal and CSC properties following treatment with genotoxictherapies (49, 50).

Elevated serum levels ofOSM are found in patients with PDAC,in line with the significant increase in OSM mRNA (19, 51). InPDAC patients, sustained OSM serum levels following gemcita-bine was considered a poor prognostic marker, consistent withour observation that OSM rendered PDAC cells highly resistant tofirst-line gemcitabine treatment. We attribute the ability of OSMto induce gemcitabine resistance with its ability to reprogramepithelial/non-CSCs intomesenchymal/CSCs. This observation isalso in line with recent studies by Zheng and colleagues andFischer and colleagues, showing that the acquisition of mesen-chymal properties led to therapeutic resistance in pancreatic andbreast cancer cells (38, 52).

While the IL6 family of cytokines can exhibit redundancy intheir biological responses due to the shared use of the gp130transmembrane receptor, in our studies, OSM has unique bio-logical functions not recapitulated by IL6 (53). Gp130/OSMRheterodimers have characteristics unique from other IL6 familycoreceptors that may account for the distinct signaling and bio-logical effects of OSM relative to other IL6 family members, suchas enhanced JAK/STAT3 and MAPK activation. When coupledwith the observation that OSM and OSMR are significantly

elevated in PDAC, and OSMR is induced by OSM, the uniquesignaling emanating from the OSMR justifies a more thoroughanalysis of OSM and the signaling cascades described here.

Downstream of OSM/OSMR, JAK/STAT3 activation is increas-ingly gaining popularity in the field of cancer therapeutics, withongoing clinical trials using the JAK inhibitor ruxolitinib, includ-ing patients with advanced metastatic pancreatic cancer. Ruxoli-tinib, given as the second-line treatment along with capecitabine,improved overall survival in patients with systemic inflammationand those resistant to first-line therapies (54). STAT3-inhibitingdrugs given in combinationwith additional targeted therapies areproving beneficial in oncogene-addicted cancer cells (such asthose expressing the active KRAS mutant) that acquire resistanceto MEK inhibitors, gemcitabine, and erlotinib by engaging afeedback activation of STAT3 (27). Furthermore, the recent devel-opment of a STAT3 inhibitor BBI-608 (55) as a CSC-selectiveagent highlights the importance of STAT3 to the CSC phenotype.Wepropose that suppressingOSM function (by neutralizingOSMin the TME or inhibiting OSMR activity) will prevent cancer cellsfrom engaging a favored escape mechanism, which is to acquireand maintain mesenchymal/CSC properties (Fig. 7A and B).Directly targeting OSM and OSMR has significant advantages, asboth OSM and OSMR protein are expressed at lower levels innormal human tissueswhen comparedwith other components ofthe OSM/OSMR/JAK/STAT3 axis (Supplementary Fig. S8). Ele-vated OSM expression occurs only in inflammatory, pathologicconditions (such as cancer, arthritis, and inflammatory heartdisease), which vastly improves the therapeutic window to target

Figure 7.

Schematic of OSM-induced mesenchymal/CSC plasticity. Elevated OSM within the pancreatic tumor microenvironment induces STAT3 activation (Y705phosphorylation), leading to transcriptional activation of ZEB1. As ZEB1 accumulates, E-cadherin and CD24 surface expression are repressed, while Snail andCD44 surface expression are increased. The resulting EMT and acquisition of CSC properties decrease gemcitabine sensitivity, and increase tumor-initiatingcapacity and metastasis. A–C, Points where the aggressive phenotypes engaged by OSM can be inhibited (including OSM-neutralizing or OSMR-blockingantibodies, chemical inhibition of activated OSMR/JAK, or ZEB1 silencing).

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OSM/OSMR. Neutralizing or blocking antibodies, and decoyreceptors that prevent OSM from engaging OSMR can amelioratearthritis or prevent inflammatory heart failure in mouse models(56). Of note, blocking antibodies targeting the extracellulardomain of OSMR reduced the OSM-mediated dedifferentiationof cardiomyocytes responsible for heart failure (57). We proposethat OSMR-targeting antibodies could be conjugated with cyto-toxic agents, resulting in more selective targeting of tumor cells,similar to current approaches linking HER2-targeting antibodieswith DM1 (58).

Finally, we found that suppression of OSMR/JAK/STAT3–mediated ZEB1 expression markedly reverted the mesenchy-mal/CSC phenotype, implicating ZEB1 as a crucial driver ofOSM-induced CSC properties. In addition to defining ZEB1 asa key downstream effector of OSMR/JAK/STAT3 activation, thesefindings demonstrated the reversible nature of OSM-inducedmesenchymal/CSC plasticity. ZEB1 links EMT and CSC featuresin pancreatic cancer cells, by inhibiting the expression of themiRNA-200 family, which represses stem cell factors and inducesepithelial differentiation. Moreover, ZEB1 and CD44s wereshown to participate in a self-sustaining loop to maintain CSCfeatures in pancreatic cancer (59). The net result of OSMR/JAK/STAT3/ZEB1 pathway activation is a reversibly induced mesen-chymal/CSC population encompassing aggressive cancer cellproperties. By gaining a better understanding of TME cytokines,such as OSM, that induce mesenchymal/CSC plasticity, one mayenvision tailoring therapies directed at the source of the cytokineor the associated signaling so as to enhance efficacy of currenttherapies and ultimately improve patient survival.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.M. Smigiel, N. Parameswaran, M.W. JacksonDevelopment of methodology: J.M. Smigiel, N. ParameswaranAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.M. Smigiel, N. ParameswaranAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.M. Smigiel, N. Parameswaran, M.W. JacksonWriting, review, and/or revision of the manuscript: J.M. Smigiel,N. Parameswaran, M.W. Jackson

AcknowledgmentsThe authors gratefully acknowledge the research support provided by the

Case Comprehensive Cancer Center (Flow cytometry, Imaging and Micro-scopy core, Athymic Animal and Xenograft core and Small-Animal ImagingResearch Center).

Grant SupportThis work was supported by grant from the Case Comprehensive Cancer

Center (P30CA43703).M.W. Jackson is supported by theNIH (R01CA138421)and the American Cancer Society (Research Scholar Award # RSG CCG-122517).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 5, 2016; revised November 21, 2016; accepted November30, 2016; published OnlineFirst January 4, 2017.

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