interleukin-6andoncostatin-msynergizewiththepi3k/akt ... · pathway to promote aggressive prostate...

Oncogenes & Tumor Suppressors

Interleukin-6 and Oncostatin-M Synergize with the PI3K/AKTPathway to Promote Aggressive Prostate Malignancy inMouse and Human Tissues

Daniel A. Smith1, Atsushi Kiba5, Yang Zong4, and Owen N. Witte2,3,4

AbstractChronic inflammation has been proposed as an etiological and progression factor in prostate cancer. In this study,

we used a dissociated prostate tissue recombination system to interrogate the role of interleukin 6 (IL6) and therelated cytokine oncostatin-M (OSM) in the initiation and progression of prostate cancer. We identified thatprostatic intraepithelial neoplasia (PIN) lesions induced by PTEN loss of function (PTENLOF) progress to invasiveadenocarcinoma following paracrine expression of either cytokine. Increased expression of OSM was also able todrive progression of benign human epithelium when combined with constitutively activated AKT. Malignantprogression in themousewas associatedwith invasion into the surroundingmesenchyme and increased activation ofSTAT3 in PTENLOF grafts expressing IL6 or OSM. Collectively, our work indicates that pro-inflammatorycytokines such as IL6 or OSM could activate pathways associated with prostate cancer progression and synergizewith cell-autonomous oncogenic events to promote aggressive malignancy.

Implications: Increased expression of IL6 or OSM synergizes with loss of PTEN to promote invasive prostatecancer.Visual Overview: http://mcr.aacrjournals.org/content/early/2013/09/02/1541-7786.MCR-13-0238/F1.large.jpg.Mol Cancer Res; 11(10); 1159–65. �2013 AACR.

IntroductionChronic inflammation is observed in nearly all forms of

cancer and may represent a contributing factor at initiation,progression, andmetastasis (1). Numerous factors have beenproposed to contribute to prostatic inflammation and it hasalso been proposed to play a role driving in the multifocalnature of prostate cancer (2). Studies using bacterial colo-nization of the prostate to elicit chronic inflammatoryconditions have observed epithelial reactive hyperplasia andprostatic intraepithelial neoplasia (PIN) lesions (3).Increased expression of inflammatory cytokines, includingmembers of the interleukin 6 (IL6) family, has been observed

in these models and could represent a functional mech-anism (4). Immunohistochemical analysis of human pros-tate biopsies have shown increased expression of IL6 andthe related OSM ligands and their cognate receptorswith prostate cancer progression (5). Therefore, we soughtto interrogate the role of inflammation by monitoringtransformation of prostate epithelial cells in a pro-inflam-matory environment induced by ectopic expression of IL6or the related OSM in the stroma of tissue recombinationexperiments.The IL6 family of cytokines consists of several related

ligands that share common protein structure and use ofGP130 signal transducer with receptor adaptors modulatingligand specificity (6). Studies have identified increased serumlevels of IL6 in patients with prostate cancer correlating toincreased clinical stage, hormone-refractory disease, andmetastasis (7, 8). Several prostate cancer cell lines have beenshown to express IL6 whereas treatment with either cytokinegenerally confers a growth advantage (9). Ectopic expressionof IL6 can promote castration-resistant conversion in cas-trate conditions, whereas OSM has been shown to activateandrogen receptor (AR) through an androgen-independentmechanism (9, 10). Antibodies targeting IL6 inhibit xeno-graft growth and castration resistance, although they haveshown poor clinical efficacy to date, indicating the need tomore fully understand how these molecules function in thetumor microenvironment (11, 12).

Authors' Affiliations: 1Molecular Biology Interdepartmental Program,2Department of Microbiology, Immunology, and Molecular Genetics, 3Eliand Edythe Broad Center for Regenerative Medicine and Stem CellResearch, 4Howard Hughes Medical Institute, David Geffen School ofMedicine, University of California, Los Angeles, California; and 5Pharma-ceutical Research Division, Takeda Pharmaceutical Company Limited,Kanagawa, Japan

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

Corresponding Author: Owen N. Witte, University of California, LosAngeles, Room 5748, MRL Building, 675 Charles E. Young Dr. S, LosAngeles, CA 90095. Phone: 310-206-0386; Fax: 310-206-8822; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0238

�2013 American Association for Cancer Research.

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We sought to interrogate the functional roles of IL6 andOSM in prostate cancer in vivo using a dissociated tissuerecombination system developed in our laboratory (13, 14).We identified that paracrine expression of either IL6 orOSM is sufficient to promote invasive progression fromintermediate PIN lesions induced by PTENLOF in mousetissues or activation of AKT in human tissues. Increasedexpression of either IL6 orOSM in the context of PTEN lossresulted in increased activation of STAT3 over PTEN lossalone, indicating a potential molecular mechanism. Thatcytokines related to IL6 exhibit similar cancer progressionbehavior indicates that highly targeted therapies aimed solelyat disrupting IL6 signaling could be less effective than thosetargeting signaling nodes common to the IL6 family.

Materials and MethodsMouse strains and regeneration assayHousing, maintenance, surgical, and experimental pro-

cedures were undertaken in compliance with the regulationsof the Division of Laboratory Animal Medicine of theUniversity of California, Los Angeles (Los Angeles, CA).Homozygous Ptenfl/fl, strain B6.129S4-Ptentm1Hwu/J, werepurchased from Jackson Laboratory and maintained in ourfacility. Lentivirus preparation, tittering and infection,and regeneration of dissociated cells were conducted aspreviously described under safety regulations for lentivirususe set by Environmental Health and Safety (EH&S) atUniversity of California, Los Angeles. Briefly, prostatetissue from 6- to 12-week-old male mice was minced,digested, and dissociated according to published protocols(13). Dissociated cells (1 � 105 to 2 � 105) were infectedwith lentivirus at a multiplicity of infection (MOI) of 50,recombined with UGSM (1� 105 to 2� 105), suspendedin a collagen plug, and engrafted under the kidney capsulefor 6 to 10 weeks. Grafts were recovered via surgicalresection of the kidney and fixed in 10% buffered formalinovernight or flash frozen. UGSM cells were prepared frompregnant C57BL/6 females on embryonic day 14 andcultured in UGSM media [Dulbeccos' Modified Eagles'Media (DMEM), 5% FBS, 5% NuSerum (BD #355504),1� selenium-transferrin-insulin (Gibco #51500-056), 2mmol/L L-glutamine].

Human prostate regenerationsFor primary human cells, we used a protocol approved

through the UCLA Office for the Protection of ResearchSubjects, and all human tissue samples were de-identified toprotect patient confidentiality. A total of 3 patient sampleswere used for this study and all specimens were processed asdescribed previously (14). Briefly, surgical prostate speci-mens were removed and cancerous areas were separated fromthe benign tissue. Basal epithelial cells were isolated byfluorescence-activated cell sorting (FACS) from dissociatedbenign tissue using phycoerythrin (PE)-conjugated CD49f(eBiosciences #12-0495-83) and allophycocyanin (APC)-conjugated Trop2 (FAB650A, R&D Systems) antibodies.Cells were stained in PrEGM supplemented with 2.5 mg/mLFungizone (Gibco) and 10 mmol/L of the p160 ROCK

inhibitor Y-27632 dihydrochloride (Tocris Bioscience#1254). Sorting was conducted on a BD FACS Aria II (BDBiosciences).

Cell lines and cytokine treatmentPEB cells were a kind gift from Dr. Lynnette Wilson

(NYU)maintained in PrEGM (Lonza, # CC-4177 and CC-3165) supplemented with 10% FBS. CaP8 cells were a kindgift from Dr. HongWu (UCLA) and maintained in UGSMmedia. Cell lines were not authenticated. Human recom-binant IL6 and OSM were purchased from Cell Signaling(#8904SF and #5367SF, respectively) and were reconsti-tuted in 1� PBS supplemented with 1% bovine serumalbumin (BSA). For cytokine treatment assays, cells wereserum starved inDMEM supplemented with 0.2% FBS and2 mmol/L L-glutamine for 6 to 18 hours and treated withcytokines for indicated times. Results presented are repre-sentative of 2 replicates.

ResultsIl6 and oncostatin-M expression promote progression toinvasive prostate cancer in mouse and human tissuesTo assess the potential role of IL6 and oncostatin-M

(OSM) transformation of both mouse and human prostateepithelium, we used a dissociated prostate regenerationsystem developed in our laboratory (13, 14). For mousetissues, we used dissociated prostate cells from adult malehomozygous Ptenfl/fl conditional knockout mice transducedwith either GFP control or Cre-GFP lentivirus that werecombined with urogenital sinus mesenchyme (UGSM)transduced with RFP control, IL6-RFP, or OSM-RFPlentivirus (Fig. 1A). Western blot analysis was used toconfirm increased expression and activity of secreted IL6and OSM in lysates and conditioned media of transducedUGSM (Fig. 1B).Control grafts exhibited regeneration of normal prostate

cellular architecture, whereas PTENLOF combined withcontrol UGSM resulted in PIN lesion formation (Fig. 1C).PTENLOF combined with UGSM expressing either IL6 orOSM exhibited a heterogeneous range of transformationstates, although graft wet weights were not significantlydifferent (Supplementary Fig. S1A–S1C and SupplementaryTable S1). IL6-expressing grafts largely exhibited high-gradePIN lesions and locally invasive growth, with a few poorlydifferentiated lesions present. OSM-expressing grafts oftenpresented as locally invasive lesions with several regions ofpoorly differentiated adenocarcinoma with anaplastic fea-tures, indicating that OSM could promote a more aggressivedisease (Supplementary Fig. S1C). Grafts displayed immu-nohistochemical features of human prostate cancer includ-ing nuclear AR expression and loss of P63-expressing basalcells in high-grade lesions (Fig. 1D and Supplementary Fig.S1D). Phenotypically similar results were achieved usingautocrine expression of OSM in donor prostate tissue withactivated AKT, although grafts with IL6 were significantlyinhibited and transformation was not observed (Supplemen-tary Fig. S2). We used immunofluorescent confocal micros-copy to qualitatively assess epithelial invasion, using E-

Smith et al.

Mol Cancer Res; 11(10) October 2013 Molecular Cancer Research1160




cadherin to identify epithelial cells with respect to thebasement membrane component collagen IV. Control andPTENLOF grafts exhibited normal E-cadherin localization tothe nuclear membrane, whereas IL6 and OSM grafts exhib-ited disorganized E-cadherin expression with epithelial cellsinvading into the surroundingmesenchyme (SupplementaryFig. S3).For human experiments, expression of constitutively

active AKT was used as a surrogate for PTENLOF. Benignhuman epithelium was isolated from radical prostatectomysamples and transduced with either RFP or AKT-RFPlentivirus. Transduced cells were then combined withUGSM transduced with GFP, IL6-, or OSM-GFP lentivi-rus, suspended inMatrigel, and injected subcutaneously intoimmunodeficient hosts (Fig. 2A). Control grafts exhibitednormal epithelial regeneration with nuclear AR expression inluminal epithelium and P63 expression in basal cells (Fig. 2Band C and Supplementary Fig. S4A). Expression of activatedAKT resulted in PIN lesions similar to those observed inmouse PTEN-knockout regenerations (Fig. 2B). Paracrineexpression of either IL6 or OSM alone dramatically inhib-ited epithelial regeneration with only a few small nests ofepithelial cells primarily composed of P63-expressing basal

cells (Supplementary Fig. S4B). Similarly, paracrine expres-sion of IL6 in grafts with AKT-infected epithelium alsoexhibited significant inhibition and no epithelial regenera-tion or transformation was observed (Fig. 2B and C). Graftsexpressing OSM with AKT exhibited small nests of dysplas-tic epithelial cells that express AR, although they retainedP63-expressing cells, indicating the lesions had not pro-gressed to clinical adenocarcinoma (Fig. 2B and C). Acti-vation and overexpression of AKT was confirmed by immu-nohistochemical analysis for phospho-serine 473 AKT levelsand total protein (Fig. 2C and Supplementary Fig. S4C).The quantification of observed phenotypes is reported inSupplementary Table S2.

Invasive tumor foci identified in grafts expressing IL6 orOSMwith loss of PTEN exhibited increased activation ofJAK/STAT pathwayLigand engagement of IL6 family members activates

constitutively Janus-activated kinases (JAK), resulting inactivation of the signal transduction and transcription factor(STAT), mitogen-activated protein kinases (MAPK), andAKT pathways (6). To interrogate whether IL6 and OSMexhibit differential activation of downstream pathways, we

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Figure 1. Paracrine expression of IL6 or OSM synergizes with epithelial loss of PTEN to promote invasive adenocarcinoma. A, diagram of regeneration andtransformation process with lentiviral vector diagrams. B, i–iii, Western blot analysis of UGSM cells infected with control, IL6, and OSM vectorsshowing heightened expression of IL6 andOSM in their respective cell lines, with amild increase in IL6 expression in OSM-infectedUGSM. Loading control isERK1/2. iv and v, secretion and activity of the IL6 and OSM cytokines was confirmed by treating serum-starved 3T3 cells with conditioned media frominfected UGSM. Increased phosphorylation of STAT3 in cells treated with IL6 and OSM conditioned media indicates functional activity. Imagedusing Licor Odyssey CLx with Image Studio software. C, representative histologic sections of prostate regeneration and transformation following 6 to8weeks in vivo. Control grafts exhibit normal prostate epithelial architecturewith PTEN-intact grafts expressing either IL6 or OSMexhibited reduced epithelialregeneration, whereas IL6- and OSM-expressing grafts exhibited occasional mild, focal hyperplasia. Grafts with loss of PTEN alone exhibited PIN lesionsformation with characteristic neoplastic growth into the luminal interior, whereas tumor foci from PTENLOF with IL6 or OSM exhibited invasion into thesurrounding mesenchyme. Scale bars: 10�, 200 mm; 40�, 100 mm. D, immunohistochemical (IHC) analysis of AR and p63 in prostate regenerations.All prostate epithelial regenerations and tumor foci retained high expression of nuclear AR. Normal regenerations exhibited P63-expressing basal cells alongthe basement membrane and were detached from the membrane in PIN lesions present in PTENLOF grafts and PTENLOF grafts with IL6. High-grade lesionspresent in PTENLOF with OSM exhibited loss of P63-expressing basal cells. Scale bars: 20�, 100 mm; 63�, 50 mm.

Invasive Carcinoma Induced by Loss of PTEN with IL6 or OSM

www.aacrjournals.org Mol Cancer Res; 11(10) October 2013 1161




treated benign PEB and tumorigenic CaP8 cell lines withhuman recombinant IL6 and OSM and assessed activationof downstream pathways by western blot analysis (Fig. 3A).Treatment with IL6 or OSM exhibited increased STAT3and AKT activation in both PEB and CaP8 cell linescompared with controls, with OSM exhibiting a greaterincrease over IL6 for both signaling pathways. Neither IL6nor OSM exhibited a dramatic increase in extracellularsignal-regulated kinase 1/2 (ERK1/2) activation over carri-er-treated cells.We used immunohistochemical analysis to interrogate

activation of pathways downstream of IL6 and OSM fromin vivo transformations using mouse tissues (Fig. 3B).Control regenerations exhibited little-to-no activation ofAKT, extracellular signal–regulated kinase ERK1/2, orSTAT3. Increased AKT activation was observed in allPTENLOF lesions and were similar across PTENLOF aloneand with IL6 or OSM. PTENLOF resulted in increasedERK1/2 activation over control grafts with similar levelsobserved in grafts from PTENLOF with IL6, whereasPTENLOF grafts with OSM exhibited mild, although con-sistently increased ERK1/2 activation. PTENLOF resulted in

increased STAT3 activation compared with control graftswith further increased levels observed in grafts from bothPTENLOF with IL6 or OSM. Immunohistochemical anal-ysis for total proteins confirmed basal expression levels incontrol grafts with all PTENLOF grafts exhibiting increasedexpression of total AKT, ERK1/2, and STAT3 protein(Supplementary Fig. S5). These results indicate that tumorprogression via IL6 and OSM is associated with increasedactivation of the STAT pathway, although OSM synergycould be also be mediated, in part, by MAPK.

DiscussionCorrelative and in vitro data strongly indicate that chronic

inflammation could act as an etiologic and progressionfactor, highlighting the need for further validation using invivo models. Our study identified that both IL6 and OSMexhibited synergy with PTENLOF and display heterogeneoustransformation phenotypes ranging from high-grade PINand locally invasive lesions to poorly differentiated adeno-carcinoma with anaplastic features. We speculate that thiscomplexity is a result of variations in the local concentration

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Figure 2. Cell-autonomous expression of AKTwith increased paracrine expression of OSM results in tumor progression of benign human prostate epithelium.A, diagram of human prostate regeneration with lentiviral constructs. B, representative histology of human prostate epithelial regenerations following8 to10weeks in vivo. Benign regenerations shownormal glandular epitheliumwhereas those transducedwithAKTshowPIN lesion formation.No regenerationor transformation was observed in grafts expressing activated AKT with IL6. Grafts with activated AKT and OSM exhibited dramatic morphologicprogression. Insets show expression of linked RFPmarker to track viral transduction. Scale bars: 20�, 100 mm; inset: 20�, 100 mm. C, immunohistochemical(IHC) analysis of AR and p63 from human prostate regenerations. All grafts and tumor foci retain nuclear expression of AR. Normal regenerationsexhibited P63-expressing basal cells along the basement membrane whereas PIN lesions induced by AKT alone exhibited P63-expressing basal cellsdetached from the basement membrane similar to murine PIN lesions. Tumor foci from AKT with OSM retain P63 expressing basal cells despite invasivemorphological characteristics. Scale bars: 20�, 100 mm; 63�, 50 mm.

Smith et al.





of IL6 or OSM and could serve as a model for the hetero-geneity commonly observed in human prostate cancer.While our work focused on the interaction of IL6 and

OSM in the context of PTENLOF, other studies indicate that

this synergy could extend to other oncogenic insults. Exog-enous expression of IL6 has been shown to transform anontumorigenic prostate epithelial cell line immortalizedwith SV40 large T-antigen, indicating potential synergy with

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Figure 3. Increased expression ofOSM in PTENLOF grafts results inincreased phosphorylation ofSTAT3 and ERK1/2 downstream ofIL6 and OSM. A, Western blotanalysis of PEB and CaP8 cellstreated with carrier or 10 ng/mL ofIL6 or OSM in DMEM with 0.2%FBS for indicated times. Treatmentwith carrier resulted in transientactivation of ERK1/2 in both celllines with a slight increase in AKTand STAT3 activation. Treatmentwith either IL6 or OSM exhibitedactivation of both AKT and STAT3pathways with OSM consistentlyexhibiting increased activationover IL6. Activation of ERK1/2above background was notconsistently observed. B,immunohistochemical (IHC)analysis of AKT, ERK1/2, andSTAT3 activation from normal,PTENLOF, and PTENLOF with IL6or OSM. All grafts with PTENLOF

exhibited increased activation ofAKT, ERK1/2, and STAT3 abovebasal levels in normalregenerations. PTENLOF grafts withIL6 exhibited increased levels ofSTAT3 phosphorylation overPTENLOF alone with no discernibleincrease in ERK1/2 activation.PTENLOF with OSM exhibitedconsistently higher levels of STAT3phosphorylation with a mildincrease in ERK1/2phosphorylation. Scale bars: 20�,100 mm; 63�, 50 mm.






inhibition of the TP53 and RB pathways (15).Work by Kanand colleagues identified that while OSM suppressed thegrowth of normal human breast epithelium, co-expression ofcMYC abrogated growth arrest and promoted transforma-tion (16).Our results support this finding as neither cytokinewas sufficient to transform mouse or human prostate epi-thelium on its own in our system.Oncogenic response to IL6and related cytokines therefore seems to be dependent on thepresence coincident mutations within the prostate.Expression of either IL6 or OSM alone strongly inhibited

epithelial regeneration in both mouse and human tissues,whereas studies using prostate cancer cell lines have shownparadoxical effects of either growth promotion or inhibitiondepending on the cell line. Growth inhibition by IL6-typecytokines in tumor cell lines occurs through a variety ofmechanisms including activation of the P27/KIP1 check-point (17). A study from Duijn and Trapman indicated thatthe PI3K/AKT pathway modulated expression of SKP2,whichmediated the downregulation of P27/KIP1 inDU145and PC3 but not LNCaP cells (18). Intriguingly, thiscorresponds with published data concerning prostate cancercell line response to IL6 (9). Activation of the PI3K/AKTpathway by IL6 or OSM in DU145 and PC3 could inhibitthe P27/KIP1 checkpoint and promote cell growth, whereasLNCaP cells do not exhibit this same behavior and thusrespond by growth inhibition and differentiation. PTENLOF

has been associated with increased levels of SKP2 anddecreased levels of P27/KIP1 in human prostate cancerbiopsy specimens, indicating that this checkpoint might bebypassed in the context of chronic inflammation withPTENLOF, allowing for increased tumor growth (19).Sequencing studies indicate that prostate cancer does not

exhibit dramatic mutation rates compared to many othercancers (20). Studies from our laboratory and others haveshown that most single oncogenes are not sufficient toinduce invasive prostate cancer, indicating that it is necessaryto activate multiple signaling pathways (14). Both IL6 andOSM have been shown to activate STAT3, AKT, andMAPK in several cell types, whereas our in vivo studiessuggest strong activation of STAT3 with a mild increase inERK1/2 (6). These pathways have been implicated in nearlyall cancers to some degree and several exhibit functionalsynergy when co-activated.We propose that chronic inflam-mation could expose tumor cells to a diverse array ofcytokines that could activate multiple oncogenic pathwaysand serve as a surrogate for direct mutation.

Current therapeutic research strategies are increasinglyusing highly targeted drugs such as antibody-based thera-peutics designed to inhibit the IL6 ligand or the IL6Rasubunit. The humanized antibody CNTO328 inhibits theconversion to androgen-independent disease and modulatesactivation of downstream pathways in prostate cancer xeno-grafts, although it has shown poor performance in clinicaltrials (11, 12).Our study indicates that othermembers of theIL6 family, and likely other cytokines, can also exhibit pro-tumorigenic functions that can even exceed those of IL6.Highly targeted therapies directed at single ligands such asIL6 could exhibit reduced efficacy due to coexpression offamily members with redundant activity. Therapies target-ing signaling nodes such as the JAK family could thereforeexhibit greater efficacy through inhibition of both relatedfamily members and shared downstream pathways such asSTAT3 and ERK1/2. Therapeutic strategies such as thiswould benefit greatly from an increased understanding ofhow cytokine expression profiles can be used as diagnosticbiomarkers.

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

Authors' ContributionsConception and design: D.A. Smith, A. Kiba, Y. Zong, O.N. WitteDevelopment of methodology: D.A. Smith, A. Kiba, Y. Zong, O.N. WitteAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): D.A. Smith, A. Kiba, Y. ZongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): D.A. Smith, A. Kiba, Y. Zong, O.N. WitteWriting, review and/or revision of the manuscript: D.A. Smith, A. Kiba, Y. Zong,O.N. WitteStudy supervision: O.N. Witte

AcknowledgmentsThe authors thank all of the members of the Witte laboratory for their helpful

comments and insightful discussion and the technicians of the UCLA TranslationalPathology Core Laboratory for their assistance in tissue processing and H&E staining.They also thank their FACS specialist Dr. Donghui Cheng andDr. Jioati Huang of theUCLA Pathology Department.

Grant SupportD.A. Smith is supported by UCLA Tumor Biology Program, US Department of

Health and Human Services, Ruth L. Kirschstein Institutional National ResearchService Award no. T32 CA009056 and is a Collegium of University Teaching Fellowwith the University of California, Los Angeles. A. Kiba was a visiting Fellow onsabbatical and is a scientist withTakeda Pharmaceutical Company Limited, Kanagawa,Japan.O.N.Witte is supported by a Prostate Cancer FoundationChallenge Award (PI:Owen Witte). Y. Zong is an associate of and O.N. Witte is an investigator for theHoward Hughes Medical Institute.

Received May 8, 2013; revised July 8, 2013; accepted July 10, 2013;published OnlineFirst July 18, 2013.

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2013;11:1159-1165. Published OnlineFirst July 18, 2013.Mol Cancer Res Daniel A. Smith, Atsushi Kiba, Yang Zong, et al. Human TissuesPathway to Promote Aggressive Prostate Malignancy in Mouse and Interleukin-6 and Oncostatin-M Synergize with the PI3K/AKT

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