An Immune-Inﬂammation Gene Expression ... - Cancer …associated prostate cancer progression,...

Molecular and Cellular Pathobiology

An Immune-Inflammation Gene ExpressionSignature in Prostate Tumors of SmokersRobyn L. Prueitt1, Tiffany A.Wallace1, Sharon A. Glynn1, Ming Yi2,Wei Tang1, Jun Luo3,Tiffany H. Dorsey1, Katherine E. Stagliano4, John W. Gillespie5, Robert S. Hudson1,Atsushi Terunuma1, Jennifer L. Shoe6, Diana C. Haines7, Harris G. Yfantis8, Misop Han3,Damali N. Martin1, Symone V. Jordan1, James F. Borin9, Michael J. Naslund9,Richard B. Alexander9, Robert M. Stephens2, Christopher A. Loffredo10, Dong H. Lee8,Nagireddy Putluri11, Arun Sreekumar11, Arthur A. Hurwitz4, and Stefan Ambs1

Abstract

Smokers develop metastatic prostate cancer more frequentlythan nonsmokers, suggesting that a tobacco-derived factor isdriving metastatic progression. To identify smoking-inducedalterations in human prostate cancer, we analyzed gene andprotein expression patterns in tumors collected from current, past,and never smokers. By this route, we elucidated a distinct patternof molecular alterations characterized by an immune and inflam-mation signature in tumors from current smokers that were eitherattenuated or absent in past and never smokers. Specifically,this signature included elevated immunoglobulin expression bytumor-infiltrating B cells, NF-kB activation, and increased chemo-

kine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects ofnicotine in humanprostate cancer cells and prostate cancer–proneTRAMPmice. These investigations showed that nicotine increasedglutamine consumption and invasiveness of cancer cells in vitroand accelerated metastatic progression in tumor-bearing TRAMPmice. Overall, our findings suggest that nicotine is sufficient toinduce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel can-didate driver underlying metastatic prostate cancer in currentsmokers. Cancer Res; 76(5); 1055–65. �2015 AACR.

IntroductionProstate cancer is a leading cause of cancer mortality among

men (1). Few environmental factors have been consistently asso-

ciated with prostate cancer (2). Although cigarette smoking maynot influence early disease development (3, 4), it increases the riskof fatal prostate cancer (3, 5, 6). This observationwas replicated instudies showing that current smokers develop distant metastasismore frequently than nonsmokers (7, 8). Because smoking ces-sation reduces the risk ofmetastasis (3, 6), a tobacco-related factorappears to induce reversible molecular alterations in prostatecancer that facilitate metastatic spread.

Here, we pursued a 2-fold hypothesis. First, we exploredwhether prostate tumors from current smokers have a geneexpression profile that differentiates them from tumors of neveror past smokers. Second, because nicotine can activate oncogenicsignaling pathways that promote cancer progression (9, 10), wealso explored its effects in human prostate cancer cell lines andprostate cancer–prone TRAMP mice to evaluate whether theyresemble the epidemiology of smoking-associated prostate cancerprogression. Using these approaches, we identified a distinctimmune and inflammation signature in prostate tumors of cur-rent smokers. We also found that physiologic concentrations ofnicotine induce Akt pathway activation and metabolic changes,and increase invasiveness of human prostate cancer cells. Finally,nicotine accelerated the onset of metastasis in TRAMP mice.

Materials and MethodsTissue collection and cell lines

Sixty-seven fresh-frozen prostate tumors were obtained fromthe NCI Cooperative Prostate Cancer Tissue Resource (CPCTR;n ¼ 37), Department of Pathology, University of Maryland(UMD; n ¼ 10), and Department of Urology, Johns HopkinsMedical Institutions (JHU; n¼ 20). Additional 69 formalin-fixed,

1Laboratory of Human Carcinogenesis, Center for Cancer Research(CCR), NCI, NIH, Bethesda, Maryland. 2Advanced Biomedical Com-puting Center, Leidos Biomedical Research/NCI, Frederick, Maryland.3Department of Urology, Johns Hopkins Medical Institutions, Balti-more, Maryland. 4Laboratory of Molecular Immunoregulation, CCR,NCI, NIH, Frederick, Maryland. 5Laboratory of Pathology and UrologicOncology Branch, CCR, NCI, NIH, Bethesda, Maryland. 6LaboratoryAnimal Sciences Program, Leidos Biomedical Research, FrederickNational Laboratory, Frederick, Maryland. 7Pathology/Histotechnol-ogy Laboratory, Leidos Biomedical Research, Frederick National Lab-oratory, Frederick, Maryland. 8Pathology and Laboratory Medicine,BaltimoreVeteransAffairsMedical Center, Baltimore,Maryland. 9Urol-ogy and Greenebaum Cancer Center, University of Maryland, Mary-land. 10Cancer Prevention and Control Program, Lombardi Compre-hensive Cancer Center, Georgetown University Medical Center,Washington, DC. 11Department of Molecular and Cell Biology, Vernaand Marrs McLean Department of Biochemistry, Alkek Center forMolecular Discovery, Baylor College of Medicine, Houston, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

R.L. Prueitt, T.A. Wallace, and S.A. Glynn contributed equally to the article.

Current address for S.A. Glynn: Sharon Glynn, Lambe Institute/TranslationalResearch Facility, School of Medicine, NUI Galway, Galway, Ireland.

Corresponding Author: Stefan Ambs, Laboratory of Human Carcinogenesis,NCI, Bldg. 37/Room 3050B, MSC Convent Drive 4258, Bethesda, MD 20892.Phone: 301-496-4668; Fax: 301-496-0497; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3630

�2015 American Association for Cancer Research.

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paraffin-embedded (FFPE) tumor specimens were collected atUMD. Tissue collection was approved by the institutional reviewboards at the participating institutions. Written informed consentwas obtained from all donors. CPCTR has been described previ-ously (11). Smoking information at the time of surgery (current,past, never) was obtained from medical records and cancerregistry entries for CPCTR and JHU. For patients from UMD, thisinformation was abstracted from an epidemiologic question-naire. The human immortalized prostate epithelial cell line,RWPE-1, and human prostate cancer cell lines (22Rv1, PC-3,LNCaP, DU145) were obtained from the ATCC between 2006and 2010. Authentication of these cell lines was performed inDecember, 2013, using a short tandem repeat analysis withGenePrint10 (9 loci þ amelogenin for sex determination). Fordetails on tissue collection and assessment of smoking status, seeSupplementary Methods.

RNA extraction from frozen bulk tissue and cell linesTissue macrodissection and isolation of total RNA from tissues

and cell lines was performed according to standard methodsdescribed in Supplementary Methods.

Affymetrix microarraysRNA labeling and hybridization were performed according to

Affymetrix standard protocols, as described previously (12).Labeled cRNA was hybridized either to Affymetrix GeneChipHG-U133A 2.0 or mouse 1.0 ST arrays. In accordance withMinimum Information About a Microarray Experiment guide-lines, we deposited the CEL files for the microarray data andadditional patient information into the Gene Expression Omni-bus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/). TheGEO submission accession number for the 47 bulk tissue tumors,which were initially analyzed, is GSE6956. GSE68138 containsthe gene expression data for the additional 20 bulk tissue tumors(JHU samples) and the laser capture microdissected tumor sam-ples (n ¼ 10), and for prostate tumors from TRAMP mice �nicotine treatment (n ¼ 10), and cell lines (22Rv1 and LNCaPcells) � nicotine treatment (n ¼ 12). For more information, seeSupplementary Methods.

RNA isolation from microdissected prostate tumorsEnriched tumor epithelium was obtained from 5 current and 5

never smokerswith laser capturemicrodissection (LCM)of frozentissue samples. These tumors were also analyzed as bulk tissues. Atotal of 5,000 to 15,000 cells per tumor were collected. RNA wasisolated using the PicoPure protocol (Arcturus). mRNA wasamplified with two linear amplification steps by in vitro transcrip-tion using the MEGAscript T7 Kit (Ambion) followed by labelingusing the BioArrayHighYield RNATranscript LabelingKit T3 fromEnzo Life Sciences. Labeled cRNA was hybridized onto arrays.

Data normalization and statistical analysis of geneexpression data

All chips were normalized using the Robust Multi-arrayAverage procedure (13). Because two sets of array data wereanalyzed for human prostate tumors, we controlled for abatch effect using the Partek Genomics Suite (www.partek.com) or the Bioconductor limma R package (www.biocon-ductor.org). To generate lists of differently expressed genes,the resulting datasets were subjected to the significance anal-

ysis of microarray procedure (14) or linear modeling featuresimplemented in limma. Supplementary Tables S7–S11describe differentially expressed genes in LCM tumor epithe-lium comparing current (n ¼ 5) versus never smokers (n ¼ 5;S7-S8), nicotine-treated (n ¼ 3) versus untreated (n ¼ 3)22Rv1 and LNCaP cells (S9-S10), and prostate tumors fromnicotine-treated (n ¼ 5) versus untreated (n ¼ 5) TRAMP mice(S11), respectively. For more information, see SupplementaryMethods.

Gene set enrichment analysisGene set enrichment analysis (GSEA) was performed as

described previously (15). For details, see SupplementaryMethods.

Additional methodsFormore informationonmethodsnot described (qPCRof gene

expression; in situ hybridization for immunoglobulin k and llight chain expression in prostate tumors and IHC; proliferation,motility, and invasion assays of nicotine-treated cells; integrin cellsurface expression and extracellular matrix protein-bindingassays; Western blot analysis of nicotine-treated cells; and mea-surement of IL8 in human plasma samples), see SupplementaryMethods.

Glutamine consumption in nicotine-treated prostate cancercells

22Rv1 and LNCaP cells were plated in T150 flasks, serumstarved, and treated with 100 nmol/L nicotine. One milliliter ofmedia was collected and cell pellets were prepared. Dried extractsof these samples were resuspended in injection solvent composedofwater:methanol (50:50) and subjected to LC/MS.Details canbefound in Supplementary Methods.

Nicotine treatment of prostate cancer–prone TRAMPmice and evaluation of lung metastasis

Male TRAMP mice were bred at the Assisted ReproductionLaboratory, Frederick National Laboratory for Cancer Research(Frederick, MD), using in vitro fertilization (B6�FVB F1). At 8 to 9weeks of age, they received either tap water or a solution of either100 or 250 mg/mL of nicotine in tap water, which is similar to aprevious described protocol (16). The three groups consisted of20 to 25 animals each. At the selected concentration, nicotinegenerates nicotine plasma concentrations comparable with thoseof active smokers and causes some weight loss (SupplementaryFig. S1). All mice were euthanized after 80 days or when theybecamemoribund because of prostate cancer. To assess the effectsof nicotine on prostate cancer development and metastasis, theprostate glands and lungs were collected and were formalin-fixedfor histologic examination by a boarded veterinary pathologist.All described animal procedures were reviewed and approved bythe NCI-Frederick Institutional Biosafety Committee (IBC regis-tration #06-060 and 11-041). NCI-Frederick is accredited byAAALAC International and follows the Public Health ServicePolicy for the Care and Use of Laboratory Animals. More detailscan be found in Supplementary Methods.

Statistical analysisStatistical analyseswere performedusing STATA (StataCorp) or

GraphPad Prism 6 (GraphPad Software). All statistical tests were

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two sided and an association was considered statistically signif-icant with P < 0.05. The Spearman rank correlation (e.g., forcontinuous B-cell numbers in never, past, current smokers) or theFisher exact tests (e.g., for nuclear p-NF-kB stratified into presentor absent in never, past, current smokers) were used to calculatePtrend. The Wilcoxon rank-sum test was used as a nonparametricstatistical test to compare two independent groups. Comparisonsamong more than two independent groups were performed withthe ANOVA and Kruskal–Wallace tests.

ResultsA smoking-associated gene expression signature in prostatetumors

We evaluated gene expression characteristics from tumorscomparing current with past and never smokers. Patients aredescribed in Supplementary Table S1: current, past and neversmokers did not differ significantly by age, race/ethnicity, orclinicopathology. Initially, we analyzed the gene expression pro-files of 47 tumors from 9 current, 21 past, and 17 never smokersusing Affymetrix GeneChipmicroarrays. This analysis revealed animmune signature in tumors from current smokers. The mostupregulated transcripts among current smokers representedimmunoglobulins (Supplementary Table S2). When we per-formed a hierarchical cluster analysis, immunoglobulin expres-sion separated tumors into two clusters (Fig. 1). Tumors fromcurrent smokers were significantly overrepresented in cluster 2,which consisted of tumors with upregulated immunoglobulinexpression. Furthermore, we applied a linear regression model toexamine whether the differences in immunoglobulin expressionby smoking status are confounded by race/ethnicity and foundthat these differences were independent of race/ethnicity.

To further investigate the immunoglobulin signature, weconducted in situ hybridization (ISH) for signature validation,and to localize expression. ISH for both k and l light chainmRNA expression was performed on additional 22 FFPEtumors (6 current, 7 past, 9 never smokers). This approachrevealed an elevated number of immunoglobulin-expressing Blymphocytes in tumors of current smokers compared with pastand never smokers (Fig. 2). The lymphocytes infiltrated thetumor stroma (Fig. 2A and B and Supplementary Fig. S2).Average number of l light chain–positive B lymphocytes per250� field increased from 3.6 (range: 0–24) among neversmokers to 5 (range: 0–16) among past smokers to 23 (range:1–86) among current smokers (Spearman rank correlation, r ¼0.51; P ¼ 0.02; Fig. 2C and D).

Because our initial analysis described only few genes otherthan immunoglobulins as being altered in tumors from currentsmokers, gene expression profiles from additional patients(JHU dataset: 7 current, 7 past, 6 never smokers) were com-bined with the discovery dataset to allow identification of moregenes that are differently expressed between current and never/past smokers. For the combined dataset (67 tumors), wegenerated two gene lists using Significance Analysis of Micro-arrays: one for differently expressed genes between current (n ¼16) and never smokers (n ¼ 23), and one for differentlyexpressed genes between current (n ¼ 16) and never/pastsmokers (n ¼ 51). The comparison of tumors from currentand never smokers yielded 98 transcripts that represented 73differentially expressed genes at a false-discovery rate �30%(Supplementary Table S3). The second comparison, current

versus never/past smokers, yielded 70 transcripts representing40 differently expressed genes (Supplementary Table S4). Nota-bly, many of the differentially expressed genes in the two listshave known immune-regulatory functions, and their expres-sion was increased in current smokers [e.g., immunoglobulins,indolamine-2,3-dioxygenase (IDO1), and chemokines]. Quan-titative (q)RT-PCR analysis confirmed overexpression of immu-noglobulins (IGH, IGK, IGL), IDO1, and several chemokines(CCL5, CXCL10, CXCL11) among current smokers in an anal-ysis of 57 tumors (15 current, 18 never, 24 past smokers) fromthe microarray study (Supplementary Fig. S3) whereas CXCL8(IL8) did not validate. Expression of these immune genestended to be low or absent in tumors from never or pastsmokers. Lastly, we generated additional gene lists for classi-fication using Bioconductor limma R (Supplementary Fig. S4).Differentially expressed genes were then assessed using thelinear modeling features implemented in limma. P values <0.05 were used to generate two gene lists for current versusnever and current versus never/smokers. Probesets with samedifferential expression in both gene lists (n ¼ 601, Supplemen-tary Table S5) were selected for classification. As shown in Fig.3, the gene expression pattern defined by these probesetsseparated the 67 tumors into two clusters with greatly differentgene expression. Upregulation of genes in immune-relatedpathways was the main characteristic of tumors from currentsmokers that differentiated them from others.

Nuclear accumulation of NF-kB in tumors and increased IL8 inblood of current smokers

Next, we tested whether increased stress signaling throughNF-kB may occur in tumors from smokers because B-cellactivation has been linked to NF-kB signaling (17). We usedIHC to determine nuclear localization of phosphorylated NF-kB, p65 subunit (Ser536), in the tumor epithelium as describedpreviously (18), to assess NF-kB activation (Fig. 4A and B).Analyzing 69 tumors, we found nuclear NF-kB pSer536 in 5/26tumors from never smokers (19%), 12/24 tumors from pastsmokers (50%), and 11/19 tumors from current smokers (58%;Ptrend ¼ 0.014; Fisher exact test). Thus, accumulation of phos-phorylated NF-kB correlated with smoking status. Because ourmicroarray analysis initially indicated that IL8 is upregulated intumors from current smokers, we also examined plasma levelsof IL8 in 97 prostate cancer patients and 89 controls to assesswhether circulating IL8 is increased in patients who are currentsmokers. The analysis showed that IL8 levels were significantlyelevated in plasma from current smokers with prostate cancer(Fig. 4C), but not among current smokers without the disease(Fig. 4D).

GSEAWe used GSEA to identify common features between the

smoking-related gene signature in prostate tumors, nicotine-induced gene signatures in human prostate cancer cells, andarchived signatures in the Molecular Signatures Database(MSIGDB) (http://www.broad.mit.edu/gsea/msigdb; ref. 15).Weaimed to identify candidate mechanisms by which smoking mayinduce gene expression alterations in the cancerous prostate, andto better define possible roles of nicotine.

Four signatures were subjected to GSEA. The first containedgenes that were differentially expressed between current andnever smokers in bulk tumors. The second signature was derived

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from the same contrast, but used microdissected tumor epithe-lium from 5 current and 5 never smokers as the source for themRNA. The last two signatures were generated from LNCaP and22Rv1 prostate cancer cells treated with 100 nmol/L nicotine(vs. untreated), which is within the physiologic concentrationrange for nicotine in current smokers (�10–500 nmol/L blood;

refs. 19, 20). Results are summarized in Supplementary Fig. S5.In short, a hepatocyte growth factor (HGF)-induced gene sig-nature in monocytes (21) and a glutamine starvation signature(22) were the two MSIGDB-archived gene signatures with sig-nificant associations among all four gene lists. The data suggestthat nicotine may influence cell metabolism, leading to

Figure 1.Immunoglobulin expression in prostate tumors from current, past, and never smokers. A, hierarchical cluster analysis separates 47 prostate tumors (9 current,21 past, and 17 never smokers) into two clusters based on immunoglobulin expression. Heatmap shows upregulated immunoglobulin expression in cluster 2 (red colorintensity). Current smokers are overrepresented in this cluster (8/21 vs. 1/26 in cluster 1, P < 0.01) whereas the "low immunoglobulin" cluster 1 was enrichedfor tumors from never smokers (13/26 vs. 4/21 in cluster 2, P ¼ 0.03). Patients' smoking status is shown for each tumor above the heatmap. Shown arearray-based expression data with corresponding probeset IDs. B–D, increased expression of immunoglobulin (Ig) heavy constant m, k constant, and l locus intumors from current smokers by qRT-PCR analysis. � , different at P < 0.05 between current smokers (n ¼ 8) and past smokers (n ¼ 10) or never (n ¼ 12).qRT-PCR was performed on a subset of tumors from the microarray analysis.

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increased glutamine consumption, and also exerts functionsthat may mimic HGF in human prostate tumors. Glutaminedeprivation can occur when glutamine is excessively metabo-lized, and it has been shown that glutamine deprivation leads toNF-kB activation (23). To validate this GSEA prediction, weexamined glutamine consumption of nicotine-treated prostatecancer cells. As shown in Fig. 5A and B, nicotine increasedglutamine consumption in these cells, resulting in glutaminedeprivation in culture medium.

Nicotine activates the Akt pathway and induces a prometastaticphenotype

Nicotine may have prometastatic properties in prostate can-cer patients. We tested this hypothesis but first examinedwhether nicotinic acetylcholine receptors (nAChR) areexpressed in the cancerous prostate. qRT-PCR showed thatvarious nAChR subunits are expressed in prostate tumors andcancer cell lines (Supplementary Fig. S6). Notably, the nAChRa7 subunit, which has been linked to PI3K-Akt pathway acti-vation and other oncogenic effects (24), was significantlyupregulated in tumors. Next, we investigated whether nicotineactivates oncogenic Akt signaling in human prostate cancer celllines and in RWPE-1 immortalized prostate epithelial cells.Treatment of 22Rv1 prostate cancer cells with 10 nmol/L and100 nmol/L nicotine led to phosphorylation of Akt (the keyactivating step for Akt) and its downstream targets, for example,GSK3b and human MDM2 (Fig. 5C–E). Akt pathway activationwas also observed in other cell lines (Supplementary Fig. S7).Both mecamylamine, an inhibitor of nAChR signaling, and the

PI3 kinase inhibitor, LY294002, blocked nicotine-induced Aktphosphorylation (Fig. 5D and Supplementary Fig. S7B). Next,we assessed whether nicotine can induce a metastatic pheno-type in prostate cancer cells and evaluated migration andMatrigel invasion in response to nicotine. Nicotine enhancedMatrigel invasion of both 22Rv1 and PC-3 cells (Fig. 6A and B),which further increased when nicotine and HGF were addedtogether (Fig. 6C). However, only nicotine-induced, but notHGF-induced, invasion was inhibited by mecamylamine (Fig.6D). Nicotine also enhanced migration of 22Rv1 cells but didnot increase it in PC-3 cells (Supplementary Fig. S8). Ourfindings suggest that nicotine may selectively enhance invasiveproperties of prostate cancer cells. This hypothesis was furthersupported by the observation that nicotine affected cell surfaceintegrin expression and extracellular matrix binding, as nico-tine increased binding of 22Rv1 cells to the bone-associatedfilaments, collagen type I and IV (Supplementary Fig. S9),which is associated with metastasis-promoting integrin signal-ing (25, 26).

Nicotine accelerates the onset of metastasis in TRAMP miceOur experiments showed that nicotine induces metastasis-

related phenotypes in cell culture. For further corroboration, weevaluated the effect of nicotine on metastasis in TRAMP mice,which develop aggressive prostate tumors at 100% penetranceand start to develop visible pulmonary metastatic lesions at24 weeks of age (27). We treated animals with 100 or 250 mg ofnicotine/mL in the drinking water (n ¼ 20–25) and assessedprimary tumor growth and metastasis to the lung after 80 days

Figure 2.Immunoglobulin expression by ISH.A and B, shown is the expression of klight chain mRNA in a representativeprostate tumor from a current smokerin two different fields. ISH depicts klight chain mRNA expression inlymphocytic infiltrates. The dark bluenitroblue tetrazolium chromogenereveals the predominant stromallocalization of the immunoglobulin-positive B lymphocytes. Counterstain,hematoxylin. C and D, analysis ofISH results. Numbers of Ig l- andIgk-positive B lymphocytes correlatedwith smoking status [Spearman rankcorrelation test (Ptrend) for current(n ¼ 6), past (n ¼ 7), never (n ¼ 9)smokers] and were increased incurrent smokers versus never/pastsmokers (Wilcoxon rank-sum test).ISH-positive lymphocytes werecounted per 250� field. Shown arebox plots with minimum andmaximum values (whiskers) and themedian as line in the box.






of treatment. Our treatment regimens yielded an average 34nmol/L (5.5 � 5.2 ng nicotine/mL, n ¼ 6) or 105 nmol/L(17.1 � 9.0 ng/mL, n ¼ 6) plasma nicotine concentrations,respectively, whereas nicotine was undetectable in untreatedanimals (P < 0.01). Nicotine did not increase the size of the

cancerous prostate (Table 1), consistent with our observationthat 100 nmol/L nicotine either did not or only modestlyenhance proliferation of RWPE-1, 22Rv1, or PC-3 cells (Sup-plementary Figs. S10 and S11). In addition, we did not observesignificant histologic differences in the cancerous prostate

Figure 3.Upregulation of genes in immune-related pathways is a characteristic oftumors from current smokers.Hierarchical cluster analysis with 601probesets representing genes thatwere differentially expressed betweencurrent versus past and current versuspast/never smokers. The expressionpattern of these genes separates the67 tumors (16 current, 28 past, and 23never smokers) into two clusters.Cluster 1 represents tumors withupregulated expression of immuneresponse–related genes as shown bythe Gene Ontology (GO) term andKyoto Encyclopedia of Genes andGenomes (KEGG) pathwayassociation. This cluster is highlyenriched for tumors from currentsmokers (P < 0.001). Overexpressedgenes in cluster 2 did not have asignificant pathway or GO termassociation.

Figure 4.Nuclear accumulation of NF-kB and increasedIL8 among prostate cancer patients who smoke.A and B, nuclear accumulation of NF-kB P-Ser536 is shown for tumors from a past (B,�200) and current (D,�100) smoker (magnifiedin the insets). Counterstain, methyl green. C,cancer patients who are current smokers haveincreased IL8 plasma levels when compared withpast or never smokers.Median increase in currentsmokerswas 1.5-fold. D, current smokers withoutthe disease did not show elevated IL8 plasmalevels. P values were calculated with the Mann–Whitney test. A Kruskal–Wallis test fordifferences between the three smoking groupsindicated existing differences among cases (P ¼0.006) but not the controls (P ¼ 0.46).

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between the treatment groups. However, the nicotine treatmentproduced metastatic lesions in the lung that were not present incontrol animals (Table 1), indicating it accelerates the onset ofmetastasis in this model. A total of 13 out of 45 nicotine-treatedanimals (29%) were positive for lung metastasis, comparedwith none of the 20 controls (P ¼ 0.006, Fisher exact test). To

gain insights on the nicotine-induced molecular alterations, weanalyzed gene expression in the cancerous prostate of untreatedand 250 mg/mL nicotine-treated TRAMP mice (n ¼ 5 eachgroup). The analysis showed that nicotine-treated tumors haveincreased expression of genes regulating synaptic signaltransduction (Supplementary Table S6). An Ingenuity pathway

Figure 5.Increased glutamine consumption andAkt activation in nicotine-treatedhuman prostate cancer cells. 22Rv1 andLNCaP cells were treated with 100nmol/L nicotine for 48 hours andglutamine levels were measured inculture medium and cell pellets. A,treatment of 22Rv1 and LNCaP cellswith nicotine depleted glutamine in theculture medium. B, nicotine treatmentof 22Rv1 and LNCaP cells significantlyincreased glutamine and glutamatelevels in their cell extracts. Glutamate isthe intracellular oxidation product ofglutamine and was not detectable incell culturemedium. Experiments weredone in triplicates. Shown is mean �SD. � , statistically different fromuntreated (two-sided t test, P < 0.05).C and D, nicotine at 10 nmol/L and100 nmol/L concentrations inducedAkt phosphorylation in 22Rv1 cells (C),which was inhibited by mecamylamine(MC), an antagonist of nicotinicacetylcholine receptors (D). E, nicotinetreatment also inducedphosphorylation of knowndownstream targets of Akt signaling,such as GSK3b, human Mdm2 (HDM2),and BAD. In D, cells were exposedto nicotine �/þ mecamylamine for2 hours.






analysis also suggested an association of the differentiallyexpressed genes with G2–M DNA damage checkpoint regula-tion (P ¼ 1.1 � 10�6), mitotic roles of Polo-like kinases (P ¼6.6 � 10�6), and the complement system (P¼ 4.7� 10�5), andtheir strongest disease association was with cancer (P ¼ 3.4 �10�13). Lastly, we did not observe an immune signature with B-cell infiltration in these tumors.

DiscussionIn this study, we describe an immune and inflammation

signature in prostate tumors from current smokers. We furtherdiscovered that nicotine increases invasiveness of human prostatecancer cells and accelerates the onset of metastases in tumor-bearing TRAMP mice. These observations point to previously

unrecognizedmechanisms by which smokingmay enhance pros-tate cancer progression. While mechanistically novel, they are inagreement with epidemiologic studies and a recent publicationdescribing inflammation in prostate tumors of current smokers(28). Also, a systematic review of the relationship betweensmokeless tobacco and cancer revealed that prostate cancer isone of the few cancers associated with the use of smokelesstobacco (29), which is a key source of nicotine and nicotine-derived nitrosamines, which both activate nAChRs (9, 10, 30).

Our analysis of prostate tumors indicated an increased pres-ence of immunoglobulin-expressing B cells in tumors of currentsmokers whereas nicotine did not increase their numbers intumors of TRAMP mice. B-cell numbers are commonlyincreased in human prostate tumors, but this increase wasnot found to correlate with standard markers of disease

Figure 6.Nicotine enhances Matrigel invasion ofhuman prostate cancer cell lines. 22Rv1(A) and PC-3 (B) cells were plated ininvasion chambers and were treatedwith nicotine. C, 22Rv1 cells weretreated with nicotine, HGF, or both, toassess whether nicotine and HGF havea synergistic effect on Matrigelinvasion. D, nicotinic acetylcholinereceptor antagonist, mecamylamine(MCA), significantly decreasednicotine-induced increase in Matrigelinvasion, but not HGF-inducedincrease in invasion, in 22Rv1 cells.Shown are mean � SD for n ¼ 3.�, significantly different from control(P < 0.05).

Table 1. Lung metastasis in nicotine-treated TRAMP mice

Treatment Tap waterNicotine100 mg/mL

Nicotine250 mg/mL

# of animals 20 22 23Lung (number examined) 20 22 23Lung metastasis (0%) 6 (28%) 7 (31%)a

Adenocarcinoma with metastasis to the lung (0%) 1 (5%) 2 (9%)Neuroendocrine carcinoma with metastasis to the lung (0%) 5 (23%) 5 (22%)Lymph node (# examined) 3 7 3Lymph node metastasis (0%) 2 (29%)b 1 (33%)b

Urogenital tract weight (mg) without seminal vesiclesc

592 � 231 513 � 195 532 � 462aP ¼ 0.046; Fisher exact test for trend.bNeuroendocrine histology.cNot significantly different between groups in both the animal weight-adjusted and unadjusted analysis (ANOVA test).

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aggressiveness (31). Although their presence in primary tumorsmay not immediately have a progression-enhancing effect, theirincrease in current smokers could be deleterious at the transi-tion to a castration-resistant prostate cancer (CRPC). It wasshown that B cells accelerate this transition in a CRPC mousemodel while B-cell depletion delayed CRPC development (32).The critical role of tumor infiltrating B cells was attributed tolymphotoxin-b secretion enhancing inflammation in theanimal study. We examined lymphotoxin-b plasma levels inprostate cancer patients but could not detect elevated lympho-toxin-b in current smokers (Supplementary Fig. S12). Perhaps,increased lymphotoxin-b is restricted to the tumor microenvi-ronment. Alternatively, B cells may require stimuli for lympho-toxin-b release that specifically arise in the environment ofCRPC.

Several studies reported that B cells enhance cancer devel-opment. In a mouse model of skin carcinogenesis, cancerprogression driven by chronic inflammation was shown to beB cell–dependent (33). Here, deposition of circulating immunecomplexes into the tumor parenchyma led to the release ofproangiogenic and prometastatic molecules (34). Similarly, weobserved NF-kB activation and increased expression of chemo-kines in prostate tumors of current smokers with high B-cellcounts, indicative of a proinflammatory tumor microenviron-ment. One of the chemokines, CCL5, has been linked to diseaseprogression of multiple cancers including prostate cancer (35).Likewise, increased NF-kB signaling predicts prostate cancerprogression in prostate (32, 36). Hence, our finding thatprostate tumors of current smokers tend to have an immunesignature and NF-kB activation is consistent with theirincreased metastatic potential.

Nicotine has oncogenic properties that meet the criteria of aprometastatic factor (10). Becausewe could not directly examinethe effects of nicotine in cancer patients, we investigated them incell culture and TRAMP mice and observed that nicotineincreases glutamine consumption of cancer cells and enhancesinvasion and metastasis. Increased glutamine consumption is ahallmark of cancer and predicts poor survival in breast cancer(37). In TRAMP mice, primary tumors from nicotine-treatedanimals showed increased expression of genes regulating syn-aptic signal transduction, whereas nicotine-driven Akt pathwayactivation was prominent in cell culture. Akt signaling enhancesprostate cancer progression (38, 39). The cancer-promotingeffects of nicotine have also been evaluated in animal modelsof lung cancer. Although one study reported that nicotinepromotes tumor growth and metastasis (40), two other studiescould not find a nicotine effect on tumor growth (41, 42). Thus,the effect of nicotine on tumor growth is controversial andmodel-dependent. Moreover, metastasis-related rather thantumor growth–related phenotypes may develop in nicotine-treated animals. We believe that the use of TRAMP mice wasjustified because this model captures a neuroendocrine differ-entiation that is also observed in CRPC (43). Nicotine maypromote progression of cancers with neuroendocrine featuressuch as a subset of castration-resistant tumors and metastaticprostate cancers, or the aggressive small-cell lung cancer (44). Inaddition, autonomic nerve development has recently beenshown to contribute to prostate cancer progression (45). Note-worthy, in this context, is our finding that nicotine increasedexpression of genes in synaptic signal transduction, thus poten-tially increasing nerve development and signaling.

Finally, we found that circulating IL8 levels are increased incurrent smokers with prostate cancer. IL8 expression can beinduced by nicotine in human neutrophils (46), and its expres-sion correlates with metastasis in prostate cancer (47, 48).Although nicotine may induce IL8 directly, upregulated IL8 insmokers may also arise from other mechanisms, for example,activation of monocytes by various smoking-related xenobiotics.Nonetheless, the finding of increased IL8 in current smokers withprostate cancer reveals another candidate mechanism by whichtobacco use following a prostate cancer diagnosis could enhancemetastasis.

In summary, our study uncovered several mechanisms bywhich smoking may increase metastasis in prostate cancerpatients. However, our study has few limitations; for example,we used TRAMPmice to show that nicotine accelerates metastasisin vivo. These animals develop mainly neuroendocrine tumorsthat aredifferent from typical adenocarcinomas.Nevertheless, ourfindings point to the need of additional mechanistic and popu-lation-based studies, to define the relative contribution of nico-tine to prostate cancer metastasis in current smokers.

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

Authors' ContributionsConception and design: R.L. Prueitt, T.A. Wallace, N. Putluri, A.A. Hurwitz,S. AmbsDevelopment of methodology: R.L. Prueitt, T.A. Wallace, S.A. Glynn, M. Yi,K.E. Stagliano, R.S. Hudson, D.C. Haines, M.J. Naslund, N. PutluriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.A. Glynn, M. Yi, J. Luo, K.E. Stagliano, J.W. Gillespie,R.S. Hudson, J.L. Shoe, D.C. Haines, S.V. Jordan, J.F. Borin, M.J. Naslund,R.B. Alexander, C.A. Loffredo, N. Putluri, A. Sreekumar, A.A. HurwitzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.L. Prueitt, T.A. Wallace, S.A. Glynn, M. Yi, W. Tang,A. Terunuma, D.C. Haines, H.G. Yfantis, S.V. Jordan, M.J. Naslund,R.M. Stephens, C.A. Loffredo, N. PutluriWriting, review, and/or revision of the manuscript: R.L. Prueitt, T.A. Wallace,S.A. Glynn, M. Yi, J. Luo, K.E. Stagliano, J.W. Gillespie, A. Terunuma,D.C. Haines, M. Han, D.N. Martin, C.A. Loffredo, N. Putluri, A. Sreekumar,A.A. Hurwitz, S. AmbsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.L. Prueitt, S.A. Glynn, M. Yi, J. Luo,T.H. Dorsey, K.E. Stagliano, D.C. Haines, D.H. LeeStudy supervision: C.A. Loffredo, A.A. Hurwitz, S. Ambs

AcknowledgmentsThe authors thank CPCTR for providing tissue specimens and supporting

data and also thank personnel at the University of Maryland and the BaltimoreVeterans Administration Hospital for their contributions with the recruitmentof subjects.

Grant SupportThis research was supported by the Intramural Research Program of the NIH,

NCI, Center for Cancer Research, and was also funded with federal funds fromthe NCI under Contract No. HHSN261200800001E. In addition, grants to A.Sreekumar and N. Putluri supported this work (W81XWH-12-1-0130 fromDOD, DMS 1161759 from NSF, NIH U01 CA167234, CPRIT MetabolomicsCore-RP120092 Alkek CMD Grants).

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 December 10, 2014; revised October 13, 2015; accepted December7, 2015; published OnlineFirst December 30, 2015.






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2016;76:1055-1065. Published OnlineFirst December 30, 2015.Cancer Res Robyn L. Prueitt, Tiffany A. Wallace, Sharon A. Glynn, et al. Tumors of SmokersAn Immune-Inflammation Gene Expression Signature in Prostate

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