tobacco carcinogen induced production of gm-csf ......signature genes upregulated by nnk are listed...

Tumor Biology and Immunology

Tobacco Carcinogen–Induced Production ofGM-CSF Activates CREB to Promote PancreaticCancerSupriya Srinivasan1, Tulasigeri Totiger1, Chanjuan Shi2, Jason Castellanos3,Purushottam Lamichhane1, Austin R. Dosch1, Fanuel Messaggio1, Nilesh Kashikar4,Kumaraswamy Honnenahally3, Yuguang Ban5, Nipun B. Merchant1,6, Michael VanSaun1,6,and Nagaraj S. Nagathihalli1,6

Abstract

Although smoking is a significant risk factor for pancreaticductal adenocarcinoma (PDAC), the molecular mechanismsunderlying PDAC development and progression in smokers arestill unclear. Here, we show the role of cyclic AMP responseelement-binding protein (CREB) in the pathogenesis of smok-ing-induced PDAC. Smokers had significantly higher levels ofactivated CREB when compared with nonsmokers. Cell linesderived from normal pancreas and pancreatic intraepithelialneoplasm (PanIN) exhibited low baseline pCREB levels com-pared with PDAC cell lines. Furthermore, elevated CREBexpression correlated with reduced survival in patients withPDAC. Depletion of CREB significantly reduced tumor burdenafter tobacco-specific nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment, suggesting a CREB-dependent contribution to PDAC growth and progressionin smokers. Conversely, NNK accelerated PanIN lesion andPDAC formation via GM-CSF–mediated activation of CREB ina PDAC mouse model. CREB inhibition (CREBi) in mice moreeffectively reduced primary tumor burden compared withcontrol or GM-CSF blockade alone following NNK exposure.GM-CSF played a role in the recruitment of tumor-associatedmacrophages (TAM) and regulatory T cell (Treg) expansion andpromotion, whereas CREBi significantly reduced TAM and Tregpopulations in NNK-exposed mice. Overall, these results suggestthat NNK exposure leads to activation of CREB throughGM-CSF, promoting inflammatory and Akt pathways. Directinhibition of CREB, but not GM-CSF, effectively abrogates theseeffects and inhibits tumor progression, offering a viable thera-peutic strategy for patients with PDAC.

Significance: These findings identify GM-CSF-induced CREB as a driver of pancreatic cancer in smokers and demonstratethe therapeutic potential of targeting CREB to reduce PDAC tumor growth.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6146/F1.large.jpg. Cancer Res; 78(21); 6146–58.�2018 AACR.

1Department of Surgery, University of Miami Miller School of Medicine, Miami, Florida.2Department of Pathology, Vanderbilt University School of Medicine, Nashville,Tennessee. 3Department of Surgery, Vanderbilt University School of Medicine, Nash-ville, Tennessee. 4Department of Pathology, University of Colorado, Denver, Colorado.5Department of Public Health, University of Miami Miller School of Medicine, Miami,Florida. 6Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida.

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

Corresponding Author: Nagaraj S. Nagathihalli, University of Miami, MillerSchool of Medicine, Sylvester Comprehensive Cancer Center, 1550 NW 10thAve, FOX 140,Miami, FL 33136. Phone: 361-720-9347; Fax: 305-243-2810, E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0579

�2018 American Association for Cancer Research.

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IntroductionPancreatic ductal adenocarcinoma (PDAC) is the third leading

cause of cancer-related death in the United States and is charac-terized by its poor prognosis and a dismal 5-year survival rateapproaching 5% (1). It is predicted to become the second leadingcause of cancer-related deaths by 2030 (1, 2). Smoking is a majormodifiable risk factor that accounts for almost 20% to 30% ofPDAC cases in the United States (3). Cigarette use increases thelikelihood of developing PDAC and adversely influences overallpatient survival (4). Nicotine and nitrosamines are the mostpotent and highly abundant carcinogens present in cigarettesmoke. These compounds have been shown to initiate a spectrumof oncogenic processes integral to the development of PDAC (5).In smokers, nicotine accumulates to high concentrations innumerous organs, including the pancreas (6, 7). Similarly, nitro-samines are present at 7 times higher levels in the pancreatic juiceof smokers (8) compared with nonsmokers. Among multipletobacco-specific nitrosamines, 4-(methyl nitrosamino)-1-(3-pyr-idyl)-1-butanone (NNK) is considered to be the most carcino-genic (9, 10). The precise molecular pathway governing theprogression from dysplasia to invasive cancer due to NNK expo-sure remains unclear.

Recent evidence suggests that nicotine exposure significantlyincreases the number of low-grade pancreatic intraepithelialneoplasms (PanIN) in LSL-KrasG12D/þ;Pdx1Cre/þ (KC) mice(10), suggesting the role of NNK in promoting tumorigenesisthrough multiple diverse oncogenic pathways (5). Binding ofNNK to the b-adrenergic receptor activates the EGFR/MAPKpathway, leading to ERK1/2 phosphorylation and thereby pro-moting cellular proliferation, migration, and angiogenesis (11).Inhibition of the EGFRpathway in smokers using Erlotinib (EGFRinhibitor) monotherapy has failed to show efficacy in a phase IIclinical trial (12), suggesting that alternate signaling pathwayscontribute to NNK-induced PDAC pathogenesis and requirefurther investigation. NNK acts as an agonist to nAChR, anda7nAChR is the most investigated subunit of nAChR, whichactivates protein kinase A (PKA) and PI3K/Akt signaling (13).

Cyclic AMP response element-binding protein (CREB) is atranscriptional coactivator that has been shown to be activatedwith nicotine use (14). CREB is activated throughphosphorylationat Ser133 (KIDdomain) by several key oncogenic pathways such asPKA, Akt/protein kinase B (PKB), MAPK, and p90 ribosomal S6kinase (p90RSK; ref. 15). ActivatedCREB (pCREB) thenbinds to itsmammalian transcriptional coactivator, theCREB-binding protein(16), enabling the recruitment of additional transcriptionalmachinery elements that drive malignant progression. Moreover,GM-CSF signaling can promote PDAC tumorigenesis (17), andGM-CSF production can induce activation of CREB throughMEK-dependent signaling pathway (18). Recently, studies in PDAChavealso been reported that GM-CSF is involved in the recruitment andpolarizationof tumor-associatedmacrophages (TAM) in the tumormicroenvironment (TME; refs. 17, 19). Therefore, wehypothesizedthat CREB could be a possible target for smoking-induced PDACprogression through its interactions with GM-CSF.

In this study, we demonstrate that NNK promotes PanINprogression and PDAC formation through GM-CSF–mediatedactivation of CREB. CREB inhibition (CREBi) effectively pre-vented this progression, suggesting CREB is a critical regulatorof NNK-induced tumor progression and a novel potential targetfor therapy in smoking-induced PDAC.

Materials and MethodsCell lines, reagents, and primers

Human pancreatic ductal epithelial cell lines (HPNE, HPNE-Kras) and PDAC cell lines such as MiaPaCa2, PANC1, SW1990,AsPC1, CAPAN1, CEPAC, HPAC, and BxPC3 were obtained fromthe ATCC andweremaintained according to the ATCCguidelines.Cell authentication was performed by using short tandem repeatDNA profiling (latest date: June 16, 2016, and July 21, 2017), andcell lines tested negative for Mycoplasma via Genetica cell linetesting using eMYCO plus kit (iNtRON Biotechnology). Cellswith relative low passage numbers (<20) were used in the study.Cells treated with NNK in Figs. 1 and 5 with relative passagenumbers (<60) were used in the study. ATCC cell lines werecharacterized and were free of Mycoplasma contamination, testedby Hoechst DNA stain (indirect) and agar culture (direct)methods.

Primary antibodies used for Western blot analysis, immuno-histochemistry, and immunofluorescence were listed in theSupplementary Table S1, and gene-specific primers used forquantitative reverse transcription PCR (qRT-PCR) are listed inSupplementary Table S2.

CREB gene knockdown by shRNASMART vector LentiviralHumanCREB1 shRNA (#V3SH11243-

00EG1385) and SMART vector Empty Vector Control(#VSC11649) were obtained from Dharmacon. Transfection wasperformed according to supplier protocol and as previouslydescribed (20). Lentiviral particles were prepared by cotransfect-ing plasmids into 293T cells. MiaPaCa2 and PANC1 cells weretransduced with lentiviral vectors at a multiplicity of infection of40, and GFP expression was confirmed by FACS Caliber flowcytometer (BDBiosciences). The cellswere then selected for 7 dayswith puromycin (1.5 mg/mL), and when cultures reached nearconfluency, cells were trypsinized and processed for FACS sortingto separate cells with highest GFP expression. To generate stableknockdown clones, these cells were plated at high dilutions in10 cm petri dishes, and colonies obtained from single cells werescreened for the expression of CREB by immunoblottingtechnique.

RNA sequencingTotal RNA was isolated from PanIN cells treated with NNK

(1 mmol/L) for 5 and 50 days and untreated cells (n ¼ 3) usingRNeasy Plus Mini Kit (Qiagen) and submitted to the MicroarrayCore Facility at Vanderbilt University. Ingenuity analysis wasperformed with help from Vanderbilt Technology for AdvancedGenomics and Research Design. The upregulated and downregu-lated gene lists for 5 days (Supplementary Tables S3 and S4) and50 days (Supplementary Tables S5 and S6) are listed. The CREBsignature genes upregulated by NNK are listed in SupplementaryTable S7. The raw RNA sequencing (RNA-seq) data have beendeposited at Gene Expression Omnibus under accession number(GSE119517).

Cytokine array analysisNNK (1 mmol/L)-exposed humanH6c7 andmouse PanIN cells

were grownon cultureflasks until 70% to 80%confluent and thencultured in serum-free medium (SFM) for 24 hours at 37�C.Conditioned medium (CM) was collected separately from thesecells and stored at �80�C. The CM was collected multiple times.

Targeting CREB in Smokers with Pancreatic Cancer

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Human and mouse collections were separately pooled. The cellswere maintained in culture with fresh media added twice weekly.CM and culture medium were concentrated using Milliporecentriprep centrifugal filters (EMDMillipore) with 10k cutoff andcentrifuged at 2,000 rpm, 4�C to concentrate. The concentratedCM and culture medium were quantified for the protein concen-tration, and the aliquots were stored at �80�C. In the followingexperiments, conditioned media protein was diluted with SFM,and the cytokine levels in the conditioned media were measuredusing human and mouse cytokine array kits (R&D systems)according to the manufacturer's directions.

Human tissue microarrayClinical materials were obtained from patients who provided

written informed consent, according to the Vanderbilt Institu-tional Review Board standards and guidelines. The studies wereconducted in accordance with recognized ethical guidelines(Declaration ofHelsinki, CIOMS, Belmont Report, U.S. CommonRule). Tissue microarrays (TMA) were designed and constructedas previously described (21). The TMA slides were simultaneouslyevaluated by a pathologist (C. Shi). Normal pancreas, well-differentiated PDAC, moderately-differentiated PDAC, andpoorly-differentiated PDAC tissue cores were used as previouslydetailed (21). The staining of the tissues was assessed usingstaining index measured as a sum of the intensity score (0, nostaining; 1þ, weak; 2þ, moderate; 3þ, strong) and distributionscore (0, no staining; 1þ, staining of <33% of cells; 2þ, between33%and66%of cells; and 3þ, staining of>66%of cells). Stainingindices were classified as follows: 2þ to 3þ or higher, strongstaining; 0 to 1þ, weak staining. Activated CREB (pCREB) wasscored as positive if any detectable staining was present.

Genetically engineered transgenic micePtf1aCre/þ;Tgfbr2flox/flox and LSL-KrasG12D/þ;Tgfbr2flox/flox mice

were provided by Dr. Hal Moses (Vanderbilt University Medical

Center, Nashville, TN). These two lines were intercrossed togenerate Ptf1aCre/þ;LSL-KrasG12D/þ;Tgfbr2flox/flox mice (PKT) on aC57Bl/6 background (21). Genotyping of alleles was per-formed using oligonucleotide primers as described previously(22, 23). Ptf1aCreER knock-in mice (provided by Dr. ChristopherWright, Vanderbilt University Medical Center, Nashville, TN)expresses CreER under endogenous Ptf1a promoter/enhancerelements (24). CreER production recapitulates endogenousPtf1a expression, with nuclear translocation within 24 hoursof tamoxifen (Tam) administration. Ptf1aCreER mice wereintercrossed with LSL-KrasG12D/þ to generate Ptf1aCreER;LSL-KrasG12D/þ (iPK) mice. LSL-KrasG12D/þ, Pdx1Cre/þandp53R273H/þ mice were intercrossed to generate indicated LSL-KrasG12D/þ; Pdx1Cre/þ (KC) and LSL-KrasG12D/þ; Trp53R172H/þ;Pdx1Cre/þ (KPC) animals. All mice were housed under a 12-hour light–dark diurnal cycle with controlled temperature(21�C–23�C) and provided with a standard rodent diet andwater ad libitum throughout all experiments.

In vivo xenograft studiesAthymic nude mice—Foxn1 nu/nu (4–5 weeks old)—were

purchased from Harlan Sprague Dawley, Inc. Subcutaneoustumorswere establishedby injecting 2�106MiaPaCa2or PANC1CREB knockdown (CREB shRNA) cells into the flank of a 6-week-old Fox1-nu/numouse (n¼5 in each group) as previously detailed(21). Treatment was initiated when the s.c. tumors reached 70 to200mm3 size. NNK (10mg/100 g body weight, 3 times/week—adosage similar to the level of NNK found in the pancreatic juice ofsmokers; ref, 25) or vehicle (saline) was administered i.p. for 29(MiaPaCa2) and 25 (PANC1) days. The s.c. tumor volume andpercent body weight change were recorded as previouslydescribed (21). Growth curves for tumors were plotted as themean volume � SD of tumors for mice from each group. At theend of the study, animals were sacrificed, and their primarytumors were removed.

Figure 1.

NNK exposure activates CREB inhuman pancreatic epithelial cells. A,Western blot demonstrating baselineexpression of activated and totalCREB levels in a panel of 11 human andpancreatic cell lines including PDACcells (left). Actin was used as a loadingcontrol. Right, densitometry analysesof pCREB normalized to total CREB.B,Western blot demonstrating time-dependent pCREB expression in H6c7and HPNE human pancreatic ductalepithelial cells (exhibiting low-basalpCREB expression) upon NNKtreatment (1 mmol/L) for up to 100days. C, Western blot demonstratingtime-dependent pCREB expressionin H6c7 cells upon NNK exposure(10 mmol/L) for 10 and 50 days,respectively.

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Treatment of PKT micePKTmicewere treatedwith vehicle, GM-CSFblockade antibody

(aGM), CREB inhibitor (666-15), or a combination of aGM and666-15. Mice in the NNK (10mg/100 g/3 day), aGM (2.5mg/kg/3 days), and 666-15 (10mg/kg/day) armwere received starting at4 weeks of age. NNK and/or aGM were administered i.p., and666-15 or vehicle was administered by oral gavage for 3 weeks,after which, mice were euthanized and dissected. Due to theirregularity of the tumor dimensions, size was determined byweighing the entire tumor. Tumor tissue was processed for furtherimmunohistochemical examination.

ImmunohistochemistryTissues were fixed and immunostained using antibodies

against cytokeratin 19 (CK19), Ki67, F4/80, FoxP3, CD3, andclaudin 18 (Supplementary Table S1). These tissues werestained for alcian blue. Stained tissues were evaluated by anexpert pathologist (C. Shi). Tissue slides were deparaffinized,antigen retrieval was carried out in citrate buffer (pH ¼ 6.0)under pressure for 15 minutes, and endogenous peroxidaseactivity was blocked by incubating with 3% H2O2 for 10minutes. The sections were stained with primary antibodiesat described concentrations and developed using DAB sub-strate (Vector). Immunostained slides were scanned, and theircorresponding staining was quantified using the Ariol SL-50platform (Leica Camera, AG). Digital slide images were adjust-ed to exclude areas containing obvious histologic artifacts,such as tissue folds or nonorganic material, from the digitalimage. Values were calculated and reported as relative expres-sion of protein staining.

Statistical analysisDescriptive statistics were calculated using Microsoft Excel and

Prism software (Graphpad). Results are shown as values ofmean � SD unless otherwise indicated. Statistical analyses ofimmunohistochemistry data were performed using the ANOVAfollowed by Tukey multiple comparisons test to determineP values. The two-sided Student t test was used for two groupscomparison, with P < 0.05 considered as statistically significant,except when indicated otherwise. Survival curves were estimatedwith the Kaplan–Meier method, and significance of the differ-ences between groups was assessed with the log-rank test.

Ethics statementAll experiments were performed in compliance with the reg-

ulations and ethical guidelines for experimental and animalstudies of the Institutional Animal Care and Use Committees atthe Vanderbilt University (Nashville, TN) and the University ofMiami (Miami, FL; Protocol # 15-057, 15-099, and 18-081).

For additional experimental procedures, please refer to Sup-plementary Materials and Methods.

ResultsNNK exposure induces pCREB expression in pancreas cells

To understand the mechanistic role of smoking in pancreaticcancer progression, we first exposed human primary pancreaticductal epithelial cells (H6c7) to NNK for up to 50 days andperformed a phosphokinase array (Supplementary Fig. S1A).Array data revealed a significant upregulation of CREB and Akt

phosphorylation upon NNK exposure. Akt inhibition withMK-2206 in these cells resulted in downregulation of pCREBexpression, demonstrating that CREB activation is dependent,at least in part, on Akt signaling (Supplementary Fig. S1B).MK-2206 is known to inhibit the activity of Akt by decreasingthe phosphorylation of serine 473 (S473) and threonine 308(T308) on Akt (26). This provided the molecular basis tofurther examine the role of NNK-induced activation of CREBin pancreas cells.

A panel of normal pancreatic ductal epithelial and PDAC celllines were screened for their respective baseline pCREB expres-sion. This analysis showed that all PDAC cell lines except SW1990exhibit robust expression of pCREB, whereas normal ductalepithelial cells (H6c7 and HPNE) had low basal expression (Fig.1A). To determine if NNK exposure results in CREB activation,H6c7 and HPNE cells were treated with NNK (1 mmol/L) andimmunoblotted for pCREB expression. NNK dosage of 1 and 10mmol/L corresponds to lowest and highest dose delivered percigarette, respectively (6). There was a significant time-dependentincrease in pCREB expression in both the cell lines upon chronicexposure to NNK (Fig. 1B). Because NNK acts as an agonist tonAChR (a7 subunit; a7nAChR), the baseline expression of thisreceptor and CSFR2a (Supplementary Fig. S2A) on a panel ofseven pancreatic cell lines was determined. As shown in Supple-mentary Fig. S2B (27), robust expression ofa7nAChRwas seen inmost pancreas cells including HPNE. Interestingly, exposure ofH6c7 cells to a high concentration of NNK (10 mmol/L corre-sponding to equivalent dose of 5–6 cigarettes/day) for 50 daysexhibited similar time-dependent CREB activation (Fig. 1C) thatwas observed with low concentration NNK exposure, suggestingthat NNK-induced CREB activation is a phenomena dependenton duration of exposure more so than concentration.

Cigarette smokers have elevated pCREB levels and decreasedsurvival in human PDAC

Smokers had a shorter median survival than nonsmokers(median survival of 16 months vs. 37 months, respectively;P ¼ 0.0291; Fig. 2A). TMA samples from patients with knownsmoking status were examined for pCREB expression (Fig. 2B andC). pCREB expressionwas significantly higher in both normal andmalignant pancreatic tissues obtained from smokers comparedwith nonsmokers. Furthermore, elevated pCREB expression inPDAC tumors was associated with worse overall survival (OS)compared with low pCREB levels (median survival of 11 monthsvs. 49 months, respectively; P < 0.0001, Fig. 2D).

NNK exposure accelerates PanIN lesion development inPtf1aCreER; LSL-KrasG12D/þ mice

To determine the role of pCREB in tumor progression, wescreened for pCREB levels in multiple cell lines derived fromPanIN, primary PDAC (PDA), and liver metastasis (LMP)lesions, which were generated from LSL-KrasG12D/þ;Trp53R172H/þ; Pdx1Cre/þ (KPC) genetically engineered mousemodel (GEMM) of PDAC (Fig. 3A). These cell lines serve as apredictive in vitro model of tumor progression as validated inour previous studies (28). Premalignant PanIN cells exhibitedlowest basal expression of pCREB compared with malignantPDA and LMP cell lines. Chronic exposure of NNK (1 mmol/L)to PanIN cells for up to 50 days revealed an elevated expressionof pCREB in a time-dependent fashion relative to their corre-sponding vehicle-treated controls (Fig. 3B), concordant with






our results seen with NNK-treated human pancreatic ductalepithelial cells (Fig. 1B).

Early stage PDAC development is characterized by the for-mation of PanINs, occurring through acinar-to-ductal metapla-sia (29). In Ptf1aCreER; LSL-KrasG12D/þ (iPK) mice, KrasG12D isspecifically activated in acinar cells using a Tam-inducible Creexpressed by the endogenous Ptf1a locus (24, 30). iPKmice caninitiate PDAC by activating oncogenic KrasG12D specificallywithin mature acinar cells, whereas KrasG12D activation inadult duct cells or centroacinar cells has little or no effect onPDAC initiation (31). These mice can normally develop ADMby 9 weeks of age and PanIN lesions at 8 to 17 months of age,which arise from acinar cells (30). The endogenous Kras in iPKmodel rely on Ptf1aCre to obtain acinar cell–specific expressionof Kras by Cre-mediated recombination of a stop cassetteplaced in the Kras locus. Cre-mediated recombination canexcise the stop codon and permit the oncogenic protein to beexpressed. The oncogenic Kras expression is for the cell's life-time because it is a genetic change. Thus, the cells targeted andall the cells that come from that cell will have the recombinedKrasG12D allele and presumably express it. Interestingly,chronic NNK exposure led to progressive increase in CK19,alcian blue–positive ductal precursor lesions (PanIN-1 andPanIN-2), and claudin 18 levels as early as 8 weeks in thepancreatic tissues of iPK mice, whereas these effects were notseen even at 24 weeks in the corresponding vehicle-treated mice(Fig. 3C and D). Chronic NNK exposure (upto 16 weeks andonward) also significantly elevated pCREB expression in thepancreatic tissues of iPK mice compared with their correspond-ing vehicle-treated control mice (Fig. 3E). Furthermore, chronic

NNK exposure upto 24 weeks also led to increase in PanIN-3lesions. These results show that NNK exposure acceleratesPDAC progression through progressive increase in number ofPanIN lesions.

CREB knockdown decreases NNK-induced pancreatic tumorgrowth

To further delineate the role of CREB in NNK-induced pancre-atic carcinogenesis, MiaPaCa2 cells were treated with a CREBinhibitor (CREBi, 666-15) with or without NNK exposure. Spec-ificity of 666-15 has been previously investigated and shown toinhibit CREB's transcription activity with an IC50 of approximate-ly 80 to 100 nmol/L (32). Our results showed 666-15 in PDACcells resulted in inhibition of cell proliferation and increase inapoptosis (Supplementary Fig. S3A and S3B), as well as it inhib-ited CREB-mediated gene transcription of FOS and EGR1 (Sup-plementary Fig. S3C). CREBi significantly reduced NNK-inducedanchorage-independent growth inMiaPaCa2 cells (Fig. 4A).Next,CREB knockdown (CREBsh) cells were generated for MiaPaCa2and PANC1 (Supplementary Fig. S4A and S4B), which showed asignificant reduction in the number of colonies compared with avector control (Fig. 4B).

MiaPaCa2 and PANC1CREB shRNA xenografts were generatedusing cell lines via s.c. flank injection in athymic nude mice (Fig.4C; Supplementary Fig. S4C). The empty vector and CREB shRNAxenografts were then treated with either NNK or vehicle (Fig. 4Cand D). Administration of NNK (10 mg/100 g body weight)administration by i.p. injection was chosen as it recapitulates thelevels of NNK found in smokers (25). CREBsh MiaPaCa2 xeno-grafts showed absence of pCREB expression when compared with

Figure 2.

Activated CREB levels in humanpancreatic tissues correlate with theirsmoking status. A, Kaplan–Meiersurvival curve comparing OS ofpatients with PDAC with neversmokers versus smokers (n¼ 129; P¼0.0291). B, Representativeimmunohistochemical analysis ofpCREB staining in TMA constructedfrom human pancreatic tissuesobtained from smokers andnonsmokers (one specimen per groupis shown). C, x2 analysis of pCREBexpression in normal pancreatic ductand tumor tissues obtained fromsmokers and nonsmokers (n ¼ 97;P < 0.0001). D, Kaplan–Meier survivalcurve comparing OS of patients withPDAC, stratified by theircorresponding pCREB staining levels(n ¼ 57; P < 0.0001).

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Figure 3.

Smoking accelerates PanIN and PDAC formation and activates CREB in the spontaneous Ptf1aCreER; LSL-KrasG12D/þ (iPK) mouse model. A, Western blotdemonstrating baseline expression of activated and total CREB levels in a panel of three mouse pancreatic cell lines including cancer cells (left). Actin wasused as a loading control. Normalized pCREB expression levels were quantified using ImageJ image analysis software (right). B, Western blot demonstratingtime-dependent pCREB expression in precancerous mouse PanIN cells (exhibiting low-basal pCREB expression) upon NNK treatment (1 mmol/L) for up to100 days. C, Representative hematoxylin and eosin (H&E), cytokeratin 19, alcian blue, and claudin 18 staining of pancreatic tissues harvested from NNK (10 ppm indrinkingwater)-treated iPKmice for up to 24weeks. Scale bar, 50mm.D,Quantification of the number of alcian blue–positive PanIN lesions (low- and high-grade) perhigh-power field (HPF) in the control and NNK-exposed iPK mice (n ¼ 8; �� , P < 0.01; �� , P < 0.001). E, Western blot demonstrating pCREB expression inpancreatic tissues harvested from NNK (10 ppm in drinking water)-administered iPK mice (left) and densitometry analyses of pCREB normalized to total CREBprotein are shown (right).






Figure 4.

CREBi effectively suppresses NNK-induced pancreatic tumor growth. A, MiaPaCa2 cells were treated with NNK (1 mmol/L) and/or CREB inhibitor (1 mmol/L) andanalyzed for number of colonies. Colony size results were calculated from eight photographs analyzed from triplicate wells. Data are represented asmean� SD. B,MiaPaCa2 and PANC1 CREB shRNA cells were analyzed for number of colonies. Colony size results were calculated from eight photographs analyzedfrom triplicate wells. C, Experimental design for NNK treatment in CREBsh MiaPaCa2 xenograft mouse model (n ¼ 5 per experimental cohort). D, CREBshMiaPaCa2 and empty vector cells were s.c. injected onto the flank of Fox1-nu/nu mice and treated with NNK at 10 mg/100 g by i.p. administration, 3 times/week for29 days. Tumor volumes were measured up to 36 days. The tumors were later dissected, and growth curves were measured and are represented as mean � SD(n ¼ 5). E, Immunofluorescence staining of pCREB expression in vehicle- or NNK-treated vector and CREBsh MiaPaCa2 flank xenografts. F, Representative Ki67staining of vehicle- or NNK-treated vector and CREBsh MiaPaCa2 flank xenograft tissues (left). Quantification of Ki67 staining data obtained fromvehicle- or NNK-treated vector andCREBshMiaPaCa2 flank xenografts (right; n¼ 3; ns, nonsignificant, P >0.05; �� ,P <0.001, Student t test). ns,P >0.05; � , P <0.05;�� , P < 0.01; �� , P < 0.001; and �� , P < 0.0001.

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Figure 5.

NNK administration differentially stimulates GM-CSF secretion in vitro and in vivo.A,RNAs obtained fromPanIN cells treatedwith NNK (1 mmol/L) or DMSO (control)for 5 and 50 days, respectively, were subjected to RNA-seq analysis. The differentially expressed genes between the control and treatment groups weredetermined using hierarchical clustering and subsequent heatmap generation of normalized gene expression in standardized units. Data analysis using IPA revealedthe upregulated genes in the treatment group (red). B, qPCR data of RNA collected from PanIN cells (top) and H6c7 cells (bottom) treatedwith NNK (1 mmol/L) or DMSO (control) for 50 days and analyzed to validate the genes upregulated in RNA-seq data (n ¼ 3). C, Human cytokine array dataobtained using conditioned media collected from NNK (1 mmol/L)-treated H6c7 cells and PanIN cells (left). Relative GM-CSF expression from the respectiveNNK-treated cells (H6c7 and PanIN) quantified using ImageJ image analysis software (right). D, GM-CSF levels in serum of Ptf1aCreER; LSL-KrasG12D/þ miceadministered with NNK (10 mg/100 g/3 days) in a dose-dependent manner, measured using ELISA (top; n ¼ 6 per group). KC (LSL-KrasG12D/þ; Pdx1Cre/þ), KPC(LSL-KrasG12D/þ; Trp53R172H/þ;Pdx1Cre/þ), andPKT (Ptf1aCre/þ; LSL-KrasG12D/þ; Tgfbr2 flox/flox)micewere injectedwith NNK at 10mg/100 g by i.p. administration every3days/week for 2weeks, andGM-CSF levels in serumweremeasured usingELISA (bottom;n¼4per group).E,Western blot demonstrating pCREBexpression levelsin MiaPaCa2 cells treated with NNK (1 mmol/L) with or without GM-CSF antibody blockade (aGM; 1 mg/mL) and CREBi (666-15; 1 mmol/L). F, Western blotdemonstrating pAkt and pCREB expression levels inMiaPaCa2 cells treatedwithMK2206 (250 nmol/L)with orwithout recombinant GM-CSF (rGM-CSF; 100 ng/mL).G, Serum GM-CSF level was measured from PKTmice treated with CREBi (666-15; 10 mg/kg/daily) for 3 weeks. ns, nonsignificant, P > 0.05; � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001.






vector control xenografts (Fig. 4E). Furthermore, CREB knock-down significantly impaired NNK-induced tumor growth andproliferation, as measured by Ki67 index, in comparison withNNK-treated vector controls (Fig. 4D and F). This effect on NNK-induced tumor growth was also observed in PANC1 CREBshxenografts (Supplementary Fig. S4C). NNK and its metaboliteshave a long retention time in vivo (33). Our results showed thatthe tumor growth rate remained unaltered in the absence ofNNK. Importantly, NNK treatment did not have any impact onmouse weight throughout the treatment period (SupplementaryFig. S4D).

GM-CSF activates CREB in vitro and in vivoTo elucidate the molecular signals that drive NNK-induced

pancreatic tumor growth, RNA-seq analysis was used to examinethe upregulated pathways in PanIN cells exposed to NNK (for 5and 50 days; Fig. 5A; Supplementary Fig. S5). Ingenuity pathwayanalysis (IPA) identified top seven cellular functions, overlappinggenes, upstream/downstreammolecules, and the top five diseases(Supplementary Fig. S5A–S5D). This analysis revealed thatCREB1, FOS, EGR1, and GM-CSF (CSF2) were the most upregu-lated genes in response to chronic (50 days) NNK exposure (Fig.5B), indicating that NNK induction activates CREB at the tran-scriptional level. In addition, FOS and EGR1, known downstreamtarget genes of CREB, were also significantly upregulated uponNNK exposure. The RNA-seq results were subsequently validatedusing qRT-PCR (Fig. 5B) for bothmouse PanIN and humanH6c7cells treated with NNK for 50 days. Given the elevated expressionof GM-CSF with NNK exposure, we then sought to delineate therole of GM-CSF in NNK-induced tumor cell growth by analyzingthe CM obtained from NNK-treated H6c7 and PanIN cells.GM-CSF secretion was significantly increased in the conditionedmedia of NNK-treated cells compared with their respectiveuntreated controls, in concordance with our RNA-seq findings(Fig. 5C). This effect was also noted in vivo, where a significantincrease in serum levels of GM-CSF in NNK-treated iPK (Fig. 5D,top),KC,KPC, andPKT (Fig. 5D, bottom)micewas observed. Thisindicated that NNK induction stimulates GM-CSF release bothin vitro and in vivo.

In order to determine the specific role of GM-CSF on pancreaticcancer cell function, we first examined the expression of theGM-CSF receptor protein CSF2Ra on five different primaryhuman pancreatic cancer cell lines and two pancreatic ductalepithelial cell lines. The GM-CSF receptor CSF2Ra was expressedin all the cell lines tested (Supplementary Fig. S2). NNK-inducedactivation of CREB was reduced by blocking GM-CSF (aGM) orCREBi or combination of aGM and CREBi in H6c7 cells (Fig. 5E).To determine if the protumorigenic effects of GM-CSF weremediated through CREB, HPNE-Kras (Supplementary Fig. S6A)and MiaPaCa2 (Supplementary Fig. S6B) cells were treated withrecombinant GM-CSF (rGM-CSF) with or without CREB inhib-itor. CREBi significantly reduced GM-CSF–mediated colony for-mation, suggesting that GM-CSF exerts its cellular functions, atleast in part, through CREB. To understand the mechanistic linkbetween GM-CSF and CREB, MiaPaCa2 cells were treated withrGM-CSF with or without Akt inhibitor MK-2206 (Fig. 5F). Cellstreated with rGM-CSF showed activation of both Akt and CREB.We observed a significant reduction in both pAkt and pCREBlevels with Akt inhibition in comparison with the controls orrGM-CSF, suggesting that GM-CSF also causes upregulation ofCREB by activating Akt signaling in PDAC cells. Furthermore,

there was a significant reduction in GM-CSF levels in the serumobtained from CREBi-treated PKT (Ptf1aCre/þ;LSL-KrasG12D/þ;Tgfbr2flox/flox)mice in comparisonwith the vehicle-treated controls(Fig. 5G). These results confirm that CREBi affects GM-CSF levels.

Targeting CREB affects the immune TMETumor-derived GM-CSF plays a critical role in regulating

inflammation and immune suppression within the tumormicroenvironment (34). Our results demonstrating anenhanced release of GM-CSF upon NNK exposure both in vitroand in vivo compelled us to further explore the potential effectsof smoking on in vivo PDAC tumor growth and its effect on theimmune TME. NNK was administered with or without GM-CSF blockade and/or CREBi in PKT mice (Fig. 6A). CREBi(666-15) dosage has been chosen as it is the dose that is well-tolerated in vivo (32). CREBi significantly reduced tumor bur-den in NNK-treated PKT mice compared with the correspond-ing vehicle-treated controls. The arm receiving additional GM-CSF blockade did not display any additional antitumor effectscompared with CREBi monotherapy in NNK-treated PKT mice(Fig. 6B), suggesting these effects are mediated primarilythrough CREB. The effect on the different immune compart-ments of an autochthonous and aggressive PKT mouse modelof PDAC was evaluated. CREBi monotherapy significantlyreduced F4/80-positive TAMs and FoxP3-positive regulatoryT cells (Treg cell population) in NNK-treated PKT mice whencompared with their corresponding vehicle, aGM, or combi-nation of CREBi and aGM-treated mice (Fig. 6C and D). Inaddition, CREBi increased the CD3þ T-cell population in theNNK-treated tumors. None of the treatmentmodalities employedhad any significant impact on mouse weights assessed duringtreatment as shown in Supplementary Fig. S6C. Analysis of theeffect of CREB on inflammation has been performed using flowcytometry (Supplementary Table S8). Results show that CREBiincreased the infiltration of CD3þ T cells while at the same timeincreasing the proportion of CD8þ T cells. This resulted inincreased CD8/CD4 T-cell ratio.

Targeting CREB affects tumor stroma and immune infiltrationTo explore the relationship between CREB signaling, stroma,

and immune infiltration, we utilized PKT mice. Tissues fromPKT mice treated with NNK with or without CREBi werestained for aSMA and trichrome blue, which showed abundanttumor stroma (Fig. 7A). Coincident with tumor formation, thepancreatic tissue of PKT mice was highly fibrotic, as reportedpreviously (35, 36). CREBi significantly reduced tumor stromain NNK-treated PKT mice compared with the correspondingcontrols. Furthermore, CREBi significantly reduced prolifera-tion in NNK-treated PKT mice compared with the correspond-ing controls (Fig. 7A). These results suggest that the underlyingreason of reduced tumor growth is due to reduced prolifera-tion of the tumor cells and invoked antitumor immunity dueto reduced infiltration of immunosuppressive cells.

DiscussionOncogenic Kras is the most frequent mutation and a critical

driver of PDAC growth and development (37). Targeting Kras hasbeen largely ineffective, leading investigators to pursue otherdownstream targets for therapeutic effect. CREB is a downstreammediator in the Kras pathway and is constitutively active in most

Srinivasan et al.





PDAC tumors. In our current study, survival outcomes are sig-nificantly worse in smokers compared with never smokers (Fig.2A), and we confirmed that activated CREB is overexpressed in

pancreatic tissues of smokers (Fig. 2B and C). In addition, acti-vated CREB levels were strongly associated with worse OS inpatients with PDAC, suggesting the potential role of cigarette

Figure 6.

Targeting CREB decreases tumor growth and alters recruitment of TAMs and Tregs in NNK-exposed PKT mice. A, Experimental design for NNK treatment with orwithout GM-CSF blockade (aGM) and/or CREBi (666-15) in PKT mice model (n ¼ 5 per group). B, Tumor weights obtained from resected tissues obtainedfrom PKTmice treated with NNK (10mg/100 g/3 days) with or without aGM (2.5 mg/kg/3 days), or with or without CREBi (666-15; 10mg/kg/daily) for 3 weeks (n¼5). C, Representative hematoxylin and eosin (H&E), F4/80, FoxP3, and CD3 staining of pancreatic tissues harvested from PKT mice treated with NNK with orwithout 666-15 and/or aGM. Scale bar, 50 mm. D, Quantification of relative expression of F4/80, FoxP3, and CD3 in the harvested PKT tumor tissues (n ¼ 5). ns,nonsignificant, P > 0.05; �, P < 0.05; �� , P < 0.01; �� , P < 0.0001.






smoking in worsening overall disease prognosis through upregu-lation of pCREB.

Our results demonstrate a predominant increase in pCREBexpression, which correlates with an accelerated progression ofADM, and low-grade and high-grade PanIN lesions during thetransformation from normal epithelium to dysplastic tissue withchronic exposure to NNK (Fig. 3B–D). Activation of CREB in iPKmice exposed toNNKwas consistentwith our in vitroobservationsin human normal ductal epithelial andmouse preneoplastic cellsexposed to NNK. In addition, we noted that pCREB levels wereelevated to a greater degree with chronic NNK exposure whencompared with acute NNK exposure (Figs. 1 and 3), indicatingthat the duration of NNK exposure is an important risk factor forPDAC development (38).

Our results provide compelling evidence that CREB activa-tion induced by NNK exposure plays a critical role in the onset(Fig. 3D) and development of PDAC as CREB knockdownsignificantly reduces NNK-induced PDAC tumor growth andproliferation in vivo (Fig. 4C and E). NNK is metabolizedinto 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)and [4-(methylnitrosamino)-1-(3-pyridyl) but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc) in vivo (39). The slowelimination of carcinogenic metabolites found in cigarettesmoke and their presence within multiple organs suggests a high

retention of NNK within host tissues including the pancreas. Thismay indicate an increased risk for PDAC development and pro-gression among former smokers even after smoking cessation.Further investigation into this phenomenon is warranted.

After delineating the strong correlation between NNK andpCREB overexpression, we successfully showed the regulatorymechanism that leads to pCREB induction after NNK exposure.The RNA-seq and subsequent qPCR data obtained from NNK-treatedH6c7 andPanIN cells showed a significant upregulation ofCREB, FOS, EGR1, and GM-CSF in comparison with their corre-sponding vehicle-treated controls. GM-CSF blockade attenuatedpancreas tumor weight gain in NNK-exposed PKT mice, suggest-ing a protumorigenic role of GM-CSF in PDAC. This is consistentwith the recent report about the role of CREB inGM-CSF secretionin lung fibroblast cells (40) and smoking-induced lung cancermodel. Our study revealed a strong correlation between pCREBlevels and serum levels of GM-CSF in both in vitro cell models andin vivo PDAC GEMMs when were treated with NNK (Fig. 5C andD). To the best of our knowledge, these data are the first report tolink GM-CSF to smoking-induced PDAC formation and progres-sion. Furthermore, we showed that CREBi completely abrogatedthe adverse oncogenic effects of GM-CSF, indicating that NNK-induced activation of CREB occurs through GM-CSF (Fig. 5E andG). Recent reports have defined the role of GM-CSF in malignant

Figure 7.

Targeting CREB decreases tumorstroma and proliferation by reducingimmunosuppressive infiltrate in NNK-exposed PKT mice. A, Tissuesobtained from PKT mice treated withNNK (10 mg/100 g/3 days) withor without CREBi (666-15;10 mg/kg/daily) for 3 weeks (n ¼ 5)were stained for Ki67, aSMA,trichrome blue, and cleaved caspase-3(left). Scale bar, 50 mm. Quantificationof relative expression of aSMA,trichrome blue, and Ki67 in theharvested PKT tumor tissues (right;n ¼ 5). ns, nonsignificant, P > 0.05;� , P < 0.05; �� , P < 0.01. B, Proposedmodel of the NNK-inducedacceleration of the PDAC tumorgrowth by activating CREB. NNKinduces GM-CSF that activates CREBthrough Akt signaling, which results inthe activation of TAMs, Tregs, tumorgrowth/proliferation, and tumorstroma, and regulates immuneinfiltration, which further acceleratedthe progression of PDAC.

Srinivasan et al.





progression (41), worse prognosis (42), and modulation ofimmune TME through recruitment and differentiation ofmyeloidprogenitor cells into immunosuppressive myeloid-derived sup-pressor cells in the surrounding stroma in PDAC (43). Moreimportantly, we show here that CREB also regulates GM-CSF(Fig. 5G), which may have an indirect effect on these suppressivecells.

We also observed that NNK-treated PKTmice exhibited elevat-edGM-CSF secretion, which corresponded to an increased tumor-al macrophage population. Other studies have suggested the roleof TAMs in ADM formation in PDAC (44). Our data suggest thatNNK exposure may mediate TAM infiltration into pancreatictissue and promote ADM through the CREB/GM-CSF pathway.It has also been shown that CREB activation promotes theinduction and maintenance of Tregs (45) and generation ofimmunosuppressivemacrophages (46).We showhere thatCREBialone can significantly reduce tumor burden and decrease infil-tration of these immunosuppressive TAMs and Tregs in NNK-exposed PKT mice, suggesting that CREB is a critical regulator ofthe immune microenvironment. Specifically, in terms of TAMs, ithas been shown that CREB can promote the M2 macrophagephenotype (47). Hence, specific contribution of CREBi in mod-ulating M1 versus M2 phenotype of TAMs in the TME should beanalyzed in the future studies. Furthermore, our results show thatCREBi also increases the infiltrationofCD3þT cells into the PDACtumors, suggesting an overall switch from immunosuppressive totumor-inhibitory TME. Further studies aimed at evaluating thedirect effects of CREBi on various immune cells should inform theextent of direct effects of CREBi on immunemicroenvironment inthe TME.

Previous reports have identified GM-CSF as a central signalingmolecule in promotion of an immunosuppressive microenviron-ment (48). Although GM-CSF may not directly affect Tregs, it hasbeen shown that increased GM-CSF levels may promote gener-ation of Tregs secondary to the induction of CD11cþCD8a�

DCs (48). Therefore, targeting the negative effects of GM-CSFpathway, which is an important component of multiple normalhomeostatic processes, may be achieved through downstreaminhibition of CREB. Therefore, CREBi further presents a noveltherapeutic pathway in regulating the PDAC immune micro-environment. It is known that GSK3 and NF-kB pathwaysregulate inflammatory cells through CREB (45, 49). It is plau-sible that CREBi may regulate inflammation indirectly throughGSK3 and NF-kB signaling pathways. Our results indicate thatthese effects proceed through CREB, thus presenting a potentialpathway that can be therapeutically targeted to improve overallimmune responses. A more detailed understanding of thecommunication between tumor and stromal cells would fur-ther aid in knowing the potential direct and indirect effects of

CREBi on immune cells and role of inflammatory cells withinthis process.

Our work demonstrates for the first time that CREB playsa significant role in smoking-induced PDAC tumor growththrough modulation of the GM-CSF pathway, mediatingAkt, affecting downstream proinflammatory factors, tumorstroma, and reducing TAMs and Treg infiltration (Fig. 7B).Furthermore, CREB depletion delays tumor growth in PDACmouse models and appears to downregulate the Akt path-ways induced by cigarette smoking. Overall, these dataprovide mounting evidence of the functional role of CREBin smoking-induced PDAC progression and provide with aunique therapeutic target in smoking-induced cancermodels.

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

Authors' ContributionsConception and design: S. Srinivasan, P. Lamichhane, N.B. Merchant,M. VanSaun, N.S. NagathihalliDevelopment of methodology: S. Srinivasan, N.B. Merchant, N.S. NagathihalliAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Srinivasan, T. Totiger, J. Castellanos, P. Lamichhane,A.R. Dosch, F. Messaggio, N. Kashikar, K. Honnenahally, N.B. Merchant,N.S. NagathihalliAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Srinivasan, T. Totiger, J. Castellanos, P. Lamichhane,A.R. Dosch, F. Messaggio, N. Kashikar, Y. Ban, N.B. Merchant, M. VanSaun,N.S. NagathihalliWriting, review, and/or revision of the manuscript: S. Srinivasan, T. Totiger,C. Shi, P. Lamichhane, A.R. Dosch, N. Kashikar, Y. Ban, N.B. Merchant,M. VanSaun, N.S. NagathihalliAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Srinivasan, T. Totiger, C. Shi, N.B. Merchant,M. VanSaun, N.S. NagathihalliStudy supervision: N.S. Nagathihalli

AcknowledgmentsThe authors thank Frank Revetta, Dr. Xizi Dai, Jennifer Barretta, and Yanhua

Xiong for their technical assistance.This work was supported by the NIH NCI R21 CA209536, American Cancer

Society IRG 98-277-13, and Stanley Glaser Foundation Research Award(UM SJG 2017-24) to N.S. Nagathihalli and R01 CA161976 and NIH T32CA211034 to N.B. Merchant. Histopathology Core Service was performedthrough the Sylvester Comprehensive Cancer Center (SCCC) support grant (toN.S. Nagathihalli).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 22, 2018; revised July 26, 2018; accepted September 10,2018; published first September 19, 2018.

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2018;78:6146-6158. Published OnlineFirst September 19, 2018.Cancer Res Supriya Srinivasan, Tulasigeri Totiger, Chanjuan Shi, et al. CREB to Promote Pancreatic Cancer

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