1 Signal TransductionQ1

2 TheQ2 Multifunctional Growth Factor Midkine Promotes3 Proliferation and Migration in Pancreatic Cancer45 Tamina Rawnaq1, Luisa Dietrich1, Gerrit Wolters-Eisfeld1, Faik G. Uzunoglu1, Yogesh K. Vashist1,6 Kai Bachmann1, Ronald Simon2, Jakob R. Izbicki1, Maximilian Bockhorn1, and Cenap G€ung€or1

7 Abstract8 Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis among solid tumors and despite increased9 knowledge of the molecular mechanisms contributing to progression and metastasis, minimal progress has been10 done in establishing new targeted therapies for this deadly disease. The expression of the multifunctional growth/11 differentiation factor midkine (MK) promotes a variety of cellular functions leading to increased angiogenesis,12 proliferation, migration, and survival. Moreover, MK is intensively discussed as a potential new-therapy target and13 as biomarker for cancer progression and chemotherapeutic resistance in multiple cancers. Therefore, the present14 study investigated the molecular role of MK in pancreatic cancer. It was found that MK is elevated in PDAC and15 differentially expressed in other histologic subtypes of pancreatic cancer, whereas normal pancreatic cells did not16 express MK, thus making it an attractive candidate for targeted therapies. As a secreted growth/differentiation17 factor, MK was investigated as a biomarker in clinical serum specimens using ELISA. In addition, knockdown18 studies of MK revealed a link to proliferation and migration status in vitro. Finally, upstream signaling pathways19 were analyzed, with TNF-a and EGF being the main inductors of MK expression in PDAC.

20 Implications: This study presents novel MK functions and new upstream signaling effectors that induce its21 expression to promote PDAC and therefore defines an attractive new therapeutic target in pancreatic cancer.Mol22 Cancer Res; 1–11. �2014 AACR.23242526 Introduction27 An unusual aggressiveness and early metastatic locor-28 egional as well as distant spread of pancreatic cancer cells29 reflects the urgent necessity of new therapeutic options for30 this deadly disease as its incidence still nearly equals31 mortality in the Western countries (1). The clinical32 treatment failure of patients is often attributed to the33 early metastatic growth, an unmet high drug resistance to34 standard therapy options, and high rates of local recur-35 rence (2–4). However, insufficient diagnostic tools and36 therapeutic options for pancreatic ductal adenocarcinoma37 (PDAC) still substantiate its ranking as fourth leading38 cause of cancer-related death (5). Therefore, a better39 understanding of newly identified and cancer-specific key40 molecules that promote proliferation, migration and sur-41 vival of cancer cells may lead to the development of more42 effective therapeutic strategies.

44Previous studies have revealed that expression of midkine45(MK) plays an important role in different aspects of tumor46progression in diverse solid tumors (6–8). We have recently47shown that high mRNA and protein expression of MK in48PDAC protected cancer cells from chemotherapy-induced49apoptosis. Moreover, we have identified that gemcitabine50dose-dependently induced expression and secretion of MK51followed by activation of Notch signaling to promote epi-52thelial–mesenchymal transition (EMT) and intrinsic as well53as acquired chemotherapy resistance through upregulation54ofNF-kB. In addition, our results explained for the first time55how MK may trigger cancer cells to become more resistant56against cell death and how it promotes cell viability (9).57Recently, we and others have shown that the secretion ofMK58in tumor cells may serve as biomarker for gastrointestinal59tumors and glioblastomas (10, 11). Therefore, the develop-60ment of MK inhibitors has been considered as an attractive61idea to prevent tumor growth and antiapoptosis in cancer62cells (12, 13).63In this study, we addressed the question of whetherMK is64also expressed in other histologic subtypes of PDAC and that65siRNA- and short hairpin RNA (shRNA)–mediated deple-66tion of MK may have dramatic effects on cancer cell67proliferation and migration in vitro. The analysis of MK68upstream signaling may provide reasonable explanations for69the observed high MK expression levels in different cancers70and therefore link therapeutic benefits with targeting of the71multifunctional growth factor MK.

Authors' Affiliations: 1Department of General, Visceral and ThoracicSurgery, Experimental Oncology and 2Institute for Pathology, UniversityMedical Center Hamburg-Eppendorf, Hamburg, GermanyQ3

Corresponding Author: Cenap G€ung€or, Department of General, Visceraland Thoracic Surgery, University Medical Center Hamburg-Eppendorf,Martinistrasse 52, 20246 Hamburg, Germany. Phone: 49-4074-1051-956; Fax: 49-4074-1053-496; E-mail: c.guengoer@uke.deQ4

doi: 10.1158/1541-7786.MCR-13-0467

�2014 American Association for Cancer Research.

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74 Materials and Methods75 Study design and patients76 The study was approved by Ethics Committee of Cham-77 ber Physicians in Hamburg, Germany. Written informed78 consent was obtained from patients using tissue and serum.79 Weanalyzed 103 pancreatic cancer tissue spots and 37 serum80 samples for MK expression, retrospectively. As healthy81 controls, 148 blood bank donors were included. A tissue82 microarray (TMA), including 56 ductal, 28 papillar adeno-83 carcinoma, and 19 neuroendocrine resected pancreatic can-84 cer was stainedwith antibodies against humanMK (Abcam).

85 ELISA86 For quantification of human MK in serum of patients87 with cancer, weQ5 used MK ELISA (Antigenix America).88 Briefly, microtiter wells precoated with anti-human MK89 antibodies were incubated with patient's serum. After90 washing, biotin-labeled anti-human MK antibody was91 added and plates were incubated for 1 hour with captured92 MK. After thorough wash, streptavidin–HRP (horseradish93 peroxidase) conjugate was added to convert substrate94 H2O2–tetramethylbenzidine.

95 TMA construction and immunohistochemistry96 Tissue samples were fixed in 4% formalin, paraffin97 embedded, and used for TMA construction as described98 elsewhere (14). Hematoxylin-eosin–stained sections were99 made from selected primary tumor blocks (donor blocks)100 to define representative tumor regions. Immunohistochem-101 istry (IHC) and scoring were judged by two independent102 board-certified pathologists (T. Rawnaq and R. Simon). For103 each tissue sample spot, the fraction of positive-immunos-104 tained tumor cells was recorded, and staining intensity was105 estimated on a four-step scale (0, 1þ, 2þ, and 3þ). A final106 score was then built from these two parameters according to107 the following score: negative scores had absence of MK108 staining; weak scores had staining intensity of 1þ in �70%109 of tumor cells or staining intensity of 2þ in� 30% of tumor110 cells; moderate scores had staining intensity of 1þ in >70%111 of tumor cells, staining intensity of 2þ in >30% but in112 �70% of tumor cells or staining intensity of 3þ in�30% of113 tumor cells; and strong scores had staining intensity of 2þ in114 >70% of tumor cells or staining intensity of 3þ in >30% of115 tumor cells.

116 Cell lines, conditions, and growth factors117 Tumor tissues for establishment of primary chemoresis-118 tant PDACcell line PaCa 5061was taken from a patientwho119 underwent total pancreaticoduodenectomy for advanced120 PDAC in 2010. The procedure of cell line establishment121 from tumor tissue was previously published (15). Primary122 cells were cultured in complete TUMQ6 medium (15). PANC-123 1 cells were cultured in Dulbecco's Modified Eagle Medium124 (DMEM; Invitrogen) supplemented with 10% fetal calf125 serum (FCS; Lonza), 200 IU/mLPen-Strep (Sigma-Aldrich)126 at 37�C, 5% CO2. Establishment of HPDE was described127 elsewhere (16). HPDE cells were cultured in K-SFM (Invi-

129trogen) supplemented with bovine pituitary extract, 5130ng/mL EGF and 5% FCS (Lonza), and 200 IU/mL Pen-131Strep (Sigma-Aldrich). All cell lines were authenticated using132the STR GenePrint System (Promega) in 2012. For MK133induction studies, cells were grown overnight (12–15 hours)134in serum-free media and were stimulated using 10% FCS135(Lonza), recombinant TNF-a (10 ng/mL), EGF (10136ng/mL), insulin growth factor I (IGF-I; 10 ng/mL), basic137fibroblast growth factor (bFGF; 10 ng/mL; R&D Systems)138and (all-trans-) retinoic acid (1 mmol/L; Sigma-Aldrich). For139analysis/quantification of secreted MK (S-MK), we used140human MK-ELISA kit (Antigenix America). Recombinant141human MK (rh-MK; R&D Systems) was used at concen-142trations of 20 ng/mL.

143siRNA/shRNA transfections and Western blot analyses144Cells were transfected with siRNAs against MK or con-145trol-siRNA (Eurofins Scientific) using Lipofectamine (Invi-146trogen). PANC-1 and PaCa 5061 cells were transfected with147indicated siRNAs. PaCa 5061 cells were transfected using148shRNA-plasmids for stable MK downregulation. Briefly,149cells were transfected either with sh-control or specific sh-150MK plasmids (RHS4533-EG4192; Open Biosystems).151After 72 hours, cells were incubated in the presence of152puromycin and positive cell clones were subcloned using153glass cylinders (Sigma-Aldrich). Viable cell clones were154subsequently lysed and analyzed using SDS-PAGE and155Western blot analysis for specific MK knockdown using156anti-MK antibody. Following transfection, cells were lysed157in RIPA buffer (Sigma-Aldrich) containing 1� protease158inhibitor cocktail (Roche). For analyses of S-MK, cell culture159media were collected and filtered (0.22 mmol/L). Super-160natants were resolved by SDS-PAGE and followed by161immunoblotting as previously described (9). Western blot162analyses and immunostainings were performed using MK163antibodies (rab. anti-MK, Abcam; mouse anti-MK,164Abnova).

165Real-time reverse transcription PCR166Real-time reverse transcription PCR (real-time RT-PCR)167was conducted to quantify gene expression or to verify168siRNA-mediated downregulation. Of note, 1 mg of total169RNA was reverse-transcribed using the Transcriptor cDNA170Kit (Roche). Data were analyzed according to the compar-171ative CT method and were normalized for cyclophilin172expression in each sample.

173Immunocytochemistry and subcellular protein174fractionation175Cells were grown on coverslips in 24-well plates. Follow-176ing fixation using paraformaldehyde (4%), cells were177blocked with 1% BSA/0.1% Triton-X. Cells were then178incubated using anti-MK antibody overnight and washed179extensively using PBS (Invitrogen). For visualization, Cy3-180coupled secondary antibodies (Invitrogen) were used. Image181acquisition was done using confocal microscopy (Leica). For182analyses of subcellular MK distribution, we stepwise sepa-183rated cytoplasmic, membrane, and nuclear soluble proteins

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186 by using the Subcellular Fractionation Kit (Pierce). As187 marker proteins for different compartments, we used anti-188 calpain, anti-calnexin, and anti-lamin antibodies (Santa189 Cruz Biotechnology).

190 Scratch and Transwell migration assay191 We performed scratch-/wound healing assay by plating192 Paca 5061 and PANC-1 wild-type, shControl- and sh-MK–193 transfected subclones to create a confluent monolayer.194 Monolayer was scraped in a straight line to create a "scratch"195 in similar size with tips. Debris was washed out using PBS.196 For image acquisition, we used phase-contrast microscopy197 (Leica) at different time points (0, 16 hours). For Transwell198 assay, corresponding knockdown clones of PaCa 5061 cells199 and PANC-1 cells were transferred to migration assay for200 indicated time points. Cell migration was analyzed using 24-201 well inserts (8 mmol/L) according to the manufacturer's202 protocol (BD Biosciences). Briefly, cancer cells remaining203 on top-side of the membrane were carefully removed,204 whereas cancer cells that had migrated to lower-side of the205 chamber were fixed and stained with crystal violet. At least 5206 randomly selected fields per insert were photographically207 documented and average counted.

208 Cell proliferation209 Cell proliferation was determined by CellTiter 96 AQue-210 ous One Solution Assay according to the manufacturer's211 protocol (Promega). Each experiment was performed in212 quadruplicate. The absorbance was measured at 490 nm.213 Values for control cells were considered as 100% viability.214 Every measurement was performed at least in three inde-215 pendent experiments.

216 Data analyses217 Expression analyses and achieved data were analyzed using218 GCOS 1.4 and scaled to default target signal value of 150.

220Absolute and comparative analyses were performed using221MAS 5.0 algorithm. Annotations were further analyzed with222interactive query analysis. Experiments presented in figures223are representative of three different repetitions. The data are224presented as the mean values � SE.

225Statistical analysis226SPSS for Windows (v21.0; SPSS Inc.) was used for227statistical analysis. Statistical significance was evaluated by228the Mann–Whitney U test and the Kruskal–Wallis test.229Significance statements refer to P values of less than 0.05.

230Results231MK expression in normal pancreatic and cancer cells232First, the MK gene and protein expression were evaluated233in PDAC cell lines PANC-1 and PaCa 5061, whereasHPDE234cells served as control. HPDE cells were previously estab-235lished from normal pancreatic tissue (16). PaCa 5061 cells236were previously established from patient-derived PDAC237tissue, operated in our clinic (15).238Because MK was already shown to be significantly over-239expressed in cancer cells compared with corresponding240normal cells (9), we investigated and quantified MKmRNA241in the investigated lines using real-time RT-PCR (Fig. 1A).242We observed a statistically significant mRNA overexpression243(P < 0.05), whereas no expression was detected in HPDE244cells. In a previously published report, we already showed245that MK expression in patients with PDAC is significantly246upregulated in more than 50% of the investigated samples247(9). To verify that the MK mRNA expression resulted in248robust protein expression, we investigated the MK protein249expression in cancer cell lines and HPDE cells using specific250antibodies (Fig. 1B). Because MK is a secreted growth factor251involved in paracrine/autocrine regulation of growth and252differentiation, we investigated whether the MK secretion is253increased in PDAC compared with HPDE cells. Indeed, we

Figure 1. MK expression in normalpancreatic and cancer cells. A,differential MK mRNA expressionin PaCa 5061 and PANC-1 cellscompared with normal pancreaticcells (HPDE) obtained by real-timeRT-PCR. MK is significantlyoverrepresented in PDACcell lines,more than 5-fold compared withHPDE cells. Results are expressedas fold over control; �, P < 0.05.B, Western blot analyses showingsteady-state MK expression (top)as well as S-MK levels (bottom) inPaCa 5061 and PANC-1 cells,compared with HPDE cells.Q7

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256 found robust released MK levels in PDAC cells, whereas no257 increasedMK expression was detectable in HPDE cells at all258 (Fig. 1B).

259 Elevated MK expression in pancreatic cancer tissue and260 serum samples261 To gain relevant insight into expression of MK in PDAC262 patient samples, we analyzed the MK protein expression263 using TMA. Because the vast majority of published infor-264 mation about MK expression in cancer cells resulted from265 evaluation of ductal adenocarcinomas, we hypothesized that266 MK may also be differentially expressed in other histologic267 subtypes. We, therefore, conducted TMA stainings from268 103 patients with pancreatic cancer and 10 normal pancre-269 atic tissue samples that were spotted on the TMA. From270 these 103 samples, 56 were ductal adenocarcinoma, 28271 intraductal papillary-mucinous tissue samples, and 19 neu-272 roendocrine tumors and all of them were stained with MK273 antibodies (Fig. 2A). Consistent with previous observations274 where MK was shown to be significantly overexpressed in275 ductal adenocarcinomas compared with normal control cells276 (9), we found moderate to strong MK expression in 36%277 (20 of 56) and 38% (21 of 56) of ductal adenocarcinomas,278 respectively. More interestingly, we found also strong MK279 expression in 64% (18 of 28) of papillary adenocarcinoma280 tissue samples as well as in 47% (9 of 19) of neuroendocrine281 patient samples (Table 1).282 Because MK is a secreted growth factor and its detection283 may serve as biomarker, we also quantifiedMK expression in

285patient's serum using ELISA. This analysis showed signif-286icantly increased serum MK levels in patients with PDAC287compared with healthy donors (Fig. 2B; Table 2).

288MK is primarily expressed in the cytoplasm of PDAC289cells290The MK expression was previously shown to promote291survival through influencing expression of apoptosis-related292proteins such as Bcl-2 (17). In addition, MK was shown to293localize to the nucleus and nucleolus of HepG2 cells (18),294whereas other studies showed different MK localizations.295The nucleolus is a known hotspot for rRNA transcription,296and the authors conducted siRNA-mediated knockdown of297MK, which resulted in lower rRNA transcription rates. The298authors claimed that nucleolar MK expression influenced299the transcription of rRNA in HepG2 cells and therefore300affecting cell proliferation and apoptosis. The intracellular301expression pattern ofMK in cancer cells is still enigmatic and302confusing. We, therefore, analyzed intracellular localization303of endogenous MK in PDAC cells. First, we performed304immunocytochemical stainings with PANC-1 and PaCa3055061 cells using specific MK antibodies. The subcellular306localization of MK was examined using laser confocal307microscope (Fig. 3A). MK was clearly expressed and local-308ized exclusively in the cytoplasm. Second, we separated309subcellular fractions from PANC-1 and PaCa 5061 cells310and analyzed MK expression in comparison with expression311of cell compartment–relevant protein markers. Strikingly,312we found that MK is predominantly expressed in the

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Figure 2. Elevated MK expressionin pancreatic cancer tissue andserum samples. A, singlerepresentative tissue spots ofMK expression in PDAC (A),pancreatic neuroendocrinecarcinoma (B), and pancreaticpapillar adenocarcinoma (C;magnification, �100). B, boxplotanalysis of S-MK proteinconcentration of healthy controls(n ¼ 148) and patients withpancreatic cancer (n ¼ 37)determined by ELISA. Statisticalsignificance was determined byMann–Whitney U test (P < 0.001).

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315 cytoplasm of pancreatic cancer cells (Fig. 3B), which is in316 contrast with previously published results (18).

317 siRNA-mediated knockdown of MK is associated with318 decreased migration of pancreatic cancer cells in vitro319 Early metastasis formation and invasion is a hallmark of320 pancreatic cancer and reflects the urgent need of new321 therapeutic strategies for this deadly disease. Because MK322 was earlier discussed to be involved in migration of embry-323 onic neuronal cells (19), we hypothesized that MK expres-324 sion is linked to the highly migratory potential of PDAC325 cells. To pinpoint whether overexpressed MK promotes326 migration of PDAC cells in vitro, we first performed tran-327 sient siRNA and stable shRNA-mediated downregulation of328 MK in PaCa 5061 (Fig. 4A) and PANC-1 cells (Fig. 4E).329 Effective MK downregulation was proven by Western blot-330 ting whole-cell lysates and cell culturemedium using specific331 antibodies (Fig. 4B and F) and real-time RT-PCR (Fig. 4C332 and G). In a second approach, knockdown and correspond-333 ing control cells were transferred to a scratch-induced migra-334 tion assay. After PANC-1 and PaCa 5061 cells had grown for335 16 hours under normal conditions, we compared the respec-336 tive cell migration. Strikingly, downregulation of MK is337 linked to markedly impaired migratory potential of both cell338 lines and the injured area is nearly unchanged after 16 hours339 (Fig. 4D and H). On the other hand, the respective control340 cells showed no impairment in migration and filled the341 injured area. In addition, we performed Transwell assays and342 quantified the migrative potential of PDAC cell lines lacking

344MK expression, compared with corresponding control345siRNA transfections (Fig. 4I). Our results clearly demon-346strate that MK is indeed linked to the high migratory347potential of PDAC cells in vitro.

348MK depletion is linked with decreased proliferation of349pancreatic cancer cells in vitro350MK is a heparin-binding growth factor and was recently351shown to promote growth of various cancer cells in vitro and352in vivo. The MK overexpression and/or extracellular treat-353ment of tumor cells with recombinant MK was shown to354promote and accelerate the growth of osteosarcoma cells355(20), neuroblastoma (21) and gastric cancer cells (22). To356examine whether MK promotes proliferation of PDAC cells357in vitro, we first downregulated MK expression through

Table 1. MK expression in pancreatic cancer TMAQ8

MK immunostaining (%)

Tumor subtype N Negative Weak Moderate Strong

Pancreas, adenocarcinoma ductal 56 5 (9) 10 (18) 20 (36) 21 (38)Pancreas, adenocarcinoma papillary 28 1 (4) 4 (14) 5 (18) 18 (64)Pancreas, neuroendocrine 19 4 (21) 2 (11) 4 (21) 9 (47)

Table 2. Elevated S-MK concentration inpancreatic cancer compared with healthycontrols

ControlsPancreascarcinoma

Total 148 37Mean age, y � SD 58 � 11 64 � 10Sex, No. (%)Male 98 (66) 19 (51)Female 50 (34) 18 (49)

S-MK, y � SD (pg/mL) 128 � 115 1,200 � 1,712S-MK, median (pg/mL)[25th and 75thpercentile]

99 [33 and 198] 721 [409and 1,197]

Figure 3. MK is primarily expressed in the cytoplasm of PDAC cells.A, intracellular localization of endogenous MK (red) in PaCa 5061 andPANC-1 cells. Cells were grown on cover slips followed by fixation,blocking, and antibody treatment. Appropriate secondary Cy3-labeledantibodieswere used.DAPI (40,6-diamidino-2-phenylindole)wasused forcostaining nuclei (blue) and immunoreactions were documented on aconfocal microscope. B, subcellular fractions from PANC-1 and PaCa5061 cells were analyzed for MK expression in comparison withexpression of cell compartment–relevant protein markers. MK ispredominantly expressed in cytoplasm of pancreatic cancer cells.

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360 transient transfections of specific siRNA or control siRNA361 into PaCa 5061 (Fig. 5A) and PANC-1 cells (Fig. 5C). We362 then analyzed consequences of depleted MK and performed363 proliferation assays. Corresponding untransfected wild-type

365cells were used as independent control. Interestingly, we366found that MK depletion is strongly linked with impaired367growth of PaCa 5061 (Fig. 5B) and PANC-1 cells (Fig. 5D).368In contrast, proliferation of control transfected cells was not

Figure 4. siRNA-mediatedknockdown of MK is associatedwith decreased migration ofpancreatic cancer cells in vitro. A,shRNA-mediated knockdown ofMK in PaCa 5061 cells efficientlydownregulated MK as shown byWestern blotting total cell lysates oftwo independent cell clones,compared with control-transfectedcells. Actin served as loadingcontrol. B, shRNA-mediatedknockdown of MK in PaCa 5061cells resulted in undetectable S-MK levels as shown by Westernblotting cell culture medium fromtwo knockdown cell clones,compared with control-transfectedcells. C, effective MKdownregulation on mRNA levelwas proven by real-time RT-PCR.D, PaCa 5061 wild-type, control-transfected, and two independentMK-knockdown cell clones weretransferred to scratch-inducedmigration assay. After placing thescratch, cells were grown foradditional 0 and 16 hours,respectively, and cell migrationwas captured using high-powerfield microscope. E, siRNA-mediated knockdown of MK inPANC-1 cells using twoindependent and specific siRNAsefficiently downregulated MK asshown by Western blotting totalcell lysates. F, siRNA-mediatedknockdown of MK in PANC-1 cellsresulted in decreased S-MK levelsas shown by Western blotting cellculture medium. G, effective MKdownregulation on mRNA levelwas proven by real-time RT-PCR.H, PANC-1 wild-type, control-transfected, and two MK-knockdown cell clones weretransferred to scratch-inducedmigration assay. After placing thescratch, cells were grown foradditional 0 and 16 hours,respectively, and cell migrationwas captured using high-powerfield microscope. I, Transwellmigration assay was performedwith PaCa 5061 and PANC-1 MKknockdown cells. The graphsindicate the average number ofcells per field at the indicated timepoints. ��, P < 0.01; �, P < 0.05.

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371 impaired. In proof-of-concept studies, we performed rescue372 experiments via exogenous treatment of MK-depleted PaCa373 5061 and PANC-1 cells with recombinant human MK374 (rh-MK; 20 ng/mL; Fig. 5B and D). Here we observed375 substantially increased proliferation in rh-MK–treated376 MK-depleted cells, compared with untreated cells and pro-377 liferation reached almost similar levels as control-transfected378 and wild-type cells. Because proliferation of PANC-1 and379 PaCa 5061 cells was strongly impaired in MK-depleted cells380 compared with control transfected and wild-type cells, these381 results suggest that high expression and secretion of MK382 positively regulate proliferation of PDAC cells in vitro.383 Interestingly, exogenous treatment of MK-depleted384 PANC-1 and PaCa 5061 cells with rh-MK rescued the385 observed impaired proliferation of MK-depleted pancreatic386 cancer cells.

387 MK expression is inducible by EGF and TNF-a in388 pancreatic cancer cells in vitro389 Although much effort has been done on catching cellular390 MK functions during embryonic development and in neo-391 plastic cells, very little information is available about392 upstream signaling that controls expression of this interest-393 ing multifunctional growth factor. A single report has394 investigated the role of various growth factors and cytokines395 that may induce MK transcription in prostate cancer cells396 (23). The authors claimed that EGF, androgen, IGF-I,397 hepatocyte growth factor (HGF), and cytokines TNF-a398 and interleukin-1beta (IL1-b) are inductors of MK expres-

400sion. To identify factors that may induce MK expression in401PDAC, we treated PaCa 5061 and PANC-1 cells with402serum, IGF-I, bFGF, TNF-a, and EGF or left them403untreated. Because MK was first identified as a retinoic404acid–inducible gene during embryonal carcinoma cell dif-405ferentiation (24, 25), we also treated cells with retinoic acid.406First, we serum starved PaCa 5061 and PANC-1 cells for 12407to 15 hours and treated cells with various growth factors/408cytokines or serum (10%) for 24 hours or left themuntreated409and analyzed MK expression by Western blotting using410specific antibodies (Fig. 6B and D). In a second attempt,411we quantified MK expression in PaCa 5061 and PANC-1412cells following growth factor/cytokine treatment using a413specific MK ELISA (Fig. 6A and C). Interestingly, we were414able to induce strong MK secretion with extracellular treat-415ment of TNF-a (10 ng/mL) and EGF (10 ng/mL) in both416cell lines. Although the MK expression in PaCa 5061 cells417was induced to a lesser content by bFGF (10 ng/mL) and418IGF-I (10 ng/mL), we detected very faint MK expression419after retinoic acid treatment (1 mmol/L) in both cell lines420(Fig. 6A). In contrast, treatment of PANC-1 cells with bFGF421showed undetectable MK expression levels. Moreover,422serum-treated PaCa 5061 and PANC-1 cells displayed423comparable MK expression levels as those resulted from424single TNF-a and EGF treatments, whereas control cells425showed almost no MK expression at all (Fig. 6C). These426results suggested that MK is indeed inducible by TNF-a,427EGF, and serum, whereas retinoic acid, IGF-I, and serum428starvation (12–15 hours) failed to induce MK expression in

Figure 5. MK-depletion is linkedwith decreased proliferation ofpancreatic cancer cells in vitro. A,PaCa 5061 cells were transientlytransfected with two independentsiRNAs against MK or with si-control for 48hours. Protein lysateswere extracted. Actin served asloading control. B, MTT assay ofPaCa 5061wild-type (WT), control-transfected, and MK knockdowncells transfected with twoindependent siRNAs. In rescueexperiments, MK-knockdown cellswere also treated with rh-MK(20 ng/mL) for indicated timepoints. C, PANC-1 cells weretransiently transfected with twoindependent siRNAs againstMK orwith si-control for 48 hours. Proteinlysates were extracted. Actinserved as loading control.D, MTT assay of PANC-1 wild-type, control-transfected, and MK-knockdown cells. In rescueexperiments, MK-knockdown cellswere also treated with rh-MK (20ng/mL) for indicated time points.

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431 PDAC cells. Similar results were also observed in Western432 blotting experiments, reflecting consistency (Fig. 6B andD).433 Moreover, it is known that the MK signaling is posttransla-434 tionally extinguished by ubiquitination and proteasomal435 degradation. Therefore, we examined whether exogenously436 applied growth factor–induced MK upregulation occurs at437 the mRNA level. Indeed, we found that MK mRNA

439expression is robustly induced by TNF-a, EGF, and serum440(10%; Fig. 6E and F).

441Discussion442There are still no satisfactory treatment options for pan-443creatic cancer. As a matter of fact, the clinical reality regarding

Figure 6. MK expression is inducible by EGF and TNF-a in pancreatic cancer cells in vitro. A, serum-starved PaCa 5061 cells were treated with various growthfactors/cytokines or treated with serum (10%) as indicated for 96 hours or left them untreated (S/f) and quantified MK expression/secretion levels byELISA. B, similar results were observed in Western blotting experiments, reflecting consistency. Actin served as loading control. C, same experimentas inAwasperformedusingPANC-1cells.D, comparable resultswereobtainedusingWesternblotting.Actin servedas loading control.MKmRNAexpressionlevels in PaCa 5061 (E) and PANC-1 cells (F) treated with indicated growth factors/cytokines were quantified using real-time RT-PCR.

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446 successful therapeutic intervention of PDAC still reflects that447 only very few patients benefit from current therapeutic448 options and the majority of patients still cannot be cured in449 90% to 95% (26). PDAC is often identified in an advanced450 stage inwhich it is no longer possible to operate. This is due to451 rapid growth and invasive phenotypes leading to early onset452 metastatic spread, accompanied by frequent occurrence of453 chemotherapeutic resistance. Even surgical complete tumor–454 resected patients develop relapse disease, which leads to death455 despite receiving chemotherapy.456 Early detection and novel therapeutic targets are urgently457 needed to improve the outcome. Therefore, an intensive458 investigation ofmolecularmechanisms contributing to tumor459 proliferation andmigration of PDAC should be conducted as460 a basis for successful establishment of new targeted therapies.461 Elevated MK expression levels have been detected in462 various tumors (27). MK is described as a potential prog-463 nostic marker in several malignancies (10, 28–32). The464 present study confirmed the frequent overexpression of MK465 in patient-derived PDAC tissue samples.More interestingly,466 analyses of histologic subclasses revealed strong MK expres-467 sion in the majority of patient-derived papillary adenocar-468 cinoma and neuroendocrine tumors. Furthermore, we dem-469 onstrated that serum MK concentrations were significantly470 elevated in patients with PDAC compared with healthy471 controls. These results encourage us to examine serum MK472 levels as a novel prognostic tumor marker for PDAC, which473 can be easily detected in peripheral blood using commer-474 cially available ELISA.Moreover, downregulation ofMK by475 shRNA and siRNA strategies resulted in substantially476 reduced proliferation rates compared with control cells.477 Interestingly, exogenous treatment of PDAC cells with478 rh-MK rescued proliferation deficiency, suggesting that MK479 may trigger extracellular signaling to accelerate proliferation480 of PDAC cells. In addition, MK is known to be a secreted481 growth factor and high expression levels may contribute to482 increased proliferation by autocrine/paracrine signaling.483 A pronounced stromal response referred to as desmoplas-484 tic reaction is a hallmark of PDAC that is characterized by a485 complex interplay between the host epithelial cells, invading486 tumor cells, stromal fibroblasts, pancreatic stellate cells,487 immune cells, proliferating endothelial cells, and an altered488 extracellular matrix. Elevated expression and secretion of489 MK by the tumor might affect the surrounding stromal cells490 that express MK receptors in a paracrine mechanism and491 thereby contribute to the formation of the latter. Because it492 was shown that the stromal compartment in PDAC nega-493 tively affects radiation therapy (33, 34) and creates a milieu494 for chemotherapy resistance (35, 36), a fundamental issue in495 PDAC treatment, the role of MK in tumor stroma interplay496 and its effects on chemotherapy resistance requires further497 investigation.498 Because several studies have already shown that MK may499 interact with various extracellular receptors (27), it is chal-500 lenging to identify the specific receptors contributing to this501 effect. The list of MK interacting receptors on cell surface is502 still growing, since we were recently able to identify the503 Notch-2 receptor as a new MK interactor in PDAC cells

505whose activation by extracellular MK ended in a highly506chemoresistant phenotype accompanied by EMT (9).507PDAC is known to have a devastating prognosis, which is508primarily due to an early locoregional and distant metastatic509spread. This process is supported by the ability of primary510cancer cells that acquire invasive properties and gain access to511blood and/or lymphatic vascular systems to establish a new512deadly niche. In this study, we could clearly show thatMK is513involved in migration processes of PDAC cells. The migra-514tive ability of PDAC cells in which MK is specifically and515stably knocked down, revealed strong migration deficiency.516The specificity of our observation was further supported by517transient siRNA transfections targeting MK in other PDAC518cell lines. In line with this, MK was recently shown to519interact with various protein members of the TGF-b path-520way in vitro, a pathway that is well accepted to be a central521mediator of EMT processes and consequently navigate522increased migration of cancer cells in vitro and in vivo (37).523On the other hand, subcellular distribution of MK in524cancer cells is still enigmatic. Therefore, we analyzed MK525localization in PDAC cells using specific antibodies and526detected MK in cytoplasm of all investigated PDAC cell527lines, whereas no nuclear staining was observed. Moreover,528we separated subcellular fractions of cultured cells and used529specific compartment markers that corroborate our findings.530This is in contrast with previously shown observations in531which MK was found primarily localized to nucleus and532nucleolus in HepG2 cells; the latter is a known hotspot for533rRNA synthesis. The authors claimed that MK might534interfere with rRNA transcription and ribosomal assembly535and ultimately with increased protein synthesis in vitro (38).536Therefore, MK intracellular distribution may be cell con-537text–dependent and need further attention in future studies.538The primary goal of this study was to gain relevant insight539into mechanisms governingMK expression in PDAC and to540further identify relevant new target structures that might541interfere with MK expression. Recently, it was shown that542growth factors EGF and TNF-a induced expression and543secretion of MK in prostate cancer cells in vitro. Of all the544cytokines and growth factors tested, TNF-a was the stron-545gest inducer of MK expression in LNCaP cells. The authors546claimed that MK expression is inducible by the NF-kB547signaling pathway and elevated MK may partially inhibit548TNF-a–induced apoptosis in prostate cancer cells (23). In549line with this, our investigations revealed that TNF-a may550also induceMK expression and secretion in all tested PDAC551cell lines. Moreover, treatment with EGF resulted again in552robust MK expression and secretion levels in an equal553manner, suggesting that MK expression is in fact primarily554inducible by TNF-a and EGF in PDAC. Because retinoic555acid seems to be the main inductor of MK expression during556mouse embryogenesis (39), our analyses showed almost no557influence on MK expression in PDAC cells in vitro.558There are accumulating evidences in the literature reflect-559ing that cellular MK act as a multifunctional growth factor560whose expression is highly linked to proliferation, migration,561survival, angiogenesis, and EMT in various neoplasms.562Interestingly, several studies have already discussed the

Relevance of MK in Pancreatic Cancer

www.aacrjournals.org Mol Cancer Res; 2014 9

565 possibility of successfully neutralizing MK by using anti-566 bodies, antisense RNA, or aptamers and thereby blocking567 tumor growth or reversing chemotherapeutic resistance (9).568 Presumably, in case of PDAC, the appliance of TNF-a569 antagonists such as infliximab might be conceivable to570 counteract elevatedMK expression in general or even during571 chemotherapeutical treatment modalities. Another strategy572 might be the utilization of tyrosine kinase inhibitors such as573 sunitinib and axitinib that have been designed to counteract574 angiogenesis. This might also reflect a new strategy to575 interruptMK-induced angiogenesis in salivary gland tumors576 (40), in which inhibition of angiogenesis may limit metas-577 tasis formation, which is, especially for PDAC, a very early578 and challenging clinical event and the primary cause of579 cancer-related death worldwide.580 In conclusion, our study showed to our best knowledge for581 the first time that MK is, beyond PDAC, frequently upre-582 gulated in other histologic and neoplastic subtypes of the583 pancreas like neuroendocrine tumors or papillary adenocar-584 cinoma. TheMK expression is robustly inducible by TNF-a585 and EGF and high expression levels are strongly linked to the586 proliferative and migrative potential of PDAC cells in vitro.587 Therefore, it is of great interest to investigate how MK may588 influence proliferation, metastasis formation, and even che-589 motherapeutic resistance of PDAC cells in a mouse model590 and how currently available treatment modalities might591 circumvent MK expression to gain prospectively a benefit592 for patients suffering from this deadly disease.

594Disclosure of Potential Conflicts of Interest Q9

595No potential conflicts of interest were disclosed.

596Authors' Contributions597Conception and design: T. Rawnaq, Q10Y.K. Vashist, K. Bachmann, J.R. Izbicki,598M. Bockhorn, C. G€ung€or599Development of methodology: T. Rawnaq, L. Dietrich, C. G€ung€or600Acquisition of data (provided animals, acquired and managed patients, provided601facilities, etc.): T. Rawnaq, L. Dietrich, F.G. Uzunoglu, Y.K. Vashist, R. Simon, J.R.602Izbicki, C. G€ung€or603Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-604tational analysis): T. Rawnaq, G. Wolters-Eisfeld, Y.K. Vashist, K. Bachmann,605C. G€ung€or606Writing, review, and/or revision of themanuscript:T. Rawnaq, G.Wolters-Eisfeld,607F.G. Uzunoglu, Y.K. Vashist, K. Bachmann, R. Simon, J.R. Izbicki, M. Bockhorn,608C. G€ung€or609Administrative, technical, or material support (i.e., reporting or organizing data,610constructing databases): T. Rawnaq, F.G. Uzunoglu, C. G€ung€or611Study supervision: T. Rawnaq, J.R. Izbicki, M. Bockhorn, C. G€ung€or

612Acknowledgments613The authors thank U. Eicke-Kohlmorgen, A. Heinecke, P. Merkert, and P.614Schr€oder for excellent technical assistance. The authors also thank Prof. Sebens615(University Kiel, Germany) for providing HPDE cells.

616Grant Support617This work was in part supported by the Roggenbuck Foundation (053/103) to618C.G€ung€or.619The costs of publication of this article were defrayed in part by the payment of page620charges. This article must therefore be herebymarked advertisement in accordance with62118 U.S.C. Section 1734 solely to indicate this fact.

622Received September 3, 2013; revised February 10, 2014; accepted February 10,6232014; published OnlineFirst xx xx, xxxx.

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Relevance of MK in Pancreatic Cancer

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Tamina Rawnaq

Luisa Dietrich

Gerrit Wolters-Eisfeld

Faik G. Uzunoglu

Yogesh K. Vashist

Kai Bachmann

Ronald Simon

Jakob R. Izbicki

Maximilian Bockhorn

Cenap G€ung€or