pancreatic cancer derived exosomes cause paraneoplastic b ... · pancreatic cancer patient blood...

Biology of Human Tumors

Pancreatic Cancer–Derived Exosomes CauseParaneoplastic b-cell DysfunctionNaureen Javeed1, Gunisha Sagar1, Shamit K. Dutta1, Thomas C. Smyrk2, Julie S. Lau1,Santanu Bhattacharya1, Mark Truty3, Gloria M. Petersen4, Randal J. Kaufman5,Suresh T. Chari6, and Debabrata Mukhopadhyay1

Abstract

Purpose: Pancreatic cancer frequently causes diabetes. Werecently proposed adrenomedullin as a candidate mediator ofpancreatic b-cell dysfunction in pancreatic cancer. How pan-creatic cancer–derived adrenomedullin reaches b cells remotefrom the cancer to induce b-cell dysfunction is unknown. Wetested a novel hypothesis that pancreatic cancer sheds adre-nomedullin-containing exosomes into circulation, which aretransported to b cells and impair insulin secretion.

Experimental Methods: We characterized exosomes fromconditioned media of pancreatic cancer cell lines (n ¼ 5) andportal/peripheral venous blood of patients with pancreaticcancer (n ¼ 20). Western blot analysis showed the presenceof adrenomedullin in pancreatic cancer-exosomes. We deter-mined the effect of adrenomedullin-containing pancreatic can-cer exosomes on insulin secretion from INS-1 b cells andhuman islets, and demonstrated the mechanism of exosomeinternalization into b cells. We studied the interaction betweenb-cell adrenomedullin receptors and adrenomedullin presentin pancreatic cancer-exosomes. In addition, the effect of adre-nomedullin on endoplasmic reticulum (ER) stress response

genes and reactive oxygen/nitrogen species generation in bcells was shown.

Results: Exosomes were found to be the predominant extra-cellular vesicles secreted by pancreatic cancer into culturemedia and patient plasma. Pancreatic cancer-exosomes con-tained adrenomedullin and CA19-9, readily entered b cellsthrough caveolin-mediated endocytosis or macropinocytosis,and inhibited insulin secretion. Adrenomedullin in pancreaticcancer exosomes interacted with its receptor on b cells.Adrenomedullin receptor blockade abrogated the inhibitoryeffect of exosomes on insulin secretion. b cells exposed toadrenomedullin or pancreatic cancer exosomes showed upre-gulation of ER stress genes and increased reactive oxygen/nitrogen species.

Conclusions: Pancreatic cancer causes paraneoplastic b-celldysfunction by shedding adrenomedullinþ/CA19-9þ exosomesinto circulation that inhibit insulin secretion, likely throughadrenomedullin-induced ER stress and failure of the unfoldedprotein response. Clin Cancer Res; 21(7); 1722–33. �2014 AACR.

See related commentary by Korc, p. 1508

IntroductionNearly 85% of pancreatic cancer patients have hyperglycemia

and themajority (45%–67%)have diabetesmellitus (1, 2), whichis frequently new onset (75%), i.e., less than 36 months in

duration (1, 3, 4). The mechanism of pancreatic cancer–induceddiabetes mellitus (PC-DM) remains unclear. Understanding thepathogenesis and mediators of PC-DM could lead to identifica-tion of novel biomarkers for early diagnosis of the cancer (5, 6).

Although theAmericanDiabetesAssociation classifiesPC-DMasa form of pancreatogenous diabetes mellitus (7), its mechanismremains unclear. PC-DM and type II diabetes mellitus share anumber of features including risk factors (1), presence of markedinsulin resistance, andhighplasma insulin levels (1, 2, 8, 10). Thesefeatures also distinguish PC-DM from chronic pancreatitis-relateddiabetes mellitus which is associated with decreased insulin levelssecondary to low b-cell mass. Unlike type II diabetes mellitus,which is ameliorated byweight loss, PC-DMdevelops in the face ofongoing, often profound, weight loss (11). Finally, in pancreaticcancer, new-onset diabetes mellitus paradoxically resolves inmorethan 50% of patients after pancreatico-duodenectomy (1). Amy-loid deposits in islets, a histologic hallmark of type II diabetesmellitus and a major contributor to b-cell dysfunction and loss(12–14) in type II diabetesmellitus, are absent in PC-DM (15, 16).Thus, PC-DM appears to be distinct from both type II diabetesmellitus and other forms of pancreatogenous diabetes mellitus.

The very high prevalence of new-onset diabetes mellitus inpancreatic cancer suggests a pancreatic cancer–specific mediatorthat dysregulates glucose homeostasis (6). We have previouslyshown that pancreatic cancer cell line–conditioned media induce

1Department of Biochemistry and Molecular Biology, Mayo Clinic,Rochester, Minnesota. 2Department of Laboratory Medicine andPathology, Mayo Clinic, Rochester, Minnesota. 3Department of Sur-gery, Mayo Clinic, Rochester, Minnesota. 4Department of HealthSciences Research, Mayo Clinic, Rochester, Minnesota. 5DegenerativeDisease Research Program, Sanford BurnhamMedical Research Insti-tute, La Jolla, California. 6Division of Gastroenterology and Hepatol-ogy, Mayo Clinic, Rochester, Minnesota.

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

S.T. Chari and D. Mukhopadhyay contributed equally to this article.

Corresponding Authors: Debabrata Mukhopadhyay, Department of Biochem-istry and Molecular Biology, Gugg 1321C, Mayo Clinic, 200 First Street SW,Rochester, MN 55905. Phone: 507-778-3581; Fax: 507-293-1058; E-mail:[email protected]; and Suresh T. Chari, Division of Gastro-enterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN55905. Phone: 507-255-6028; Fax: 507-284-5486; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-2022

�2014 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 21(7) April 1, 20151722

on September 8, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 29, 2014; DOI: 10.1158/1078-0432.CCR-14-2022





http://clincancerres.aacrjournals.org/



b-cell dysfunction in both the rat insulinoma b-cell line (INS-1)and mouse islets (17). In previous studies, glucose intolerancecould be induced in mice homozygous for the scid mutation byrepeated intraperitoneal injection of pancreatic cancer–condi-tioned media (18) or by xenografting human pancreatic cancer(17). The weight of epidemiologic, clinical, and laboratory evi-dence suggests that PC-DM is a paraneoplastic phenomenoncaused by tumor-secreted factors (6).

In recent years, membrane-derived extracellular vesicles haveemerged as important conduits for cell-to-cell communication(19). Both normal and malignant cells shed microparticles ofvarying sizes into the surrounding extracellular space (20). Extra-cellular vesicles contain components of the cells' membrane andcytoplasm, which affect recipient cells by transferring their bio-logically active cargoes (20). Extracellular vesicles are produced byat least three distinct mechanisms: inward or reverse budding ofmultivesicular bodies leading to formation of nanoparticle-sized(30–100 nm) exosomes, outwardmembrane blebbing producing100 to 1,000 nm sized microvesicles, and outward blebbing ofapoptotic cell membranes producing large (500–2,000 nm) apo-ptotic bodies (21). The morphologic and functional character-istics of extracellular vesicles shed by pancreatic cancer have notbeen previously delineated. Here, we show that the majority ofextracellular vesicles isolated from pancreatic cancer cell line–conditioned media and in the plasma of patients with pancreaticcancer are exosomes. This finding led us to study the in vitrobiologic action of exosomes shed by pancreatic cancer on insulinsecretion. We hypothesize that b-cell dysfunction in pancreaticcancer ismediated by exosomes carrying b-cell toxic cargo that areshed into blood circulation.

Microarray analysis of commercially available cell lines thatcaused b-cell dysfunction led us to identify adrenomedullin as acandidate mediator of PC-DM (17). Adrenomedullin is the mosthighly expressed gene in pancreatic cancer cell lines grown underharsh conditions of low glucose and low oxygen (22). Althoughthis ubiquitous polypeptide inhibits insulin secretion, adreno-medullin has no known physiologic role in glucose metabolism(23). We showed that inhibition of insulin secretion by condi-tioned media from pancreatic cancer cell lines is abrogated byinhibiting adrenomedullin expression using shRNA (17). How-ever, how adrenomedullin is transported from pancreatic cancer

to b cells and the molecular mechanism of adrenomedullin-induced b-cell dysfunction are unknown.

In this study, we show that the predominant extracellularvesicles shed by pancreatic cancer, not only in conditioned mediabut also in peripheral and portal venous blood of patients, areexosomes. We show that pancreatic cancer exosomes (PC-Exo),containing adrenomedullin andCA19-9, readily enter INS-1b cellsand human islets by both caveolin-mediated endocytosis andmacropinocytosis, and inhibit insulin secretion. We demonstratethat pancreatic cancer exosomal adrenomedullin interacts withadrenomedullin receptors [ADMR; calcitonin receptor–like recep-tors (CRLRs)] on b cells with the inhibitory effect of PC-Exo oninsulin secretionbeing reversedbyadrenomedullin receptor block-ade. Finally, we show that adrenomedullin causes upregulation ofgenes associated with endoplasmic reticular (ER) stress (Bip,Chop)which promotes increased ROS/RNS production, due to failure ofthe unfolded protein response (UPR) leading to increased b-celldeath. Adrenomedullin receptor blockade abolished these effects.Taken together, our studies provide insight into a novel molecularmechanism of paraneoplastic diabetes in pancreatic cancer.

Materials and MethodsStudies pertaining to human subjects, including the study of

exosomes in human plasma, were approved by the Mayo ClinicInstitutional Review Board.

Cell cultureINS-1 cells were cultured in RPMI medium containing 10%

FBS, 10 mmol/L HEPES, 2 mmol/L L-glutamine, 1 mmol/Lsodium pyruvate, and 50 mmol/L b-mercaptoethanol. Primarypancreatic cancer patient–derived xenograft cell lines developedin the Mukhopadhyay laboratory were cultured in DMEM/F-12medium supplemented with 10% FBS and 1% antibiotic–anti-mycotic solution (Life Technologies) on plates coatedwith rat tailcollagen type I. Whole human islets purchased from ProdoLaboratories were cultured for 48 hours in islet completemedium(Prodo Laboratories) before studies on glucose responsiveness.

Isolation of exosomesExosomes were isolated by differential centrifugation of con-

ditionedmedia collected from human umbilical vein endothelialcells (HUVEC), human pancreatic ductal epithelial (HPDE),PANC-1, primary patient-derived xenograft cell lines, or humanplasma. Cells were grown in their respectivemedia to 70% to 80%confluency. The medium was then replaced with medium con-taining FBS deprived of microparticles by differential centrifuga-tion (60minutes at 100,000� g). After a 72-hour incubation, theconditioned media were initially cleared of cellular debris/deadcells with two sequential spins at 3,000 rpm for 10minutes at 4�C.The resulting supernatants were then spun at 100,000 � g for 70minutes at 4�C. The exosome pellet was washed with 1� PBSsolution and centrifuged again at 100,000� g for 70minutes. Thefinal exosome pellet was resuspended in 1� PBS. To isolateexosomes from peripheral or portal blood, samples were centri-fuged at 3,000 rpm for 10minutes to separate the plasma from thered blood cells. The collected plasma was centrifuged again at3,000 rpm for 10 minutes and then isolated with high-speedcentrifugation, as described above. The resulting exosome pelletwas washed and resuspended as described. Protein quantificationof exosome preparations was determined by the BicinchoninicAcid Assay (Pierce).

Translational Relevance

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and is projected to moveto the secondpositionby the year 2015. Pancreatic cancer has adismal prognosis (�5% 5-year survival) mainly because 85%are diagnosed when the cancer is already at an advanced stage.New development of diabetes is the only harbinger of pan-creatic cancer before cancer symptoms occur as it develops innearly 50% of patients with pancreatic cancer an average of12 months before the development of cancer symptoms. Ourprevious work strongly suggests that the new diabetes associ-atedwithpancreatic cancer is causedby cancer-secreted factors.In this current study, we looked at pancreatic cancer exosomesas mediators of b-cell dysfunction, which can help to under-stand the mechanism and mediators of pancreatic cancer-induced diabetes, leading to early diagnosis of the cancer.

Pancreatic Cancer Exosomes Cause Paraneoplastic Diabetes

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1723




NanoTracker analysisIsolated exosome fractions frompancreatic cancer cell lines and

pancreatic cancer patient blood plasma were analyzed on theNanoSight NS300. Fractions were diluted accordingly and 5 60-second movies were taken for each sample. Analysis of the datawas done using the software supplied with the machine. Graph-ical analysis shows particle size distribution of the microparticlesin each fraction, and a concentration was reported as particlesper milliliter.

Western blot analysisExosomes were isolated by the procedure mentioned above

fromHUVEC,HPDE, PANC-1, four primary pancreatic cancer celllines, and peripheral and portal venous pancreatic cancer bloodsamples. After isolation, exosomes were quantified by Bicincho-ninic Acid Assay (Pierce), and tergitol-type NP-40 (Boston Bio-Products) was added to 20mg of exosomes from each preparation.Samples were run on a Western blot according to standard

protocols, and probed with CA19-9 antibody (Abcam), or adre-nomedullin antibody (Phoenix Pharmaceuticals) and either Alix(Cell Signaling Technology) or TSG101 (Abcam) to detect exo-somes in the fractions.

For insulin and proinsulin Western blotting, INS-1 cells weregrown in a 6-well plate to approximately 70% confluency. Themedia were replaced with media containing varying concentra-tions of adrenomedullin peptide and incubated for 48 hours.Cells were glucose stimulated for 4 to 6 hours and cell lysates wereisolated using Tergitol-type NP-40 (Boston BioProducts). Twentymicrograms of proteinwas loaded and blots were probedwith theinsulin antibody (Santa Cruz Biotechnology Inc.) or proinsulinantibody (Novus Biologicals) with b-actin (Sigma-Aldrich) as theloading control.

Electron microscopic imaging of exosomesExosomes suspended in water from the PANC-1 cell line were

resuspended in Trump fixative solution [4% formaldehyde: 1%

Figure 1.Pancreatic cancer sheds CA19-9 and adrenomedullin (AM)-containing exosomes that readily enter b cells. A, NanoTracker analysis (average of 5 60-secondmovies)of size distribution of microparticles isolated from conditioned medium of a pancreatic cancer patient–derived cell line (left), PANC-1 cell line (center), andplasma fromapancreatic cancer patient (right). Redbars indicate�1 SEM. B,Western blot analysis for CA19-9 in 15mgof exosomes fromHUVEC andHPDE (controls),and pancreatic cancer patient plasma (PC1–5). C andD,Western blot analysis probing for adrenomedullin in 20 mg of exosomes. C, PC-Exo isolated fromPANC-1 andHPDE cell lines and PC1-PC4 (pancreatic cancer patient–derived xenograft cell lines). D, PC-Exo isolated from peripheral venous blood (pev) or portal venousblood (pov) of patients with pancreatic cancer. E, electron microscopy of exosomes isolated from PANC-1 cell line. Scale bar, 200 mm. F, confocal microscopy aftercoincubation of PKH67 PANC-1-dyed exosomes with human islets for 48 hours. Scale bar, 50 mm.

Javeed et al.

Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research1724




glutaraldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.2)].Multiple rinses occurred in this sequence: 0.1 mol/L sodiumphosphate buffer (pH 7.2), 1% osmium tetroxide in 0.1 mol/Lsodium phosphate buffer, distilled water, 2% uranyl acetate,distilled water, ethanol, and 100% acetone. Exosomes were thenplaced on a transmission electron microscopy grid, and imageswere taken using a Philips Tecnai T12 transmission electronmicroscope.

Exosome internalization and confocal analysisExosomes were isolated from the PANC-1 cell line and dyed

with greenfluorescent linker PKH67 (Sigma-Aldrich) according tothe manufacturer's protocol. Fifty micrograms of PKH67-dyedexosomes were incubated with cultured human islets in a 35-mmculture dish (culture conditions as described above). Confocalmicroscopy images were obtained after 24 and 48 hours using aZeiss LSM 780 confocal microscope.

Insulin secretion assayINS-1 or human islet medium was replaced with micropar-

ticle-free medium containing no glucose. Fifty micrograms ofexosomes were incubated with the cells for 24 hours to allowuptake. Cells were glucose stimulated with 15 mmol/L glucose

(INS-1) or 16.7 mmol/L glucose (islets) for 4 to 6 hours. For thedose–response curve, varying amounts of PC-Exo were incu-bated with INS-1 cells for 24 hours. Cells were stimulated with15 mmol/L glucose for 4 to 6 hours. The supernatants werecollected and assayed using a rat insulin ELISA Kit (CrystalChem), and insulin values were normalized to total proteincontent. Protein samples were quantified using the Bicincho-ninic Acid Assay (Pierce).

Duolink assay systemINS-1 cells were treated with varying amounts of PKH67-dyed

(as per the manufacturer's protocol; Sigma-Aldrich) PC-Exo orPC-Exo with 10 nmol/L adrenomedullin inhibitor (AM 22-52;AnaSpec, Inc.) for 24 hours. Cells were washed with 1� PBS andfixed with 4% paraformaldehyde. Duolink in situ kits were pur-chased from Olink Biosciences. Antibodies for adrenomedullin(Phoenix Pharmaceuticals) andCRLR (SantaCruzBiotechnology,Inc.) were used for assessing adrenomedullin/ADMR interactions.Antibodies for Bip (Abcam) and proinsulin (Novus Biologicals)were used to detect Bip/proinsulin in the presence of increasingPC-Exo. Conjugation of proximity ligation assay (PLA) probes,rolling circle amplification, and detection procedures were doneaccording to themanufacturer's instructions. Cells were mounted

200

150

100

50

0

200

150

100

50

0

500

400

300

200

100

0

200

150

100

50

0

150

100

50

0

150

100

50

0

Nor

mal

ized

insu

lin s

ecre

tion

Nor

mal

ized

insu

lin s

ecre

tion

Nor

mal

ized

insu

lin s

ecre

tion

Nor

mal

ized

insu

lin s

ecre

tion

Nor

mal

ized

insu

lin s

ecre

tion

Nor

mal

ized

insu

lin s

ecre

tion

A B C

D E F

−Exo

−Exo

Islet

only

+Exo

+Exo

+Exo

−Exo

+Exo

+Exo

+AM

inhib

+Exo

+AM

inhib −E

xo −Exo

12.5

µg25

µg50

µg

100

µg

200

µg

+Exo

+Exo

+AM

inhib

+Exo

+AM

inhib

+Exo

+AM

inhib

Exo-fr

ee m

edia

Exo-fr

ee m

edia

Exo-fr

ee m

edia

Figure 2.PC-Exo decrease insulin secretion with the effect abrogated by ADMR blockade. Glucose-stimulated insulin secretion in INS-1 cells (A, C–E) and humanislets (B) coincubated with PC-Exo (þExo) compared with control culture medium (�Exo), PC-Exo with ADMR inhibitor [þExo þ adrenomedullin (AM) inhib] andpancreatic cancer–conditioned medium depleted of exosomes (Exo-free medium). Exosomes were isolated from a pancreatic cancer patient–derived cell line(A and B), PANC-1 (C), and plasma from two pancreatic cancer patients (D and E). F, INS-1 cell response to insulin secretion by PC-Exo in a dose-dependent manner.� , P < 0.05; �� , P < 0.001; �� , P < 0.0001.






using 40,6-diamidino-2-phenylindole (DAPI)-containing mount-ing media (Vector Labs). Images were taken on a Zeiss LSM 780confocal microscope.

Pharmacologic inhibition and quantification of exosomeinternalization

INS-1 cells were grown in 4-well chamber slides (800,000 cells/well) with optimal treatment concentrations and timings adaptedfrom Bhattacharya and colleagues (ref. 24; Supplementary TableS1). After treatment, cells were washed three times with 1� PBSand MV-free media were added to the cells with the addition of5 mL PKH67-dyed PANC-1 exosomes. Exosomes were incubatedwith cells for 5 hours to allow internalization, then washed with1� PBS two times, fixed with 4% PFA, and mounted with DAPI-containing mounting media (Vector Labs). Images were takenusing a Zeiss LSM 780 confocal microscope. Quantification ofgreen fluorescence (exosome) internalization was performedusing KS400 Image Analysis software (Zeiss). For each image,the nuclei were counted and the total green fluorescence for theentire imagewas divided by the nuclei (cell) count to obtain greenfluorescence per cell.

Real-time PCRINS-1 cells were treated with varying concentrations of adre-

nomedullin peptide (Phoenix Pharmaceuticals) or PC-Exo(50 mg) for 24 or 48 hours. Total RNA was isolated using theRNeasy Kit (Qiagen), and cDNAwas reverse transcribed from1 mgof RNAusing the iScript cDNA kit (Bio-Rad). RT-PCR analysis wasperformed using the ABI 7500 Real-Time PCR System and theSYBRGreenMaster Mix (Applied Biosystems). Primers for insulin

and ER stressmarkers (Bip, Chop, and Xbp-1s)were adapted fromLipson and colleagues (25).

Annexin V stainingINS-1 cells were plated in a 6-well plate (4 � 106 cells/well) in

microparticle-free medium. Fifty micrograms of PC-Exo wereincubated with the cells for 48 hours. Cells were trypsinized,collected, and resuspended in 1� binding buffer supplied withthe Annexin V-FITC Apoptosis kit (BioVision, Inc). Annexin V–propidium iodide staining was completed according to the man-ufacturer's protocol. Flow cytometry analysis was completed todetect early and late apoptotic cells.

Cellular ROS/superoxide detectionINS-1 cellswere seeded in 4-well chamber slides (800,000 cells/

well). Cells were pretreated with 1 or 20 pmol/L adrenomedullinpeptide for 24 hours, then glucose stimulated for 4 hours with 15mmol/L glucose. Cellular ROS/Superoxide Detection Assay Kit(Abcam) was used for ROS/RNS detection. Treatment of cells fordetection was adapted by this protocol including for the positiveand negative controls. Cells were then washed in 1� wash buffer(supplied with kit), and mounted with DAPI-containing mount-ing media. Images were obtained using a Zeiss LSM 780 confocalmicroscope.

Statistical analysisStatistical comparisons between 2 groups were performed

using a two-sample t test. Comparisons across more than twogroups were performed using ANOVA with a Dunnett posttest tocompare all columns to the control. All analyses were conducted

AM/CRLR PKH67 DAPI Merge AM/CRLR PKH67 DAPI Merge

−−Exo −Exo + I

5 μL 5 μL + I

10 μL + I

15 μL + I

10 μL

15 μL

A B

Figure 3.PC-Exo deliver adrenomedullin (AM) to INS-1 cells and increase adrenomedullin/ADMR interaction with the effect abrogated by ADMR blockade. A and B,the Duolink Assay System was used to assess the interaction of adrenomedullin/ADMR in INS-1 cells treated with increasing amounts (0, 5, 10, or 15 mL)of PKH67-dyed PC-Exo in the absence (A) and presence (B) of adrenomedullin 22-52, an ADMR antagonist. Scale bar, 10 mm.

Javeed et al.





using GraphPad software. P values less than 0.05 were consideredstatistically significant.

ResultsExtracellular vesicles in pancreatic cancer are predominantlyexosomes

We isolated all microparticles (microvesicles and exosomes)shed by pancreatic cancer using differential centrifugation. Withthe NanoSight NS300 system (Malvern Instruments Ltd), wedetermined that the predominant microparticles in pancreaticcancer cell lines andpatient plasmawere of exosomal size (modes,�100 nm; Fig. 1A; Supplementary Fig. S1). However, this did nothold true for all pancreatic cancer cell lines, as exemplified inSupplementary Fig. S1B, where the majority of the microparticleswere ofmicrovesicular size. In addition, scanning electronmicros-copy revealed that microparticles from PANC-1–conditionedmedium are of exosomal size and morphology, thereby confirm-ing the NanoTracker results (Fig. 1E).

PC-Exo readily enter human isletsHuman islets were incubated with PKH67-dyed exosomes

isolated from the PANC-1 cell line. Using confocal microscopy,we identified PKH67-dyed exosome internalization into isletswithin 48 hours of coincubation (Fig. 1F).

Exosomes from pancreatic cancer–conditioned media andplasma of pancreatic cancer patients contain CA19-9 andadrenomedullin

Western blot analysis revealed the presence of CA19-9 in allpancreatic cancer patient exosome samples (n¼ 11) with TSG101serving as a marker of exosomes (Fig. 1B; five representativesamples). In contrast, exosomes isolated from normal pancreaticcell lines HPDE and HUVEC showed no CA19-9 and TSG101expression (Fig. 1B). This suggests that the content and amount ofnormal exosomes being produced differ in comparison withtumor-derived exosomes and that isolated exosome fractionsfrom pancreatic cancer patient plasma are in fact derived fromthe pancreatic tumor. We next looked for the presence of adre-nomedullin throughWestern blotting in exosomes shedbyHPDE(control cell line), PANC-1, and 4 pancreatic cancer patient–derived xenograft cell lines. All revealed the presence of adreno-medullin; however, exosomes from the HPDE cell line may beproduced less because of the lack of TSG101 expression found in20 mg of exosomes (Fig. 1C). Western blotting of exosomesisolated from peripheral (n ¼ 18) and portal venous (n ¼ 2)blood of 20 patients with pancreatic cancer showed the presenceof adrenomedullin (Fig. 1D; representative samples shown).Although exosome bioavailability and t1/2 studies were not con-ducted to determine the rate of PC-Exo uptake in peripheralorgans, this result suggests that a steady production of PC-Exois secreted from the tumor into circulation which contains met-abolically active cargoes.

PC-Exo inhibit insulin secretionIn a recent study, we reported that conditioned media from

various pancreatic cancer cell lines decreased insulin secretionin both a rat INS-1 b-cell line and mouse islets (17). Wehypothesized that the inhibitory effect of conditioned mediaresided in exosomes. The addition of PC-Exo from a patient-derived pancreatic cancer cell line showed a decrease in insulinsecretion in both INS-1 cells and human islets (Fig. 2A and B).

In addition, PC-Exo isolated from the PANC-1 cell line and 2pancreatic cancer patient plasma samples showed a similareffect of decreasing insulin secretion from b cells (Fig. 2C–E).Adrenomedullin receptor abrogation blocked the insulin inhib-itory effect of PC-Exo with values exceeding past the baselinedue to the blocking of endogenous (free) adrenomedullin toADMRs (Fig. 2A–E). In addition, exosome-depleted media hadno effect on insulin secretion, confirming that the inhibitoryeffect is caused by exosomes and not any other soluble factor(s)secreted into the media (Fig. 2A–C). In addition, this insulininhibitory effect was seen in PC-Exo isolated from additionalpancreatic cancer cell lines and plasma of patients with pan-creatic cancer (Supplementary Fig. S2). Finally, increasing con-centrations of PC-Exo isolated from pancreatic cancer patientplasma were incubated with INS-1 cells for 48 hours. Theresults showed that PC-Exo can inhibit insulin secretion in adose-dependent manner (Fig. 2F).

PC-Exosomal adrenomedullin binds to cell surface receptorson b cells to inhibit insulin secretion

The Duolink Assay System (Olink Biosciences) was used toassess the interaction between exosomal adrenomedullin and itsreceptor ADMR. This method allows for determination of pro-tein–protein interactions by using specialized fluorescent PLAprobes that attach to the primary antibodies for adrenomedullinand the ADMR. ADMRs require activation of both co-receptorsCRLR and either RAMP2or 3; therefore, we chose to use a primaryantibody for CRLR as this co-receptor is consistently activatedeach time there is an adrenomedullin/ADMR interaction. Rollingcircle amplification amplifies this region, and punctate dotsindicate ligand–receptor interactions. INS-1 cells were incubatedfor 24 hours with increasing PKH67-dyed PC-Exo. Confocalmicroscopy was used to visualize adrenomedullin/ADMR inter-actions with increasing PC-Exo as indicated by the diffuse cyto-plasmic red dots (Fig. 3A). In addition, quantification of theinteractions revealed an overall increase in adrenomedullin/ADMR interactions with increasing addition of PC-Exo (data notshown). Conversely, ADMR inhibition with AM 22-52 in thepresence of PC-Exo decreased adrenomedullin/ADMR interac-tions, as indicated by less punctate red staining (Fig. 3B). Thesedata indicate that PC-Exo containing adrenomedullin are able toshuttle the peptide efficiently to recipient cells and that compet-itively inhibiting adrenomedullin in the presence of PC-Exoresults in less adrenomedullin/ADMR interaction.

Exosome internalization is mediated through caveolin-dependent endocytosis and macropinocytosis

The results from the Duolink Assay suggest that adrenomedul-lin/ADMR interactions occur intracellularly as indicated by thediffuse red cytoplasmic dots (Fig. 3). We sought to gain anunderstanding of the mechanism of exosome entry and subse-quent adrenomedullin delivery to the cell. Pharmacologic inhi-bition of various endocytic mechanisms was used to determinethe exact method of exosome internalization. Chlorpromazine(clathrin inhibitor), nystatin (caveolin inhibitor), amiloride(inhibitor of macropinocytosis), and nocodazole (inhibitorof endosomal trafficking) were tested in INS-1 cells using theconcentrations and incubation timings listed in SupplementaryTable S1. The internalization of PKH67-dyed PC-Exo (green) wassignificantly inhibited with amiloride and nystatin treatmentsas indicated by less green fluorescence (Fig. 4A and D).






PKH67 DAPI Merge

PKH67 DAPI Merge

Control

25 μμmol/Lamiloride

50 μmol/Lamiloride

25 μg/mLnystatin

50 μg/mLnystatin

100 μmol/Lamiloride

Control 100 μmol/L amiloride

Control 50 μg/mL nystatin

Control

Gre

en f

luo

resc

ence

/cel

l 30,000

20,000

10,000

0

Gre

en f

luo

resc

ence

/cel

l 30,000

20,000

10,000

0

Control

25 μ

mol/L

50 μ

mol/L

100 μm

ol/L

Control

25 μ

g/mL

50 μ

g/mL

A

B

C

D E

F

Javeed et al.





Quantification of green fluorescence/cell showed a 42.5%decrease in internalized exosomes with the addition of 100mmol/L amiloride, and 20.5% and 59.5% decreases in exosomeinternalizationwith25mg/mLand50mg/mLnystatin, respectively(Fig. 4C and F). Conversely, varying treatment concentrations ofchlorpromazine and nocodazole did not have an inhibitory effecton internalizing PC-Exo (Supplementary Fig. S3). Taken together,these data show that PC-Exo internalize into INS-1 cells throughboth caveolin-dependent endocytosis and macropinocytosis.

Adrenomedullin deregulates theUPR pathway leading tob-celldysfunction

As in many other target cells, adrenomedullin has beenreported to cause an increase in cAMP levels in islets (26) whichis normally associated with increased insulin secretion (27–29).This is mediated through increases in insulin exocytosis as well asinsulin production (30). Indeed, we found a modest increase ininsulin mRNA in response to adrenomedullin (SupplementaryFig. S4). ER stress is a physiologic b-cell response to increasedinsulin production, while an efficient UPR safely resolves ERstress. However, the question arises whether the paradoxicalinhibition of insulin secretion by adrenomedullin, despite anincrease in cAMP, could be due to an independent adrenome-dullin-induced unresolved ER stress resulting from a failed UPR.

Classical characteristics of a failed UPR response are increasedBip (ER chaperone protein) coupling to proinsulin in the ER, andactivation of Chop (inducer of apoptosis; ref. 31). To assesswhether the presence of adrenomedullin in the cell can activatethese ER stress markers, we looked at Bip and Chop gene expres-sions in the presence of adrenomedullin. INS-1 cells were treatedwith varying concentrations of the adrenomedullin peptide for 48hours and then glucose stimulated for 4 to 6 hours. Real-time(RT)-PCR analysis showed upregulation of ER stress genes Bip andChop, with only modest upregulation of Xbp-1s (ER stress markerinvolved in the normal UPR) with the addition of increasingconcentrations of adrenomedullin (Fig. 5A–C). The addition ofthe adrenomedullin inhibitor (AM 22-52) with varying concen-trations of adrenomedullin peptide caused mRNA levels of allthree genes to subside to endogenous levels (Fig. 5A–C). Themodest increase in Xbp-1s suggests that normal UPR functionmaybe activated slightly in the presence of adrenomedullin; however,the evident upregulation of Bip and Chop in the presence ofadrenomedullin suggests failure of this response. In addition,50 mg of PC-Exo increased both markers (Fig. 5D and E). As ChopmRNA levels were elevated, annexin V staining was done todetermine cellular apoptosis. Fifty micrograms of PC-Exo wereincubated with INS-1 cells for 48 hours; a higher percentage ofcells were observed undergoing early apoptosis compared withthe control (untreated) cells (Fig. 5F).

Another defining characteristic of a failed UPR in b cells is theincreased coupling of proinsulin to Bip in the ER (31). Proinsulinprotein levels were assessed in INS-1 cells with the addition of

varying concentrations of adrenomedullin. Our results showedincreased proinsulin in the presence of increasing adrenomedul-lin (Fig. 5G). In addition, theDuolinkAssay System (as previouslynoted)was utilized to assess the Bip/proinsulin interactions in situwith increasing amounts of PC-Exo. Indeed, INS-1 cells incubatedwith increasing amounts of adrenomedullin-containing PC-Exoshowed increasing Bip/proinsulin interactions as indicated byincreased red punctate dots (Fig. 6A).

Overwhelming ER stress can also induce oxidative stress (31).We therefore examined whether adrenomedullin can invokereactive oxygen/nitrogen species (ROS/RNS) production inINS-1 cells. With the addition of 1 and 20 pmol/L adrenome-dullin, ROS (green filter) and superoxide (orange filter) produc-tion increased compared with control (untreated) cells (Supple-mentary Fig. S5). This collective body of evidence suggests thatadrenomedullin and PC-Exo containing adrenomedullin is capa-ble of deregulating the UPR pathway, which inevitably leads tomarked b-cell dysfunction/death.

DiscussionCompelling clinical, epidemiologic, and experimental evidence

strongly supports the notion that new-onset diabetes mellitus inpancreatic cancer is a paraneoplastic phenomenon caused bytumor-secreted products (7). We observed that pancreatic cancersheds nanoparticle-sized exosomes that are abundantly present inpancreatic cancer–conditionedmedia and in portal and peripheralvenous blood of patients with pancreatic cancer. There is growingevidence that exosomes are important mediators of cell–cell com-munication (20). Their ability to influence adjacent cells andstroma is now increasingly recognized as a critical cellular function.We observed that the PC-Exo derived from in vitro and in vivosources were morphologically and functionally indistinguishableand exosomes from both sources readily entered b cells andinhibited insulin secretion. We also show that the inhibitory effectof pancreatic cancer cell line conditioned media on insulin secre-tion ismediated by exosomes as exosome-freemedia had no effecton insulin secretion. Thus, our studies not only show that exo-somes are the predominant extracellular vesicles shedbypancreaticcancer, both into culture media and plasma, but also demonstratetheir ability to dysregulate insulin secretion.

We have previously proposed adrenomedullin as a candi-date mediator of b-cell dysfunction in pancreatic cancer as itis overexpressed in pancreatic cancer and inhibits insulinsecretion (17). We have also reported increased plasma adre-nomedullin concentrations in patients with PC-DM (22.9 �10.7 fmol/L) compared with patients with pancreatic cancerbut normal fasting glucose (18.3 � 7.0 fmol/L), noncancerpatients with new-onset type II diabetes (14.8 � 10.7 fmol/L),and noncancer subjects with normal fasting glucose levels(12.9 � 6.6 fmol/L; ref. 17). However, because plasma con-centrations were only modestly elevated this raises the

Figure 4.Pharmacologic inhibition of the endocytic pathway blocks exosome uptake into INS-1 cells. A, INS-1 cells were treated with varying concentrations of amiloride (25,50, and 100 mmol/L) for 15 minutes. PANC-1 exosomes were dyed with PKH67 and incubated with the cells after amiloride treatment for 5 hours to allowexosome uptake. Scale bars represent 10 mm. B, magnified images (10.5�) of exosome internalization into INS-1 cells in the presence or absence of 100 mmol/Lamiloride. Scale bar represents 5 mm. C, quantification of green fluorescence/cell in all treatment groups. D, INS-1 cells were treated with 25 mg/mL or50 mg/mL nystatin for 30minutes. PKH67-dyed exosomeswere incubated with the cells for 5 hours to allow internalization. Scale bars represent 10 mm. E,magnifiedimages (10.5�) of cells treated with 50 mg/mL nystatin compared with control (untreated) cells show decreased PKH67-dyed exosome internalization.Scale bar represents 5 mm. F, green fluorescence quantification of internalized exosomes in the presence of absence of nystatin. �� , P < 0.0001.






question of whether adrenomedullin is acting as a hormone.Our current work suggests that adrenomedullin is carried frompancreatic cancer to b cells as exosomal cargo. After Kitamuraand colleagues first reported isolating adrenomedullin fromhuman phaeochromocytoma in 1993 (32), adrenomedullinwas proposed to be a circulating hormone regulating systemicand pulmonary blood pressure (33). In healthy individuals,circulating adrenomedullin levels are in the low picomolar

range. Physiologic increases in adrenomedullin levels are seenduring pregnancy, with the source being the placenta (34). Path-ologic conditions associated with increased adrenomedullinlevels include diabetes, severe hypertension, chronic renal failure,heart failure, pulmonary hypertension, subarachnoid hemor-rhage, sepsis, hyperthyroidism, and during cardiac surgery.

It has been debated whether these elevations simply reflectexcess tissue adrenomedullin production or whether excess

2.5

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0

2.0

1.5

1.0

0.5

0.0

2.0

1.5

1.0

0.5

0.0

15

10

5

0

Rel

ativ

e ge

ne e

xpre

ssio

n

Rel

ativ

e ge

ne e

xpre

ssio

n

Rel

ativ

e ge

ne e

xpre

ssio

n

Rel

ativ

e ge

ne e

xpre

ssio

n

Rel

ativ

e ge

ne e

xpre

ssio

n

% o

f Ann

exin

V+ c

ells

Bip mRNA expression: 48 h

Bip mRNA expression: 48 h

Chop mRNA expression: 48 h

Chop mRNA expression: 48 h

Xbp-1s mRNA expression: 48 h

AM (pmol/L)

Proinsulin

β-Actin

− 1 20 40

−AM

−Exo

+Exo

−Exo

+Exo

−Exo

+Exo

−AM

+1 p

mol/

L AM

+1 p

mol/

L AM

+40

pmol/

L AM

+40

pmol/

L AM

+20

pmol/

L AM

+20

pmol/

L AM

+20

pmol/

L AM

+ In

hib

+20

pmol/

L AM

+ In

hib

+1 p

mol/

L AM

+ In

hib

+1 p

mol/

L AM

+ In

hib

+40

pmol/

L AM

+ In

hib

+40

pmol/

L AM

+ In

hib −AM

+1 p

mol/

L AM

+40

pmol/

L AM

+20

pmol/

L AM

+20

pmol/

L AM

+ In

hib

+1 p

mol/

L AM

+ In

hib

+40

pmol/

L AM

+ In

hib

A B C

D E

F G

Figure 5.Adrenomedullin (AM) and PC-Exosomes increase expression of ER stress markers. A–C, mRNA expression of ER stress markers Bip (A), Chop (B), and Xbp-1s (C) inINS-1 cells after treatment with varying concentrations of adrenomedullin peptide, or peptide þ adrenomedullin inhibitor (AM 22-52). D and E, mRNAexpression of ER stress markers Bip (D) and Chop (E) in INS-1 cells after treatment with 50 mg of PC-Exo (þExo) from a pancreatic cancer patient–derivedcell line compared with control culture medium (�Exo). F, percentage of apoptotic cells determined by annexin V staining of INS-1 cells treated with 50 mg ofpancreatic cancer-Exo (þExo) compared with control (untreated) cells (�Exo). G, Western blot analysis for proinsulin protein levels with the addition ofadrenomedullin peptide (1, 20, or 40 pmol/L adrenomedullin) versus untreated INS-1 cells. � , P < 0.05; �� , P < 0.001; �� , P < 0.0001.

Javeed et al.





circulating adrenomedullin has systemic biologic effects. How-ever, infusion of adrenomedullin into humans (35) at a rate thatquadrupled physiologic levels of adrenomedullin had no effecton heart rate and blood pressure. Extremely high doses thatachieved a concentration over 40 times normal circulating adre-nomedullin levels did significantly decrease blood pressure (35).In another study, high dose adrenomedullin infusion affectedhemodynamic parameters, but not low-dose adrenomedullininfusion (36). Thus, the circulating concentration of adrenome-dullin necessary to affect blood pressure greatly exceeds thatobserved in healthy volunteers and in patients with a range ofphysiologic (notably pregnancy) and pathologic (notably cancer,heart failure, vasculopathies) conditions.

In relation to our work, neither low-dose nor high-doseadrenomedullin infusion had any effect on blood glucose(35). Thus, the elevation of plasma adrenomedullin is unlikelyto be the cause of the biologic effects of adrenomedullin inpancreatic cancer. We are not aware of any mechanism forintrapancreatic transport of molecules through the length of thepancreas to explain global b-cell dysfunction leading to diabe-tes mellitus in pancreatic cancer. Therefore, we studied a novelhypothesis that adrenomedullin was being transported frompancreatic cancer to islets distant from the cancer in exosomesshed into portal circulation and eventually reaching the isletsthrough systemic circulation.

In PC-DM, there are both b-cell dysfunction and increasedinsulin resistance that resolve with tumor resection (1, 4, 8,18, 38). Insulin levels in pancreatic cancer are high due to insulin

resistance. Our data suggest that b-cell dysfunction leads toinadequate insulin response to insulin resistance. We believe itis this dual hit that promotes a consistent destabilization ofglucose homeostasis in pancreatic cancer, resulting in hypergly-cemia. Thus far, the mechanism and mediators of insulin resis-tance in pancreatic cancer remain unknown, and the role of PC-Exo, adrenomedullin, and other factors contributing to insulinresistance needs further study.

Through the use of the Duolink Assay System, we showed thatadrenomedullin contained in PC-Exo can interact with ADMRs(CRLR) intracellularly with the absence of PC-Exo showing littleto no interaction in INS-1 cells (Fig. 3). The exact mechanism ofhow adrenomedullin in PC-Exo interacts with ADMRs is notknown. We hypothesize that PC-Exo internalize along withADMRs in the early endosomes as this location is a potential siteof adrenomedullin release from the exosomes, which leaves thepeptide in close proximity to ADMRs internalized into earlyendosomes (Fig. 6B). Further studies are necessary to confirmthis exact mechanism.

We show that exosomes inhibit insulin secretion and adreno-medullin receptor blockade abrogates the effect of exosomes oninsulin secretion. On the basis of our studies, we propose amechanism for b-cell dysfunction in PC-DM in that the effect ofadrenomedullin and exosomal adrenomedullin is mediated viaupregulation and eventual failure of the UPR due to hyperstim-ulation of the cAMP-dependent pathway. We found hallmarkfeatures of a failed UPR, including increased mRNA expression ofER stress markers Bip and Chop (Fig. 5). We observed increased

Figure 6.Adrenomedullin (AM)-containing PC-Exo can increase Bip/proinsulin interactions in b cells. A, the Duolink Assay System was used to assess Bip/proinsulininteractionswith increasing amounts (0, 5, 10, or 15 mL) of PKH67-dyed PC-Exo. Scale bar represents 10mm. B, the potential mechanism for exosomal adrenomedullinrelease and signaling differs from the conventional mechanism shown in (1) in which adrenomedullin binds to cell surface adrenomedullin receptors (ADMRs)and activates the cAMP-dependent pathway. Instead, PC-Exo internalize through either caveolin-mediated endocytosis or macropinocytosis thus fusing tothe early endosome, which is also the site of endocytosed ADMRs (2). The early endosome/multivesicular body could be another site of adrenomedullin/ADMRinteraction. Once the pathway is activated adenyl cyclase activation of cAMP releases the catalytic subunits of PKA, which translocate to the nucleus andsubsequently activates insulin gene transcription. Excessive exosomal adrenomedullin activates ER stress proteins that regulate degradation of misfolded orunfolded ER proteins. However, overproduction of insulin due to excessive adrenomedullin pathway activation eventually leads to failure of the UPR marked byincreased Bip/proinsulin coupling in the ER, increased ROS/RNS production, and an increase in Chop, an inducer of apoptosis, leading to a decrease in insulinsecretion due to b-cell death.






association of Bip with proinsulin in the presence of increasingamounts of PC-Exo, another classic feature of a failed UPRresponse in b cells (Fig. 6A). We also showed increased b-cellapoptosis after exposure to adrenomedullin, further confirmingChop-mediated apoptosis (Fig. 5F). The upregulation of Bip andChop was abolished by adrenomedullin receptor blockade, sug-gesting that the effects seen were due to adrenomedullin–ADMRinteractions and not a nonspecific toxic effect of adrenomedullinon b cells. We conclude from these data that adrenomedullinleads to excessive activation and subsequent failure of the UPRleading to eventual b-cell death (Fig. 6B). Further work will beneeded to understand themolecularmechanismof the failedUPRin response to exosomal adrenomedullin.

Our studies thus far have focused specifically on the role thatadrenomedullin plays on b-cell dysfunction in PC-DM. However,it is possible that adrenomedullin is not the only mediator ofb-cell dysfunction, but rather it may be working in concert withother key factors, such as tumor necrosis factor a, to promoteb-cell dysfunction (38). Therefore, further studies will investigatewhether these factors are contained in PC-Exo and their potentialinteraction with adrenomedullin. Understanding all the keymodulators that cause exosome-mediated b-cell dysfunction, andthe interplay between them, will give us a more in-depth under-standing of the pathogenesis of PC-DM.

Our long-term programmatic goal is to develop a rational,evidence-based strategy to screen for sporadic pancreatic cancer.Our work has shown that new-onset diabetes mellitus is a har-binger of pancreatic cancer (39). We reported that subjects withnew-onset diabetes mellitus are eight times more likely than thegeneral population to be diagnosed with pancreatic cancer within3 years of meeting the criteria for diabetes mellitus (40). On thebasis of these observations,we are conducting thefirst clinical trialof screening for pancreatic cancer in new-onset diabetes mellitus(NCT02001337). However, what is urgently needed in the field is

a reliable marker to distinguish PC-DM from the more commontype II diabetes mellitus.

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

Authors' ContributionsConception and design: D. Mukhopadhyay, N. Javeed, S. Bhattacharya,S.T. ChariDevelopment of methodology:D. Mukhopadhyay, N. Javeed, S. Bhattacharya,R.J. Kaufman, S.T. ChariAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Javeed, S.K. Dutta, T. Smyrk, J.S. Lau, S. Bhatta-charya, M.J. Truty, G.M. Petersen, S.T. ChariAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Javeed, T. Smyrk, S.T. ChariWriting, review, and/or revision of the manuscript: D. Mukhopadhyay,N. Javeed, T. Smyrk, J.S. Lau, M.J. Truty, G.M. Petersen, R.J. Kaufman, S.T. ChariAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.T. ChariStudy supervision: D. Mukhopadhyay, R.J. Kaufman, S.T. ChariOther (conducted Western blot analyses for the manuscript): G. Sagar

AcknowledgmentsThe authors thank the Pancreatic Cancer SPORE, James Tarara and Kristin

Mantz of the Mayo Clinic Core Microscopy facilities for assistance in confocalmicroscopy, and Drs. Gregory Gores and Petra Hirsova for suggestions andassistance on using the NanoSight NS300.

This work was partly supported by CA78383 and CA150190 (to D. Mukho-padhyay), CA100685 (to S.T. Chari), P50 CA102701 (to G.M. Petersen andS.T. Chari), 1T32 CA148073 (to G. Sagar), and T32 CA148073 and MayoGraduate School (to N. Javeed).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 6, 2014; revised September 25, 2014; accepted October 11,2014; published OnlineFirst October 29, 2014.

References1. Pannala R, Leirness JB, Bamlet WR, Basu A, Petersen GM, Chari ST.

Prevalence and clinical profile of pancreatic cancer-associated diabetesmellitus. Gastroenterology 2008;134:981–7.

2. Permert J, Ihse I, Jorfeldt L, von Schenck H, Arnqvist HJ, Larsson J.Pancreatic cancer is associated with impaired glucose metabolism. Eur JSurg 1993;159:101–7.

3. Chari ST, Klee GG, Miller LJ, Raimondo M, DiMagno EP. Islet amyloidpolypeptide is not a satisfactory marker for detecting pancreatic cancer.Gastroenterology 2001;121:640–5.

4. Chari ST, Leibson CL, Rabe KG, Timmons LJ, Ransom J, de Andrade M,et al. Pancreatic cancer-associated diabetes mellitus: prevalence andtemporal association with diagnosis of cancer. Gastroenterology 2008;134:95–101.

5. Pannala R, Basu A, Petersen GM, Chari ST. New-onset diabetes: a potentialclue to the early diagnosis of pancreatic cancer. Lancet Oncol 2009;10:88–95.

6. Sah RP, Nagpal SJ, Mukhopadhyay D, Chari ST. New insights into pan-creatic cancer-induced paraneoplastic diabetes. Nat Rev GastroenterolHepatol 2013;10:423–33.

7. Diagnosis and classification of diabetes mellitus. Diabetes Care 2012;35Suppl 1:S64–71.

8. Chari ST, Zapiach M, Yadav D, Rizza RA. Beta-cell function andinsulin resistance evaluated by HOMA in pancreatic cancer subjects withvarying degrees of glucose intolerance. Pancreatology 2005;5:229–33.

9. Permert J, Ihse I, Jorfeldt L, von Schenck H, Arnquist HJ, Larsson J.Improved glucosemetabolismafter subtotal pancreatectomy for pancreaticcancer. Br J Surg 1993;80:1047–50.

10. Permert J, Larsson J, Westermark GT, Herrington MK, Christmanson L,Pour PM, et al. Islet amyloid polypeptide in patients with pancreatic cancerand diabetes. N Engl J Med 1994;330:313–8.

11. Hart PA, Kamada P, Rabe KG, Srinivasan S, Basu A, Aggarwal G, et al.Weight loss precedes cancer-specific symptoms in pancreatic cancer-asso-ciated diabetes mellitus. Pancreas 2011;40:768–72.

12. Butler AE, Janson J, Soeller WC, Butler PC. Increased beta-cell apoptosisprevents adaptive increase in beta-cell mass in mouse model of type 2diabetes: evidence for role of islet amyloid formation rather than directaction of amyloid. Diabetes 2003;52:2304–14.

13. Hoppener JW, Ahren B, Lips CJ. Islet amyloid and type 2 diabetes mellitus.N Engl J Med 2000;343:411–9.

14. Hull RL, Westermark GT, Westermark P, Kahn SE. Islet amyloid: a criticalentity in the pathogenesis of type 2 diabetes. J Clin Endocrinol Metab2004;89:3629–43.

15. SarucM, Iki K, Pour PM.Morphometric studies in humanpancreatic cancerargues against the etiological role of type 2 diabetes in pancreatic cancer.Histol Histopathol 2010;25:423–32.

16. Li D. Diabetes and pancreatic cancer. Mol Carcinog 2012;51:64–74.17. Aggarwal G, Ramachandran V, Javeed N, Arumugam T, Dutta S, Klee GG,

et al. Adrenomedullin is up-regulated in patients with pancreatic cancerand causes insulin resistance in beta cells and mice. Gastroenterology2012;143:1510–7.

18. Basso D, Brigato L, Veronesi A, Panozzo MP, Amadori A, Plebani M.The pancreatic cancer cell line MIA PaCa2 produces one or more factorsable to induce hyperglycemia in SCID mice. Anticancer Res 1995;15:2585–8.


Javeed et al.




19. Andaloussi SE, Mager I, Breakefield XO, Wood M. Extracellular vesicles:biology and emerging therapeutic opportunities. Nat Rev Drug Discov2013;12:347–57.

20. Shifrin DA Jr, Demory Beckler M, Coffey RJ, Tyska MJ. Extracellularvesicles: communication, coercion, and conditioning. Mol Biol Cell 2013;24:1253–9.

21. Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles impor-tant in intercellular communication. J Proteomics 2010;73:1907–20.

22. Natsuizaka M, Ozasa M, Darmanin S, Miyamoto M, Kondo S, Kamada S,et al. Synergistic up-regulation of Hexokinase-2, glucose transporters andangiogenic factors in pancreatic cancer cells by glucose deprivation andhypoxia. Exp Cell Res 2007;313:3337–48.

23. Lopez J, Cuesta N. Adrenomedullin as a pancreatic hormone. Microsc ResTech 2002;57:61–75.

24. Bhattacharya S, Roxbury D, Gong X, Mukhopadhyay D, Jagota A. DNAconjugated SWCNTs enter endothelial cells via Rac1 mediated macropi-nocytosis. Nano Lett 2012;12:1826–30.

25. Lipson KL, Fonseca SG, Ishigaki S, Nguyen LX, Foss E, Bortell R, et al.Regulation of insulin biosynthesis in pancreatic beta cells by an endo-plasmic reticulum-resident protein kinase IRE1. Cell Metab 2006;4:245–54.

26. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al.Regulation of insulin secretion and blood glucose metabolism by adre-nomedullin. Endocrinology 1996;137:2626–32.

27. Lemaire K, Schuit F. Integrating insulin secretion and ER stress in pancreaticbeta-cells. Nat Cell Biol 2012; 14:979–81.

28. Takhshid MA, Poyner DR, Chabot JG, Fournier A, Ma W, Zheng WH, et al.Characterization and effects on cAMP accumulation of adrenomedullinand calcitonin gene-related peptide (CGRP) receptors in dissociated ratspinal cord cell culture. Br J Pharmacol 2006;148:459–68.

29. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, et al.International Union of Pharmacology. XXXII. The mammalian calcitoningene-related peptides, adrenomedullin, amylin, and calcitonin receptors.Pharmacol Rev 2002;54:233–46.

30. Knoch KP, Meisterfeld R, Kersting S, Bergert H, Altkruger A, Wegbrod C,et al. cAMP-dependent phosphorylation of PTB1 promotes the expressionof insulin secretory granule proteins in beta cells. Cell Metab 2006;3:123–34.

31. Scheuner D, Kaufman RJ. The unfolded protein response: a pathway thatlinks insulin demand with beta-cell failure and diabetes. Endocr Rev2008;29:317–33.

32. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H,et al. Adrenomedullin: a novel hypotensive peptide isolated from humanpheochromocytoma. Biochem Biophys Res Commun 1993;192:553–60.

33. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, et al.Plasma levels of adrenomedullin, a newly identified hypotensive peptide,in patients with hypertension and renal failure. J Clin Invest 1994;94:2158–61.

34. Senna AA, Zedan M, el-Salam GE, el-Mashad AI. Study of plasma adre-nomedullin level in normal pregnancy and preclampsia. Medscape J Med2008;10:29.

35. Miyao Y, Nishikimi T, Goto Y, Miyazaki S, Daikoku S, Morii I, et al.Increased plasma adrenomedullin levels in patients with acute myocardialinfarction in proportion to the clinical severity. Heart 1998;79:39–44.

36. Piquard F, Charloux A,Mettauer B, Epailly E, Lonsdorfer E, Popescu S, et al.Exercise-induced increase in circulating adrenomedullin is related tomeanblood pressure in heart transplant recipients. J Clin Endocrinol Metab2000;85:2828–31.

37. Chari ST, Leibson CL, Rabe KG, Ransom J, de Andrade M, Petersen GM.Probability of pancreatic cancer following diabetes: a population-basedstudy. Gastroenterology 2005;129:504–11.

38. Jatoi A, Sideras K, Nguyen PL. Tumor necrosis factor-alpha as a treatmenttarget for the cancer anorexia/weight loss syndrome. Support Cancer Ther2004;1:237–42.

39. Muniraj T, Chari ST.Diabetes andpancreatic cancer.MinervaGastroenterolDietol 2012;58:331–45.

40. Hudson TJ, Anderson W, Artez A, Barker AD, Bell C, Bernabe RR, et al.International network of cancer genome projects. Nature 2010;464:993–8.






Correction

Correction: Pancreatic Cancer-DerivedExosomes Causes Paraneoplastic b-cellDysfunction

In this article (Clin Cancer Res 2015;21:1722–33), which was published in the April1, 2015, issue of Clinical Cancer Research (1), funding was omitted from the GrantSupport section. It should read as follows: "This work was partly supported byCA78383 and CA150190 (to D. Mukhopadhyay), CA100685 (to S.T. Chari), P50CA102701 (to G.M. Petersen and S.T. Chari), 1T32 CA148073 (to G. Sagar), T32CA148073 and Mayo Graduate School (to N. Javeed), and NIH grants DK042394,DK088227, and HL052173 (to R.J. Kaufman)." The authors regret this error.

Reference1. Javeed N, Sagar G, Dutta SK, Smyrk TC, Lau JS, Bhattacharya S, et al. Pancreatic cancer-derived

exosomes causes paraneoplastic b-cell dysfunction. Clin Cancer Res 2015;21:1722–33.

Published online October 1, 2015.doi: 10.1158/1078-0432.CCR-15-1524�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 4495

2015;21:1722-1733. Published OnlineFirst October 29, 2014.Clin Cancer Res Naureen Javeed, Gunisha Sagar, Shamit K. Dutta, et al. Dysfunction

-cellβDerived Exosomes Cause Paraneoplastic −Pancreatic Cancer

Updated version

10.1158/1078-0432.CCR-14-2022doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2014/11/04/1078-0432.CCR-14-2022.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/21/7/1722.full#ref-list-1

This article cites 40 articles, 5 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/21/7/1722.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/21/7/1722To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-14-2022

http://clincancerres.aacrjournals.org/content/suppl/2014/11/04/1078-0432.CCR-14-2022.DC1

http://clincancerres.aacrjournals.org/content/21/7/1722.full#ref-list-1

http://clincancerres.aacrjournals.org/content/21/7/1722.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/21/7/1722


pancreatic cancer derived exosomes cause paraneoplastic b ... · pancreatic cancer patient blood...

Documents