UBC Faculty Research and Publications

Effects of insulin on human pancreatic cancer progression modeled in vitro Chan, Michelle T; Lim, Gareth E; Skovsø, Søs; Yang, Yu H C; Albrecht, Tobias; Alejandro, Emilyn U; Hoesli, Corinne A; Piret, James M; Warnock, Garth L; Johnson, James D Nov 6, 2014

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-12885_2014_Article_4996.pdf [ 1.82MB ]
JSON: 52383-1.0221339.json
JSON-LD: 52383-1.0221339-ld.json
RDF/XML (Pretty): 52383-1.0221339-rdf.xml
RDF/JSON: 52383-1.0221339-rdf.json
Turtle: 52383-1.0221339-turtle.txt
N-Triples: 52383-1.0221339-rdf-ntriples.txt
Original Record: 52383-1.0221339-source.json
Full Text

Full Text

RESEARCH ARTICLEEffects of insulin on humaiCadBackgroundThe incidence of pancreatic cancer is increasing, inand to develop preventative measures [1-3]. Multipleepidemiological studies have drawn a positive linkChan et al. BMC Cancer 2014, 14:814http://www.biomedcentral.com/1471-2407/14/814pro-survival effects in cells within the pancreas [8]. ItCanadaFull list of author information is available at the end of the articleparallel with the obesity and type 2 diabetes epidemics.Despite intense research efforts, the average 5-yearsurvival rate for pancreatic cancer remains below 5%,which underscores the need to identify key risk factorsbetween high levels of insulin and an increased risk ofpancreatic cancer [1,4,5]. Obesity and early stage type2 diabetes are both associated with elevated insulinlevels, known as basal hyperinsulinemia [6]. Given thatinsulin is a powerful mitogen and that its levels likelyvary physiologically within the pancreas [7], it is possiblethat sustained increases in local insulin levels within thepancreas provide increased growth advantages and* Correspondence: James.D.Johnson@ubc.ca1Department of Cellular and Physiological Sciences, University of BritishColumbia, Vancouver, BC, Canada2Department of Surgery, University of British Columbia, Vancouver, BC,AbstractBackground: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorlyunderstood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise ofhyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, theactions of insulin at the various stages of pancreatic cancer progression remain poorly defined.Methods: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in threehuman cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells,the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells weretreated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTTassays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting.Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to thesurvival of each cell model.Results: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we foundthat there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the celltypes. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cellproliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, wefound that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were moredependent on RAF/ERK signalling.Conclusions: Our data suggest that excessive insulin signalling may contribute to proliferation and survival inhuman immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult humanpancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired duringpancreatic cancer progression.Keywords: Hyperinsulinemia, Pancreatic cancer, PANC1, HPDE, Diabetes, PDAC, Pancreatic ductal adenocarcinoma,AKT, ERKprogression modeled in vMichelle T Chan1, Gareth E Lim1, Søs Skovsø1, Yu HsuanCorinne A Hoesli3,4, James M Piret3, Garth L Warnock2 an© 2014 Chan et al.; licensee BioMed Central LCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open Accessn pancreatic cancertrorol Yang1, Tobias Albrecht1, Emilyn U Alejandro1,James D Johnson1,2*td. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Chan et al. BMC Cancer 2014, 14:814 Page 2 of 12http://www.biomedcentral.com/1471-2407/14/814is therefore imperative to investigate the effects of insulinon different stages of pancreatic cancer progression.The molecular mechanisms by which hyperinsulinemiamay affect pancreatic cancer progression remain incom-pletely understood, but several studies have demonstratedthe importance of the RAS-MEK-ERK pathway and thePI3K-AKT pathway. Over 90% of human pancreaticadenocarcinoma cases involve the KRASG12D gain-of-function mutation, and this mutation is sufficient tolead to pre-cancerous lesions and rare tumours in mousemodels [9]. The KRasG12D mutation leads to constitutiveactivation of RAF-MEK-ERK and PI3K-AKT cascades todrive uncontrolled growth, proliferation and survival ofcancer cells [10]. KRas-driven transformations can beinhibited by expression of dominant-negative Raf-1, MEKor ERK, which all lie downstream of Ras [11,12]. It hasbeen established that Raf-1 can promote the initiation,transformation and maintenance of neoplastic lesions insome cancer models [13,14]. Constitutively active AKTcan also transform normal mouse pancreatic duct cellsinto malignant pancreatic cancer cells in vivo [15], butthe inability of PI3K-AKT inhibition to affect severalRas-driven cancers suggests that KRas acts on multiplepathways in oncogenesis [10,16,17].In the present study, we examined the effects andmechanisms of insulin in three in vitro cell models de-signed to mimic the progression of pancreatic cancerin vivo. These cell models were: pancreatic ductal cellcultures, an immortalized human ductal epitheliumcell line (HPDE), and an advanced metatstatic humanpancreatic ductal cancer cell line (PANC1). We foundthat high levels of insulin accelerated the proliferationof immortalized and metatstatic pancreatic ductal cellsbut not primary ductal cells. Furthermore, the molecularsignalling mechanisms activated by insulin were distinctin each model, suggesting that these processes may berewired during the progression of pancreatic cancer.These studies reveal potential mechanisms of insulin-mediated growth and survival effects and provide a betterunderstanding in the etiology of hyperinsulinemia-associated pancreatic cancer.MethodsHuman mixed pancreatic exocrine and ductal cell culturePrimary pancreatic exocrine cells that would normallybe discarded were obtained from the Vancouver GeneralHospital (Vancouver, BC) as part of the Human IsletTransplant Program, from cadaver organ donors who hadpreviously provided informed consent. Dr. Warnock’sorgan retrieval protocols are approved by the University ofBritish Columbia Clinical Research Ethics Board. Tissueswere from 7 donors, males and females between the agesof 32 and 58. Procedures involved in the culturing, dissoci-ating and sorting of primary mixed exocrine and ductaltissue were adapted from published protocols, with minoralterations [18,19]. Briefly, human ductal cell culture wasperformed as follows. First, unsorted primary cells,after being dispersed by shaking incubation for 1 hourand trituration with trypsin, were plated (10 × 106cells) in T-150 flasks, to allow preferential adhesionand removal of fibroblasts. Then, fibroblast-depletedcell suspensions were then seeded in 6-well plates atcell density of 1.5 × 106 cells per well for further treat-ments. For immunoblot analysis, dissociated mixed-pancreatic exocrine-ductal cells were used. For cellproliferation and cell survival assays, sorted ductalcells were used (CD90 negative population). Prior toinsulin treatments, cells were cultured in basal media(CMRL1066, 0.5 mg/L transferrin, 10 mM nicotina-mide, 5 μg/L sodium selenium, 0.5% BSA, 2 mM glutam-ine) for 6 hours, then treated with 0.2, 2, 20, 200 nM ofhuman recombinant insulin (Sigma Aldrich, Missouri,USA), 5 μM GW5074 (Life Technologies, California,USA), or 100 nM Akti-1/2 (EMD Biosciences, Darmstadt,Germany).HPDE and PANC1 cell culture and treatmentHPDE cells were kindly provided by Dr. Ming Tsao.HPDE cells between passages 7 to 15 were used, andwere cultured in KSF medium as previously described[20], but switched to DMEM for the experiments be-cause KSF medium contains 779.1 ± 87.43 nM insulin asmeasured by radioimmunoassay. PANC1 cells (ATCC,Manassas, USA) were cultured in DMEM as previouslydescribed [21]. For treatments, cells were washed withPBS and starved in 1 mg/ml glucose DMEM for for6 hours (HPDE cells), or 24 hours (PANC1 cells).Thereafter, the cells were treated with insulin, IGF-1,DMSO, 10 μM GW5074, 10 μM U0126 (Cell Signaling,USA), 200 nM Akti-1/2 or 1 μM wortmannin (EMDBiosciences). These concentrations were chosen basedon the literature and were shown to block signalling inPANC1 cells.Cell counting and cell survival assaysThe number of cells, live-stained with a concentration ofHoechst-33342 (50 ng/ml) that does not affect viability[22], was measured over time using ImageXpressMICRO highcontent imaging systems (Molecular Devices, Sunnyvale,California, USA). Images were analyzed with Acuity Xpress2.0 (Molecular Devices). Cell death was measured by quan-tifying the percentage of cells incorporating propidiumiodide (Sigma-Aldrich, 0.5 μg/ml) [23-25]. Cell viability,as indicated by metabolic capacity, was also quantifiedusing the XTT kit (ATCC). Bromodeoxyuridine (BrdU)incorporation (Roche, Basel, Switzerland) was also used todetermine proliferation in primary cells as previously de-scribed [19,26].Chan et al. BMC Cancer 2014, 14:814 Page 3 of 12http://www.biomedcentral.com/1471-2407/14/814Immunoblotting and protein analysisCells were lysed and subjected to immunoblotting aspreviously described [27]. Polyclonal mouse and rabbitsecondary antibodies, monoclonal antibodies for insulinreceptor, ERK1/2, p-ERK1/2(T202/Y204), AKT, p-AKT(S473), and cleaved caspase 3 were obtained from CellSignaling. Mouse monoclonal beta-actin antibody wasobtained from Novus Biologicals (Littleton, Colorado,USA). Chemiluminescence of the blots was imaged onfilms that were subsequently scanned. The density ofindividual bands was quantified using the histogramfunction of using Adobe Photoshop CS5 after inversionand auto-contrast functions were applied to the wholeimage. Protein levels were expressed as the fold changerelative to control.Statistical analysisAll data were analyzed by paired sample t-test, or one-way or two-way ANOVA, followed by post-hoc tests(Dunnett’s or Bonferroni analysis) with Prism (GraphPad,La Jolla, California, USA). Results are presented asmean ± SEM, and are considered significant if the p-valuewas less than 0.05.ResultsBaseline abundance of insulin signalling proteins inhuman primary pancreatic ductal cells, human HPDE cellsand human PANC1 cellsPancreatic ductal adenocarcinoma originates in the exo-crine pancreas and progresses to a highly invasive state.In the present study, we attempted to model three statesin this progression: normal pancreatic exocrine ductalcells to represent the baseline, HPDE cells to represent aproliferative but non-invasive stage [20,28,29], andPANC1 cells to represent a metastatic stage [30,31]. As afirst step in comparing these cell models, we sought toanalyze the protein levels of insulin receptor β, IGF1R,AKT and ERK in a small initial pilot western blot study.Notably, protein abundance of insulin receptors appearedto be clearly higher in primary ductal cells than in HPDEor PANC1 cells, even when a fraction of the lysate wasloaded (Figure 1). On the other hand, the IGF1R was mosthighly abundant in HPDE cells (Figure 1). The baselineabundance of downstream signaling proteins, AKT andERK, was more similar between the models. The totalamount of AKT protein appeared to be slightly higherin PANC1 cells. Most cell batches exhibited negligiblebaseline phosphorylation of AKT on serine 473 (Figure 1).The total amount of ERK tended to be slightly higher inthe HPDE cell line, whereas the baseline phosphorylationstatus of ERK on T402/Y204 was consistently higher inPANC1 cells (Figure 1). While none of these resultsshould be considered quantitative, due to the small natureof the pilot study and the use of antibodies, they doprovide some context for the subsequent comparisons ofAKT and ERK signaling in response to insulin and IGF1ligands.Insulin signaling in primary human exocrine and ductalpancreas cellsTo set a baseline for our in vitro model of pancreaticcancer progression, we next sought to establish the effectsof insulin on normal human pancreatic exocrine-ductalcells. Primary pancreatic exocrine-ductal cells were ex-posed to a range of insulin doses for 5 minutes (acute)and 24 hours (chronic) and examined for the activation ofAKT and ERK signalling. Rapid rises in the phosphoryl-ation of ERK-T402/Y204 and AKT-S473 were detectedafter acute insulin treatment, most notably with 20 nMand 200 nM insulin treatment (Figure 2A,B). Chronic in-sulin treatments led to an increase in AKT phosphoryl-ation but not ERK (Figure 2C,D). Proliferative effects ofinsulin were not observed in sorted primary pancreaticductal cells (Figure 2E,F). Higher levels of insulin elicitedprotective effects in sorted primary cells (Figure 2G).Phase contrast microscopy revealed that high doses of in-sulin altered the granularity, shape, and distribution in ofhuman primary ductal cells in culture (Figure 2H).The importance of two of the major insulin signallingkinases, ERK and AKT, was evaluated by treatingunstimulated cultures with small molecule inhibitorstargeting AKT (Akti-1/2) or RAF1 (GW5074), an up-stream kinase of ERK. Inhibition of AKT caused a signifi-cant increase in PI-positive cells, whereas blocking ERKsignalling did not promote cell death (Figure 2I). Thesedata suggest that AKT signalling is critical for the survivalof human pancreatic ductal cells, while RAF1/ERK signal-ling is dispensable, under these basal conditions.Insulin signalling in HPDE cellsHPDE cells are human pancreatic ductal cells that wereimmortalized by transfection of E6E7 protein from humanpapilloma virus 16 [20,28,29]. Unlike other pancreatic car-cinoma cell lines, which commonly reveal homozygousp16 gene deletion, HPDE cells express normal p16 geno-type [29]. As compared to other pancreatic carcinoma celllines, HPDE cells express relatively lower levels of EGFR,erbB2, TGF-α, HGFR, VEGF and KGF [29]. However, theresponse profiles of this cell line to insulin and IGF1 havenot been reported. This human ductal epithelial cell linehas been proposed as an important tool to study pre-cancer or early stages of pancreatic cancer [20]. Here, weused them as a model of proliferating, but not yet cancer-ous, pancreatic cells. Similar to primary pancreatic ductalcells, HPDE cells displayed responsiveness to insulin, asseen by AKT and ERK phosphorylation (Figure 3A,B).In the absence of serum, insulin as low as 2 nM exhib-ited protective effects on cell survival in HPDE cellsnptoasChan et al. BMC Cancer 2014, 14:814 Page 4 of 12http://www.biomedcentral.com/1471-2407/14/814Figure 1 Baseline abundance of insulin signalling proteins in humaPANC1 cells. Relative expression of Insulin receptor β (InsRβ), IGF1 recephosphorylated ERK (p-ERK), and total ERK (ERK) were examined under b(Figure 3C). Similar results were observed with IGF1,which activates receptors with 75% structural hom-ology. Activation of both insulin and IGF1 receptorshas been implicated in pancreatic cancer progressionand chemotherapy resistance [32,33]. Interestingly,HPDE cells were more sensitive to IGF1 than to insulin(Figure 3A,B), but differences in cell survival effectswere not observed between these two ligands (Figure 3C).In the absence of serum or exogenous insulin or IGF1, in-hibition of RAF1 with GW5074 dramatically decreasedHPDE cell viability after only 23 hours (Figure 3D,E). Con-trary to what was observed in primary human sorted cells,inhibition of the PI3K-AKT pathway had no effect onHPDE cell viability (Figure 3D-F). Thus, the RAF1 pathway,and not the PI3K/AKT pathway, is required for themaintenance of HPDE cell survival under these basalconditions.Insulin signalling in PANC1 cellsThe PANC1 cell line was originally isolated from a pan-creatic adenocarcinoma containing the constitutivelyactive KRASG12D mutation, a homozygous p16 deletionand an inactivating p53R273H mutation [30,31]. This cellline is routinely used to study the late stages of pancreaticcancer. Acute and chronic treatment of PANC1 cells withinsulin revealed striking differences in the kinetics andindependent primary ductal cells samples, four HPDE cells samples and fouloading of primary ductal samples as indicated by the actin loading controevery effort was made to load an equal amount of protein into each lane.primary pancreatic ductal cells, human HPDE cells and humanr (IGF1R), phosphorylated AKT at S473 (p-AKT), total AKT (AKT),al conditions. From the left to right, there are three biologicaldose–response profiles of AKT and ERK phosphorylation.Several concentrations of insulin tested elicited acuteAKT and ERK phosphorylation in these experiments(Figure 4A,B). On the other hand, insulin treatment for24 hours resulted in maximal AKT activation at the 20nM dose, without further stimulation by 200 nM insulin.Notably, 24 hours of insulin treatment was only capable ofactivating ERK at lower doses (Figure 4C,D). We have pre-viously found that lower doses of insulin can be more ef-fective at activating RAF1/ERK and related pathways inpancreatic endocrine cells [25,26,34-38] and our recentmathematical model suggests that such low concentra-tions are present in the human pancreas [7]. Proliferativeand protective effects were only observed at higher insulindoses (Figure 4E,F). In PANC1 cells treated for 120 hours,insulin was more effective at promoting cell viability thanIGF1. The increase in proliferation induced by insulin wasconfirmed with BrdU incorporation (Figure 4C). No dif-ferences were observed between insulin and IGF1 on cellsurvival (Figure 4H-I). To the best of our knowledge, thisis the first direct comparison of the effects of insulin andIGF1 in pancreatic cancer cells.Next, we assessed the requirement for RAF1/ERK ver-sus PI3K/AKT signalling on the viability of PANC1 cells.Inhibition of RAF1 significantly increased cell death(Figure 5A-C) and reduced cell viability (Figure 5D,E) inr PANC1 cells samples from different passages. Note the unevenl, which prevents quantitative comparisons. Other than lanes 1 and 2,Chan et al. BMC Cancer 2014, 14:814 Page 5 of 12http://www.biomedcentral.com/1471-2407/14/814PANC1 cells. A more modest delayed effect on cell via-bility and cell death was also observed after MEK1/2inhibition by U0126 (Figure 5A,D,E), similar to thefindings in the HPDE cells. AKT inhibition was muchFigure 2 Effects of insulin on AKT and ERK phosphorylation and cell vAKT and ERK were measured in primary pancreatic exocrine cultures treate24 hours (C, D) (n =3-4) Fold refers to the fold change of sample relative tcell-counting studies employing live-cell imaging of Hoechst-labeled cell custaining of treated relative to untreated over 3 days (n =4). (G) Quantificatiiodide (PI) labeled, over 60 hours relative to non-treated cells. (n =3). (H) H3 days. Bright-field images are representative of 3 cultures. (I) Effects of inhor AKT signalling with 100 nM Akti1/2 on human primary pancreatic exocriANOVA analyses with Bonferroni’s post-test were performed. *Represents sless effective at inducing PANC1 cell death as assessedby cell counting, PI incorporation, and cleaved caspase3 levels (Figure 5A-E). These observations indicate thatthe RAF1/ERK pathway, and not the PI3K/AKT pathway,iability in primary human pancreatic duct cells. Phosphorylatedd with the indicated concentrations of insulin for 5 minutes (A, B) ando control at the same time point. (E) Quantification of automatedltures over 60 hours. (n =3). (F) Quantification of proliferation by BrdUon of the average number of dying/dead treated cells, propidiumuman exocrine cells were exposed to 0, 0.2, 2, 20, 200 nM insulin foribition of RAF1/ERK signalling on PI incorporation with 10 μM GW5074ne cell viability (n =3). SF denotes serum free. Repeated Measurestatistical significance of p < 0.05 when compared to DMSO control.Chan et al. BMC Cancer 2014, 14:814 Page 6 of 12http://www.biomedcentral.com/1471-2407/14/814may play a more important role in the maintenance ofPANC1 cell survival under these basal conditions.Effects of three insulin analogs on PANC1 cellsSome studies, but not all, have reported that individualsusing long-acting insulin analogs have increased risk ofcancer [39]. As an adjunct to our studies on the effectsof insulin in pancreatic cancer cells, we compared nativeinsulin to a short-acting insulin analogue (Lispro™) and along-acting insulin analogue (Glargine™) on the viabilityof PANC1 cells. Acute treatment of PANC1 cells withrecombinant insulin, Lispro and Glargine significantlyincreased AKT phosphorylation (Figure 6A). No statis-tical difference in AKT phosphorylation was observedbetween the Lispro and native insulin, although ourstudies (n = 16) were not powered to detect very subtledifferences. Glargine was found to induce slightly moreAKT phosphorylation in PANC1 cells when comparedto the other insulin analogues. No differences in ERKFigure 3 Effects of insulin on AKT and ERK phosphorylation and cell vmeasured in HPDE cells treated with a range of insulin and IGF-1 concentrassessed by XTT assay. Briefly, cells were treated and the activated XTT reagand A660nm) was measured 6 hours post-addition. Insulin or IGF1 was not aassessed by propidium iodide (PI) incorporation in Hoechst-positive cells. Fto control after 23 hours of treatment (n =3). (A-C). Two-tailed paired sampBonferroni post-test were performed.phosphorylation were observed (data not shown). Not-withstanding these modest changes in signalling, wefound that recombinant insulin, Lispro and Glargine ledto similar levels of PANC1 cell viability (Figure 6B).Interestingly, the viability of PANC1 cells was augmentedwith low doses of Glargine (Figure 6B). Together, thesedata indicate that all forms of insulin tested were capableof similar effects on PANC1 cell survival and proliferation,although Glargine exhibited a shift in potency. Cautionshould be exercised when extrapolating these in vitroconditions to the in vivo clinical situation, since highnanomolar doses of insulin are not physiologically orpharmacologically relevant.DiscussionInsulin and IGF1 are growth factors with putative regula-tory roles in proliferation, survival and cancer progression[40]. Given that hyperinsulinemia has been identified as anindependent risk factor for pancreatic cancer [1,2,39,41], itiability in HPDE cells. (A, B) Phosphorylated AKT and ERK wereations for 5 minutes (n =10, 8). (D-E) Proliferation of HPDE cells wasent was added at designated time, and the absorbance of Δ(A475nmdded in these studies (n =3). (C, F) Quantification of cell death wasold refers to the number of PI positive cells in treatment group relativele t-tests were performed. (D-E) One-way ANOVA analyses withChan et al. BMC Cancer 2014, 14:814 Page 7 of 12http://www.biomedcentral.com/1471-2407/14/814is imperative to understand how changes in insulin signal-ling may promote cancer progression. To date, not muchis known about the action of insulin on normal humanpancreatic exocrine and ductal cells. Furthermore, directcomparisons of insulin signalling effects across modelsof different stages of pancreatic cancer have not beenFigure 4 Effects of insulin on AKT and ERK phosphorylation and cell vin PANC1 cell cultures treated with the indicated concentrations of insulincellular viability was also assessed by XTT at 24 hours or 5 days of incubatiorelative to control (n =5-6). Insulin was not added in these studies (G) PANwas not added. (H) PANC1 cell death measured by propidium iodide incorcaspase 3 was measured after 24 hours (n =5). (A-F, H-I) Two-tail paired sap < 0.05 when compared to control (0 nM Insulin). # in Figure 4F denotes stwo-tailed paired sample t-test was performed.reported. In the present study, we demonstrated thatpancreatic cancer progression is associated with changesin insulin signalling pathways that underlie cell survival,proliferation and viability. We found that primary humanductal cells are responsive to insulin and exhibit re-duced cell viability when AKT signalling is disrupted.iability in PANC1 cells. Phosphorylated AKT and ERK were measuredfor 5 minutes (A, B) and 24 hours (C, D) (n =7-12). (E, F) PANC1n, and expressed as fold change in mean absorbance treatmentC1 cell proliferation measured after 24 hours using BrdU (n =6) Insulinporation after 48 hours (n =5). Insulin was not added. (I) Cleavedmple t-test were performed. *Represents statistical significance oftatistical significance between 200 nM IGF1 and 200 nM insulin whenChan et al. BMC Cancer 2014, 14:814 Page 8 of 12http://www.biomedcentral.com/1471-2407/14/814Immortalized HPDE ductal cells were also responsiveto insulin, but less so than to IGF1, perhaps due to anabundance of IGF1 receptors. In contrast to the primarycells, HPDE cells required MAPK signaling and not AKTFigure 5 RAF1/ERK signalling is preferentially required for PANC1 celdifferent small molecule inhibitors on propidium iodide (PI) incorporation (PI)percent of PI and Hoechst co-positive cells over total Hoechst positive cells atserum-free control by two-way ANOVA (n =3) Data points that have been shanon-treated conditions at that time point. # Indicates statistical significance in(B) Average number of PI positive cells over time of each treated group in FigGW5074 exhibited statistical significance, where as other treatments did not yWort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors onimmunoblot of three independent biological replicates (n =3). (D-E) PANC1 c10 μM U0126, 200 nM Akti-1/2 and 1 mM wortmannin (wort.) for 24 hours anfold change of the treated relative to control. (C-E) One-way ANOVA analysissignificance of p < 0.05 where treated groups are compared to control (−) insignaling to survive. The metastatic PANC1 cell modelresponded to insulin, more so than to IGF1, and also hada strong dependence on MAPK signalling and not AKTsignalling. Collectively, our results imply a re-wiring ofl survival in the absence of exogenous insulin. (A) Effects ofin PANC1 cells were tracked and expressed as the fold change in thethat hour relative to t =0 hour. Kinetic data were analyzed relative toded solid black represent statistical significance when compared tocells treated with Akti1/2 when compared to control at that time point.ure 5A is shown as a histogram expressed in arbitrary units (AU).ield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395,cleaved caspase 3 protein levels in PANC1 cells. This is a representativeells were serum starved and treated with either DMSO, 10 μM GW5074,d 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as thewith Bonferroni post-test was performed. *Represents statisticalthe post-hoc test.ecst rtraTwChan et al. BMC Cancer 2014, 14:814 Page 9 of 12http://www.biomedcentral.com/1471-2407/14/814ductal cell dependence on the MAPK signalling axis forcell survival. Further understanding of how cells favor onepathway over another in pancreatic cancer progressionmay lead to novel approaches to halt early carcinogenesisand improve the long-term survival of pancreatic cancerpatients.In the present study, we found that these cell modelsderived from exocrine tissue required higher doses ofinsulin to elicit responses when compared to our previousexperience with pancreatic exocrine cells that respond tophysiological insulin doses in the high picomolar range[6,26,34,35,37,38,42]. This finding suggests the possibilityFigure 6 Effects of insulin analogues on PANC1 cell viability. (A) Effphosphorylation after 60 minutes (n =16). Two-tailed paired sample t-tephosphorylation than recombinant insulin at the 200 nM insulin concenassay on PANC1 cells treated with insulin analogues for 24 hours (n =8).when compared to non-treated condition.that the exocrine cells and their cancerous descendantsmay be somewhat refractory to low concentrations insulinand may require sustained hyperinsulinemia to acceleratecancer progression. Multiple epidemiological studies havedemonstrated that the hyperinsulinemic states of obesityand recent onset type 2 diabetes are associated with differ-ent types of cancer [43,44], and this has been replicated insome animal models. For example, elevated insulin levelshave been implicated in in vivo mouse models of breastcancer [45,46]. The metabolic changes that result fromboth conditions make it difficult to discern causal factorsthat promote carcinogenesis. Hyperinsulinemia can pre-cede and lead to the development of obesity [6], whichsuggests that it may contribute to carcinogenesis indirectlyas well. Indeed, high levels of circulating insulin have beenassociated with increased risk of breast cancer in post-menopausal women [47,48]. Given the association be-tween hyperinsulinemia and pancreatic cancer [1], ithas been suggested that excessive secretion of insulin bypancreatic β-cells required to maintain glucose homeostasismay directly influence pancreatic carcinogenesis in at-riskindividuals.The mitogenic actions of insulin have been well de-scribed in vitro and in vivo [49], but little is known ofinsulin’s proliferative effects on the endocrine and exo-crine compartments of the pancreas. We previouslydemonstrated that insulin, even at physiological pico-molar doses [7], promotes the proliferation of pancre-atic endocrine β-cells [26], but whether similar effectsoccur on the exocrine compartment was not known. Inthe present study, we did not observe any proliferativeeffects of insulin in primary ductal cells or transformedHPDE cells. Instead, we found that insulin and closelyrelated IGF1 promoted cell viability and survival ints of recombinant insulin, insulin Lispro, and insulin Glargine on AKTevealed insulin Glargine promoted greater stimulation of AKTtion denoted by # ( p < 0.05). (B) Cell viability assessed by XTTo-tailed t-test were performed, and * denotes statistical significancemultiple models of pancreatic cancer progression. Collect-ively, these findings suggest that the oncogenic propertiesof insulin may be due to its effects on survival as opposedto its mitogenic effects. The downstream mechanisms ofinsulin action in these three models remain unclear. How-ever, a recent report has suggested that HPDE prolifera-tion depends on Pdx1 [50], which we have shown is ananti-apoptotic transcription factor controlled by low dosesof insulin [42,51]. Additional studies are warranted to fullyelucidate the mechanisms.ConclusionsThe aim of the present study was to determine whetherthe response to insulin was different between primaryhuman pancreatic ductal cells, an immortalized pancre-atic ductal cell line (HPDE), and an advanced pancreaticcancer cell line (PANC1). Indeed, we uncovered someinteresting differences, which may hold clues to the roleof insulin and insulin signalling at different cancerstages. Our data support a working model (Figure 7)whereby primary pancreatic duct cells respond to insu-lin (mostly via AKT signalling), but do not respond withChan et al. BMC Cancer 2014, 14:814 Page 10 of 12http://www.biomedcentral.com/1471-2407/14/814increased proliferation or survival. On the other hand,proliferative and cancerous pancreatic ductal cells respondvia both AKT and ERK signalling, with the ERK pathwaybeing the predominant pathway controlling survival. Therole of insulin during cancer progression has been debated[52-54]. The present study examined the actions of insulinon cell viability across different stages of pancreatic cancerin vitro. If the cell models chosen in this study faithfullyrecapitulate the natural progression of the disease, our ex-perimental data may suggest that hyperinsulinemia maynot play a role in initiating pancreatic cancer, but highlevels of insulin may accelerate the cancer progression viaincreased RAF1/ERK-dependent cell survival. The studiesdescribed in this manuscript have the caveats of employ-ing only a single cell line to represent dividing duct andadenocarcinomas and of being entirely in vitro. Comple-mentary in vivo studies are urgently needed to assess therole of insulin and insulin signalling on pancreatic cancerprogression.Figure 7 Working model of insulin’s effects at different stages of panduct cells respond to insulin (mostly via AKT signalling), but do not increascancerous pancreatic ductal cells respond via both AKT and ERK signalling,Competing interestsThe authors declare that they have no competing interests with respect tothis manuscript.Authors’ contributionsMTC performed the majority of the experiments and drafted the manuscript.GEL helped conceive and design experiments, supervised the studies andedited the manuscript. SS helped design and perform experiments, andedited manuscript. TA helped design and perform experiments, and editedmanuscript. YHCY helped design experiments, and edited manuscript. EUAhelped design and perform experiments, and edited the manuscript. CHhelped design and perform experiments, and edited manuscript. JMPsupervised studies. GLW provided human pancreas cells and securedfunding for some of the experiments. JDJ conceived the studies, supervisedthe research, secured funding, co-wrote the manuscript and is the guarantorof this work. All authors read and approved the final manuscript.AcknowledgementsThe authors thank Caitlin Der, Ling Mu, Qinya Zhang, Roger Kiang, andothers in the Johnson laboratory for their efforts throughout this project. Wethank Dr. Sylvia Ng (University of British Columbia) and Dr. Ming Tsao(University of Toronto) for the HPDE cell line. This study was supported by agrant from the Cancer Research Society to J.D.J. and a grant from theVancouver Hospital Foundation to G.L.W and J.D.J.creatic cancer. Our data support a model whereby primary pancreatice proliferation or survival. On the other hand, proliferative andwith cell survival predominantly controlled by the ERK pathway.Chan et al. BMC Cancer 2014, 14:814 Page 11 of 12http://www.biomedcentral.com/1471-2407/14/814Author details1Department of Cellular and Physiological Sciences, University of BritishColumbia, Vancouver, BC, Canada. 2Department of Surgery, University ofBritish Columbia, Vancouver, BC, Canada. 3Department of Chemical andBiological Engineering, University of British Columbia, Vancouver, BC, Canada.4Present address: Département de génie chimique | Department of ChemicalEngineering, Université McGill University, 3610 University Street, WongBuilding, Room 4230, Montréal H3A 0C5, Canada.Received: 7 March 2014 Accepted: 27 October 2014Published: 6 November 2014References1. 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(2):504–511.2. Pisani P: Hyper-insulinaemia and cancer, meta-analyses of epidemiologicalstudies. Arch Physiol Biochem 2008, 114(1):63–70.3. Mihaljevic AL, Michalski CW, Friess H, Kleeff J: Molecular mechanism ofpancreatic cancer–understanding proliferation, invasion, and metastasis.Langenbecks Arch Surg 2010, 395(4):295–308.4. Calle EE, Murphy TK, Rodriguez C, Thun MJ, Heath CW Jr: Diabetes mellitusand pancreatic cancer mortality in a prospective cohort of United Statesadults. Cancer Causes Control 1998, 9(4):403–410.5. Huxley R, Ansary-Moghaddam A, Berrington de Gonzalez A, Barzi F, WoodwardM: Type-II diabetes and pancreatic cancer: a meta-analysis of 36 studies.Br J Cancer 2005, 92(11):2076–2083.6. Mehran AE, Templeman NM, Brigidi GS, Lim GE, Chu KY, Hu X, Botezelli JD,Asadi A, Hoffman BG, Kieffer TJ, Bamji SX, Clee SM, Johnson JD:Hyperinsulinemia drives diet-induced obesity independently of braininsulin production. Cell Metab 2012, 16(6):723–737.7. Wang M, Li J, Lim GE, Johnson JD: Is dynamic autocrine insulin signalingpossible? A mathematical model predicts picomolar concentrations ofextracellular monomeric insulin within human pancreatic islets. PLoS One2013, 8(6):e64860.8. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell2011, 144(5):646–674.9. Kopp JL, von Figura G, Mayes E, Liu FF, Dubois CL, Morris JP, Pan FC, Akiyama H,Wright CV, Jensen K, Hebrok M, Sander M: Identification of Sox9-dependentacinar-to-ductal reprogramming as the principal mechanism for initiation ofpancreatic ductal adenocarcinoma. Cancer Cell 2012, 22(6):737–750.10. Gysin S, Salt M, Young A, McCormick F: Therapeutic strategies fortargeting ras proteins. Genes Cancer 2011, 2(3):359–372.11. Schaap D, van der Wal J, Howe LR, Marshall CJ, van Blitterswijk WJ: Adominant-negative mutant of raf blocks mitogen-activated proteinkinase activation by growth factors and oncogenic p21ras. J Biol Chem1993, 268(27):20232–20236.12. Westwick JK, Cox AD, Der CJ, Cobb MH, Hibi M, Karin M, Brenner DA:Oncogenic Ras activates c-Jun via a separate pathway from theactivation of extracellular signal-regulated kinases. Proc Natl Acad SciU S A 1994, 91(13):6030–6034.13. White MA, Nicolette C, Minden A, Polverino A, Van Aelst L, Karin M, WiglerMH: Multiple Ras functions can contribute to mammalian celltransformation. Cell 1995, 80(4):533–541.14. Ehrenreiter K, Kern F, Velamoor V, Meissl K, Galabova-Kovacs G, Sibilia M,Baccarini M: Raf-1 addiction in Ras-induced skin carcinogenesis. CancerCell 2009, 16(2):149–160.15. Elghazi L, Weiss AJ, Barker DJ, Callaghan J, Staloch L, Sandgren EP, Gannon M,Adsay VN, Bernal-Mizrachi E: Regulation of pancreas plasticity and malignanttransformation by Akt signaling. Gastroenterology 2009, 136(3):1091–1103.16. Ihle NT, Lemos R Jr, Wipf P, Yacoub A, Mitchell C, Siwak D, Mills GB, DentP, Kirkpatrick DL, Powis G: Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866whereas oncogenic Ras is a dominant predictor for resistance. CancerRes 2009, 69(1):143–150.17. Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, Maira M,McNamara K, Perera SA, Song Y, Chirieac LR, Kaur R, Lightbrown A,Simendinger J, Li T, Padera RF, Garcia-Echeverria C, Weissleder R, MahmoodU, Cantley LC, Wong KK: Effective use of PI3K and MEK inhibitors to treatmutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med2008, 14(12):1351–1356.18. Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, Aye T, Sharma A: Thepancreatic ductal epithelium serves as a potential pool of progenitorcells. Pediatr Diabetes 2004, 5(Suppl 2):16–22.19. Hoesli CA, Johnson JD, Piret JM: Purified human pancreatic duct cellculture conditions defined by serum-free high-content growth factorscreening. PLoS One 2012, 7(3):e33999.20. Ouyang H, Mou L, Luk C, Liu N, Karaskova J, Squire J, Tsao MS: Immortalhuman pancreatic duct epithelial cell lines with near normal genotypeand phenotype. Am J Pathol 2000, 157(5):1623–1631.21. Liehr RM, Melnykovych G, Solomon TE: Growth effects of regulatorypeptides on human pancreatic cancer lines PANC-1 and MIA PaCa-2.Gastroenterology 1990, 98(6):1666–1674.22. Yang YH, Johnson JD: Multi-parameter single-cell kinetic analysis revealsmultiple modes of cell death in primary pancreatic beta-cells. J Cell Sci2013, 126(Pt 18):4286–4295.23. Luciani DS, Gwiazda KS, Yang TL, Kalynyak TB, Bychkivska Y, Frey MH, JeffreyKD, Sampaio AV, Underhill TM, Johnson JD: Roles of IP3R and RyR Ca2+channels in endoplasmic reticulum stress and beta-cell death. Diabetes2009, 58(2):422–432.24. Jeffrey KD, Alejandro EU, Luciani DS, Kalynyak TB, Hu X, Li H, Lin Y,Townsend RR, Polonsky KS, Johnson JD: Carboxypeptidase E mediatespalmitate-induced beta-cell ER stress and apoptosis. Proc Natl Acad SciU S A 2008, 105(24):8452–8457.25. Alejandro EU, Johnson JD: Inhibition of raf-1 alters multiple downstreampathways to induce pancreatic beta-cell apoptosis. J Biol Chem 2008,283(4):2407–2417.26. Beith JL, Alejandro EU, Johnson JD: Insulin stimulates primary beta-cellproliferation via Raf-1 kinase. Endocrinology 2008, 149(5):2251–2260.27. Alejandro EU, Lim GE, Mehran AE, Hu X, Taghizadeh F, Pelipeychenko D,Baccarini M, Johnson JD: Pancreatic β-cell Raf-1 is required for glucosetolerance, insulin secretion, and insulin 2 transcription. FASEB J 2011,25(11):3884–3895.28. Furukawa T, Duguid WP, Rosenberg L, Viallet J, Galloway DA, Tsao MS:Long-term culture and immortalization of epithelial cells from normaladult human pancreatic ducts transfected by the E6E7 gene of humanpapilloma virus 16. Am J Pathol 1996, 148(6):1763–1770.29. Liu N, Furukawa T, Kobari M, Tsao MS: Comparative phenotypic studies ofduct epithelial cell lines derived from normal human pancreas andpancreatic carcinoma. Am J Pathol 1998, 153(1):263–269.30. Githens S: The pancreatic duct cell: proliferative capabilities, specificcharacteristics, metaplasia, isolation, and culture. J Pediatr GastroenterolNutr 1988, 7(4):486–506.31. Deer EL, Gonzalez-Hernandez J, Coursen JD, Shea JE, Ngatia J, Scaife CL,Firpo MA, Mulvihill SJ: Phenotype and genotype of pancreatic cancer celllines. Pancreas 2010, 39(4):425–435.32. Appleman VA, Ahronian LG, Cai J, Klimstra DS, Lewis BC: KRAS(G12D)- andBRAF(V600E)-induced transformation of murine pancreatic epithelialcells requires MEK/ERK-stimulated IGF1R signaling. Mol Cancer Res 2012,10(9):1228–1239.33. Awasthi N, Zhang C, Ruan W, Schwarz MA, Schwarz RE: BMS-754807, asmall-molecule inhibitor of insulin-like growth factor-1 receptor/insulinreceptor, enhances gemcitabine response in pancreatic cancer. MolCancer Ther 2012, 11(12):2644–2653.34. Alejandro EU, Kalynyak TB, Taghizadeh F, Gwiazda KS, Rawstron EK, Jacob KJ,Johnson JD: Acute insulin signaling in pancreatic beta-cells is mediatedby multiple Raf-1 dependent pathways. Endocrinology 2010,151(2):502–512.35. Johnson JD, Alejandro EU: Control of pancreatic beta-cell fate byinsulin signaling: The sweet spot hypothesis. Cell Cycle 2008,7(10):1343–1347.36. Johnson JD, Ford EL, Bernal-Mizrachi E, Kusser KL, Luciani DS, Han Z, Tran H,Randall TD, Lund FE, Polonsky KS: Suppressed insulin signaling andincreased apoptosis in CD38-null islets. Diabetes 2006, 55(10):2737–2746.37. Luciani DS, Johnson JD: Acute effects of insulin on beta-cells fromtransplantable human islets. Mol Cell Endocrinol 2005, 241(1–2):88–98.38. Johnson JD, Misler S: Nicotinic acid-adenine dinucleotide phosphate-sensitivecalcium stores initiate insulin signaling in human beta cells. Proc Natl Acad SciU S A 2002, 99(22):14566–14571.39. Bodmer M, Becker C, Meier C, Jick SS, Meier CR: Use of antidiabetic agentsand the risk of pancreatic cancer: a case–control analysis. Am JGastroenterol 2012, 107(4):620–626.40. Novosyadlyy R, Leroith D: Insulin-like growth factors and insulin: at thecrossroad between tumor development and longevity. J Gerontol A BiolSci Med Sci 2012, 67(6):640–651.41. Osorio-Costa F, Rocha GZ, Dias MM, Carvalheira JB: Epidemiological andmolecular mechanisms aspects linking obesity and cancer. Arq BrasEndocrinol Metabol 2009, 53(2):213–226.42. Johnson JD, Bernal-Mizrachi E, Alejandro EU, Han Z, Kalynyak TB, Li H, BeithJL, Gross J, Warnock GL, Townsend RR, Permutt MA, Polonsky KS: Insulinprotects islets from apoptosis via Pdx1 and specific changes in the humanislet proteome. Proc Natl Acad Sci U S A 2006, 103(51):19575–19580.43. Rousseau MC, Parent ME, Pollak MN, Siemiatycki J: Diabetes mellitus andSubmit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionChan et al. BMC Cancer 2014, 14:814 Page 12 of 12http://www.biomedcentral.com/1471-2407/14/814cancer risk in a population-based case–control study among men fromMontreal, Canada. Int J Cancer 2006, 118(8):2105–2109.44. Coughlin SS, Calle EE, Teras LR, Petrelli J, Thun MJ: Diabetes mellitus as apredictor of cancer mortality in a large cohort of US adults. Am JEpidemiol 2004, 159(12):1160–1167.45. Novosyadlyy R, Lann DE, Vijayakumar A, Rowzee A, Lazzarino DA, Fierz Y,Carboni JM, Gottardis MM, Pennisi PA, Molinolo AA, Kurshan N, Mejia W,Santopietro S, Yakar S, Wood TL, LeRoith D: Insulin-mediated accelerationof breast cancer development and progression in a nonobese model oftype 2 diabetes. Cancer Res 2010, 70(2):741–751.46. Ferguson RD, Gallagher EJ, Scheinman EJ, Damouni R, LeRoith D: Theepidemiology and molecular mechanisms linking obesity, diabetes, andcancer. Vitam Horm 2013, 93:51–98.47. Gunter MJ, Hoover DR, Yu H, Wassertheil-Smoller S, Rohan TE, Manson JE, Li J,Ho GY, Xue X, Anderson GL, Kaplan RC, Harris TG, Howard BV, Wylie-Rosett J,Burk RD, Strickler HD: Insulin, insulin-like growth factor-I, and risk of breastcancer in postmenopausal women. J Natl Cancer Inst 2009, 101(1):48–60.48. Kabat GC, Kim M, Caan BJ, Chlebowski RT, Gunter MJ, Ho GY, Rodriguez BL,Shikany JM, Strickler HD, Vitolins MZ, Rohan TE: Repeated measures ofserum glucose and insulin in relation to postmenopausal breast cancer.Int J Cancer 2009, 125(11):2704–2710.49. Tran TT, Naigamwalla D, Oprescu AI, Lam L, McKeown-Eyssen G, Bruce WR,Giacca A: Hyperinsulinemia, but not other factors associated with insulinresistance, acutely enhances colorectal epithelial proliferation in vivo.Endocrinology 2006, 147(4):1830–1837.50. Liu SH, Patel S, Gingras MC, Nemunaitis J, Zhou G, Chen C, Li M, Fisher W,Gibbs R, Brunicardi FC: PDX-1: demonstration of oncogenic properties inpancreatic cancer. Cancer 2011, 117(4):723–733.51. Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, Fujita J, Misler S, EdlundH, Polonsky KS: Increased islet apoptosis in Pdx1+/− mice. J Clin Invest2003, 111(8):1147–1160.52. Davidson JK, Eddleman EE: Insulin resistance; review of the literature andreport of a case associated with carcinoma of the pancreas. AMA ArchIntern Med 1950, 86(5):727–742.53. Johnson JA, Carstensen B, Witte D, Bowker SL, Lipscombe L, Renehan AG:Diabetes and cancer (1): evaluating the temporal relationship betweentype 2 diabetes and cancer incidence. Diabetologia 2012, 55(6):1607–1618.54. Renehan AG, Yeh HC, Johnson JA, Wild SH, Gale EA, Moller H: Diabetes andcancer (2): evaluating the impact of diabetes on mortality in patientswith cancer. Diabetologia 2012, 55(6):1619–1632.doi:10.1186/1471-2407-14-814Cite this article as: Chan et al.: Effects of insulin on human pancreaticcancer progression modeled in vitro. BMC Cancer 2014 14:814.Submit your manuscript at www.biomedcentral.com/submit


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items