Oncogenic Ras/Src cooperativity in pancreatic neoplasia

ORIGINAL ARTICLE

Oncogenic Ras/Src cooperativity in pancreatic neoplasia

DJ Shields1, EA Murphy1, JS Desgrosellier1, A Mielgo1, SKM Lau1, LA Barnes1, J Lesperance1,M Huang1, C Schmedt2, D Tarin1, AM Lowy3 and DA Cheresh1

1Department of Pathology, Moore’s UCSD Cancer Center, San Diego, CA, USA; 2Genomics Institute of Novartis ResearchFoundation, San Diego, CA, USA and 3Department of Surgery, Moore’s UCSD Cancer Center, San Diego, CA, USA

Pancreas cancer is one of the most lethal malignancies andis characterized by activating mutations of Kras, presentin 95% of patients. More than 60% of pancreatic cancersalso display increased c-Src activity, which is associatedwith poor prognosis. Although loss of tumor suppressorfunction (for example, p16, p53, Smad4) combined withoncogenic Kras signaling has been shown to acceleratepancreatic duct carcinogenesis, it is unclear whether elevatedSrc activity contributes to Kras-dependent tumorigenesisor is simply a biomarker of disease progression. Here, wedemonstrate that in the context of oncogenic Kras,activation of c-Src through deletion of C-terminal Srckinase (CSK) results in the development of invasivepancreatic ductal adenocarcinoma (PDA) by 5–8 weeks.In contrast, deletion of CSK alone fails to induceneoplasia, while oncogenic Kras expression yields PDAat low frequency after a latency of 12 months. Analysis ofcell lines derived from Ras/Src-induced PDA’s indicatesthat oncogenic Ras/Src cooperativity may lead to genomicinstability, yet Ras/Src-driven tumor cells remain depen-dent on Src signaling and as such, Src inhibitionsuppresses growth of Ras/Src-driven tumors. Thesefindings demonstrate that oncogenic Ras/Src cooperateto accelerate PDA onset and support further studies ofSrc-directed therapies in pancreatic cancer.Oncogene (2011) 30, 2123–2134; doi:10.1038/onc.2010.589;published online 17 January 2011

Keywords: pancreatic cancer; Src; Ras; oncogeniccooperativity

Introduction

Almost a century ago, Peyton Rous reported thediscovery of a ‘transmissible tumor-causing particle’that produced sarcomas when injected into chickens(Rous, 1911). This early description of the Roussarcoma virus set the stage for the identification of thefirst oncogene, v-Src, some 60 years later. Studies byVarmus, Bishop, Vogt and colleagues in the 1970’sestablished that expression of the Src retroviral

oncoprotein was sufficient to transform avian cells andalso promote sarcoma formation in chickens (Martin,2001). Subsequently, the cellular counterpart, c-Src wascharacterized as the founding member of the Src familyof protein tyrosine kinases (SFKs) (Stehelin et al., 1976;Hunter and Sefton, 1980). Since that time, Src kinasehas been intrinsically linked with cellular transformationand changes in its activity impact proliferation, invasionand migration (Bromann et al., 2004). More recently, anumber of studies utilizing in vivo orthotopic transplan-tation models have not only underscored the importanceof Src activity during tumorigenesis, but also providedcompelling support for Src as a therapeutic target(Yezhelyev et al., 2004; Rucci et al., 2006; Trevinoet al., 2006; Koreckij et al., 2009; Vitali et al., 2009). Inaddition to its direct effects on the properties of a tumorcell, Src can also indirectly modulate neoplasia throughits role in tumor-associated processes such as angiogen-esis and vascular permeability, yet the precise role of Srcduring the development of malignancy remains unclear(Eliceiri et al., 1999).

Overexpression or activation of Src kinase has beendescribed in many human cancers including those of thecolon, breast, lung, liver, head and neck, and brain andpancreas (Irby and Yeatman, 2000; Ishizawar andParsons, 2004). Furthermore, Src kinase activity has beenshown to increase as a function of tumor progression,implicating Src in the transition to malignancy (Talamontiet al., 1993). As Src activity is tightly controlled post-translationally, deregulation at this level can lead to anincrease in oncogenic potential. Src activation can occurfollowing a loss of regulation at its inhibitory C-terminaltyrosine residue (Tyr-529 in mouse, Tyr-530 in humans)due to reduced C-terminal Src kinase (CSK) abundance,elevated phosphatase activity or altered CSK-bindingprotein function (Irby and Yeatman, 2000). Loss of Tyr-529 phosphorylation leads to autophosphorylation at Tyr-418 (mouse)/Tyr-419 (human) and activation of Src.Alternatively, Src can be activated as a consequence ofelevated signaling from the upstream receptor tyrosinekinases, epidermal growth factor receptor, RON, IGF-1Rand c-Met, as well as the chemokine receptor, CXCR4 andintegrins such as avb3, each of which have been implicatedin carcinogenesis (Barton et al., 1991; Billadeau et al.,2006; Thomas et al., 2007; Desgrosellier et al., 2009;Ricono et al., 2009).

Pancreatic ductal adenocarcinoma (PDA) is one ofthe most lethal malignancies, causing more than 200 000

Received 24 April 2010; revised 1 November 2010; accepted 2 December2010; published online 17 January 2011

Correspondence: Dr DA Cheresh, Department of Pathology, Moore’sUCSD Cancer Center, Room 2344, 3855 Health Sciences Drive,MC 0803, La Jolla, San Diego, CA 92093-1503, USA.E-mail: [email protected]

Oncogene (2011) 30, 2123–2134& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11

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deaths worldwide annually. Despite advances in ourunderstanding of the genetic basis of PDA, the 5-yearsurvival rate for patients diagnosed with the disease in2009 remains less than 5% (Warshaw and Fernandez-del Castillo, 1992; Jemal et al., 2009). Elevated Src levelshave been reported in more than 70% of patients withductal adenocarcinoma of the pancreas and more than60% of pancreatic tumors show increased Src activity(Lutz et al., 1998; Morton et al., 2010). Furthermore,increases in Src protein and activity are associated withvascular invasion, lymph node positivity and diminishedsurvival (Morton et al., 2010). To test the hypothesisthat heightened Src activity may contribute to PDAprogression, we used floxed CSK mice as a tool toenable deletion of the inhibitory kinase, CSK, resultingin concomitant activation of the Src family kinases inthe embryonic pancreas. We were particularly interestedin the consequences of Src activation in the context ofoncogenic Kras signaling as B95% of PDA patientsharbor Kras mutations at diagnosis (Almoguera et al.,1988). Previous studies have documented that loss oftumor suppressors such as p16, p53, Smad4 or TGF-beta receptor 2, dramatically accelerates the develop-ment of Kras-dependent PDA in mice (Aguirre et al.,2003; Hingorani et al., 2005; Bardeesy et al., 2006; Ijichiet al., 2006; Izeradjene et al., 2007). Here, we show thatCSK deletion and activation of c-Src dramaticallyaccelerates both the onset of precursor lesions initiatedby oncogenic Kras signaling as well as the progression toinvasive adenocarcinoma. These findings support a rolefor Src kinase activity in KrasG12D-dependent pancreaticneoplasia, and provide a model system to study the roleof Src in pancreatic tumorigenesis.

Results

Activation of Src kinase is an early event in pancreascancerTo ascertain the activation status of the SFKs duringthe development of PDA, we performed immunohisto-chemistry analysis on primary tumor specimens fromPDA patients using an antibody that recognizes thephosphorylated (Y419 in human, Y418 in mouse), activeform of each member of the Src-family kinases. Strongphospho-SFK (Y419) staining, denoting an active SFKenzyme, was evident in the neoplastic ducts of morethan 60% (14/21) of the patient tumor specimens(Figure 1), but undetectable or present at low levels inthe adjacent non-neoplastic ductal epithelium (Figures 1aand b). We noted strong pSFK(Y419) staining not onlyin the primary tumor (Figures 1e and f), but also inadjacent pancreatic intraepithelial neoplasia (Figures 1aand d), which suggests that SFK activation may bean early event in pancreatic tumorigenesis. Thesedata corroborate a recent study describing elevated Srcactivity in human pancreas cancer (Morton et al., 2010).Given that Kras mutations are the earliest identifiablegenetic alterations in PDA, we became particularlyinterested in the question of whether elevated SFK

signaling might cooperate with oncogenic Kras topromote pancreatic neoplasia.

Dual Ras/SFK activation cooperate to promote PDATo determine the role of activated SFK signaling in theonset and progression of KrasG12D initiated neoplasia, wetargeted oncogenic KrasG12D expression and homozygousdeletion of the SFK inhibitor, CSK to the embryonicpancreas using Pdx1-Cre-driven recombination (Figure 2a).Allele-specific genotyping confirmed the genetic compo-sition of the compound LSL-KrasG12D;CSKf/f;Pdx1-Cremice (Supplementary Figure S1). Activation of the Srcfamily kinases concurrent with pancreatic KrasG12D

expression led to visible changes in the pancreas by 3weeks and precursor lesions were detectable by histo-logical examination, even at this early age, with apenetrance of B40% (n¼ 63 compound mutant mice)(Figures 2b, 3b and d). Disease progression was rapidwith mice developing PDA by 5–8 weeks of age (Figures 3eand f). In the 40% of compound mice that developedtumors, median survival was 6.0 weeks (n¼ 26) demon-strating that activation of SFKs not only acceleratesKrasG12D-driven tumor initiation but also promotes rapidprogression to lethal invasive adenocarcinoma (Figures 3eand f, Supplementary Figure S2). The remaining 60% oftumors did not develop tumors or present withprecursor lesions by 18 months of age. The reasons forincomplete penetrance of the phenotype have not beendetermined at this time. By comparison, mice thatexpress KrasG12D only, in the context of wild-type levelsof Src activity, do not develop pancreatic intraepithelialneoplasia until 2–5 months of age and only 10%ultimately develop PDA by 12 months (Hingoraniet al., 2003). Deletion of pancreatic CSK alone did notimpair pancreatic development indicating that CSK isnot required for normal development of this organ.Most significantly, none of the CSKf/f;Pdx1-Cre animalsharbored neoplastic lesions at the time of necropsy (upto 60 weeks of age, n¼ 25 mice; Supplementary Figure S3).These findings demonstrate that CSK deletion aloneis insufficient to initiate neoplasia and that oncogenic Krasand SFK activation can cooperate to accelerate thedevelopment of PDA in mice.

Immunohistochemical analysis of tumors fromLSL-KrasG12D;CSKf/f;Pdx1-Cre mice confirmed the absenceof CSK and the activation of the Src family kinases,as measured by phospho-SFK (Y418) staining in theneoplastic ducts (Figure 4a). Activation of the MAPkinase pathway was demonstrated by strong phospho-Erk staining in the ductal epithelium (Figure 4a). Bycontrast, Src and Erk are not activated in the non-neoplastic pancreas (Figure 4a). Src, Yes, Fyn and Lynhave each been implicated in neoplastic transformationof sites such as the pancreas, prostate and breast. Todefine the SFKs that are activated in this model, wedeveloped cell lines from the murine PDA’s (Figure 4b).Src was the predominant active SFK in the lysates oftumor-derived cell lines, with Yes displaying minimallevels of activity (Figure 4b). Lyn and Fyn activity wereundetectable (data not shown).

Oncogenic Ras/Src cooperativityDJ Shields et al

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Ras/Src derived PDA displays features of the humandiseaseTo further characterize the oncogenic cooperativitybetween KrasG12D and activated c-Src, we investigatedthe kinetics of tumor formation and the stromal/epithelial organization in the pancreata of compoundmutant mice. Ductal hyper-proliferation was evidentthroughout the pancreas as early as the suckling-weaning transition (3 weeks) (Figure 3c). By 5 weeks,the pancreata were characterized by multiple foci ofcarcinoma, most of which were poorly differentiated,but which also included some moderately to welldifferentiated neoplastic ducts (Figures 3c and f).

Neoplastic cells in the Ras/Src-driven tumors ex-pressed the ductal marker, cytokeratin 19 (Figure 5a),and scattered areas of tumor cells were found thatsecrete acidic (Alcian blue positive, Figure 5b) andneutral (periodic acid Schiff positive, Figure 5c) mucins,indicating aberrant differentiation in the spontaneoustumors. Papillary structures were regularly found in theductal lesions as was epithelial bridging between residualislands of in situ carcinoma, two frequently observedhistological features in the human disease (Hrubanet al., 2004) (Figures 3g–i). At necropsy, mice typicallypresented with locally advanced pancreatic tumors thatimpinged on the stomach, spleen, liver and intestines.

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Figure 1 SFK activation is evident at the earliest stages of pancreatic neoplasia. Immunohistochemical staining of activated SFK(pY419) in primary human pancreatic tumor specimens. (a) Large atypical ductal structure with mucinous epithelium (large arrow)surrounded by fibrosis. Ductal cells display heterogeneous pSFK(419) staining. An adjacent small atrophic non-neoplastic duct isindicated (red arrowhead). (b) Early pancreatic intraepithelial neoplasia (PanIN-1, (large arrow) with mucinous change and anadjacent non-neoplastic duct (red arrowhead) are shown. Strong pSFK staining is evident on the apical surface of some of the mucin-filled ductal epithelium. (c) Atypical ductal structure with strong junctional staining of pSFK, surrounded by cellular stromal tissue(asterisk). Note, distorted glandular shapes, intra-luminal papilla and variable height of epithelium. (d) Pancreatic intraepithelialneoplasia-2/3 with strong junctional pSFK staining surrounded by cellular stroma (asterisk), which contains numerous pSFK-positivecells (possibly inflammatory cells). (e, f) Pancreatic ductal adenocarcinoma exhibiting marked pSFK staining on the luminal surfaceand to a lesser extent in the basal cytoplasm. Scale bar represents 100mM, all images � 200.


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We did not detect visceral metastases to the liver orlungs, which could be due to a requirement foradditional genetic lesions and/or the locally aggressivenature of the disease denying time for the formation ofdistant disease.

Marked stromal hyperplasia and fibroblast infiltra-tion was observed in the Ras/Src-driven tumors, aknown feature of human tumors (Hernandez-Munozet al., 2008) (Figure 3) and in fact, the surroundingstroma commonly featured a higher number of pro-liferating cells than the neoplastic ductal epithelium(Figure 5d). This led to highly fibrous tumors that werefrequently palpable through the abdominal wall and hada firm consistency at the time of necropsy. This could beattributed, at least in part, to the significant levels ofcollagen deposition throughout the tumor, as visualizedby multispectral, birefringence imaging (Figure 5e). Thesurrounding stroma was also characterized by high

levels of desmin expression, distinguishing it clearlyfrom the invading carcinomatous glands (Figure 5f).

Oncogenic Ras/Src cooperativity and genomic instabilityNext, we sought to characterize the underlying mechan-isms of Src/Ras-driven tumorigenesis in this model. Oneof the hallmarks of PDA is genomic instability (Hezelet al., 2006), and in our analysis of the tumor-derivedKras/Src cells, we noted multiple examples of abnormalmitotic spindles and centrosomal amplification, asobserved by immunofluorescence analysis of a-tubulinand g-tubulin, respectively (Supplementary Figure S4A-B). Furthermore, LaminB staining of the nuclearenvelope in these cells enabled the identification ofdiscrete micronuclei (MN) (Figure 6a), which areestablished markers of genomic instability resultingfrom chromosomal malsegregation (Fenech, 2000). Todetermine if dual Ras/Src activation correlated with MNincidence, we quantified the numbers of MN’s in Kras/Src cells (activated Kras and Src), and Kras cells(activated Kras only) derived from the LSL-KrasG12D/Pdx1-Cre spontaneous model of PDA. Kras/Src cellscontained significantly higher numbers of MN’s thanKras cells (12.6% vs 0.5% respectively, Po0.05)(Figures 6a and b). To determine if combinatorial Src/Ras signaling is sufficient to promote MN formation, wetransiently expressed activated Src kinase (SrcA) in Krascells. Expression of activated Src in Kras cells causeda significant increase in the appearance of MN’s ascompared with cells transfected with vector control (5.5vs 0.5% respectively, Po0.05) (Figure 6c). Thesefindings suggest that in combination with oncogenicKras, Src activation can promote the onset of genomicinstability, a hallmark of malignant disease.

Tumor cells exhibit signs of Src dependencyAs combined Src/Ras activation may promote geneticinstability, which in turn may lead to differential tumorsuppressor loss, the question arises as to whethersecondary genetic lesions become the drivers of tumori-genicity in the Kras/Src cells, or if the cells remaindependent on activated Src signaling. To address thisquestion, we reintroduced CSK to Kras/Src cells andperformed anchorage-independent growth assays. Im-portantly, reconstitution with CSK significantly reducedcolony formation in soft agar whereas a kinase-deadversion of CSK had no effect on colony formation inthis assay (Figure 7a). In contrast, expression of eitherwild-type CSK or the kinase-dead derivative did notimpair the colony-forming ability of Kras cells(Figure 7a), which express oncogenic Kras in the contextof wild-type (non-activated) Src. These findings indicatethat the Kras/Src cells exhibit a dependence on Src andmight be sensitive to pharmacological inhibitors of Src.

Dasatinib is a small molecule Src/Abl inhibitor thatwas originally approved to treat imatinib (Gleevec)-resistant chronic myeloid leukemia patients (Lombardoet al., 2004) and is currently in trials against a plethoraof solid tumors. To assess the sensitivity of the Kras/Srctumor-derived cell lines to Src blockade, we initially

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expression in the mouse pancreas. (a) Both CSK alleles areconditionally deleted and the endogenous KrasG12D allele isactivated in the pancreas of mice expressing Cre recombinaseunder the control of the Pdx1 promoter in KrasG12D; CSKf/f; Pdx1-Cre mice. (b) Upper: gross pathological images of a non-neoplasticpancreas from a control littermate (LSL-KrasG12D;CSKf/f) (left);and enlarged tumor-bearing pancreas in a 7-week-old compoundmouse (right). Lower: corresponding hematoxylin and eosinstained sections showing normal pancreatic acini and an islet (asdenoted by asterisk) (left). Dual Src/Ras activation leads to asignificant reduction in acinar cell content and increased numbersof disorderly proliferating ducts (large arrow) surrounded by richcellular stroma (arrowhead) (middle).


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Figure 3 Dual Ras/Src activation causes rapid development of invasive pancreatic ductal adenocarcinoma. (a–f) Histological featuresin LSL-KrasG12D;CSKf/f;Pdx1-Cre mice: (a) low power image of adenocarcinoma infiltrating and replacing islands of acinar cells(arrow). Residual uninvolved intra-pancreatic lymph node is indicated with asterisk. (b) Pancreatic intraepithelial neoplasia (arrow) aredetectable in the pancreata at the suckling-weaning transition (3 weeks). Fibrosis is evident in the surrounding stroma (asterisk).(c) Adenocarcinoma (arrow) including papillary structures and epithelial bridging between regions of in situ carcinoma. Mitotic figuresare indicated (arrowhead). (d) Moderately differentiated infiltrating ductal carcinoma adjacent to an entrapped non-neoplastic duct(arrow). (e) Invading distorted glandular structures (arrow) surrounded by cell-rich stroma. Individual invading tumor cells can be seenin the stroma (arrowhead). (f) Clump of invading pleiomorphic, poorly differentiated adenocarcinoma cells (arrow) surroundedby fibrotic stroma (asterisk). The spontaneous tumors contain histological features commonly found in the human disease including(as denoted by arrows) papillary structures (g) epithelial bridging (h) and cribriform structures (i).


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exposed the cells to varying doses of dasatinib in vitro.Dasatinib impaired tumor cellular proliferation in vitro(Supplementary Figure S5, IC50¼ 47.6 nM), at a doserequired to achieve inhibition of Src kinase signaling inthese cells (Supplementary Figure S6). Next, to evaluatethe consequences of Src kinase inhibition on tumorigen-esis in vivo, Kras/Src cells were orthotopically injectedinto the pancreas of 8-week-old nu/nu mice. After 7 daysof tumor growth, animals were systemically treated withdasatinib (25mg/kg, p.o., qd� 14) and tumors wereresected and analyzed on day 21. Dasatinib treatmentcaused a significant reduction (50%) in tumor burden(P¼ 0.0019, n¼ 9 mice/treatment group) relative totumors in vehicle-treated mice (Figure 7b). Significantly,dasatinib had no effect on the growth of tumors derivedfrom Kras cells, which express KrasG12D in the context ofsignificantly lower levels of activated Src kinase thanKras/Src cells (Figures 4b and 7b). Dasatinib treatmentcaused a significant reduction in Src activation in theKras/Src tumors (Figure 7c, upper), and while immu-nohistochemical analysis of the orthotopic tumorsdemonstrated that Src activity was most markedlyreduced in the neoplastic ducts, a reduction in Srcactivity in the surrounding stroma was also observed(Figure 7c, lower). These studies suggest that Srcactivity, in concert with oncogenic Kras, has a key role

in the growth of these tumors, and provide support forthe inherent Src dependency of these cells.

Discussion

A novel role for Src activation in pancreatic neoplasiaAccumulating evidence from xenograft and orthotopicstudies has strongly implicated Src in tumorigenesis andmetastasis. However, data from genetically engineeredmouse models had indicated that Src activation alonemay be insufficient for tumor initiation. For example,overexpression of oncogenic v-Src in astrocytes leads tothe development of astrocytomas, but the latency is long(65 weeks) and the penetrance is low as only 14% ofmice develop tumors (Maddalena et al., 1999). Similarly,overexpression of SrcA(Y530F), wild-type c-Src ordeletion of CSK in keratinocytes each promotesepidermal hyperplasia, but a secondary insult such aswounding, or exposure to a combination of carcinogens7,12-dimethylbenz(a)anthracene (DMBA) and promot-ing agents 12-O-tetradecanoylphorbol-13-acetate (TPA)is required for carcinoma development (Matsumotoet al., 2004). In a more recent study, global over-expression of the human c-Src gene yielded neoplasticlesions after 20 months, but again, only 15% of the mice

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Figure 4 Conditional CSK deletion promotes activation of specific Src-family kinases. (a) Pdx1-Cre mediated recombination of thefloxed CSK alleles was evidenced by the lack of CSK immunohistochemical staining in the neoplastic ducts. Strong pSFK (Y418) andpErk staining demonstrated activation of the Src- and Ras-driven signaling cascades in the ductal epithelium. In contrast, activation ofSrc and Erk was not observed in normal pancreata. (b) Characterization of cell lines derived from pancreatic intraepithelial neoplasiaand spontaneous tumors in LSL-KrasG12D/Pdx1-Cre (Kras) and LSL-KrasG12D/CSKf/f (Kras/Src) mice, respectively. Immunoblotanalysis confirms CSK deletion, concurrent loss of CSK-mediated inhibitory phosphorylation (Y529) on the C-terminal tail of c-Srcand activation of Src (pY418).


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were affected (Kline et al., 2008). Intercrossing with p21null mice increased the incidence to 26% at 14 months(Kline et al., 2008), suggesting that additional geneticevents may enable the oncogenic potential of Src kinase.

Cooperative Kras/Src signaling in the developmentof PDAHere, we show that cooperative signaling betweenKrasG12D and activated Src promotes rapid developmentof aggressive pancreatic cancer. Although oncogeniccooperativity has been documented previously betweenproteins such as c-Myc and Bcl-2 in lymphoid tumors

(Fanidi et al., 1992), as well as between mutant Kras andc-Myc in mammary carcinogenesis (Podsypanina et al.,2008), this is, to our knowledge, the first demonstrationthat oncogenic cooperation between activated Kras/Srccan promote neoplasia in the mouse. Importantly,oncogenic Ras/Src cross-talk had previously been demon-strated in Drosophila (Vidal et al., 2006), suggesting thatcooperative signaling between these two classical onco-proteins may not be restricted to pancreatic carcinomas.

Deletion of CSK alone was insufficient to initiatecarcinogenesis, but combined Ras activation and CSKdeletion leading to activation of Src led to thedevelopment of PDA by 5–8 weeks of age, as compared

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Figure 5 The epithelial and stromal sub-compartments of LSL-KrasG12D;CSKf/f ;Pdx1-Cre tumors share many features in commonwith human pancreatic carcinoma. Both infiltrating carcinoma and precursor lesions display strong expression of the ductal marker,CK-19 (a). Acidic and neutral mucin content of infiltrating neoplastic ducts (large arrows) was demonstrated by alcian blue (b) andperiodic acid Schiff (c) staining, respectively. Proliferative cells were evident in both the stroma (asterisk) and neoplastic epithelium(arrow) of invasive carcinomas as demonstrated by Ki-67 staining (d). Collagen deposition (green) can be detected by multispectralbirefringence imaging (e), whereas strong desmin staining also identified the abundant fibroblast content (f).


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with 12–14 months for Kras alone, establishing that inthe context of oncogenic Kras, activated Src kinasesignaling can act as a promoter of tumor progression.Loss of CSK function can lead to the activation of eachof the Src family kinase members but in this model,c-Src was the only SFK member activated in tumorswith oncogenic Kras expression and CSK loss. How-ever, this does not rule out a possible role for other asyet unidentified CSK substrates in the progression ofKras-dependent PDA. Similarly, CSK loss may impactthe function of key interactants, such as the p53 bindingprotein ASPP. Additional studies will be necessary todefine the precise role of CSK and Src in pancreascancer progression.

In recent years, a number of other spontaneous mousemodels of pancreatic cancer have been generated basedon the known genetic lesions in PDA patients. Althoughmutational activation of oncogenic Kras has beenestablished as a key initiating event, additional driverevents that contribute to disease progression include thesilencing or deletion of Ink4a/Arf (80–85% of clinicalPDA specimens), mutational inactivation of p53 (65%)and the homozygous deletion of Dpc4/Smad4 (55%)(Hezel et al., 2006). So, how might changes in Srcactivity interplay with these other key genetic lesions inpancreatic neoplasia? v-Src was the first identifiedoncogene, but activating mutations of c-Src are ex-tremely rare in human cancers and none have beendetected in PDA. Despite this, Src activity is increased in60% of PDA’s and this is likely due to increased Srcprotein abundance or elevated signaling from activatedRTK’s such as ErbB-2 (Her2-Neu) and RON (Thomaset al., 2007; Ricono et al., 2009). In fact, members of theepidermal growth factor receptor family are over-expressed in up to 70% of PDA’s (Hall et al., 1990),and upregulation of ErbB-2 is believed to be one of theearliest events in neoplastic conversion of the pancreas(Hruban et al., 2000). Genetic ablation of Ink4a/Arf inthe context of oncogenic Kras signaling leads to amurine model of metastatic PDA that recapitulatesfeatures of the human disease (Aguirre et al., 2003).Intriguingly, this combination of mutations also leads toa significant upregulation of epidermal growth factorreceptor and Her2 expression in the neoplastic ducts,each of which can activate Src kinase. More recently,analysis of tumors from the LSL-KrasG12D; LSL-p53R172H;Pdx1-Cre mouse model, which closely recapitu-lates many clinical features of the disease, demonstratedthat Src activity is elevated in the pancreatic intrae-pithelial neoplasia lesions and continues to increase as afunction of disease progression in this model (Mortonet al., 2010). Furthermore, progression to advancedmetastatic disease was attenuated by systemic Srcinhibition (Morton et al., 2010).

A model of Src dependencyConversely, data from our model suggest that Rasactivation combined with CSK deletion may actuallypromote genetic instability, which, in turn, could lead toloss of key tumor suppressors such as p16, p53 and

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Figure 6 Cooperative Ras/Src signaling promotes MN formationin PDA cells. (a) Immunofluorescence of LaminB staining to denotethe nuclear envelope in Kras cells (left) and Kras/Src cells (right).Discrete MN (indicated by yellow arrowhead) that form duringchromosomal malsegregation are visualized as extranuclear DNA-containing structures that are demarcated by a LaminB-positivemembrane. (b) Quantification of the incidence of MN-containingcells in the Kras and Kras/Src PDA cell lines. Data are expressed asa percentage of the total cells that were assessed (mean of threeseparate experiments±s.e.m). Kras/Src cells had a significantlyhigher incidence of MN than Kras cells. The * symbol indicatesPo0.05 compared with MN incidence in Kras cells; unpaired t-test.(c) Quantification of the incidence of MN-containing Kras cells thatwere transiently transfected with an activated Src (SrcA) or controlplasmid. Cells were stained and MN incidence was quantified48h post transfection. Transient expression of activated Src kinasecaused a significant increase in the incidence of MN in Kras cells.The symbol * indicates Po0.05 compared with Kras cellstransfected with control vector; unpaired t-test.


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Smad4. These findings are consistent with previousreports showing that Src/MAPK signaling promotesgenomic instability through activation of Aurora Bkinase (Kabil et al., 2008), and that SFKs have been

proposed to regulate cell proliferation through abroga-tion of p53 function (Broome and Courtneidge, 2000).Interestingly, combined Src/MAPK signaling is alsorequired for abscission during cytokinesis and cell

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Figure 7 Kras/Src tumor cells exhibit Src dependency. (a) Effect of Src inhibition on anchorage-independent growth. Kras and Kras/Src cells were infected with adenovirus expressing wild-type CSK or the kinase dead derivative, or empty adenovirus. CSK, but notCSK-KD expression caused a significant reduction in colony growth of the Kras/Src, but not Kras cells on soft agar. A representativeexperiment is shown. In independent experiments n¼ 3. The symbol * indicates Po0.05, as compared with Kras/Src cells infected withCSK or empty virus; analysis of variance with post-hoc test. (b) Effect of Src inhibition on tumor burden in an orthotopic model ofPDA. Cells (5� 105) derived from LSL-KrasG12D;Pdx1-Cre tumors (activated Kras, wild-type Src; ‘Kras’) and LSL-KrasG12D;CSKf/f;Pdx1-Cre tumors (activated Kras and activated c-Src; ‘Kras/Src’) were implanted into the tail of the pancreas in 6–8-week-old malenu/nu mice. Tumors were allowed to establish for 7 days and then mice were administered vehicle or dasatinib (25mg/kg, p.o., qd� 14 d).At 21d, tumors were resected and weighed. Dasatinib treatment led to a significant reduction in burden of the Kras/Src, but not theKras tumors; the symbol * indicates P¼ 0.0019 compared with vehicle-treated mice; paired t-test (n¼ 9 mice/treatment group).(c) Immunoblot analysis of activated Src levels in Kras/Src tumors following dasatinib treatment. Immunohistochemical analysis ofp(Y418) Src (activated enzyme) demonstrated a reduction in Src activity in both the neoplastic ducts and surrounding stroma. Tumor-bearing mice were dosed as in b above and the tumors were resected on day 14 at 1 h post dose for analysis by immunoblot andimmunohistochemistry.


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proliferation (Kasahara et al., 2007). Irrespective of theimpact of dual Ras/Src signaling on suppressors ofKras-mediated tumorigenesis, these tumors exhibit anongoing dependence on the hyperactivated Src signalingcascade. Accordingly, genetic or pharmacological Srcinhibition attenuates the tumorigenicity of neoplasticcells from Ras/Src derived PDA’s. In contrast, Mortonet al. recently demonstrated that Src inhibition attenu-ated metastatic disease but not primary burden in theRas/p53 mouse model (Morton et al., 2010), suggestingthat Src may have different roles in tumorigenesis ormetastasis, depending on the underlying mutational con-text. Elucidation of the molecular basis for the differencesin Src dependency could enable the identification of patientsubsets that respond to therapeutic intervention with agentstargeting the Src signaling pathways.

Materials and methods

Mouse strainsCSKf/f (floxed), Lox-Stop-Lox (LSL)-KrasG12D and Pdx1-Cremouse strains have been described (Schmedt et al., 1998;Jackson et al., 2001; Hingorani et al., 2003). These strains wereinterbred to generate the compound triple mutant animals;KrasLSL-G12D/þ ; CSKf/f; Pdx1-Cre (KCC). All animal proce-dures were conducted in accordance with the appropriateregulatory standards under protocol S05018 as approved bythe UCSD IACUC.

Histological and immunohistochemical analysisAll histological processing of fixed tumor specimens andstaining (hematoxylin and eosin, periodic acid Schiff,alcian blue) of paraffin embedded sections was performed atthe Histology Core in Moores UCSD Cancer Center.Histological analysis was performed by Dr David Tarin(Moores UCSD Cancer Center) and Dr Greg Boivin (WrightState University, Dayton, OH, USA). Immunostaining wasperformed according to the manufacturer’s recommendations(Vector Labs, Burlingame, CA, USA), on 5mM sections ofparaffin-embedded tumors from the spontaneous mouse modelor from human patients diagnosed with PDA (as approved bythe Institutional Review Board at University of California,San Diego—Project#090407X). For pSFK(Y418/Y419) im-munohistochemistry, antigen retrieval was performed inEDTA buffer pH 8.0. Antigen retrieval for all other antibodieswas performed in citrate buffer, pH 6.0, at 95 1C for 20min.Sections were treated with 0.3% H2O2 for 30min, blocked innormal goat serum, phosphate-buffered saline Tween 20 (PBS-T)-for 30min followed by Avidin-D and then incubated over-night at 4 1C with primary antibody diluted in blockingsolution as follows: CSK (AbCam, Cambridge, MA, USA,1/100), pErk (Cell Signaling, Danvers, MA, USA, 1/500),CK19/TromaIII (Hybridoma bank, Iowa City, IA, USA,1/1000), Ki67 (AbCam, 1/1000), Desmin (Neomarkers, Fremont,CA, USA, 1/500), Insulin (Invitrogen, Carlsbad, CA, USA,1/10 000), Glucagon (Santa Cruz, Santa Cruz, CA, USA,1/1000), Amylase (Sigma, St Louis, MO, USA , 1/100). Tissuesections were washed and then incubated with biotinylatedsecondary antibody (1:500, Jackson ImmunoResearch, WestGrove, PA, USA) in blocking solution for 1 h. Sections werewashed and incubated with Vectastain ABC (Vector Labs) for30min. Staining was developed using a Nickel-enhanceddiamino-benzidine reaction (Vector Labs) and sections werecounter-stained with hematoxylin.

Immunofluorescence analysisCells were transfected with activated Src (SrcA) or controlplasmids using Lipofectamine, and seeded directly on 22mmcoverslips in 6-well plates at a density of 1� 105 per well.Following incubation at 37 1C for 48 h, cells were fixed in coldmethanol at �20 1C for 10mins (a-tubulin, g-tubulin) or 4%paraformaldehyde/PBS, pH 7 at room temperature for 10min.Following permeabilization with 0.2% Triton X-100/PBS for2min, cells were blocked in 3% bovine serum albumin/PBS for1 h at room temperature, and then incubated with the primaryantibodies (a-tubulin, g-tubulin, both AbCam; LaminB, SantaCruz; all 1/300 dilution in 3% bovine serum albumin/PBS) atroom temperature for 2 h. Cells were subsequently incubatedfor 1 h at room temperature with the appropriate species AlexaFluor secondary antibodies (488 or 568). Nuclei werevisualized following DraQ5 staining. Coverslips were mountedonto slides with Vectashield and images were acquiredusing laser scanning confocal microscopy under � 60/1.4 NAoil objective (Nikon C1si, Nikon Instruments Inc., Melville, NY,USA). Quantification of MN was achieved by capturing imagesof multiple high-power fields and scoring the number of cells thatcontained discrete MN with a LaminB demarcated boundary.A minimum of 1000 cells were quantified per condition in eachexperiment and all experiments were performed three times. Allimages presented are single sections in the Z plane.

Imaging of tumor collagen contentImages of collagen content in hematoxylin and eosin-stainedprimary Kras/Src tumor sections were acquired with Cri’sprototype ‘Nubrio’ imaging system, which enables the multi-spectral detection of birefingent structures such as collagen intumor specimens. After setting the image acquisition para-meters and taking appropriate background images for correctretardance and flat-fielding purposes, subsequent multimodal,multilayer images were acquired.

Immunoprecipitation and immunoblot analysisTumors/pancreata were homogenized by mechanical disrup-tion. Both tissues and tumor cell lines were lysed in a modifiedradioimmuno precipitation assay buffer containing 50mM TrispH 7.5 at 41C, 150mM NaCl, 1mM EDTA, 50mM NaF, 5mM

sodium pyrophosphate, 10mM b-glycerophosphate, 1% NP-40, 1mM Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS,phosphatase and protease inhibitors (Roche, Indianapolis, IN,USA). Lysates were cleared by centrifugation and the proteinconcentration of the cleared lysate was determined by thebicinchoninic acid method. Immunoblots were performedusing the following antibodies: pSFK-Y418 (Cell Signaling),Erk2, Fyn (Santa Cruz), Yes (BD Biosciences, San Diego, CA,USA), Src, pY-4G10 (Millipore, Billerica, MA, USA). Immuno-precipitations were performed with protein A/G beads and theSrc, Yes and Fyn antibodies listed above.

Pancreatic tumor cell linesPDA cell lines were isolated as described (Aguirre et al., 2003)from primary tumors in the Kras/Src model. PDA lines used inthis study from other spontaneous mouse models of pancreaticcancer have been described previously (Murphy et al., 2008;Thomas et al., 2008). The PDA lines were maintained understandard culture conditions in RPMI medium 1640 supple-mented with penicillin and streptomycin, 2mM glutamine and10% fetal bovine serum. Proliferation rate of cell lines wasdetermined by XTT assay (Promega, Madison, WI, USA),following 48 h incubation in complete media with vehicle(dimethyl sulfoxide) or the indicated dasatinib concentration.


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Anchorage independent growth assaysAfter 48 h of infection with adenovirus (10 multiplicity ofinfection) expressing wild-type or kinase-dead CSK, or controlvirus, cells were suspended in 0.3% agar/complete media andgrown on top of a bottom layer of 1% agar/complete media in48 or 24-well plates. Additional media was overlayed and cellswere cultured for 7–10 days before counting colonies consist-ing of at least five cells from fields or whole wells.

Orthotopic pancreatic carcinoma modelThe orthotopic pancreatic carcinoma model has been de-scribed (Grimm et al., 2003). Briefly, 6–8-week-old nu/nu micewere injected with 5� 105 cells derived from tumors in LSL-KrasG12D;Pdx1-Cre mice (Kras cells) or LSL-KrasG12D;CSKf/f

;Pdx1-Cre mice (Kras/Src cells) in the tail of the pancreas.Tumors were allowed to establish for 7 days and then animalswere administered vehicle or dasatinib (25mg/kg, p.o.,qd� 14). On day 21, mice were harvested, primary tumorswere resected and tumor burden was assessed by weight.

Statistical analysisData presented are means±s.e.m. Statistical analyses wereperformed with Prism (GraphPad, La Jolla, CA, USA).Statistical differences for one factor between two groups or

more than two groups were determined with an unpairedStudent’s t-test or an analysis of variance (ANOVA) with apost-hoc test, respectively. Statistical significance for theorthotopic animal studies was determined by a pairedStudent’s t test. Statistical significance was defined as Po0.05.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We thank Charles Yi and Dana Wu for excellent technicalassistance. We acknowledge Richard Aspinall, Lisette Aceve-do, Josh Greenberg and David Tuveson for valuable discus-sions, Greg Boivin for histological analysis, as well as RichardLevenson and Kristin Lane for NuBrio imaging. This workwas supported by NIH grants R21CA104898, P01-CA078045(DAC) and Collaborative Translational Research grants fromMoores UCSD Cancer Center (DAC, DJS and AML).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)


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Oncogenic Ras/Src cooperativity in pancreatic neoplasia