inverse correlation of stat3 and mek signaling mediates...

Translational Science

Inverse Correlation of STAT3 and MEK SignalingMediates Resistance to RAS Pathway Inhibition inPancreatic CancerNagaraj S. Nagathihalli1,2, Jason A. Castellanos3, Purushottam Lamichhane1,Fanuel Messaggio1, Chanjuan Shi4, Xizi Dai1, Priyamvada Rai2,5, Xi Chen2,6,Michael N. VanSaun1,2, and Nipun B. Merchant1,2

Abstract

Major contributors to therapeutic resistance inpancreatic ductal adenocarcinoma (PDAC) includeKras mutations, a dense desmoplastic stroma thatprevents drug delivery to the tumor, and activationof redundant signalingpathways.Wehavepreviouslyidentified amechanistic rationale for targeting STAT3signaling to overcome therapeutic resistance inPDAC. In this study, we investigate the molecularmechanisms underlying the heterogeneous responseto STAT3 and RAS pathway inhibition in PDAC.Effects of JAK/STAT3 inhibition (STAT3i) or MEKinhibition (MEKi) were established in Ptf1acre/þ;LSL-KrasG12D/þ; and Tgfbr2flox/flox (PKT) mice andpatient-derived xenografts (PDX). Amphiregulin(AREG) levels were determined in serum fromhuman patients with PDAC, LSL-KrasG12D/þ;Trp53R172H/þ;Pdx1Cre/þ (KPC), and PKT mice.MEKi/STAT3i–treated tumors were analyzed forintegrity of the pancreas and the presence of cancerstem cells (CSC). We observed an inverse correlationbetweenERK and STAT3 phosphorylation.MEKi resulted in an immediate activation of STAT3,whereas STAT3i resulted in TACE-induced, AREG-dependent activation of EGFR and ERK. Combined MEKi/STAT3i sustained blockade of ERK, EGFR, and STAT3signaling, overcoming resistance to individual MEKi or STAT3i. This combined inhibition attenuated tumor growth in PDX andincreased survival of PKT mice while reducing serum AREG levels. Furthermore, MEKi/STAT3i altered the PDAC tumormicroenvironment by depleting tumor fibrosis, maintaining pancreatic integrity, and downregulating CD44þ and CD133þ

CSCs. These results demonstrate that resistance to MEKi is mediated through activation of STAT3, whereas TACE-AREG-EGFR–dependent activationof RAS pathway signaling confers resistance to STAT3 inhibition. CombinedMEKi/STAT3i overcomes theseresistances and provides a novel therapeutic strategy to target the RAS and STAT3 pathway in PDAC.

Significance: This report describes an inverse correlation between MEK and STAT3 signaling as key mechanisms of resistancein PDAC and shows that combined inhibition of MEK and STAT3 overcomes this resistance and provides an improvedtherapeutic strategy to target the RAS pathway in PDAC.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6235/F1.large.jpg. Cancer Res; 78(21); 6235–46.�2018 AACR.

© 2018 American Association for Cancer Research

STAT3 inhibition

Therapeutic resistance Therapeutic resistance Overcome therapeutic resistancetumor growth inhibition

TACEAREG

AZD1480(STAT3i)

AZD6244(MEKi) AZD6244

(MEKi)

pSTAT3 pSTAT3pERK

Collagen fiberCancer cell Cancer stem cells

EGFR

TACEAREG

AZD1480(STAT3i)

pSTAT3 pERK

EGFR

AREG

pERK

EGFR

MEK inhibition MEK and STAT3 inhibition

Combined MEK/STAT3 inhibition overcomes STAT3- and MEK-mediated drug resistance and provides an improved therapeuticstrategy to target the RAS pathway in PDAC.

1Department of Surgery, University of Miami Miller School of Medicine, Miami,Florida. 2Sylvester Comprehensive Cancer Center, University of Miami MillerSchool ofMedicine,Miami, Florida. 3Department of Surgery, VanderbiltUniversitySchool of Medicine, Nashville, Tennessee. 4Department of Pathology, VanderbiltUniversity School of Medicine, Nashville, Tennessee. 5Department of Medicine,University of Miami Miller School of Medicine, Miami, Florida. 6Department ofPublic Health, University of Miami Miller School of Medicine, Miami, Florida.

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

Corresponding Author: Nipun B. Merchant, University of Miami Miller School ofMedicine, Sylvester Comprehensive Cancer Center, 1120 NW 14th Street, CRB410, Miami, FL 33136. Phone: 305-243-4902; Fax: 305-243-4907; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0634

�2018 American Association for Cancer Research.

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IntroductionPancreatic ductal adenocarcinoma (PDAC) is currently the

third leading cause of cancer mortality in the United States andestimated to become the second leading cause of cancer-relateddeath by 2020 (1).Despite advances in understanding the biologyof PDAC, the clinical course is still hampered by therapeuticresistance and lack of progress in improving survival outcomes.Activating Kras mutations, present in over 90% of PDAC tumors(2), are difficult to target due to the interdependence of redundantsignaling pathways and feedback loops. Another hallmark ofPDAC is the presence of a dense desmoplastic reaction composedof stromal cells, including stellate cells, immune cells, and cancer"stem" cells (CSC) that support the tumor microenvironment(TME), acts as barrier to drug delivery and also contributes totherapeutic resistance of PDAC (3, 4). There remains an incom-plete understanding of interactions among the different TMEcomponents that support cancer growth and promote resistanceto therapy.

In PDAC, Kras-activating mutations promote proliferationand inhibit apoptosis through the RAF/MEK/ERK and PIK3/AKT pathways (5–7). Because targeting RAS has remainedelusive, efforts have focused on targeting downstream effectorsof RAS through MEK inhibition (5, 8). The clinical success ofMEK inhibitors in Kras-mutant melanoma and lung cancerimplicate their therapeutic potential in PDAC (9, 10). Unfor-tunately, clinical trials of MEK inhibition (MEKi), have beenunsuccessful in PDAC (7, 9, 11), likely due to the activation ofresistance pathways.

Shedding of EGFR ligands from PDAC cells results inautocrine feedback and subsequently facilitates Kras activationto promote the onset of pancreatic neoplasia (12, 13). EGFRligands amphiregulin (AREG) andTGFa reside as transmembraneglycoproteins in their precursor form and are enzymatically shedvia metalloproteinase TNFa-converting enzyme (TACE,ADAM17) to regulate EGFR signaling (12, 14–16). We haveshown that TACE and AREG, but not TGFa, are overexpressedin colorectal cancer and PDAC (14). AREG appears to be theprimary ligand for promoting TACE-mediated activation of EGFR,MAPK, and STAT3 signaling.

The poor response of patients with PDAC to targeted andsystemic therapies is also partially due to the dense desmo-plastic reaction, which impedes drug delivery into tumors (17–20). We have shown that targeted inhibition of STAT3 (STAT3i)in combination with gemcitabine enhances drug delivery intotumors by remodeling collagen fibers and increasing micro-vessel density (19, 20). Therapeutic resistance in PDAC is alsoconferred by increased percent of CSCs in the TME followingchemotherapy (21).

In this study, we have provided proof that STAT3 activation is anovelmolecularmechanismunderlying the therapeutic resistanceof PDAC to RAS pathway inhibition. We show a direct inversecorrelation between ERK and STAT3 signaling in PDAC andprovide evidence that a major resistance mechanism impairingMEKi occurs through TACE-induced, AREG-mediated EGFR path-way activation. We further show combined MEKi/STAT3i resultsin sustained inhibition of both ERK and STAT3 signaling, over-coming therapeutic resistance associated with the AREG-EGFR–mediated parallel feedback. In addition, we show MEKi/STAT3iinhibits tumor fibrosis and downregulates CSCs to enhancetherapeutic response in PDAC. Furthermore, elevated serum

AREG levels are decreased with MEKi/STAT3i in PKT mice, sug-gesting a role for serum AREG as a prognostic biomarker oftherapeutic response as well as a biomarker of therapeutic resis-tance to EGFR, MEK, and STAT3 inhibition.

Materials and MethodsCell lines and chemicals

Mouse pancreatic intraepithelial neoplasia (PanIN), primaryPDAC (PDA), and liver metastatic (LMP) cell lines were derivedfrom the LSL-KrasG12D/þ; Pdx1Cre/þ (KC) and LSL-KrasG12D/þ;Trp53R172H/þ; Pdx1Cre/þ (KPC) genetically engineered mousemodels (GEMM) of PDAC (kindly provided byDr. Andrew Lowy,University of California San Diego, San Diego, CA; refs. 22, 23)and maintained as described previously (20). Authenticatedhuman PDAC cell lines MiaPaCa2, PANC1, CFPAC, Capan2,Capan1, AsPC1, SW1990, Panc04, Panc02, Panc10, and BxPC3were obtained andmaintained according to ATCCguidelines. ThePC-13 and PC-17 cell lines were derived from patient-derivedxenografts (PDX; kindly provided by Dr. Anthony Capobianco,University of Miami, Coral Gables, FL) with known mutationalstatus (PC-13: KrasQ61H/þ/PIK3CAM/þ/p53R72P/þ; PC-17: APCM/M/KrasG12D/þ/p53R72P/R72P).

Cell authentication was performed by using short tandemrepeat DNA profiling (latest date: June 16, 2016) and cell linestested negative forMycoplasma via Genetica cell line testing usingeMYCOplus Kit (iNtRONBiotechnology). Cells with relative lowpassage numbers (<20) were used in the study.

Primary antibodies used for Western blot analysis (Supple-mentary Table S1), IHC (Supplementary Table S2), and flowcytometry analysis (Supplementary Table S3) were summarizedin the Supplementary Tables S1–S3. The chemical agents used arepresented at Supplementary Table S4.

Tissue microarray of human pancreatic tissuesPreviously constructed tissue microarray (TMA; ref. 20) slides

were concurrently evaluated by 2 of the authors (C. Shi and N.B. Merchant). Nuclear and cytoplasmic staining was scored asfollows: staining index was considered as the sum of theintensity score (0, no staining; 1þ, weak; 2þ, moderate; 3þ,strong) and the distribution score (0, no staining; 1þ, stainingof <33% of cells; 2þ, between 33% and 66% of cells; and 3þ,staining of >66% of cells). Staining indices were classified asfollows: 3þ or higher, strong staining; 1þ to 2þ, weak staining;and 0, negative staining. pERK and pSTAT3 were scored aspositive if any detectable nuclear or cytoplasmic staining waspresent.

MiceFemale athymic nude mice [Foxn1 nu/nu (4–5 weeks old)]

were purchased from Harlan Sprague Dawley, Inc. Ptf1acre/þ;Tgfbr2flox/flox and LSL-KrasG12D/þ;Tgfbr2flox/flox mice were provid-ed by Dr. Hal Moses (Vanderbilt University Medical Center,Nashville, TN). These two lines were intercrossed to generatePtf1acre/þ;LSL-KrasG12D/þ;Tgfbr2flox/flox mice (PKT) on a C57Bl/6background. Genotyping of alleles was performed using oligo-nucleotide primers as described previously (20, 24). LSL-KrasG12D/þ, Pdx1Cre/þand p53R273H/þmice were intercrossed togenerate indicated LSL-KrasG12D/þ; Trp53R172H/þ; Pdx1Cre/þ

(KPC) animals (22, 23).

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Xenograft modelsSubcutaneous tumors were established by injecting 2 � 106

PANC1, MiaPaCa2, or BxPC3 cells into the flank of 6-week-oldFox1-nu/numouse [n¼4 (PANC1)orn¼3 (MiaPaCa2, BxPC3) ineach group]. Treatment was initiated when the subcutaneoustumors reached 75–100 (MiaPaCa2 or PANC1) or 200–250(BxPC3) mm3 size. Drug treatment was initiated at the same timepoint. AZD1480 (30mg/kg/day), AZD6244 (25mg/kg/day), bothdrugs together or vehicle (hydroxypropyl methyl cellulose/Tween80) was administered by oral gavage for 41 (MiaPaCa2), 49(PANC1), and 27 (BxPC3) days. The data were obtained asdescribed in the Supplementary Materials and Methods.

Treatment of Ptf1acre/þ;LSL-KrasG12D/þ;Tgfbr2flox/flox (PKT) micePKT mice were treated with vehicle, AZD1480 (JAK/STAT3

inhibitor), AZD6244 (MEK inhibitor), or a combination ofAZD6244 and AZD1480. Mice in the AZD1480 (30 mg/kg/day)andAZD6244 (25mg/kg/day) arm receivedbyoral gavage 5days/week, starting at 4 weeks of age. Mice were euthanized anddissected after 2 weeks unless they were part of the survival arm.Because of the irregularity of the tumor dimensions, size wasdetermined by weighing the entire tumor. Tumor tissue wasprocessed for further IHC examination. Overall survival wasdetermined by log-rank analysis using statistical software packageR (version 3.3.2).

Statistical analysisDescriptive statistics were calculated using Microsoft Excel and

Prism software (Graphpad Software Inc.). Results are shown asvalues of mean � SD unless otherwise indicated. Statisticalanalyses of IHC data were performed using the ANOVA followedby Tukey multiple comparisons test to determine P values. Thetwo-sided Student t test was used for statistical analysis, with P <0.05 taken as significant, except when indicated otherwise.Kaplan–Meier survival analysis was performed, and survival dif-ferences between groups were assessed with the log-rank test.Pearson correlation coefficient or Pearson r analysis was con-ducted using Prism software.

Study approvalAll animal experiments were carried out using protocols

approved by the Institutional Animal Care and Use Committeesat the Vanderbilt University Medical Center (Nashville, TN) andthe University of Miami (Miami, FL; #15-099).

For additional experimental procedures please refer to Supple-mentary Materials and Methods.

ResultsInverse correlation of ERK and STAT3 signaling in PDAC

A direct inverse correlation in expression levels of phosphor-ylated ERK (pERK) and STAT3 (pSTAT3) was observed both invitro and in vivo (Fig. 1). Inverse baseline expression of pERK andpSTAT3 was confirmed in 11 human PDAC cell lines (Fig. 1A,left). Cell lines with low or minimal pERK expression showedhigh levels of pSTAT3 expression, whereas cell lines with highpERK expression showed little or no pSTAT3 expression. Thispattern was also seen in a PanIN-derived cell line from the LSL-KrasG12D/þ; Pdx1Cre/þ (KC) GEMM, and from PDA and LMP celllines derived from the LSL-KrasG12D/þ; Trp53R172H/þ; Pdx1Cre/þ

(KPC) GEMM (Fig. 1A, right; ref. 22). Results from a human

tumor TMA confirmed that the majority of patient tumors withstrong pERK expression had low pSTAT3 expression, whereastumors with high pSTAT3 expression had low pERK expression(Fig. 1B; Supplementary Fig. S1A). Knockdown of STAT3 inPANC1 cells (sh-STAT3; ref. 20) showed increased expression ofpERK when compared with sh-scrambled cell lysate (Fig. 1C). Inaddition, pancreatic tumors from Ptf1acre/þ;LSL-KrasG12D/þ;Tgfbr2flox/flox (PKT)mice (Fig. 1D; Supplementary Fig. S1B) treatedwith the JAK/STAT3 inhibitor (STAT3i) AZD1480 showedincreased pERK levels, whereas mice treated with MEKi AZD6244showed increased pSTAT3 expression. Both STAT3i and MEKishowed increased pEGFR expression (Fig. 1D).

Treatment with the MEK inhibitors, AZD6244 or MEK162,effectively suppressed pERK while inducing pSTAT3 activationin PDAC cells in a time-dependent manner (Fig. 1E). Conversely,STAT3 inhibitors, Stattic and AZD1480, resulted in pERK activa-tion in a time- (Fig. 1F; Supplementary Fig. S2) and dose-depen-dent manner (Supplementary Fig. S3A–S3C). Even SW1990 cells,which have very low basal levels of pSTAT3 (Fig. 1A, left),increased pSTAT3 and pEGFR levels with MEKi in a time-(Fig. 1G) anddose-dependentmanner (Supplementary Fig. S3D).

To determine the effects of cytotoxic chemotherapy on STAT3or ERK signaling, we treated MiaPaCa2 cells with gemcitabinewith or without AZD1480 (Supplementary Fig. S4A and S4B) orerlotinib (Supplementary Fig. S4C). Gemcitabine treatmentalone resulted in a dose-dependent activation of both pSTAT3and pERK. Cells treated with both gemcitabine and AZD1480exhibited similar effects on STAT3 and ERK signaling comparedwith AZD1480 (STAT3i) alone, demonstrating that gemcita-bine did not alter the reciprocal activation of STAT3 or ERKsignaling. EGFR inhibition with erlotinib attenuated EGFRphosphorylation.

CombinedMEKi and STAT3i inhibits pancreatic tumorigenicityTo confirm this reciprocal activation of MEK and STAT3 as a

mechanism of resistance, we targeted both STAT3 and MEK inPDAC cell lines. MiaPaCa2 (Fig. 2A) cells treated in combinationwith U0126, a highly selective inhibitor of MEK1/2 and Stattic, asmallmolecule that specifically inhibits the SH2domainof STAT3or PANC1 and MiaPaCa2 (Fig. 2B) with combined AZD6244/AZD1480 showed sustained inhibition of both ERK and STAT3activation. CombinedMEKi/STAT3i inMiaPaCa2 cells resulted inenhanced apoptosis (Fig. 2C), reduced colony formation (Fig. 2D,left), decreased cell invasion (Fig. 2D, right), and reduced spher-oid growth (Fig. 2E). Furthermore, spheroid growth of PDX cells,PC-13 and PC-17 (Fig. 2F), were significantly reduced withcombined STAT3i/MEKiwhen comparedwith control cells. Theseresults show that combined MEKi/STAT3i suppresses pancreatictumorigenicity.

STAT3i results in ERK activation through TACE-dependentshedding of AREG and EGFR activation

RAS signaling pathway is a known mediator of tumor progres-sion and survival that is activated downstream of the EGFR (25).Having shown, both in vitro and in vivo, reciprocal reactivation ofpERK or pSTAT3 upon STAT3i or MEKi, respectively (Fig. 1C–G),we hypothesized that STAT3i would also result in EGFR activa-tion, upstream of RAS signaling. As seen in Fig. 3A, STAT3i withAZD1480 resulted in pSTAT3 inhibition, with subsequent acti-vation of pERK and pEGFR in both time- and dose-dependentmanner (Fig. 3A; Supplementary Fig. S3C). Interestingly, over

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Figure 1.

Inverse correlation of ERK and STAT3 in PDAC. A, Expression of activated and total levels of ERK and STAT3 in cell lines from human PDAC (left) or cellsgenerated from PanIN, PDA, and LMP lesions from LSL-KrasG12D/þ;Pdx1Cre/þ (PanIN) and LSL-KrasG12D/þ;Trp53R172H/þ;Pdx1Cre/þ (PDA and LMP) mice (right) aredemonstrated. Densitometry analyses of pSTAT3 and pERK normalized to tSTAT3 and tERK respectively were shown in the bottom of A. B, Selected humanPDAC tumor samples were stained for pERK and pSTAT3 expression. Pearson correlation showed negative correlation for pERK and pSTAT3 expression.r¼�0.8833; P�0.0001.C, sh-STAT3 and sh-scrambled (sh-Scram) control cell lysateswere analyzed for pSTAT3 and pERK expression byWestern blot analysis.D,Tumors tissues from PKT mice treated with STAT3 inhibitor, AZD1480 (left), or MEK inhibitor, AZD6244 (right), were analyzed for pERK, pEGFR, or pSTAT3expression respectively by IHC and analyzed using ImageJ. E, Western blot analysis of lysates from MiaPaCa2 cells were treated with AZD6244 or MEK162 (MEKinhibitor) in time-dependent manner. F, Western blot analysis of lysates from MiaPaCa2 cells were treated with STAT3 inhibitors Stattic (left) or AZD1480(right) in time-dependentmanner.G,Western blot analysis of lysates fromSW1990 cells treatedwithMEKi (AZD6244) in time-dependentmanner (left) and analyzedfor the levels of pERK, pSTAT3, and pEGFR (right). Densitometry analyses of pERK, pSTAT3, and pEGFR normalized to tERK, tSTAT3, and tEGFR, respectively.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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time, pSTAT3 becomes reactivated following activation of EGFReven with STAT3i. Again, as pSTAT3 is reactivated, pERK levelsdecrease, further confirming the inverse correlation of pSTAT3and pERK signaling. These data suggest STAT3i-mediated ERKactivation may be dependent on EGFR signaling. To furtherexplore themechanism bywhich STAT3i promotes EGFR tyrosinephosphorylation, we examined the upstream activators of EGFRsignaling.

Previously, we andothers have shown that TACE is regulated byG protein–coupled receptors (GPCR) that act as mediators ofEGFR transactivation (14, 26, 27). TACE, when activated, inducescleavage and shedding of soluble forms of the EGFR ligands,AREG, and TGFa. Importantly, we have shown that TACE andAREG, but not TGFa, are overexpressed in pancreatic tumorswhen compared with normal human pancreatic tissues and areinvolved in EGFR/ERK/STAT3 signaling activation (14). First, we

confirmed whether EGFR inhibition with erlotinib activatesSTAT3 phosphorylation (Supplementary Fig. S4C). To determinethe mechanism by which STAT3i leads to EGFR activation, weanalyzed the effects of STAT3i or MEKi on TACE phosphorylation(Fig. 3B). Interestingly, STAT3i increased TACE phosphorylationin a time-dependent manner. Similarly, MEKi also showedincrease in TACE phosphorylation. Combined MEKi/STAT3iattenuated TACE activation (Fig. 3C), suggesting that TACE phos-phorylation mediates the reciprocal activation of STAT3 and ERKthrough EGFR. We then assessed MEKi/STAT3i effects on AREGshedding in conditioned media (CM) from BxPC3, PANC1, andMiaPaCa2 cells (Fig. 3D). STAT3i with AZD1480 and MEKi withAZD6244 resulted in significantly increased AREG levels in theCM from all three cell lines. This observation further confirmsAREG's involvement in MEKi and STAT3i-mediated activation ofEGFR signaling.

Figure 2.

Combined MEKi and STAT3iabrogates tumorigenicity, spheroidgrowth, and cell invasion whileenhancing apoptosis in PDAC cells.Expression of pERK and pSTAT3 inhuman MiaPaCa2 cells treated withMEK inhibitor (U0126) and STAT3inhibitor (Stattic; A) or PANC1 andMiaPaCa2 (B) cells treated withAZD6244 (MEK inhibitor) andAZD1480 (JAK/STAT3 inhibitor)in time-dependent manner for up to24 hours. C, MiaPaCa2 cells weretreated with AZD1480 with orwithout AZD6244 for 24 hours,stained with Annexin V–FITC andpropidium iodide (left) or propidiumiodide (right) and analyzed by flowcytometry. Cells with AZD1480 andAZD6244 treatment resulted in anincrease in apoptosis (Annexin-positive and propidium iodide-negative) and sub-G0 level(propidium iodide-positive).D, MiaPaCa2 cells treated with thecombination of AZD6244 andAZD1480 showed a decreasednumber of colonies (left) and cellinvasion (right). E, Combination ofAZD1480 and AZD6244 drugtreatment decreased spheroid size inMiaPaCa2 cells when compared witheither control treatment or AZD1480.F, Combination of AZD1480 andAZD6244 decreased spheroid size inPDXs PC-13 (left) and PC-17 (right)cells. �, P < 0.05; ��, P < 0.01;�� , P < 0.0001; ns, nonsignificant,P > 0.05.






Figure 3.

STAT3i results in ERK phosphorylation, which occurs through TACE-AREG-EGFR signaling. A, Western blot analysis of human MiaPaCa2 cells treated with JAK/STAT3 inhibitor (AZD1480, 100 nmol/L) and analyzed for the expression of pERK, pSTAT3, and pEGFR in time-dependent manner. B, MiaPaCa2 cells weretreatedwith AZD1480 (250 nmol/L; left) or AZD6244 (250 nmol/L; right) for up to 360minutes, immunoblotted for TACE and pTACE (T735). Densitometry analysesof pTACE normalized to tTACE are shown at the bottom ofB.C,MiaPaCa2 cellswere treated with AZD1480 (250 nmol/L) and/or AZD6244 (250 nmol/L) for up to 12hours, immunoblotted for pTACE (left). Densitometry analyses for the immunoblots from pTACE after normalization to total TACE protein are shown (right).Densitometry analyses of pTACE normalized to tTACE are shown at the bottom of C. D, PDAC cells were treated with AZD1480 (100 nmol/L) and/or AZD6244(250 nmol/L) for 48 hours and analyzed for AREG release in the culture media (concentrations per 1 � 106 cells). Each value represents the mean and SD(n¼ 3).E,MiaPaCa2 cellswere treatedwith AZD1480 (100 nmol/L)with orwithout cetuximab (C225),mAb to EGFR (4mg/mL; left) or EGFR kinase inhibitor, erlotinib(1 mg/mL; right) for up to 24 hours, lysed and immunoblotted for pSTAT3 and pERK. F, Serum AREG (pg/mL) levels from normal and patients withPDAC were analyzed (left). Serum AREG (pg/mL) levels from WT and KPC mice were analyzed (right). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

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We then sought to determine whether AREG is the ligand thatactivates EGFR signaling in this process. First, we confirmedwhether AREG activates ERK phosphorylation (SupplementaryFig. S5). To further delineate the role of STAT3 in AREG-mediatedEGFR–ERK activation, MiaPaCa2 cells were treated withAZD1480 and cetuximab (C225), a mAb that inhibits EGFRligand binding (Fig. 3E, left) or erlotinib, a tyrosine kinaseinhibitor of EGFR (Fig. 3E, right). Combined treatment withAZD1480 and C225 or erlotinib completely inhibited ERK andSTAT3 activation along with EGFR signaling. Taken together,these data suggest that EGFR tyrosine kinase activity mediatesSTAT3-dependent ERK activation.

Because TACE activation correlates with AREG-dependent ERKactivation (14), we sought to address the role of AREG releaseduring in vivo PDAC progression. Serum was collected frompatients with PDAC and normal individuals (Fig. 3F, left). Serumfrom patients with PDAC had significantly higher AREG levelswhen compared with normal patients, suggesting increased TACEactivity in patients with PDAC as we have previously shown.(14)Furthermore, we compared AREG serum levels between KPC andwild-type (WT) mice (Fig. 3F, right). Serum AREG levels weresignificantly higher in KPCmice compared with WTmice, furthercorroborating the roles of AREG and activated TACE in PDACprogression. Collectively, these data show TACE-AREG–mediatedEGFR pathway activation is a major resistance mechanism thatimpairs the efficacy of individual MEKi or STAT3i. These resultssuggest that combined EGFR and STAT3 or MEK and STAT3inhibition may be therapeutically effective in PDAC. Further-more, AREG may serve as a potential circulating biomarker ofresistance to MEK or STAT3 inhibition.

Combined MEKi and STAT3i reduces tumor burden, retainspancreatic integrity, and improves survival in PKT mice

To determine the effects of MEKi/STAT3i on tumor growthin vivo, PANC1 (Fig. 4A) and MiaPaCa2 (Fig. 4B) xenografts weresubjected to vehicle, AZD1480, AZD6244, or combined treatmentby daily oral gavage. Combined MEKi/STAT3i significantly sup-pressed tumor growth compared with vehicle or individual agenttreatment. We did not observe any significant changes in animalbody weight, suggesting there is no added toxicity from thecombined treatment (Supplementary Fig. S6A and S6B). IHCstaining of xenograft tumors showed decreased Ki67 (Fig. 4C)and increased cleaved caspase-3 (Fig. 4D) staining in the com-bined treated tumors compared with treatment with either agentalone.

To determine therapeutic efficacy in a GEMM of PDAC, wetreated PKT mice with the same treatment regimen (vehicle,AZD1480, AZD6244, or combination) beginning at 4 weeks ofage when pancreatic tumors are firmly established in this GEMM(Fig. 4E–G). There was a significant reduction in pancreatic tumorweight with combined AZD1480/AZD6244 treatment comparedwith vehicle or treatment with either agent alone (Fig. 4E).Furthermore, while pancreata of vehicle or monotherapy-treatedmice were completely replaced with tumor burden, mice treatedwith combined MEKi/STAT3i had little or no tumor burden andmaintained their pancreatic integrity (Fig. 4F and G). Again, therewere no significant changes in animal body weight, suggesting noadded toxicity from the combined treatment (Supplementary Fig.S7A). To further analyze pancreatic integrity, PKT tumors wereassessed for cytokeratin 19 (CK-19), alcian blue, collagen 1, andKi67 staining by IHC (Fig. 4G). Vehicle-treated PKT mice had

abundant staining with the proliferation marker Ki67, extracel-lular matrix protein collagen type 1 and alcian blue, as well as CK-19 staining for adenocarcinoma. Pancreata of PKT mice treatedwith AZD1480 or AZD6244 alone had a slight reduction instaining of these markers compared with vehicle-treated mice.However, PKT mice treated with combined AZD1480/AZD6244had significantly more normal pancreatic architecture and histol-ogy, demonstrating a staining profile similar to their WT litter-mates (Fig. 4G). These results show that combined MEKi/STAT3isignificantly reduced tumor burden, reduced tumor cell prolifer-ation, and retained pancreatic integrity in this aggressive PDACGEMM.

In a separate study, PKT mice were analyzed for survival underthe same treatment arms. Again, tumor growth was significantlyreduced in PKT mice treated with combined MEKi/STAT3i (Fig.5A). Furthermore, survival of PKTmice treated withMEKi/STAT3iwas significantly improved when compared with vehicle-treatedand STAT3i-treatedmice (53 vs. 85 or 63 vs. 85 days respectively; P¼ 0.0002, log-rank test). Although there was an improvement insurvival of mice treated with combined MEKi/STAT3i comparedwithMEKi-treatedmice (85 vs. 76 days, Fig. 5B), these results werenot statistically significant. Western blot analysis of whole tumorlysates showedhighpERKandpSTAT3, but lowpEGFRexpressionin vehicle-treated mice (Fig. 5C). Although STAT3i completelyinhibited pSTAT3 expression, it increased expression of bothpERK and pEGFR. MEKi inhibited pERK expression, but hadlimited effect on pSTAT3 expression. In contrast, combinedMEKi/STAT3i significantly reduced the expression of both pSTAT3and pERK without activating pEGFR. IHC staining showed asignificant decrease in Ki67 staining (Fig. 5D) and enhancedcleaved caspase-3 (Fig. 5E) expression in tumors treated withcombined MEKi/STAT3i, compared with tumors treated withvehicle or single agents alone. These results confirm the enhancedin vivo effects of combined MEKi/STAT3i in overcoming thera-peutic resistance in an aggressive PDAC GEMM.

Combined MEKi/STAT3i attenuated TACE activation (Fig. 3C;Supplementary Fig. S7B), suggesting that TACE phosphorylationmediates the reciprocal activation of STAT3 and ERK throughEGFR. Furthermore, to assess the effect of MEKi/STAT3i onsystemic AREG levels, we measured AREG in the serum of treatedPKT mice. Serum AREG levels were negligible and significantlylower in PKT mice treated with combined MEKi/STAT3i com-pared with treatment with single agents alone (Fig. 5F). Theseresults further corroborate the effect of combinedMEKi/STAT3i inovercoming AREG-EGFR–mediated resistance to RAS and STAT3signaling inhibition in PDAC.

Combined MEKi/STAT3i decreases CSCs in PDACCSCs are self-renewing cells in the TME that are resistant to

cytotoxic and targeted therapies and are often the cause oftumor recurrence despite initial therapeutic response. STAT3signaling is active in tumor-associated CSCs (3, 28, 29). Todetermine whether MEKi/STAT3i affects CSCs in PDAC, PKTmouse tumors treated with MEKi and/or STAT3i were evaluatedfor CSC cell markers by flow cytometry. Analysis showedsignificantly decreased EpCAMþCD44þ (Fig. 6A) andEpCAMþCD133þ (Fig. 6B) CSCs in tumors treated with com-bined MEKi/STAT3i when compared with vehicle treatment.These results indicate that the enhanced in vivo effects ofcombined MEKi/STAT3i further overcome therapeutic resis-tance by targeting CSCs in PDAC. To further assess the impact






Figure 4.

Combined AZD1480/AZD6244 treatment decreases tumor growth, tumor burden, and proliferation while retaining the pancreatic integrity. Tumor growth rate ofPDAC cells, PANC1 (A) and MiaPaCa2 (B) flank xenografts in Fox 1-nu/nu mice treated with vehicle, AZD1480 (30 mg/kg/day), AZD6244 (25 mg/kg/day), orAZD1480/AZD6244. Error bars indicate SD of mean; n ¼ 4 (PANC1) or n ¼ 3 (MiaPaCa2) per group. C and D, IHC of pancreatic tumor xenograft tissues showdecreased Ki-67 (C) and increased cleaved caspase-3 (Cl Casp 3) expression with MEKi/STAT3i treatment and analyzed using ImageJ (bottom). E–G,PKTmice receivedAZD1480, AZD6244, or AZD1480/AZD6244 by oral gavage 5 days/week, starting at 4weeks of age for 2weeks. E, Total pancreatic tumorweightwas measured at the end of treatment at 6 weeks of age. Treatment of MEKi and MEKi/STAT3i showed a significantly reduced weight compared withvehicle-treatedmice.F andG, The relative tumor area (F)wasmeasured and the histologic sections of pancreatic tissuewere stained for cytokeratin 19 (CK-19), alcianblue, collagen 1, and Ki67 (G, left), and analyzed for the PDAC tumor integrity. The expression of the CK-19, alcian blue, collagen 1, and Ki67 stains werecalculated and analyzed using ImageJ (right). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001; ns, nonsignificant, P > 0.05.

Nagathihalli et al.





Figure 5.

Combined MEKi/STAT3i treatment improves survival of PKT mice. For survival study, PKT mice were treated with vehicle, AZD1480 (30 mg/kg/day), AZD6244 (25mg/kg/day), or combinedAZD1480/AZD6244by oral gavage5 days/week, starting at 4weeks of age.A, Tumorweight in theAZD1480/AZD6244–treatedmicewassignificantly decreased compared with vehicle-treated controls. B, Kaplan–Meier survival analysis shows significantly improved overall survival with AZD1480/AZD6244 (median 85 days) compared with vehicle control (median 53 days, P ¼ 0.0002, log-rank test). C, Western blot analysis of whole tumor lysatesdemonstrated decreased expression of pSTAT3 and pERK in mice treated with AZD1480/AZD6244 compared with vehicle or single drug–treated mice (left)and analyzed using ImageJ (right). D, Proliferation (Ki67 staining) was significantly decreased and apoptosis (cl, caspase 3; E) was significantly increasedwith AZD1480/AZD6244 treatment when compared with either vehicle- or AZD6244-treated mice. F, AREG amount was significantly decreased in theserum of AZD1480/AZD6244–treated mice when compared with either AZD1480- or AZD6244-treated mice. � , P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001; ns, nonsignificant, P > 0.05.






on CSCs, BxPC3 xenografts were treated with vehicle or com-bined MEKi/STAT3i for 27 days and then treatment wasstopped and tumor growth was assessed. There was regrowthof tumors from day 6 to day 17 after stopping MEKi/STAT3itreatment (Supplementary Fig. S7C), suggesting that MEKi/STAT3i suppress CSCs, but does not completely eliminate theCSC population.

DiscussionA major therapeutic challenge in PDAC is its oncogene-

driven innate and acquired chemoresistance. KRAS is the keyoncogenic driver of tumorigenesis and malignant progression.Due to the molecular characteristics of RAS protein, directinhibition of KRAS has yet to be achieved. We and others havedeveloped strategies to inhibit RAS indirectly by targeting itsdownstream effectors in the RAF/MEK/ERK signaling cascade.MEK is a key node in this axis and serves as the primarymediator for KRAS signal transduction. However, MEK inhibi-tion (MEKi) has not achieved clinical efficacy in PDAC due toredundant signaling pathways and molecular crosstalk thatinduce compensatory survival signaling. Furthermore, we havealso previously identified activated STAT3 signaling as a keybiomarker of resistance in PDAC (20, 30).

This study helps to define the heterogeneous response andtherapeutic resistance of PDAC to RAS and STAT3 pathwayinhibition. We have identified a novel molecular mechanismwhereby STAT3i leads to reciprocal activation of MEK–ERK sig-naling, whereas MEKi results in activation of STAT3 signaling.Furthermore, we show that STAT3i results in TACE-AREG-EGFR–dependent activation of ERK. These results provide a mechanisticrationale of acquired resistance to downstream RAS and STAT3pathway inhibition. This mechanism of ERK reactivation is rel-evant to other studies that have shown that ERK signaling reac-tivation plays a critical role in primary and acquired resistance totargeted therapies (8, 9, 31). Preclinical studies have identifieddistinct mechanisms by which cells acquire resistance to MEKi,including amplification of mutant BRAF (31, 32), PI3K upregula-tion (8, 33), or EGFR activation (8, 34). Dual inhibition of thesepathways has provided benefit in some patients (10, 25, 35).

STAT3 activation has been observed in a variety of humantumors (20, 28) through engagement of cytokine or growth factor

receptors. We have previously shown that STAT3i with gemcita-bine remodels the tumor stroma and enhances in vivo drugdelivery to the tumor, thereby improving survival in PKT mice(20). However, STAT3i results in reciprocal activation of ERKsignaling, thereby preventing a sustained therapeutic response.The results of this study support targeting two components of theEGFR-RAS-MEK-JAK/STAT3 pathway to overcome primary andacquired resistance of targeted monotherapies.

CombinedMEKi/STAT3i results in sustained inhibition of bothERK and STAT3 signaling. Importantly, the combination ofMEKi/STAT3i induces robust apoptosis in vitro and tumor regressionin vivo in PDAC tumors. In PDAC cells, EGFR is activated uponSTAT3i, perhaps as part of an initial effort by the cell to escapeapoptosis or reengage the cell cycle. Furthermore, EGFR activationis suppressed only when STAT3 is inhibited in combination withERK. With combined MEKi/STAT3i, the cellular escape mechan-isms are shut down and growth of PDAC cells is effectivelysuppressed.

TACE and AREG play a primary role in activating EGFRsignaling and are overexpressed during PDAC progression(14). The pro-EGFR ligand, AREG, has been implicated inresistance to other therapies (36, 37) and is required to induceEGFR-mediated ERK signaling (14, 27, 36). We have previouslyshown that knockdown of AREG prevents STAT3 and ERKactivation in response to deoxycholic acid in PDAC cells(14). Previous studies have also suggested inhibiting AREGactivity may be necessary to overcome resistance to EGFR-targeted therapies in NSCLC and hepatocellular carcinoma(38, 39), which is consistent with recent reports showing thatinhibition of AREG is associated with a better response tocetuximab therapy in colorectal cancer (40, 41).

Our results show AREG levels are increased following STAT3itreatment of PDAC cells, leading to EGFR phosphorylation andsubsequent reactivation of ERK and STAT3 signaling. We foundthat STAT3i results in increased EGFR activity through TACEactivation (Fig. 3A–C). Sera from KPC mice and human patientswith PDAC have significantly elevated levels of AREG (Fig. 3F),suggesting that serum AREG levels may serve as a noninvasiveprognostic biomarker. Furthermore, treatmentwithMEKi/STAT3iresults in a significant decrease in serum AREG levels, suggestingits role as a biomarker of therapeutic response to EGFR-, MEK-, orSTAT3-targeted therapies. We now demonstrate that STAT3i incombination with specific blockade of the AREG-mediated EGFRreceptor using cetuximab or erlotinib completely inhibits ERKreactivation. Therefore, our study supports the potential of target-ing TACE-AREG-EGFR axis as a potent approach to overcomeresistance to MEKi or STAT3i.

Combined MEKi/STAT3i results in enhanced therapeutic effi-cacy not only through sustained inhibition of the RAS pathway–associated redundant feedback loop reactivation, but also bysuppressing the TME through reducing CD44þ and CD133þ

CSCs. The interplay between tumor cells and surrounding sup-portive cells, such as fibroblasts, immune cells, and CSCs, clearlyindicates that targeting a single component will not result insustained inhibition of tumor growth. Targeting multiple signal-ing pathways and different components of the TME is the meansof overcoming either primary or acquired resistance to targetedmonotherapy. This approach increases the likelihood of a sus-tained response by concurrently affecting multiple diversemechanisms of action associated with cancer development andtherapeutic resistance.

Figure 6.

Effects of MEKi and STAT3i on CSCs in PKTmice. Single-cell suspension isolatedfrom pancreatic tumors of PKT mice treated with AZD1480, AZD6244, orAZD1480/AZD6244 was analyzed for CSC population by flow cytometry. A,CD44þ tumor cell populations (CD45NegEpCamþCD44þ) were significantlydecreased in the combined MEKi/STAT3i treatment compared with STAT3ialone or the vehicle groups. B, CD133þ tumor cell populations(CD45NegEpCamþCD133þ) were significantly decreased in the combinationof MEKi/STAT3i compared with the vehicle-treated group. �, P < 0.05;�� , P < 0.01; ns, nonsignificant, P > 0.05.


Nagathihalli et al.




We have identified a novel mechanism associated with resis-tance to RAS–MEK–ERKpathway inhibition through activation ofSTAT3 signaling and resistance to STAT3 pathway inhibitionresulting in TACE-AREG–mediated EGFR and ERK reactivation.We provide strong evidence supporting the role of targeting twocomponents of the AREG–EGFR–MEK–STAT3 pathway to over-come therapeutic resistance associated with RAS pathway reacti-vation in PDAC. In addition, combinedMEKi and STAT3i inhibitsCSCs and maintains pancreatic integrity, providing a secondarymechanisms by which this combination therapy may enhanceantitumor response and prevent recurrence of disease. In addi-tion, our results suggest that serumAREG levelsmay serve as a keycirculating prognostic biomarker of PDAC and a potential bio-marker of therapeutic resistance and response to EGFR, MEK, andSTAT3 inhibition.

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

Authors' ContributionsConception and design: N.S. Nagathihalli, J. Castellanos, M.N. VanSaun,N.B. MerchantDevelopment ofmethodology:N.S. Nagathihalli, J. Castellanos, M.N. VanSaun,N.B. MerchantAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.S. Nagathihalli, J. Castellanos, P. Lamichhane,F. Messaggio, M.N. VanSaun, N.B. MerchantAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):N.S.Nagathihalli, J. Castellanos, F.Messaggio, X.Dai,X. Chen, N.B. Merchant

Writing, review, and/or revision of the manuscript: N.S. Nagathihalli,J. Castellanos, P. Lamichhane, C. Shi, X. Dai, P. Rai, M.N. VanSaun,N.B. MerchantAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.S. Nagathihalli, P. Lamichhane, X. Dai,M.N. VanSaun, N.B. MerchantStudy supervision: N.S. Nagathihalli, N.B. MerchantOther (pathology and IHC): C. Shi

AcknowledgmentsThe authors thank Mr. Frank Revetta, Ms. Jennifer Barretta, Dr. Alexander

Gaidarski III, and Ms. Yanhua Xiong for their technical assistance and Dr.Nilesh Kashikar (pathologist) for evaluating stained tissues. This work wassupported by the NIH R01 CA161976, Pancreatic Cancer Action Network(PanCan)-AACR Translational Research Grant (15-65-25-MERC), NIH T32CA211034, James & Ether King Biomedical Research Program, FloridaDepartment of Health 8JK08, and Sylvester Comprehensive Cancer Center(to N.B. Merchant); NIH NCI R21 CA209536, American Cancer Society IRG98-277-13, and Stanley Glaser Foundation Research Award (UM SJG 2017-24; to N.S. Nagathihalli); and American Cancer Society IRG 98-277-13 (toM.N. VanSaun). Flow Cytometry Core service was performed through theSylvester Comprehensive Cancer Center (SCCC) support grant (to N.B.Merchant and N.S. Nagathihalli).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 27, 2018; revised July 5, 2018; accepted August 22, 2018;published first August 28, 2018.

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2018;78:6235-6246. Published OnlineFirst August 28, 2018.Cancer Res Nagaraj S. Nagathihalli, Jason A. Castellanos, Purushottam Lamichhane, et al. Resistance to RAS Pathway Inhibition in Pancreatic CancerInverse Correlation of STAT3 and MEK Signaling Mediates

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