oncogenic kras targets muc16/ca125 in pancreatic ductal ... · as reproduction and proliferation,...

Oncogenes and Tumor Suppressors

Oncogenic KRAS Targets MUC16/CA125 inPancreatic Ductal AdenocarcinomaChen Liang1,2,3, Yi Qin1,2,3, Bo Zhang1,2,3, Shunrong Ji1,2,3, Si Shi1,2,3, Wenyan Xu1,2,3,Jiang Liu1,2,3, Jinfeng Xiang1,2,3, Dingkong Liang1,2,3, Qiangsheng Hu1,2,3, Quanxing Ni1,2,3,Jin Xu1,2,3, and Xianjun Yu1,2,3

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a devastatingdisease with the 5-year survival rate less than 6%. Previousresults indicated that serum levels of CA125 (encoded byMUC16) could be used to predict which groups of pancreaticcancer patients may benefit from surgery. However, the under-lying mechanism remains elusive. Herein, using the CancerGenome Atlas and clinicopathologic data obtained from ourcenter, we demonstrate that high CA125 serum levels andexpression levels of MUC16 are predictive of poor prognosis.MUC16 is also validated as a downstream target of KRAS, andtheir expression strongly correlated with each other in vitro andin vivo. Mechanistically, the KRAS/ERK axis induced upregula-

tion of MUC16 and shedding of CA125 via its effector c-Myc inSW1990 and PANC-1 pancreatic cancer cells. Notably, proto-oncogene c-Myc could bind to the promoter of MUC16 andtranscriptionally activate its expression. Taken together, thesedata establish CA125 as a prognostic marker for pancreaticcancer, and mechanistic studies uncovered the KRAS/c-Myc axisas a driving factor for upregulation of MUC16.

Implications: The current study uncovers the contribution ofoncogenic KRAS to serum marker CA125 production through amechanism that involves the ERK/c-Myc axis.Mol Cancer Res; 15(2);201–12. �2016 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) makes up the 90%

of all pancreatic malignancies and is characterized by poor overallsurvival. Although significant progress has been made in diagno-sis and treatment, PDAC is still a devastating diseasewith extreme-ly high lethal rate, which is equal to the incidence of the disease(1–3). Thus, there is an urgent need for developing strategies inprediction of resectability, assessing the effect of chemotherapyand radiotherapy (4). Althoughmedical image examinations likeCT and ultrasoundmay provide much important information fortumor, it is still not sufficient to obtain complete data preoper-atively. The detection of serum tumormarkers is routinely used ina variety of tumor patients, because it involves noninvasive teststhat facilitate the early detection, prediction of outcomes, andassessment of response to therapy (5).

CA19-9 is a specific tumor marker of digestive system tumors,especially pancreatic cancer, and is widely used in the diagnosis

and monitoring of pancreatic cancer (6). It reflects the tumorburden and activity, and has predictive role for postoperativeoutcomes (7–9). CA125was firstly detected in ovarian cancer andhad been reported to be positive in 80% of ovarian epithelialtumor patients (10). In addition, the positive observation ofCA125 is also seen in breast cancer, pancreatic cancer, gastriccancer, and some other tumors (11). Carcinoembryonic antigen(CEA) is a nonspecific tumor marker and is commonly used fordetection of colorectal cancer (12). Moreover, CEA could alsoreflect the existence of a variety of tumors, such as pancreaticcancer, lung cancer, and gastric cancer (13, 14). Our previousstudies reported that by combination use of CA19-9 with serummarkers of CA125 and CEA, we could predict postoperativeoutcomes, and patients with baseline CEAþ/CA125þ/CA19-9� 1,000 U/mL were prone to larger tumors, lymph node metas-tases, and high tumor–node–metastasis stage (15). Thus, under-standing the underlying mechanisms for the expression of thesetumors markers will help to uncover novel strategies for theprediction and treatment for pancreatic cancer. Our most recentstudy demonstrated that CA125 could compensate CA19-9 indiagnosis and prognosis of Lewis-negative patients with pancre-atic cancer, and received much attention for research into itsunderlying regulatory mechanism.

Circulating CA125 was the cleaved product of MUC16 on thecell surface, the largestmembrane glycoprotein known.MUC16 isbelieved to play important roles not only in normal contexts suchas reproduction and proliferation, but also in pathologic statesincluding cancer and mucosal infections. It exerts its function incancer by many mechanisms such as migration, invasion, cellcycle control and metabolism reprogramming (16, 17). Since itsdiscovery, CA125 is usually used as a tumor marker, and manyefforts have been given to its contribution to tumormalignancies,

1Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center,Shanghai, China. 2Department of Oncology, Shanghai Medical College, FudanUniversity, Shanghai, China. 3Pancreatic Cancer Institute, Fudan University,Shanghai, China.

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

C. Liang, Y. Qin, and B. Zhang contributed equally to this article.

Corresponding Authors: Xianjun Yu, Fudan University, 270 Dong An Road,Shanghai 200032, China. Phone: 86-21-64175590; Fax: 86-21-64031446; E-mail:[email protected]; or Jin Xu, [email protected]

doi: 10.1158/1541-7786.MCR-16-0296

�2016 American Association for Cancer Research.

MolecularCancerResearch

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on September 27, 2020. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 31, 2017; DOI: 10.1158/1541-7786.MCR-16-0296

http://mcr.aacrjournals.org/

yet little is known about its regulation. The importance ofMUC16/CA125 in pancreatic cancer diagnosis and progressiondemands an urgent need for research on the expression regulationof this molecule.

The activating pointmutation ofKrasoncogene on codon-12 ofexon-2 induces replacement of the GGT sequence (encoding forglycine) by the GAT sequence (aspartic acid–G12D–c35 G>A),which remains the major event in pancreatic cancer (18). It isfound in approximately 95% of pancreatic intraepithelial neo-plasias (PanIN), which is considered to be the initiating geneticevent for PDAC progression (19, 20). The activation of KrasG12D

inhibits intrinsic guanosine triphosphatase (GTPase) activity ofRAS and permanently activates downstream signaling pathways.Thus, the signaling networks engaged by oncogenic KrasG12D arehighly complex and characterized by the activation of a series ofeffectors leading to proliferation, progression, and changes instroma interaction, immune system, andmetabolic state (21, 22).However, the contribution of Kras to serum tumor markersproduction has seldombeendiscussed. Based on the contributionof Kras to malignant behavior maintenance of pancreatic cancerand the critical role of CA125 in cancer diagnosis and prognosis,we sought to uncover the correlation and underlying mechanismbetween Kras and MUC16/CA125 in the present study. In depth,mechanism analysis of this correlation may present novel oppor-tunities for patient stratification, personalized therapies, andtherapeutic targets.

Materials and MethodsTissue specimens

The PDAC tissue microarrays used in this study were obtainedfrom 110 patients diagnosed with pancreatic cancer at FudanUniversity Shanghai Cancer Center (FUSCC). Prior patient con-sent and approval from the Institutional Research Ethics Com-mittee were obtained. Strict pathologic diagnoses and postoper-ative follow-ups were performed for all patients. Animal tissueslides derived from P48-Cre;KrasLSL-G12D;Ink4a/ArfF/F mice weregenerously provided by Professor Paul J. Chiao from MD Ander-son Cancer Center.

Immunohistochemical stainingImmunohistochemical staining of paraffin-embedded tissues

with antibodies for MUC16, phosphorylation of ERK (p-ERK),c-Myc, and FBW7 were performed and scored to determine theproteins expression according to standard procedures describedpreviously (23).

The Cancer Genome Atlas dataset analysisThe Cancer Genome Atlas (TCGA)-Pancreatic adenocarcinoma

(PAAD) onRNA expression (Level 3) of pancreatic cancer patientsin terms of RNA-seq by expectation–maximization was down-loaded from the Cancer Genomics Brower of the University ofCalifornia, Santa Cruz (UCSC; https://genome-cancer.ucsc.edu/).In total, 160primary pancreatic cancer samples frompatientswithdetailed expression data were chosen from the updated TCGAdatabase according to parameters mentioned.

Cell cultureThe human pancreatic cancer cell lines PANC-1, MiaPaCa-2,

CFPAC-1, Capan-1, BxPC-3, and SW1990were obtained from theATCC and cultured according to standard ATCC protocols. The

cell lines have passed the conventional tests of cell line qualitycontrol and have DNA profiling of short tandem repeats that areconsistent with those reported by the ATCC (Cell Bank & TypeCulture Collection, Chinese Academyof Sciences). iKras cell lines,which express KrasG12D upon doxycycline (doxy) treatment, andhuman pancreatic nonmalignant epithelial HPNE cells weregenerously provided by Professor Paul J. Chiao. PANC-1 andSW1990 cell lines with FBW7 overexpression were generatedpreviously (24).

Protein extraction and Western blot analysisCells were washed twice with ice-dole PBS and lysed in RIPA

buffer for 10 minutes. Cell debris was removed by centrifugationat 12,000 rpm for 20minutes at 4�C.Note that 20 mg total proteinlysate was subjected to electrophoresis in denaturing 10% SDS-polyacrylamide gel, and then transferred to a membrane forsubsequent blotting with specific antibodies. b-Actin and Krasantibodies were purchased from Proteintech. Antibodies againstp44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), and p44 MAP kinase (ERK1) were purchasedfromCell Signaling Technology. c-Myc antibodywas produced byAbcam company.

RNA isolation and quantitative real-time PCRTotal RNA was extracted by using TRIzol reagent (Invitrogen).

To obtain cDNA, the TaKaRa PrimeScript RT reagent Kit was usedfor reverse transcription. Expression status of candidate genes andb-actin were determined by quantitative real-time PCR using anABI 7900HT Real-Time PCR system (Applied Biosystems). Allreactions were run in triplicate. Primer sequences are listed asSupplementary Table S1.

Flow cytometry analysisSurface expression of MUC16 on cells was assessed by single-

color immunofluorescence and flow cytometry using primaryanti-MUC16 mAb (X306) with appropriate FITC-conjugated sec-ondary antibodies. Background levels were determined by incu-bating cell with properly matched FITC-conjugated isotype con-trol antibodies.

Transfection of siRNAAll siRNAs were purchased from Biotend company and trans-

fected by using Lipofectamine 2000 (Invitrogen), according to themanufacturer's instructions. The sequences of siRNA oligos usedin this study were as listed in Supplementary Table S2.

Transwell migration assay and invasion assayA 24-well Transwell chamber with an 8-mm-pore PET mem-

brane (BD Biosciences) was used to conduct the migration andinvasion assays. The lower chamber was filled with 800 mLmedia containing 10% FBS. Subsequently, approximately 6 �104 cells were seeded in 200 mL medium without serum in thetop chamber for migration assay, and 10 � 104 cells wereseeded into top chamber with a Matrigel-coated membrane(BD) for invasion assays. The cells were allowed to migrate at37�C with 5% CO2 over 24 hours. After removal of the non-migrating or noninvading cells, the remaining cells werewashed, fixed, and stained with crystal violet. We counted thenumber of migrating and invading cells in six fields randomlyselected at �100 magnification. Experiments were performed atleast in triplicate.

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Mol Cancer Res; 15(2) February 2017 Molecular Cancer Research202




Measurement of CA125 in cell culture supernatantsCA125 levels in cell culture supernatants were quantitatively

measured using Human CA125 ELISA Kit SimpleStep (Abcam),according to the manufacturer's instruction. Briefly, the super-natants were added into the Pre-Coated 96 Well Microplate andthen Antibody Cocktail was added into each well. The plate wasincubated for 1 hour at room temperature on a plate shaker.Next, each well was washed with 1 � Wash Buffer PT to addTMB Substrate and incubated for 10 minutes. Lastly, at theendpoint, Stop Solution was added into each well to record theOD at 450 nm.

Analysis of promoter activity with dual luciferase assayPromoters of mouse MUC16 and human MUC16 promoter

regions, spanning from�2,500 to 200of the transcription startingsite, were cloned into pGL3-Basic vector. Cells were plated on 96-well culture plates and transfected with pGL3 constructs andRenilla luciferase expression vectors by using Lipofectamine2000 (Invitrogen). Next, the cells were assayed for both fireflyand Renilla luciferase activities using a dual-luciferase system(Promega), as described in the manufacturer's protocol.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) was performed

according to the instructions of the Magna ChIP A/G ChromatinImmunoprecipitation Kit (Merck Millipore Corporation). Thenuclear DNA extracts were amplified using two pairs of primers(Supplementary Table S3) that spanned the MUC16 promoterregion.

Statistical analysesStatistical analyses were performed by SPSS software (version

17.0, IBM Corp.) using independent Student t test (two-tailed) orone-way ANOVA. Pearson correlation analysis was used to deter-mine the correlation between Kras andMUC16 expression levels.The Fisher exact test was used to determine the correlationbetween MUC16 and clinicopathologic characteristics in theTCGA and FUSCC cohorts. Statistical significance was based ontwo-sided P values of <0.05.

ResultsCA125/MUC16 expression positively correlated with Kras inpancreatic cancer

CA125 is a well-established marker in ovarian cancer. To assessits contribution to prognosis of pancreatic cancer, we examinedthe serum levels of CA125 in samples from FUSCC. In consistentwith our previous reports, higher serum CA125 levels predictedpoor prognosis (Fig. 1A).Next, we examined the expression statusof MUC16 in the TCGA dataset and analyzed its contribution toprognosis. Clinical information regarding the samples is pre-sented in Supplementary Table S4, and the level of MUC16 wasan independent prognostic factor in pancreatic cancer by analysisof TCGA (Fig. 1B; Supplementary Table S5). To further validatethis observation, we performed immunohistochemical stainingto examine the MUC16 expression in PDAC tissue microarrayfrom FUSCC as a validation cohort (Fig. 1C and SupplementaryTable S6). In consistent with the results from the TCGA cohort,higher levels ofMUC16 significantly correlatedwith poor survival(Fig. 1D and Supplementary Table S7). Kras expression andmutation is a decisive genetic event that associated with PDAC.

To assess the regulatory function of Kras in MUC16 expressionand serumCA125 level, we examined the correlation of mutationstatus in Kras with serum level of CA125 in relevant patients. Asobserved, serum level of CA125 was significantly higher in Kras-mutant patients than that in Kras wild-type patients (Fig. 1E). Byusing the TCGA cohort, we analyzed the expression correlationbetween Kras withMUC16, as described, Kras expression stronglycorrelated with MUC16 expression (Fig. 1F). Moreover, in con-sistent with our observation concerning the correlation of muta-tion status in Kras with MUC16 expression in FUSCC cohort, wefound in the TCGA cohort thatMUC16 expression was significanthigher in Kras-mutated patients in the TCGA dataset (Fig. 1G).

To confirm the observation in the TCGA cohort and the FUSCCcohort, we examined the contribution of Kras mutation toMUC16 expression in vitro. Upon doxy induction in iKras cells,we observed that MUC16 expression significantly upregulated,alongwithKrasG12D induction (Fig. 2A andB).Given thatMUC16is a membrane bondmacromolecule, we used the flow cytometrywith MUC16 antibody to examine its level in the cell membranein pancreatic cancer cell lines and HPNE cells (Fig. 2C). Throughexamination of the Kras mutation status of the cell lines used, wefound that membrane-bound MUC16 levels were correlated tothe metastatic potential and Kras status (SW1990 > CFPAC-1 >Capan-1 > PANC-1 > MiaPaca-2 > BxPC-3�HPNE; Fig. 2D).Moreover, in samples from Kras-driven transgenic pancreaticcancer mice, we observed that MUC16 was higher in pancreaticcancer samples than that in normal control samples, furtherconfirming our observation that Kras mutation contributed toMUC16 expression (Fig. 2E).

MUC16 was involved in the KrasG12D-mediated migration andinvasion

MUC16 is a membrane protein that correlated with migrationand invasion capacity of various cancer cells. To assess its role inKras mutation–driven invasiveness, Kras expression significantlycontributed the acquisition of migration and invasive capacity iniKras cells upon doxy treatment (Fig. 3A and B). Given thatMUC16 expression significantly increased upon KrasG12D induc-tion, we hypothesized that MUC16 was involved in Kras-inducedmetastasis. Next, we silenced MUC16 expression with siRNAoligos (Fig. 3C) and observed that silencing MUC16 inhibitedKras-induced migration (Fig. 3D and E). Furthermore, MUC16inhibition also attenuated Kras-induced invasion capacity (Fig. 3Fand G). These results suggested that mutant Kras-induced upre-gulationofMUC16 contributed to themigration and invasivenessin pancreatic cancer cells.

ERK activation is required for MUC16 expression in pancreaticcancer cells

In iKras cells, doxy induction could effectively induce theactivation of ERK (Fig. 4A). Oncogenic Kras-induced ERK activa-tion is vital for the Kras-induced malignant properties formationand maintenance. We speculated that ERK activation mightaccount for MUC16 expression in pancreatic cancer cells. To testthis hypothesis, wefirstly inhibited ERK activation in iKras cells bytreatment of the cellswithUO126. As observed,UO126 treatmentattenuated KrasG12D-induced expression of MUC16 (Fig. 4B).Next, in KrasG12D-mutated SW1990 and PANC-1 cells, weobserved that the expression of MUC16 decreased significantlyin the presence of UO126 treatment, suggesting that ERK activa-tion is required for MUC16 expression (Fig. 4C and D). Secreted

Oncogenic KRAS Regulates MUC16/CA125

www.aacrjournals.org Mol Cancer Res; 15(2) February 2017 203




CA125 levels in SW1990 and PANC-1 cell culture medium alsodecreasedwhen cellswere treatedwithUO126, further supportingthe role of ERK activation inMUC16 expression control (Fig. 4E).Furthermore, we silenced ERK1 expression in SW1990 andPANC-1 cells to assess the regulatory role of ERK in MUC16 expression(Fig. 4F). In ERK1-silenced SW1990 and PANC-1 cells, themRNAlevel of MUC16 decreased significantly (Fig. 4G). What is more,silencing ERK1 expression led to decrease of secretedCA125 levelsin SW1990 and PANC-1 cells (Fig. 4H). Taken together, theseresults suggested that KrasG12Dmutation–induced ERK activationis required for MUC16 expression and CA125 levels in SW1990and PANC-1 cells.

c-Myc is the oncogenic KrasG12D effector that is responsible forMUC16 expression

c-Myc, a well-accepted Kras effector, as confirmed in our study(Fig. 5A), is a proto-oncogene and functions as a transcriptionfactor that regulated the expression of many tumor-promotinggenes. In iKras cells, JQ-1 treatment effectively lowered the level ofc-Myc protein (Fig. 5B). Subsequently, we observed that JQ-1effectively decreased KrasG12D-induced MUC16 expression, indi-cating that c-Myc might regulate MUC16 expression as a tran-

scription factor (Fig. 5C). In KrasG12D-mutated SW1990 andPANC-1 cells, treatment with JQ-1 significantly decreased c-Mycexpression, and the concomitant decrease in MUC16 expression(Fig. 5D and E). Furthermore, JQ-1 treatment significantlydecreased CA125 level in cultured medium of SW1990 andPANC-1 cells (Fig. 5F). Next, we silenced c-Myc expression inSW1990 and PANC-1 cells to further explore its role in MUC16expression (Fig. 5G). Silencing c-Myc significantly loweredMUC16 expression and shedding of CA125 in SW1990 andPANC-1 cells, indicating its promoting role inMUC16 expression(Fig. 5H and I). The similar results were also obtained in SW1990and PANC-1 cells with c-Myc overexpression (Supplementary Fig.S1A and S1B).

Next, we analyzed the promoter region of MUC16 and foundtwo putative c-Myc–binding E-box elements (Fig. 5J), and per-formed promoter luciferase assay to examine the role of c-Myc inMUC16promoter activity regulation. As observed, JQ-1 treatmentor manipulation of c-Myc expression altered MUC16 promoteractivity (Fig. 5K). Furthermore, we performed ChIP assay todetermine whether c-Myc occupied the E-boxes in the MUC16promoter region. As shown, c-Myc enriched in the two E-boxes inthe promoter region, further supporting its role in MUC16

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MUC16 was involved in Kras mutation to predict a poor prognosis. A, High level of serum CA125 has a shorter overall survival of patients with pancreaticcancer. B, TCGA analysis for the association between tissue MUC16 and prognosis of pancreatic cancer. C, Immunohistochemical analysis for the tissue MUC16expression in the FUSCC cohort. D, High level of tissue MUC16 predicts a poor prognosis to pancreatic cancer in the FUSCC cohort. E, The correlationbetween Kras status and serum CA125 level in patients with pancreatic cancer. F, TCGA analysis for the association between Kras and MUC16 expression inpancreatic cancer. G, TCGA analysis for the correlation between Kras status and MUC16 expression in pancreatic cancer.

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expressional regulation (Fig. 5L). In supporting the clues obtainedfrom in vitro experiments with cell lines, we analyzed the expres-sional correlation between tissue c-Myc and MUC16 levels inpancreatic cancer samples. We observed that c-Myc level stronglycorrelated withMUC16 levels in pancreatic cancer tissue samples,and MUC16 level was significantly higher in c-MycHigh samples(Fig. 5M and N).

ERK/c-Myc axis regulated the expression of MUC16Based on the above observations, we speculated that the

ERK/c-Myc axis might be responsible for KrasG12D-inducedMUC16 expression. We analyzed the promoter region in mouseMUC16 genome and found six putative c-Myc–binding E-boxelements (Fig. 6A). We cloned the mouse MUC16 promoterinto pGL3-basic vector for subsequent analysis. In iKras cells,UO126 or JQ-1 treatment significantly attenuated doxy-induced MUC16 promoter activity (Fig. 6B and C). Moreover,UO126 treatment or silencing ERK1 decreased the c-Myc pro-tein level (Supplementary Fig. S2A and S2B), resulting ininhibiting the human MUC16 promoter activity in SW1990and PANC-1 cells (Fig. 6D). These results suggested the con-tribution of ERK activation and c-Myc upregulation in MUC16transcriptional expression. Furthermore, we observed that ERKinhibition by UO126 treatment or siRNA silencing decreasedthe occupancy of c-Myc in MUC16 promoter region (Fig. 6Eand F; Supplementary Fig. S3A and S3B). To further validate thecontribution of ERK/c-Myc axis in serum CA125 levels control,we examined the correlation between activation status of ERKand c-Myc level with serum CA125 level in pancreatic cancerpatients samples. Results demonstrated that serum CA125 levelwas significantly higher in c-MycHigh groups of patients,

although it was not statistically significant between p-ERKHigh

and p-ERKLow groups (Fig. 6G and H).

FBW7 mediated ERK/c-Myc axis in regulation of MUC16expression

FBW7 is an E3 ubiquitin ligase, and our previous studydemonstrated that Kras/ERK/FBW7 signaling pathway partici-pated in pancreatic cancer malignancies control including pro-liferation and metabolic reprogramming by targeting c-Myc.Previously, a high-throughput gene expression profiling arraywas performed and the microarray data were deposited inGEO under accession numbers GSE76443. Interestingly, wehave found that MUC16 was one of these differentiallyexpressed genes altered by FBW7 overexpression. Comparedwith control cells, the MUC16 expression in cells with FBW7overexpression has decreased by 2-fold. Thus, it is naturally toask whether FBW7 participated in MUC16 expression, andanswers to this issue may provide intervention strategies inimprovement of pancreatic cancer prognosis prediction andtreatment. In SW1990 and PANC-1 cells with FBW7 overexpres-sion, we observed a significant decrease in MUC16 expression(Fig. 7A). Moreover, the extracellular CA125 levels in FBW7-overexpressing SW1990 and PANC-1 cells were lower than therelative control cells (Fig. 7B). When FBW7 was silenced bysiRNA, this down-regulatory phenomenon could be reversed(Supplementary Fig. S4A and S4B). To further test this hypoth-esis, we examined the correlation of serum CA125 level withFBW7 expression in pancreatic cancer patients. As illustrated,serum CA125 levels were higher in patients with lower FBW7expression, further supporting the negative correlation betweenFBW7 and MUC16 expressions (Fig. 7C).

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Ras increased the MUC16 expression in PDAC. A, iKras cell was induced to increase the level of KrasG12D by treatment with doxycycline (doxy). B, Activationof Kras-induced by doxy treatment increased MUC16 mRNA expression. C, Flow cytometry analysis for the surface MUC16 expression on pancreatic cell lines.D, The characteristics in the term of metastatic potential and Kras status of pancreatic cancer cells and nonmalignant cells. E, Immunohistochemicalanalysis for MUC16 expression in transgenic KrasG12D mice and normal mice.






Next, we introduced FBW7 mutants to examine its role inMUC16 expression. As shown, introduction of wild-type FBW7and its constitutively active mutant FBW7T205A significantlydecreased MUC16 promoter activity, whereas silencing FBW7expression or exogenous introduction of dominant FBW7R465H

mutant, a dominant-negative mutant that lost interaction withc-Myc, dramatically increased MUC16 promoter activity (Fig.7D). Next, we assessed the role of FBW7 and the relativemutants in MUC16 expression and extracellular CA125 levelsin SW1990 and PANC-1 cells. In consistent with the promoteractivity assay results, introduction of FBW7 or the constitutivelyactive mutant FBW7T205A significantly decreased MUC16expression and extracellular CA125 concentration in SW1990and PANC-1 cells (Fig. 7E and F). On the contrary, exogenousexpression of FBW7R465H dominant-negative mutant increasedMUC16 expression and extracellular CA125 concentration inSW1990 and PANC-1 cells (Fig. 7E and F). To validate the invitro results with cells lines, we assessed the contribution ofFBW7 in combination with MUC16 in predicting prognosis.Patients with high CA125 and low FBW7 levels exhibited the

worst overall survival, whereas CA125Low/FBW7High groups ofpatients shown a better survival (Fig. 7G).

In summary, we have identified expression and serum levels ofMUC16/CA125 as a prognosismarker for overall survival by usingthe TCGAdataset cohort andFUSCCcohort. Indepth,mechanismanalysis uncovered that KrasG12D/ERK/FBW7/c-Myc axis isrequired for the transcriptional regulation of MUC16 expressionin PDAC, and the existence of this axis has been further validatedby using TCGA dataset analysis and clinicopathologic dataobtained fromFUSCC(Fig. 7H). These resultswill help todevelopnovel predictive and treatment targets for PDAC.

DiscussionIn the present study, we demonstrated that MUC16/CA125 is a

downstream target of mutant KrasG12D, and the expression levelsof MUC16 and serum levels of CA125 positively correlated withKras expression and mutation status. Further mechanism studiesindicated that Kras-induced upregulation of CA125 depended onthe activation of ERK kinase and the downstream effector protein

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MUC16 was involved in Kras-induced enhancement of the migration and invasion of pancreatic cancer cells. A, Kras activation increased the cell migrationand invasion (magnification, �100). B, The relative cell number of migration and invasion was quantitated and shown as means � SD. C, The iKras cells wereknockdownof CA125 by transfection of siRNA against CA125.D,Knockdown of CA125 could reverse the capacity ofmigration induced by Kras (magnification,�100).E, The relative cell number of migration was quantitated and shown as means � SD. F, Knockdown of CA125 could reverse the capacity of invasion inducedby Kras (magnification, �100). G, The relative cell number of migration was quantitated and shown as means � SD.

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c-Myc. Moreover, FBW7 played a vital role in mediating Kras-induced MUC16 expression. Our studies provided novel predic-tive and treatment targets for pancreatic cancer.

The Kras gene encodes the protein of p21 RAS, which is a smallGTPase that acts as amolecular switch by coupling cellmembranegrowth signals to intracellular signaling pathways and transcrip-tion factors to control multiple cellular processes (25, 26). Onco-genically mutated RAS proteins fail to cycle "off" from the active,GTP-bound state to the inactive GDP-bound state, and therebyaccumulated in the constitutive "on" configuration (27). Kras isthemost commononcogene that has been found tobemutated inpancreatic cancer. Oncogenic mutation of Kras resulted in con-stitutive activation of RAS proteins which permanently bound toGTP and led to subsequent activation of signaling pathways, suchas the PI3K/AKT/mTOR or RAF/MEK/ERK signaling pathway. Theactivation of these pathways was involved in transformation,uncontrolled proliferation, invasion, andmetastasis of pancreaticcancer cells (28). Detection of the Kras mutation status could beused to assess prognosis of pancreatic cancer (29–31). Moreover,the downstream targets of mutant Kras could also be used in theassessment of prognosis of pancreatic cancer (32). However, thecombination of Krasmutation status with serum tumorsmarks in

predicting prognosis, and the underlying mechanism has seldombeen reported.

CA125 is a well-accepted tumor marker for many cancers. Itsclinical role in pancreatic cancer received much attentionrecently (33, 34). Our previous study described a unique rolefor CA125 levels in diagnosis and treatment for pancreaticcancer. Higher CA125 levels specifically reflected the metasta-sis-associated tumor burden of pancreatic cancer in patientswith advanced disease, as well as the presence of occult metas-tasis in patients with clinically localized tumors. Incorporationroutine analysis of serum CA125 levels with CA19-9 and CEA inclinical examination both before and after pancreatic cancertreatments may help to improve therapeutic decisions andpatient survival (15, 35). CA125 is the cleaved product ofMUC16, a molecule with heavy molecular weights. The con-tribution of MUC16 to cancer malignancy has been studied inmany cancer types. Silencing MUC16 studies in ovarian, breast,and pancreatic cancer cells impairs tumorigenic and metastaticproperties (36–38). Moreover, MUC16 rendered antiapoptoticproperties to cancer cells, leading to chemotherapy resistance(39). One recent study demonstrated that MUC16 promotedWarburg effect in pancreatic cancer by reprogramming glucose

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Activation of ERK increased the MUC16 expression in PDAC. A, Doxy treatment increased the phosphorylation level of ERK. B, Inducible activation of Krasincreased the MUC16 expression, while this upregulation was inhibited by treatment with UO126, an inhibitor of MEK1/2. C, UO126 decreased the phosphorylationlevel of ERK in the SW1990 and PANC-1 cells. D, Inhibition of ERK kinase by UO126 decreased the expression of MUC16 in SW1990 and PANC-1 cells.E, Inhibition of ERK kinase decreased the shedding of CA125 in SW1990 and PANC-1 cells. F, ERK was decreased by siRNA-mediated silencing in SW1990 andPANC-1 cells. G, Knockdown of ERK decreased the expression of MUC16 in SW1990 and PANC-1 cells. H, The decreasing expression of ERK inhibited theshedding of CA125 in pancreatic cancer cells.






metabolism (40). Taken together, these observations identifiedMUC16/CA125 as an appropriate marker for prognosis andtarget for treatment in pancreatic cancer. However, the mech-

anism that accounts for MUC16/CA125 remains elusive andneeds in-depth study. Answers to this question will help todevelop novel predictive and treatment targets.

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c-Myc was involved in the MUC16 transcriptional expression in PDAC. A, Doxy treatment increased the level of c-Myc protein. B, JQ-1 decreased the level ofc-Myc protein in iKras cell. C, c-Myc inhibition decreased the Kras-induced MUC16 expression. D, JQ-1 decreased the level of c-Myc protein in PANC-1 and SW1990cells. E, Treatment of PANC-1 and SW1990 with JQ-1 decreased the MUC16 expression in PANC-1 and SW1990 cells. F, c-Myc activity affected the sheddingof CA125 in the SW1990 and PANC-1 cells.G, c-Myc was decreased by siRNA-mediated silencing in SW1990 and PANC-1 cells. H, Knockdown of c-Myc decreased theMUC16 expression in SW1990 and PANC-1 cells. I, Knockdown of c-Myc decreased the shedding of CA125. J, The position of the c-Myc–binding site in thehuman MUC16 promoter. K, c-Myc expression and activity affected MUC16 promoter activity in PANC-1 and SW1990 cells. L, c-Myc occupies the E-box of theMUC16 promoter region, as measured by ChIP assay. M, The correlation between tissue CA125 and MUC16 expression derived from clinical specimen. N, Thepopulation of patients with different levels of c-Myc and MUC16 expressions.

Liang et al.





Based on the decisive role ofKrasmutation and the contributionofMUC16/CA125 inpancreatic cancer, weproposed that these twofactorsmight have underlying correlations. Through analysis of theTCGA dataset, we observed that MUC16 expression was higher inKras-mutant pancreatic cancer specimens than in wild-type Kraspatients. The results were further confirmed in samples from ourcenter, suggesting that MUC16 might be a mutant Kras target.Mounting evidence has pointed out c-Myc to be a Kras target(41). Moreover, we also reported that in pancreatic cancer, Krasmutation activated ERK, leading to destabilization of tumor sup-pressor FBW7, which ultimately led to c-Myc upregulation (24).These observations prompted us to consider MUC16 as a c-Mycdownstream transcriptional target. Our further investigations sup-ported this hypothesis and indicated that theERK/FBW7/c-Mycaxis

was responsible for the upregulation of MUC16. The observationsreported in the present study established a novel link betweengenetic variations with serum level of biomarkers in pancreaticcancer and provided underlying mechanism. Application of thesefindingwill help to improve the efficacy of pancreatic cancer overallsurvival prediction and treatment. For example, CA19-9 is a tumormarker that reflects tumor burden and could be used to assess theefficacy of surgical resection, chemotherapy, and radiotherapyresponse in gastrointestinal tumors, such as pancreatic cancer andcolorectal cancer (42, 43). The novel findings of the present studymay point out further applications of utilizing Kras mutation andassessing CA125 level for measuring pancreatic cancer survival andresponse to traditional chemotherapy and radiotherapy. For exam-ple, endoscopic ultrasound-guided fine-needle aspiration biopsy is

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Kras/ERK axis increased the transcriptional expression of MUC16. A, The position of the c-Myc–binding site in the mouse MUC16 promoter. B, UO126treatment decreased the Kras-induced MUC16 promoter activity. C, c-Myc inhibition decreased the Kras-induced MUC16 promoter activity. D, Relative MUC16promoter activity in PANC-1 and SW1990 cells cotransfected the MUC16 promoter with an siRNA against ERK or UO126, respectively. E, The activity and expressionof ERK affected occupation of c-Myc to the E-box of the MUC16 promoter region examined by primer pair 1. F, The activity and expression of ERK affectedoccupation of c-Myc to MUC16 promoter region examined by primer pair 2. G, The serum levels of CA125 in the patients with high and low p-ERK. H, The serumlevels of CA125 in the patients with high and low c-Myc.






a useful tool in the diagnosis of pancreaticmasses. Combination ofgenetic analysis of these samples with serum levels of tumormarkers like CA19-9 could increase the sensitivity and specificityof diagnosis (44). However, no similar result has been reported byusing Kras mutation status by conjugation with CA125 levels inimproving sensitivity and specificity of diagnosis in pancreaticcancer, and this warrants further investigations. Moreover, evenoncogenic Kras is considered as an undruggable target, andmount-ing efforts still have been put to develop strategies in targeting Krasand Kras-related signaling pathways in the hope of opening atherapeutic window for Kras-driven cancers (45). And once thesetrailsbecomesuccessful, the serumlevelsofCA125 couldbeusedasan indicator for assessing efficacy.

In summary of the whole article, our studies uncovered a novellink between Kras mutation and serum marker of CA125, andprovided the mechanistic explanations. These results will help todevelop novel diagnostic, predictive, and treatment targets forpancreatic cancer.

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

Authors' ContributionsConception and design: Y. Qin, S. Ji, Q. Ni, X. YuDevelopment of methodology: Y. Qin, B. Zhang, W. Xu, J. Xu

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FBW7 destabilized c-Myc protein to inhibit the MUC16 transcription. A, FBW7 overexpression significantly decreased the mRNA expression of MUC16 in SW1990and PANC-1 cells. B, FBW7 overexpression decreased the shedding of CA125 in the PANC-1 and SW1990 cells. C, The serum levels of CA125 in the patientswith high and low FBW7. D, Transfection of FBW7T205A or its wild-type counterpart decreased the MUC16 promoter activity, whereas FBW7R465H and knockdown ofFBW7 exerted little impact on MUC16 promoter activity compared with its wild-type counterpart. E, Transfection with wide type and mutant type of FBW7,T205A, and R465H into PANC-1 and SW1990 cells altered the MUC16 expression. F, The function and expression of FBW7 affect the shedding of CA125 in the PANC-1and SW1990 cells. G, Kaplan–Meier analysis of the overall survival rate of patients with pancreatic cancer, according to FBW7 expression combined with thelevel of serum CA125. H, Proposed model of the mechanism of Kras-mediated regulation of metastasis via the c-Myc/MUC16 axis in pancreatic cancer.

Liang et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Liang, Y. Qin, B. Zhang, W. Xu, J. Liu, J. Xiang,D. Liang, J. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Liang, Y. Qin, J. Liu, J. Xiang, D. Liang, Q. HuWriting, review, and/or revision of the manuscript: Y. QinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Liang, S. Shi, Q. Hu, J. XuStudy supervision: S. Shi, Q. Ni, X. Yu

AcknowledgmentsWe thank Huanyu Xia for assistance in collecting the patient data and

Dr. Chen Yue-lei from Cell Bank and Type Culture Collection (Chinese Acad-emy of Sciences) for his help in testing the authentication of the cell lines.

Grant SupportThis workwas jointly funded by theNational Science Fund forDistinguished

Young Scholars of China (81625016), theNational Natural Science Foundationof China (81372651, 81502031 and 81602085), Ph.D. Programs of theFoundation of the Ministry of Education of China (20120071120104), andShanghai Sailing Program (16YF1401800).

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 September 1, 2016; revised October 10, 2016; accepted October 31,2016; published OnlineFirst November 21, 2016.

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2017;15:201-212. Published OnlineFirst January 31, 2017.Mol Cancer Res Chen Liang, Yi Qin, Bo Zhang, et al. AdenocarcinomaOncogenic KRAS Targets MUC16/CA125 in Pancreatic Ductal

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