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Cancer Therapy: Clinical

The Novel KLF4/MSI2 Signaling PathwayRegulates Growth and Metastasis of PancreaticCancerKun Guo1,2, Jiujie Cui2, Ming Quan2,3, Dacheng Xie3, Zhiliang Jia2, Daoyan Wei2,Liang Wang2, Yong Gao3, Qingyong Ma1, and Keping Xie2

Abstract

Purpose: Musashi 2 (MSI2) is reported to be a potentialoncoprotein in cases of leukemia and several solid tumors.However, its expression, function, and regulation in pancreaticductal adenocarcinoma (PDAC) cases have yet to be demonstrat-ed. Therefore, in the current study, we investigated the clinicalsignificance andbiologic effects ofMSI2 expression in PDAC casesand sought to delineate the clinical significance of the newlyidentified Kr€uppel-like factor 4 (KLF4)/MSI2 regulatory pathway.

Experimental Design: MSI2 expression and its associationwith multiple clinicopathologic characteristics in human PDACspecimens were analyzed immunohistochemically. The biolog-ical functions of MSI2 regarding PDAC cell growth, migration,invasion, and metastasis were studied using gain- and loss-of-function assays both in vitro and in vivo. Regulation of MSI2expression by KLF4 was examined in several cancer cell lines,

and the underlying mechanisms were studied using molecularbiologic methods.

Results: MSI2 expression was markedly increased in bothPDAC cell lines and human PDAC specimens, and high MSI2expression was associated with poor prognosis for PDAC. ForcedMSI2 expression promoted PDAC proliferation, migration, andinvasion in vitro and growth and metastasis in vivo, whereasknockdown of MSI2 expression did the opposite. TranscriptionalinhibitionofMSI2 expressionbyKLF4occurred inmultiple PDACcell lines as well as mouse models of PDAC.

Conclusions: Lost expression of KLF4, a transcriptional repres-sor of MSI2 results in overexpression of MSI2 in PDACs, whichmay be a biomarker for accurate prognosis. A dysregulated KLF4/MSI2 signaling pathway promotes PDAC progression and metas-tasis. Clin Cancer Res; 23(3); 687–96. �2016 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC), generally known

as pancreatic cancer, is a deadly disease. Specifically, it is theseventh leading cause of cancer-related deaths worldwide (1).In the United States, the incidence of PDAC is increasing, withan estimated 48,960 new cases and 40,560 PDAC-relateddeaths in 2015 (2). Recently, surgical resection of and radio-

therapy, chemotherapy, and immunotherapy for PDAC haveimproved greatly, but its 5-year survival rate is still less than 5%(3). Thus, further studies of the molecular mechanism of PDACdevelopment and progression and identification of new ther-apeutic targets for PDAC are urgently needed.

Musashi 2 (MSI2) is one of the two members of the Musashifamily of RNA-binding proteins, which participate in post-transcriptional regulation by binding to specific mRNAs (4, 5).Researchers have shown that the two MSIs play critical roles inproliferation and differentiation of stem cells of the hemato-poietic and nervous systems (4, 6). Elevated expression ofMSI2 in cancer cells predicts poor prognosis for both chronicmyelogenous leukemia (CML) and acute myelogenous leuke-mia (7, 8). Recent studies further demonstrated the roles ofMSI2 in development and progression of solid tumors. Forexample, MSI2 promoted the invasion and progression ofhepatocellular carcinoma by interacting with the Wnt/beta-catenin pathway and inducing epithelial–mesenchymal tran-sition (4, 9). Furthermore, MSI2 is highly expressed in intes-tinal stem cells and colorectal adenocarcinomas, and gain offunction of MSI2 promotes transformation of the intestinalepithelium (10). MSI2 expression also is reported to be ele-vated in solid pseudopapillary tumors of the pancreas. How-ever, mechanisms of the expression of MSI2 and MSI20s rolesin PDAC development and progression have yet to bedemonstrated.

Kr€uppel-like factor 4 (KLF4), a zincfinger transcription factor, isexpressed primarily in postmitotic and terminally differentiated

1Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'anJiaotong University, Xi'an, Shaanxi, P.R. China. 2Department of Gastroenterol-ogy, Hepatology and Nutrition, The University of Texas MD Anderson CancerCenter, Houston, Texas. 3Department of Oncology, Shanghai East Hospital,Shanghai Tongji University, Shanghai, P.R. China.

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

K. Guo, J. Cui, and M. Quan share first authorship.

Corresponding Authors: Keping Xie, The University of Texas MD AndersonCancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-834-6685; Fax: 713-745-1163; E-mail: [email protected]; or Qingyong Ma,Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'anJiaotong University, 277 West Yanta Road, Xi'an, Shaanxi, 710061, People'sRepublic of China. Phone: 86-29-85323171; E-mail: [email protected]; orYong Gao, Department of Oncology, Shanghai East Hospital, Shanghai TongjiUniversity, Shanghai, People's Republic of China. Phone: 86-13651797919; E-mail:[email protected].

doi: 10.1158/1078-0432.CCR-16-1064

�2016 American Association for Cancer Research.

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epithelial cells in organs, including the skin, lungs, and those inthe gastrointestinal tract (11, 12). Accumulating studies haveprovided clinical and experimental evidence of the roles andmechanisms of KLF4 in cancer development and progression,and it is considered a potential tumor suppressor (13–15). Ourprevious studies demonstrated that KLF4 regulated the develop-ment and progression of PDAC via transcriptional regulation ofthe expression of p27Kip1 and Forkhead Box M1 (16–18).Furthermore, KLF4 participated in the regulation of aerobicglycolysis of PDAC by suppressing the expression of lactatedehydrogenase A (19). However, the underlying molecularmechanism of the tumor-suppressive role of KLF4 in PDACsmust be demonstrated further. In the current study, we soughtto determine the expression of MSI2 in PDACs, determine theroles of MSI2 in PDAC growth and metastasis, and identify themechanism of regulation of the expression of MSI2 by KLF4 inPDACs. We found that MSI2 was highly expressed in PDACsand promoted PDAC growth and metastasis. Furthermore,KLF4 transcriptionally suppressed MSI2 expression in PDACs.Finally, the novel KLF4/MSI2 signaling pathway critically reg-ulated PDAC growth and metastasis.

Materials and MethodsHuman tissue specimens and immunohistochemical analysis

The expression of MSI2 and KLF4 was analyzed using tissuemicroarrays (TMA) containing 130 primary PDAC, 10 adjacentnormal pancreatic tissue, and 20 normal pancreatic tissue speci-mens (US Biomax). Immunohistochemical analyses of thesespecimens were conducted with anti-MSI2 (ab50829; Abcam)and anti-KLF4 (sc-20691; Santa Cruz Biotechnology) antibodiesas described previously (20).

Cell linesThe human PDAC cell lines PANC-1, MIA PaCa-2, AsPC-1,

BxPC-3, Capan-1, Capan-2, Hs766T, and PATU-8902 were pur-chased from the ATCC. The PDAC cell lines MDA Panc-28 andMDA Panc-48 were gifts fromDr. Paul J. Chiao (The University of

Texas MD Anderson Cancer Center, Houston, TX). The humanPDAC cell line FG was obtained from Dr. Michael P. Vezeridis(The Warren Alpert Medical School of Brown University; ref. 21).All of these cell lines weremaintained in plastic flasks as adherentmonolayers in Eagle minimal essential medium supplementedwith 10% FBS, sodium pyruvate, nonessential amino acids,L-glutamine, and a vitamin solution (Flow Laboratories). TheATCCperforms characterization or authentication of the cell linesit provides using short tandem repeat profiling, and the cell linesthey provided were passaged in our laboratory for fewer than 6months after reception.

Immunofluorescent stainingPDAC cells were seeded on chamber slides overnight prior to

experimentation. Cells werefixedwith 4%paraformaldehyde andpermeabilized in 0.1% Triton X-100 in PBS and sequentiallyblocked with 3% BSA for 30 minutes. After overnight incubationwith primary antibodies against MSI2, these cells were furtherincubated with appropriate secondary antibodies and subjectedto nuclear staining.

Plasmids and small interfering RNAsThe plasmid pCMV-MSI2 (pMSI2) was generated by Gene-

Copoeia, and Flag-tagged KLF4 (Flag-KLF4) was describedpreviously (22). siRNAs for MSI2 (siMSI2#1, 50-gcgaacacagua-guggaagauguaa-30; siMSI2#2, 50-uuacaucuuccacuacuguguucgc-30) were synthesized by Life Technologies (2, 4), and KLF4siRNAs consisted of pools of three to five target-specific 19- to25-nt siRNAs designed to knock down KLF4 expression (SantaCruz Biotechnology) as demonstrated in our previous studies(8). A negative control siRNA (Invitrogen) and control pCMVvector were used. Transfection of plasmids and siRNAs intoPDAC cells was performed using Lipofectamine 2000 CDtransfection reagent (Invitrogen). For transient transfection,cells were transfected with plasmids or siRNA at different dosesas indicated for 48 hours before performance of functionalassays. PDAC cells treated with transfection reagent alone wereincluded as mock controls.

Western blot analysisStandard Western blotting was carried out using whole-cell

protein lysates and primary antibodies against MSI2 (ab50829;Abcam) and KLF4 (sc-20691; Santa Cruz Biotechnology) and asecondary antibody (anti-rabbit IgG or anti-mouse IgG; SantaCruz Biotechnology). Equal protein sample loading was moni-tored using an anti–a-tubulin antibody.

Colony formation assayTwo hundred cells of each type were plated in 24-well plates

and allowed to grow for 14 days in culture medium, which waschanged twice a week. Cells were then fixed with 4% paraformal-dehyde and stained with 0.1% crystal violet solution for 10minutes. Colonies (>20 cells) were counted using an invertedmicroscope at �40 magnification. The results were calculated asthe percentages of proper controls.

AnimalsFemale pathogen-free athymic nude mice were purchased

from the National Cancer Institute. The animals were main-tained in facilities approved by the Association for Assessmentand Accreditation of Laboratory Animal Care International in

Translational Relevance

We used pancreatic ductal adenocarcinoma (PDAC) spe-cimens and molecular biologic and animal models of PDACto evaluate the activation and function of the Kr€uppel-likefactor 4 (KLF4)/Musashi 2 (MSI2) pathway in humanPDACs. Our clinical and mechanistic findings indicated thatMSI2 is a direct transcriptional target of KLF4 and thatfrequently dysregulated KLF4 expression frequently leadsto aberrant MSI2 expression. Moreover, expression of bothKLF4 and MSI2 regulated the expression of differentiation-associated markers and PDAC cell differentiation, whereasknockdown of expression of MSI2 and KLF4 did the oppo-site, suggesting a novel molecular basis for the critical role ofloss of KLF4 expression in PDAC development and progres-sion. Moreover, overexpression of MSI2 in primary PDACscorrelated with reduced survival durations. Therefore, ourfindings may have a significant effect on clinical manage-ment of PDAC.

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accordance with the current regulations and standards of the U.S. Department of Agriculture and Department of Health andHuman Services.

Tumor growth and metastasisTumor cells (1� 106) in 0.1mL of Hank balanced salt solution

were injected subcutaneously into the right scapular regions ofnude mice. The sizes of the resulting tumors were measured everyweek. Tumor-bearing mice were killed when they became mor-ibundor onday 35 after injection, and their tumorswere removedand weighed.

To quantitatively measure metastasis, an experimental livermetastasis model was used. Specifically, tumor cells (1 � 106)were injected intravenously into another group of mice viathe ileocolic vein. The mice were killed on day 21 afterinjection or when they appeared to be moribund. Their liverswere then removed, and surface metastases on the livers werecounted after dissecting them into their individual lobes.Every surface metastasis was examined by two investigatorswho were unaware of the experimental protocol and scoredseparately (23).

Tumor cell migration/invasion assayBoth cell scratch-wound (horizontal migration) and modified

Boyden chamber (vertical migration and invasion) assays wereperformed to determine the migratory ability and invasiveness ofPDAC cells with altered MSI2 expression as described previously(16). For the cell scratch-wound assay, cells were grown in 6-wellplates until confluent. A wound was generated on the surface ofthe resulting cell monolayer via scraping with the 10-mL tip of apipette, the cells in the wounded monolayer were photographedat different time points, and cell migration was assessed bymeasuring gap sizes in multiple fields. For the Boyden chamberassay, 24-well tissue culture plates with 12-cell culture inserts(Millipore) were used. Each insert contained an 8-mm pore sizepolycarbonate membrane with a precoated thin layer of a base-ment membrane matrix [ECMatrix.; for the invasion assay) orwithout a coated matrix (for the migration assay). Ten percentFBS-containing medium was placed in the bottom chambers toact as a chemoattractant. Cells (5 � 105) in a 300-mL volume ofserum-free medium were placed in the top chambers and incu-bated at 37�C for 48 hours. Cells on the lower surface of thepolycarbonate membrane, which had invaded the ECMatrix andmigrated through the membrane, were stained, counted, andphotographed under a microscope.

Quantitative real-time reverse transcription-PCRQuantitative real-time reverse transcription-PCR analysis of

MSI2 mRNA expression was performed using total RNA and theSYBR Green reagent with an ABI Prism 7000HT sequence detec-tion system (Applied Biosystems; ref. 24). The PCR primersequences were as follows: MSI2, 50-ttcgcagacccagcaagtg-30 (for-ward) and 50-tcgcagataacccgcctac-30 (reverse); glyceraldehyde-3-phosphate dehydrogenase, 50-acagtccatgccatcactgcc-30 (forward)and 50-gcctgcttcaccaccttcttg-30 (reverse).

Construction of MSI2 promoter reporter plasmids andmutagenesis

A 1302-bp fragment DNA containing MSI2 50 sequences from�1,200 to þ102 relative to the transcription initiation site was

subcloned into the pGL3-basic vector (Promega). The resultingfull-length reporter plasmid, which contained multiple KLF4-binding sites, was designated pLuc-MSI2-1302. Deletion muta-tion reporters for this plasmid (pLuc-MSI2-769, pLuc-MSI2-424,and pLuc-MSI2-184) then were generated. All of the constructswere verified by sequencing the inserts and flanking regions ofthe plasmids.

Promoter reporter and dual luciferase assayPDAC cells were transfected with MSI2 promoter reporters,

KLF4 siRNA, or an expression plasmid. The MSI2 promoteractivity in these cells was normalized via cotransfection of ab-actin/Renilla luciferase reporter containing a full-length Renillaluciferase gene. The resulting luciferase activity in the cells wasquantified using a dual luciferase assay system (Promega) 24hours after transfection.

Chromatin immunoprecipitation assayPDAC cells (2 � 106) were prepared for a chromatin immu-

noprecipitation (ChIP) assay using a ChIP assay kit (Millipore)according to the manufacturer's protocol. The resulting precip-itated DNA specimens were analyzed using PCR to amplify a246-bp region of the MSI2 promoter with the primers 50-gtgcaggagggtctccgccat-30 (forward) and 50-ccggacctgggagaa-gacgcc-30 (reverse). The PCR products were resolved electro-phoretically on a 2% agarose gel and visualized using ethidiumbromide staining.

Statistical analysisThe two-tailed c2 test or Fisher exact test was used to determine

the significance of differences among covariates. All in vitroexperiments were performed in triplicate and at least three times.Datawerepresented either asmeans� SD fromone representativeindependent experiment of three with similar results or means �SEM from three independent experiments. The significance of thein vitro and in vivo data was determined using the Student t test(two-tailed) or one-way ANOVA. In all of the tests, P values lessthan 0.05 were considered statistically significant. The SPSS soft-ware program (version 17.0; IBM Corporation) was used forstatistical analysis.

ResultsDirect association of elevated expression of MSI2 withpathologic features in PDACs

To determine the roles of MSI2 in PDAC pathogenesis, wefirst investigated MSI2 protein expression in the 130 primaryPDAC specimens, 10 tumor-adjacent tissue specimens, and20 normal pancreatic tissue specimens in the TMAs. The clin-icopathologic characteristics of the TMAs are listed in Supple-mentary Table S1. We observed MSI2-positive staining in boththe cytoplasm and nuclei in tumor cells in PDAC specimens(Supplementary Fig. S1), with minimal staining in the non-malignant specimens (P ¼ 0.002; Fig. 1A and B). Also, we didnot find any significant differences in MSI2 expression betweenthe normal pancreatic and tumor-adjacent normal tissue speci-mens (P ¼ 0.769; Fig. 1B). Furthermore, Western blottingconfirmed the expression of MSI2 in paired normal pancreatictissue and PDAC specimens. The MSI2 protein expression inPDAC specimens was much higher than that in normal tissuespecimens (Fig. 1C–E). We confirmed the localization of MSI2

KLF4/MSI2 Signaling in PDA Progression

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in PDAC cells using immunofluorescent staining and Westernblotting and found that MSI2 was mainly localized in thecytoplasm in MIA PaCa-2, AsPC-1, BxPC-3, PANC-1, andPATU-8902 cells, but in the nuclei in MDA Panc-28 cells(Supplementary Fig. S2). We also analyzed the relationshipsamong clinicopathologic parameters and MSI2 expression inPDAC specimens. Total MSI2 expression was positively asso-ciated with disease pT classification (P ¼ 0.002), regionallymph node metastasis (P ¼ 0.049), and TNM stage (P <0.001) and negatively correlated with tumor differentiation(P < 0.001; Fig. 1D; Supplementary Table S2). We then inves-tigated the distribution of nuclear expression of MSI2 in groupswith weak, moderate, or strong expression and found nostatistically significant differences (P ¼ 0.173; SupplementaryFig. S1B). These findings indicated that MSI2 plays critical rolesin PDAC development and progression and may be a valuablebiomarker for this disease.

Promotion of PDAC growth by MSI2 in vitro and in vivoTo determine the effect of MSI2 on PDAC growth, we

analyzed its expression in PDAC cell lines. MSI2 was highlyexpressed in AsPC-1, BxPC-3, FG, MDA Panc-48, and MIAPaCa-2 cells but expressed at relatively low levels in Capan-2, Hs766T, MDA Panc-28, and PATU-8902 cells (Fig. 1F). Weinduced overexpression of MSI2 in PANC-1, MDA Panc-28, andPATU-8902 cells and knocked down expression of it in AsPC-1,FG, and BxPC-3 cells. As shown in Fig. 2A, pMSI2 effectivelyinduced overexpression of MSI2, and siMSI2#1 and siMSI2#2markedly knocked down expression of MSI2. We then inves-

tigated the effect of MSI2 on PDAC biology using pMSI2 andsiMSI2#1. We found that overexpression of MSI2 in MDA Panc-28 cells led to increased PDAC cell monolayer growth andcolony formation, whereas knockdown of expression of MSI2resulted in decreased cell growth and colony formation (Fig. 2Band C). In animal models, transfection with pMSI2#1 consis-tently promoted the growth of MDA Panc-28 cells in thesubcutis (Fig. 2D), and knockdown of MSI2 expression inhib-ited the growth of AsPC-1 cells in vivo (Fig. 2E). These datademonstrated that MSI2 promoted the growth of PDAC in vitroand in vivo, supporting that MSI2 may function as an oncogenein PDAC cases.

Promotion of PDAC invasion and migration in vitro andmetastasis in vivo by MSI2

To determine the effect of MSI2 expression on PDAC migra-tion and invasion, we transfected MDA Panc-28 and AsPC-1cells with pMSI2 and siMSI2#1, respectively, for 48 hours. Wehad wounded the transfected cells via scratching and main-tained the cells for 12 hours. The results demonstrated thatforced expression of MSI2 strongly promoted the flattening andspreading of MDA Panc-28 cells (Fig. 3A), whereas downregu-lation of expression of MSI2 attenuated the flattening andspreading of AsPC-1 cells (Fig. 3B). The Boyden chamber assayconfirmed these results. Both the migratory ability and inva-siveness of MSI2-transfected MDA28 cells were much greaterthan those of control cells (Fig. 3A), whereas those of siMSI2-transfected AsPC-1 cells were markedly attenuated (Fig. 3B). Wethen confirmed the in vitro results in orthotopic models of

Figure 1.

ExpressionofMSI2 in and its associationwith clinicopathologic features ofPDAC. TMA PDAC specimens wereimmunostained with a specific anti-MSI2 antibody. A, Representativeimages of MSI2 expression in a PDACspecimen and an adjacent normalpancreatic tissue specimen. B, Graphsdemonstrating markedly higher MSI2expression in PDAC (TT) than inadjacent normal pancreatic tissue (TN)and normal pancreatic tissue (NN)specimens but no difference in MSI2expression between adjacent normaland normal tissue specimens. C andD, Positive association of MSI2expression with TNM stage and tumordifferentiation grade in PDACs.E, Western blot analysis verifying theexpression of MSI2 protein in pairednormal pancreatic tissue and PDACspecimens. F, Western blot analysisshowing expression of MSI2 protein inPDAC cell lines.

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PDAC, finding that enforced expression of MSI2 markedlyincreased liver metastasis of MDA Panc-28 cells (Fig. 4A),whereas knockdown of expression of MSI2 abrogated livermetastasis of AsPC-1 cells (Fig. 4B). These data further con-firmed the oncogenic role of MSI2 in PDAC development andprogression.

Close relationship between the expression of KLF4 and that ofMSI2 in PDAC cells

To identify the mechanisms underlying MSI2 overexpres-sion, we first investigated the effects of altered KLF4 expressionon MSI2 expression in PDAC cells. As shown in Fig. 5A and B,overexpression of KLF4 in AsPC-1, BxPC-3, FG, and PANC-1cells led to decreased expression of both MSI2 protein andmRNA, whereas knockdown of expression of KLF4 markedlyupregulated that of MSI2 in MDA Panc-28 cells. To furtherconfirm the regulatory effect of KLF4 on MSI2 expression, we

investigated KLF4 expression with the TMA used to analyzeMSI2 expression described above. The results demonstratedthat the PDAC specimens in the TMA, which lost expression ofKLF4, had high expression of MSI2 (Fig. 5C). Statistical anal-ysis revealed that the expression of MSI2 was negativelycorrelated with the expression of KLF4 in the PDAC specimens(r ¼ �0.460, P < 0.001; Fig. 5D). To further confirm thenegative regulatory effect of KLF4 on the expression of MSI2,we knocked down the expression and induced overexpressionof KLF4 in colon and gastric cancer cells. The results demon-strated that overexpression of KLF4 in HCT116 and GT-5 cellsled to decreased expression of MSI2, whereas knockdown ofexpression of KLF4 in SW480 and SNU-1 cells resulted inincreased expression of MSI2 (Supplementary Fig. S3A andS3B). We then further analyzed the expression of KLF4 andMSI2 in colon and gastric tissue specimens obtained frommouse models described previously (25, 26). Unlike in tissue

Figure 2.

Effect of altered MSI2 expression onPDAC growth in vitro and in vivo. A,Verification of the efficiency of MSI2overexpression vectors and siRNAs inPDAC cell lines. PANC-1, PATU-8902,and MDA Panc-28 cells weretransfected with pMSI2 or controlvectors, and AsPC-1, FG, and BxPc-3cells were transfected with siMSI2#1 orcontrol siRNAs. CTRL, control. B,Assessment of the cell growth in A viacell counting at the indicated timepoints. C, Colony formation assayperformed using 24-well plates andnumbers of AsPC-1 and MDA Panc-28colonies counted 14 days aftertransfection. D and E,MDA Panc-28 (D)and AsPC-1 (E) cells transfected withpMSI2#1 were injected subcutaneouslyinto the right scapular regions of nudemice (1 � 106 cells/mouse, 5 mice/group). Gross tumors in the mice (D1and E1), tumor growth curves (D2 andE2), and tumor weights (D3 and E3) areshown; � , P < 0.05.






specimens from control mice, the specimens with knockout ofKLF4 were negative for KLF4 but were positive for MSI2(Supplementary Fig. S3C and S3D). Collectively, these datastrongly demonstrated that KLF4 negatively regulated theexpression of MSI2 both in vitro and in vivo and furtherindicated that the regulation most likely occurred at thetranscriptional level.

Transcriptional inhibition of MSI2 expression by KLF4 inPDAC cells

To further explore the mechanism of regulation of MSI2expression by KLF4, we analyzed the MSI2 promoter sequencefor the potential KLF4-binding elements 50-CACCC-30 and 50-(G/A)(G/A)GG(C/T)G(C/T)-30, which were described previ-ously (27, 28). We identified 11 putative KLF4-binding motifsin the MSI2 promoter region. We then generated the full-length MSI2 promoter pMSI1302 and deletion mutants of it(Fig. 6A). To determine whether KLF4 regulates MSI2 expres-

sion at the transcriptional level, we co-transfected the deletionmutant reporters with or without KLF4 expression vectors into293T cells. As shown in Fig. 6A, KLF4 inhibited the activity ofall of the mutant reporters. To further determine whetherKLF4 regulates MSI2 promoter transcriptional activity inPDACs, we co-transfected the pLuc-MSI2-424 reporter withKLF4 expression vectors or siRNA into PDAC cells. As shownin Fig. 6B, increased KLF4 expression in AsPC-1 and BxPC-3cells attenuated the MSI2 promoter activity, whereas knock-down of KLF4 expression in MDA Panc-28 and PATU-8902cells activated MSI2 promoter activity. In ChIP assays, wefound that KLF4 bound directly to the putative binding sitesin AsPC-1, BxPC-3, and FG cells (Fig. 6C and D). To furtherconfirm this result, we transfected AsPC-1 cells with Flag-KLF4or control vectors and performed a ChIP assay using an anti-Flag antibody. As shown in Fig. 6E, Flag-KLF4 bound to thepromoter region of MSI2, whereas control vectors did not.These data strongly suggested that KLF4 bound to the MSI2

Figure 3.

Influence of MSI2 expression on PDACcell migration and invasion. A and B,MDA Panc-28 (A) and AsPC-1 (B) cellswere transfected with pMSI2 or siMSI2and control vectors and siRNAs (Mock)for 48 hours. For a cell scratch-woundassay, cells in each groupwere placed in6-well plates and wounded viascratching and maintained at 37�C for12 or 18 hours. Cell cultures werephotographed, and cell migration wasassessed by measuring the cell-freeareas in multiple fields (the insertnumbers indicate the percentage meangap areas � SD in triplicate). Themigration and invasion of MDA Panc-28and AsPC-1 cells were determined asdescribed inMaterials andMethods. Thedata represent means� SD in triplicatefrom one representative experiment ofthree with similar results. � , P < 0.05in comparisons of pMSI2- andsiMSI2-treated groups and mock andcontrol groups (Student t test).

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promoter in PDAC cells and transcriptionally regulated MSI2expression.

DiscussionIn this report, we found that lost expression of KLF4, a

transcriptional repressor of MSI2, results in overexpression ofMSI2 in PDACs, which may be a biomarker for accurateprognosis. A dysregulated KLF4/MSI2 signaling pathway pro-motes PDAC progression and metastasis. MSI2 is a translation-al suppressor that binds to the 30-untranslated regions of itstarget mRNAs and blocks translation by hindering access of thepoly(A)–binding protein to the elongation initiation complex(29). In most studies of MSI2, researchers examined it in thehematopoietic system. Elevated MSI2 expression predicts poorprognosis for B-cell acute lymphoid leukemia and CML in blastcrisis (30–32). Recent studies also revealed that expression ofMSI2 was deregulated during tumorigenesis in different somat-ic tumors, including glioblastoma and breast, esophageal,pulmonary, colon, liver, gastric, and bladder cancer (4, 33–38). However, expression and the function of MSI2 in PDAChave yet to be demonstrated.

In the current study, we found that MSI2 expression wasmarkedly increased in PDAC cell lines and specimens. Furtheranalysis of the relationship between TMA clinicopathologiccharacteristics of the PDAC patients from whom the TMAspecimens were obtained and MSI2 expression levels demon-strated that MSI2 expression was positively associated withPDAC pT classification, regional lymph node metastasis, TNMstage, and tumor differentiation grade. Studies of MSI2 expres-sion in hepatocellular carcinoma cases demonstrated thatMSI2 was localized to the nuclei and cytoplasm of cancer

cells, primarily the former (4, 9). Our IHC results demonstrat-ed that MSI2 localized to the nuclei and cytoplasm of thePDAC cells and that the differences in the distribution ofnuclear expression of MSI2 in weak, moderate, and strongexpression groups were not statistically significant. Further-more, using immunofluorescent staining and Western blotting,we found that MSI2 mainly localized in the cytoplasm of MIAPaCa-2, AsPC-1, BxPC-3, PANC-1, and PATU-8902 cells but inthe nuclei of MDA Panc-28 cells. Interestingly, nuclear expres-sion of MSI2 appeared to be associated with increased tumordifferentiation grade. These results indicated that MSI2 mayfunction as an oncogene and play important roles in PDACdevelopment and progression. However, determining the sig-nificance and mechanism of MSI2 protein subcellular locali-zation and translocation in the cytoplasm and nucleus requiresfurther study.

In biologic studies, we found that elevated expression of MSI2promoted PDAC proliferation, migration, and invasion in vitroand growth andmetastasis in vivo, whereas knockdown of expres-sion of MSI2 markedly decreased PDAC growth and metastasisin vitro and in vivo. These results strongly supported the notionthat MSI2 functions as an oncogene in PDAC cells. However,we did not determine the underlying mechanism of MSI20spromotion of PDAC growth and metastasis. Researchers havedemonstrated that elevated expression of MSI2 could interactwith and cause the retention of efficient translation of a series ofoncogenic factors, including MYC, HOXA9, cyclin D1, andCdk2 (5, 39). Also, MSI2 is reported to activate Wnt and Notchsignaling pathways (9, 31). How MSI2 regulates the expressionand signaling functions of its downstream targets, includingC-Myc, in PDAC cells is currently unknown and warrantsfurther investigation.

Figure 4.

MSI2-promoted PDAC cell metastasis invivo. A and B, MDA Panc-28 cells withMSI2 overexpression (A) or AsPC-1 cellswith knockdown of MSI2 expression (B)were injected intravenously into theileocolic vein in nudemice (1� 106 cells/mouse, 5 mice/group). The tumor-bearing mice were killed 21 days afterinjection. Gross liver metastases andhematoxylin- and eosin-stainedsections of livers obtained from themice (A1, A2, B1, and B2; arrows indicatemetastatic nodules, and tumor areasare outlinedwith dashed lines; T, tumor)and their numbers of liver surfacemetastases (A3 and B3) are shown.






Expression of MSI2 is elevated in cases of PDAC and severalother types of cancer, but the mechanism of increased expres-sion of MSI2 in PDACs during tumorigenesis has been seldomstudied. In cases of CML in blast crisis, authors reported thatchromosomal translocations that fuse MSI2 with HOXA9 orTTC40 resulted in increased expression of MSI2 (40, 41).However, these types of chromosomal translocations are rarein PDAC cases.

KLF4, also known as gut-enriched KLF, is an importantmember of the KLF family. KLF4 has long been known to bea tumor suppressor. Our previous studies demonstrated that inmouse models, Klf4 knockout in gastric progenitor cellsresulted in the formation and progression of tumors in theantrum (25). Also, we reported that expression of KLF4 wasdecreased and even lost in PDACs (17–19, 42–44). Herein, weprovide evidence that elevated expression of MSI2 in PDACcells owes at least in part to decreased expression of KLF4 andthat MSI2 is a transcriptional target of KLF4. First, the expres-sion of MSI2 was negatively correlated with that of KLF4 inPDAC specimens. Second, overexpression of KLF4 resulted indecreased expression of MSI2, whereas knockdown of expres-sion of KLF4 led to increased expression of MSI2. Third, KLF4directly bound to the promoter region of MSI2 and transcrip-

tionally suppressed its promoter activity. Fourth, we verifiedthe related expression of KLF4 and MSI2 in colon and gastriccancers and found that overexpression of KLF4 in colon andgastric cancer cell lines resulted in decreased expression ofMSI2, whereas knockdown of expression of KLF4 led toincreased expression of MSI2. Furthermore, we analyzed theexpression of KLF4 and MSI2 in colon and gastric tissuespecimens obtained from KLF4-knockout mouse models andfound that KLF4-knockout specimens had high expression ofMSI2. All of these results demonstrated that MSI2 expressionwas transcriptionally suppressed by KLF4.

In summary, we obtained both clinical and experimentalevidence identifying MSI2 as an important oncogene in PDACsand found that its expression was frequently increased in bothPDAC cell lines and specimens. Biologically, MSI2 expressionpromoted PDAC proliferation, migration, and invasion in vitroand growth andmetastasis in vivo. Mechanistically, the expressionof MSI2 in PDACs was transcriptionally inhibited by KLF4, animportant tumor-suppressive transcriptional factor in thesetumors. Thus, this study is fundamentally important, as we notonly identified a novelmolecularmechanism of PDACmetastasisand progression but also identified the aberrant KLF4/MSI2signaling pathway as a promising new molecular target for the

Figure 5.

Co-expression of MSI2 and KLF4 inPDAC cells. AsPC-1, BxPC-3, FG, andPANC-1 cells were transfected with aFlag-KLF4 vector or control vectors,and MDA Panc-28 cells weretransfectedwith KLF4-siRNA or controlsiRNA for 48 hours. A and B, Total RNAand protein lysateswere harvested, andthe expression of MSI2 and KLF4 wasdetermined using Western blotting (A)and real-time PCR (B). GAPDH,glyceraldehyde-3-phosphatedehydrogenase; CTRL, control. C,Immunohistochemical stains of thesame TMA PDAC sections for MSI2 witha specific anti-KLF4 antibody.Representative images of KLF4-negative and MSI2-positive PDACsamples are shown (�100 and �400magnification in the inserts). D,Assessment of negative correlationbetween KLF4 and MSI2 expression inPDAC specimens (n ¼ 130) usingPearson correlation coefficient analysis.Some of the dots on the graphrepresent more than one specimen.

Guo et al.





design of novel therapeutic modalities that inhibit PDAC meta-stasis and progression.

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

Authors' ContributionsConception and design: K. Guo, J. Cui, M. Quan, Q. Ma, K. XieDevelopment of methodology: K. Guo, J. Cui, M. QuanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Guo, J. Cui, M. Quan, D. Xie, D. WeiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):K.Guo, J. Cui,M.Quan,D.Xie, Z. Jia, L.Wang, Y.Gao,Q. Ma, K. XieWriting, review, and/or revision of the manuscript: K. Guo, J. Cui, M. Quan,L. Wang, Q. Ma, K. XieAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Guo, J. Cui, M. Quan, Z. Jia, D. Wei, L. Wang,Y. Gao, Q. Ma, K. XieStudy supervision: J. Cui, Q. Ma, K. Xie

AcknowledgmentsWe thank Don Norwood for editorial assistance.

Grant SupportThis work was supported by grants R01-CA129956, R01-CA148954,

R01CA152309, R01CA172233, and R01CA195651 from the National Can-cer Institute, NIH (to K. Xie), the Fundamental Research Funds for theCentral Universities, grant 81502083 from the National Natural ScienceFoundation of China (to K. Guo), and the grant PWZxq2014-04 from KeyDisciplines Group Construction Project of Pudong Health Bureau of Shang-hai (to Y. Gao).

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 April 26, 2016; revised June 24, 2016; accepted July 13, 2016;published OnlineFirst July 22, 2016.

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Guo et al.




2017;23:687-696. Published OnlineFirst July 22, 2016.Clin Cancer Res Kun Guo, Jiujie Cui, Ming Quan, et al. Metastasis of Pancreatic CancerThe Novel KLF4/MSI2 Signaling Pathway Regulates Growth and

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