Activation of Vitamin D Receptor Signaling Downregulates the Expression … · Biology of Human Tumors Activation of Vitamin D Receptor Signaling Downregulates the Expression of Nuclear

Biology of Human Tumors

Activation of Vitamin D Receptor SignalingDownregulates the Expression of Nuclear FOXM1Protein and Suppresses Pancreatic Cancer CellStemnessZhiwei Li1,2,3, Zhiliang Jia2, Yong Gao4, Dacheng Xie4,5, Daoyan Wei2, Jiujie Cui2,5,Lopa Mishra2, Suyun Huang3, Yanqiao Zhang1, and Keping Xie2

Abstract

Purpose: Dysregulated signaling of nuclear transcription fac-tors vitamin D receptor (VDR) and Forkhead box M1 (FOXM1)plays important roles in transformation and tumorigenesis. Inthis study, we sought to determine whether VDR signaling caus-ally affected FOXM1 signaling in and pathogenesis of pancreaticductal adenocarcinoma (PDAC).

Experimental Design:Genetic and pharmacologic approacheswere used to manipulate VDR signaling. The impacts of alteredVDR signaling on FOXM1 expression and function in PDAC cellswere determined using molecular and biochemical methods,whereas those on PDAC cell biology and tumorigenicity weredetermined using in vitro and in vivo experimental systems. Theclinical relevance of our findings was validated by analyzinghuman PDAC specimens.

Results: There was a striking inverse correlation betweenreduced expression of VDR and increased expression of FOXM1in human PDAC cells and tissues. Treatment of PDAC cells with1,25-dihydroxyvitamin D3 (1,25D), its synthetic analogueEB1089 (EB), and VDR transgenics drastically inhibited FOXM1signaling and markedly suppressed tumor stemness, growth, andmetastasis. Mechanistically, 1,25D and EB repressed FOXM1transcription and reduced the expression level of nuclear FOXM1protein.

Conclusion: Inactivation of Vitamin D/VDR signaling is acritical contributor to PDAC development and progression viaelevated expression and function of FOXM1 and enhancedPDAC cell stemness, invasion, and metastasis. Clin Cancer Res;21(4); 844–53. �2014 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the lead-

ing causes of cancer deaths in industrialized countries, with amortality rate near 75%within 1 year after diagnosis and a 5-yearsurvival rate of less than6%. (1) Furthermore, the incidence of thisdisease appears to be increasing (2). The dismal prognosis forpancreatic cancer is attributable to its tendency toward late

presentation, early metastasis, and resistance to therapy. (3,4)Better understanding the mechanisms underlying the aggres-siveness of and dismal prognosis for PDAC would help developnovel, effective prevention and therapeutic modalities to save thelives of patients with PDAC (5–7).

Forkhead boxM1 (FOXM1) is a transcription factor in the FOXprotein superfamily (8–9). FOXM1 essentially regulates multipleaspects of tumor cell biology (10–12). Overexpression of FOXM1occurs frequently in a wide variety of human tumors and con-tributes to human cancer pathogenesis (12–14), including that ofPDAC (15,16). However, the molecular mechanisms underlyingFOXM1 dysregulation and its impact on PDAC pathogenesisremain unclear (12). Interestingly, both FOXM1 and VitaminD receptor (VDR) interact with b-catenin and regulate cellularfunctions (17–20). However, whether the expression and func-tion of VDR and FOXM1 are causally related and whether dysre-gulation of their crosstalk if exists affects cellular transformationand tumorigenesis remain unknown.

VDR, which belongs to the family of transacting transcriptionalregulatory factors and exhibits sequence similaritywith the steroidand thyroid hormone receptors, binds to the active form ofVitamin D, 1,25-dihydroxyvitamin D3 (1,25D; ref. 21). Also,Vitamin D binds to nuclear VDR, which activates the receptor toform a heterodimerwith the retinoid X receptor and interacts withthe VitaminD response element (VDRE). Transcription repressorsoccupying the VDRE are then replaced by transcription activatorsto initiate transcription of targeted genes (21). Microarray anal-yses have identified many genes with VDREs in their promoter

1Department of Gastrointestinal Oncology, The Harbin Medical Uni-versity Cancer Hospital, Harbin, People's Republic of China. 2Depart-ment of Gastroenterology, Hepatology & Nutrition, The University ofTexas MD Anderson Cancer Center, Houston, Texas. 3Department ofNeurosurgery, The University of Texas MD Anderson Cancer Center,Houston, Texas. 4Department of Oncology and Tumor Institute,Shanghai East Hospital, Tongji University School of Medicine, Shang-hai, People's Republic of China. 5Department of Oncology, ShanghaiJiaotong University Affiliated First People's Hospital, Shanghai, Peo-ple's Republic of China.

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

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]; and Yanqiao Zhang,Department of Gastrointestinal Medical Oncology, Harbin Medical UniversityCancer Hospital, Harbin, The People's Republic of China. Phone: 86-451-8629-8278; Fax: 86-451-8629-8222. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2437

�2014 American Association for Cancer Research.

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regions, all of which are potential targets of the Vitamin D/VDRcomplex (22–24). Vitamin D directly alters patterns of geneexpression via the VDR as well as VDR-independent mechanisms(25, 26), and regulates transcriptome (27–30), and exerts anti-tumor effects (31–35). These genomic effects can result from theclassical mechanism of VDR recruitment of coactivators to VDREsand nonclassical interactions of VDR with activated b-catenin onother promoters (24). VDR can also influence the level of nuclearb-catenin in colon cancer cells and can therefore attenuate theimpact of oncogenic mutations that activate the Wnt/b-cateninpathway (18). Thus, Vitamin D deficiency may play an importantrole in cancer development and progression and that Vitamin Dand its synthetic analogues may have therapeutic potential (36–40). However, in a pancreatic cancer clinical trial, EB1089 (EB orSeocalcitol, a synthetic analogue of 1,25D) is well tolerated buthas no objective antitumor activity in advanced disease (41),while the underlying mechanisms for this refractory nature isunknown.

In the present study, we sought to determine whether VDRsignaling causally regulates the expression and function ofFOXM1 in and pathogenesis of PDAC. We demonstrated thatinactivation of Vitamin D/VDR signaling critically affected PDACcell stemness and invasive andmetastatic phenotypes via elevatedexpression and function of FOXM1.

Materials and MethodsCell lines and culture conditions

The humanPDAC cell lines AsPC-1, BxPC-3, CaPan-1, CaPan-2,FG, Hs766T, MiaPaCa-2, mPanc96, PANC-1, MDA-28, MDA-48,and PA-TU-8902 and human embryonic kidney 293 (HEK293)cells were purchased from the American Type Culture Collection(ATCC) or obtained as described previously (16, 42). All of thecancer cell lines were maintained in plastic flasks as adherentmonolayers in DMEM supplemented with 10% FBS, sodiumpyruvate, nonessential amino acids, L-glutamine, and a vitaminsolution. The immortalized normal human pancreatic ductalepithelial cell line HPDE (provided by Dr. Ming-Sound Tsao,Ontario Cancer Institute) was maintained in keratinocyteserum-free medium supplemented with epidermal growth factor

and bovine pituitary extract (Invitrogen). The cell lines wereobtained directly from the ATCC that performs cell line character-izations or authentication by the short tandem repeat profiling andpassaged in our laboratory for fewer than 6 months after receipt.

Lentiviral VDR expression vector construction and transfectionFor generation of a lentiviral VDR expression vector, the PCR

primers 50-ctagtgaattcggtaccgaggagatctgccgc-30 and 50-tcgcgggatccc-gtttaaaccttatcgtcgtc-30wereused inaPCRwithpCMV6-VDRusedasa template. The PCR product was subcloned into the EcoRI andBamH1 sites of a pLVX-Puro vector, and the resultant vector wasused to package lentiviral particles using the Lenti-X HT packagingsystem (Clontech Laboratories). All vector constructs were con-firmed using DNA sequence analysis. For generation of stable celllines, 5� 105 PANC-1ormPanc96 cellswere incubated for 5 hourswith 1 � 106 particles of either L-EGFP or L-VDR in 2 mL ofcomplete DMEM in the presence of polybrene (4 mg/mL;refs. 16, 42). Cells infected with the lentivirus were subjected toselection in 5 mg/mL puromycin for 10 days before use.

Measurement of cell proliferation, migration, invasion, andspheroid colony formation

Pancreatic cancer cellswere treatedwithdifferent dosesof 1,25D(Sigma-Aldrich), EB (Tocris Bioscience), or a vehicle control(EtOH) in DMEM containing 4% FBS for 2 to 6 days. Cellproliferation,migration, invasion, and spheroid colony formationwere measured using procedures described previously (16, 42).

Immunocytochemical analysisPANC-1 and mPanc96 cells were seeded in Falcon chamber

slides (Becton Dickinson) at 1 � 105 cells per well in DMEMsupplemented with 10% FBS for overnight culture. The cells werethen treated with Vitamin D or EB (100 nmol/L) or a vehiclecontrol (EtOH) in complete DMEM containing 4% FBS for 48hours. Cells were then fixed in a 4% paraformaldehyde solutionfor 8 minutes. After being washed twice with PBS, the cells wereincubated with a specific anti-FOXM1, anti–b-catenin, or anti–E-cadherin antibody and then incubated with a Texas red–labeledsecondary antibody. Nuclear staining of the cells was accom-plished via incubation in a solution containing 10 mg/mL 40,6-diamidino-2-phenylindole (Sigma-Aldrich). Fluorescent imagingof cell cultures was performed using an Axiophoto 2 microscopeand the Photoshop CS4 software program.

Western blot analysisStandard Western blotting was performed using whole-cell

protein lysates or cytoplasmic andnuclear protein lysates; primaryantibodies against FOXM1, b-catenin, c-Myc, cyclin D1, Skp2,histone H1, a-tubulin, GAPDH (Santa Cruz Biotechnology), p27(Cell Signaling Technology), E-cadherin (BD Biosciences), andVDR (OriGene Technologies, Inc.); and a secondary antibody(anti-rabbit IgG or anti-mouse IgG; Santa Cruz Biotechnology).Equal protein-sample loading was monitored using an anti-GAPDH antibody (Santa Cruz Biotechnology).

SiRNARNA interference was performed using synthetic siRNA oligos

to VDR (Santa Cruz Biotechnology). Briefly, PANC-1 ormPanc96cells were seeded to 80% confluence in six-well plates in triplicateand transiently transfected with VDR siRNA or a control siRNA(100 pmol/well) using Lipofectamine 2000CD (Invitrogen). Cell

Translational Relevance

We used a pancreatic tumor tissue microarray, molecularbiology, and animal models to evaluate the inactivation andfunction of the Vitamin D receptor (VDR)/Forkhead box M1(FOXM1) pathway in pancreatic cancer cells. Our clinical andmechanistic findings indicated that FOXM1 is a direct tran-scriptional target of VDR and that dysregulation of VDRexpression, which occurs frequently, leads to aberrant FOXM1expression. Moreover, VDR negatively regulated stemness,growth, and metastasis of pancreatic cancer cells, suggestinga novelmolecular basis for the critical role of VDR inactivationin pancreatic cancer progression. It also suggests that dysre-gulated VDR/FOXM1 signaling is a promising new moleculartarget for designing novel preventive and/or therapeutic strat-egies to control this malignancy. Therefore, our findings mayhave a major effect on clinical management of pancreaticcancer.

VDR–FOXM1 Signaling in Pancreatic Cancer

www.aacrjournals.org Clin Cancer Res; 21(4) February 15, 2015 845




and protein samples were harvested 48 hours after transfectionand processed for Western blot analysis.

Flow cytometry analysisSingle-cell suspensions of PDAC cells were prepared in Dul-

becco's PBS (DPBS)/3% FBS at a concentration of 1 to 5 � 106

cells/mL. Anti–CD44-PE antibody (BD Biosciences Pharmingen)was added to the suspensions, which were then incubated on icefor 30 minutes. After being washed twice with DPBS/3% FBS, thecells were resuspended in DPBS/3% FBS and analyzed using aFACSCalibur flow cytometer equipped with the CellQuest soft-ware program (Becton Dickinson). In some experiments, single-cell suspensionswere prepared after treatmentwith EBor a vehiclecontrol and then analyzed using flow cytometry.

Real-time reverse transcriptase PCRTotal RNA was isolated from cell cultures using TRIzol reagent

(Invitrogen) and reverse-transcribed into cDNA using the iScriptcDNA synthesis Kit (Bio-Rad Laboratories). The cDNA productswere used in quantitative PCR analysis of indicated gene expres-sion using the FOXM1 and b-catenin PCR primers and probe sets(Applied Biosystems). Each real-time reverse transcriptase PCRexperiment was performed in triplicate. The relative quantitationof gene expression was determined by using the comparative CT

(DDCT) method, and the relative level of expression of an indi-vidual target gene was normalized to both the HPRT1 gene (assayID: Hs99999909_m1, Applied Biosystems) and a calibratorsample that was run on the same plate. Relative RNA expressioncalculations were performed using a commercially available soft-ware program (SDS, version 1.2; Applied Biosystems).

Human tissue samples and immunohistochemical analysisExpression of VDR and FOXM1 was analyzed using human

pancreatic tumor and normal tissue microarrays (PA2081; USBiomax, Inc.). The pancreatic tumor microarray contained 42ductal adenocarcinoma, 3 adenosquamous carcinoma, 1 islet cellcarcinoma, 6 metastatic carcinoma, 10 islet cell tumor, 2 hyper-plasia, 10 inflamed tissue, 20 adjacent normal tissue, and 10normal tissue specimens obtained at autopsy. Use of the tissuesamples was approved by The University of Texas MD AndersonCancer Center Institutional Review Board. Standard immunohis-tochemical procedures were carried out using an anti-VDR (Ori-Gene Technologies, Inc.) or anti-FOXM1 (Sigma-Aldrich) anti-body. The staining results were scored by two investigatorsblinded to the clinical data as described previously (16, 42). Asnegative controls, the primary antibodies were omitted andreplaced with a related strain of IgG used as a negative control.

Mouse model of pancreatic tumor growth and metastasisPathogen-free female athymic BALB/c nude mice were pur-

chased from the NCI (Bethesda, MD). The animals were main-tained in facilities approved by the Association for Assessmentand Accreditation of Laboratory Animal Care International inaccordance with the current regulations and standards of the U.S.Department of Agriculture and U.S. Department of Health andHumanServices. Toproduce pancreatic and subcutaneous tumorsor experimental liver and lungmetastases, 1�106 cells in 100 ul ofHank's balanced salt solution were injected into the pancreas,Subcutis, ileocolic or tail veins of 7- to 8-week-old nudemice. Theanimals were killed 4 to 5 weeks after inoculation, and their

primary tumors were harvested, weighed, and processed forfurther analysis of related molecular marker expression.

VDR expression vector and gene transfectionThe plasmid pCMV6-Myc-DDK-VDR (OriGene Technologies,

Inc.) and the control vector pcDNA3.1 were transfected intoPDAC cells using Lipofectamine 2000CD (Invitrogen). Function-al assays for cell proliferation and migration were carried out 48hours or as indicated after transfection.

FOXM1 promoter reporter activity assayA 2.496-kb fragment of FOXM1 containing 50 FOXM1

sequences from �2430 to þ66 bp relative to the transcriptioninitiation site was subcloned into the Asp718 and XhoI sites of apGL3-basic vector (Promega). The final resulting full-lengthreporter plasmid designated as pFOXM1-2496. All constructswere verified by sequencing the inserts in and flanking regionsof the plasmid. Pancreatic cancer cells were transfected withpromoter reporters and then treated with 1,25D and/or EB, asynthetic analogue of 1,25D, or cotransfected with the indicatedpCMV6-Myc-DDK-VDR–specific gene expression plasmids. TheFOXM1 promoter activity in these cells was normalized viacotransfection of a b-actin/Renilla luciferase reporter containinga full-length Renilla luciferase gene (42). The luciferase activity inthe cells was quantified using a dual luciferase assay system(Promega) 48 hours after transfection.

Statistical analysisAll the in vitro and in vivo experiments were repeated at least

once, whereas one experiment of two or three with similar resultswas represented. The significance of the data onpatient specimenswas determined using the two-tailed x2 test and Fisher exact test.The significance of the in vitro and in vivo data was determinedusing the Student t test (two-tailed), theMann–Whitney test (two-tailed), or one-way ANOVA. P values less than 0.05 were consid-ered significant. The SPSS software program (version 12.0; IBMCorporation) was used for all statistical analyses.

ResultsInverse correlation of VDR expression with FOXM1 expressionin pancreatic tissue specimens and association withclinicopathologic features of PDAC

Previous studies have shown an elevated expression of FOXM1in human PDAC (16, 17). To determine the potential regulationof FOXM1 expression byVitaminD/VDR and its clinical relevanceof VDR–FOXM1 signaling to PDAC pathogenesis, we first soughttomeasure the expression of VDR in 46 primary pancreatic tumor,6 metastatic pancreatic tumor, and 10 normal pancreatic tissuespecimens in a tissue microarray. We observed VDR-positive orweak VDR-positive staining in the nuclei of normal pancreaticcells, whereas we observed VDR-negative staining in pancreatictumor cells. However, expression of FOXM1 occurred predomi-nantly in tumor cells (Fig. 1A and B). We detected a pronouncedinverse correlation between the levels of VDR and FOXM1 expres-sion in PDAC specimens (Fig. 1B and C). Moreover, the levels ofVDR expression correlated with tumor differentiation, as therewas a significant difference between well (grade 1) and poorly(grade 3) differentiated tumors (Fig. 1D). These clinical findingssupport that VDR interacts with FOXM1 and critically affects

Li et al.

Clin Cancer Res; 21(4) February 15, 2015 Clinical Cancer Research846




PDAC pathogenesis and that VDR and FOXM1 are potentiallyvaluable biomarkers.

Downregulation of the expression of FOXM1 and itsdownstream target genes by activation of VDR signaling

To determine whether Vitamin D/VDR causally regulates theexpression of FOXM1, we pharmacologically activated VDR sig-naling or genetically overexpressed VDR in PDAC cells by usingactive form of Vitamin D, i.e., 1,25D and its synthetic analogue,EB1089, i.e., EB. Treatment of mPanc96, MDA-28, and PANC-1cells with 1,25D and EB suppressed the expression of FOXM1 andits downstream target genes, including Cyclin D1, Skp2, c-Myc,CD44, and c-Met (Fig. 2A). This downregulation of FOXM1protein expression was consistent with suppression of FOXM1promoter activity and mRNA expression (Fig. 2B and C).

Next, we either knocked down the expression or inducedoverexpression of FOXM1 in mPanc96 and PANC-1 cells andthen exposed the cells to EB. The treatment resulted in furtherreduction of expression of FOXM1 and its downstream targetgenes caused by FOXM1 knockdown (Fig. 2D), whereas over-expressionof FOXM1attenuated downregulation of expression ofFOXM1 target genes caused by treatment with EB (Fig. 2D). Thissuggests an essential role for FOXM1 in EB-mediated downregu-lation of Cyclin D1, c-Myc, and CD44 expression.

Furthermore, overexpression of VDR induced by either genetransfection or lentiviral gene transfer caused downregulation ofFOXM1 and its downstream targets, whereas VDR knockdown

increased the expression of FOXM1 and its downstream targets(Fig. 3A). Downregulation of FOXM1 protein expression wasconsistent with decreased FOXM1 mRNA expression (Fig. 3B).VDR transfection led to the suppression of FOXM1 promoteractivity (Fig. 3C).

Although the constitutive expression levels of VDR did notsignificantly correlate with those of FOXM1 (Supplementary Fig.S1A), knockdown of VDR increased FOXM1 promoter activities(Supplementary Fig. S1B) and clearly attenuated the 1,25D- andEB-mediated repression of both FOXM1 protein expression (Fig.3C) and promoter activity (Supplementary Fig. S1C), indicatingthat expression of VDR is necessary for 1,25D- and EB-mediatedrepression of the expression of FOXM1 and its downstreamtargets. In contrast, overexpression of VDR sensitized the sup-pression of FOXM1 expression to treatment with EB (Supplemen-tary Fig. S1D).

Suppression of expression of nuclear FOXM1 by activation ofVDR signaling

FOXM1 contains a functional nuclear localization signaldomain and shuttles between the cytoplasm and the nucleus.FOXM1 also is a downstream component of the Wnt signalingpathway and is critical for b-catenin's transcriptional functionin tumor cells. Specifically, FOXM1 binds directly to b-cateninand enhances its nuclear localization and transcriptional activ-ity (42). We therefore examined whether the VDR signalingplays a role in nuclear FOXM1 expression. Treatment with

Figure 1.Inverse correlation between theexpression of VDR and FOXM1 inhuman PDAC specimens. Theexpression of VDR and FOXM1 proteinwas determined in tissue microarraysof paired normal pancreatic and PDACspecimens. A, expression of VDRprotein was higher in normalpancreatic tissue (10 cases) than inpancreatic tumors (46 cases). The x

2

test demonstrated significantdifferences in VDR and FOXM1 proteinexpression in normal tissue and tumorspecimens (P < 0.05). B,representative photographs of VDRand FOXM1 protein expression. C, asignificant inverse correlationbetween the levels of VDR and FOXM1expression was observed in PDACspecimens (P < 0.01). D, increasedVDR expression correlated withincreased tumor differentiation and asignificant difference between well(grade 1) and poorly (grade 3)differentiated tumors (P < 0.05).






1,25D and EB (Fig. 4A) and VDR transfection (Fig. 4B) weresufficient to decrease nuclear FOXM1 and b-catenin expressionin PDAC cells. Further experiments using double-cell immu-nofluorescence staining demonstrated that treatment with1,25D and EB decreased the expression of FOXM1 and b-cate-

nin and especially the nuclear FOXM1 and b-catenin in PDACcells (Fig. 4C; Supplementary Fig. S2).

Increased FOXM1 expression induced by transfection pre-vented downregulation of b-catenin expression caused by VDRoverexpression in PDAC cells (Supplementary Fig. S3A), whereasknockdown of FOXM1 expression potentiated the downregula-tion of b-catenin expression caused by VDR overexpression (Sup-plementary Fig. S3B).

Moreover, treatment with 1,25D significantly decreased theexpressionofb-catenin but slightly decreased that of E-cadherin inPDACcells (Supplementary Fig. S2). Inuntreated cells, E-cadherinwas predominantly expressed in nonnuclear compartments, e.g.,cytosolic and/or membrane-bound, whereas b-catenin wasexpressed in both nuclear and nonnuclear compartments (Sup-plementary Fig. S2). In contrast, treatment with 1,25D decreasedthe expression of b-catenin in both nuclear and nonnuclearcompartments, predominantly the nuclear b-catenin (less so fornonnuclear b-catenin). Consistently, cell fractionation experi-ments showed that EB treatment and VDR transfection decreasedthe expression levels of nuclear FOXM1, b-catenin, and E-Cad-herin, while the levels of nonnuclear E-Cadherin were relativelystable (Fig. 4A and B). These data suggested that interactionsamong b-catenin, FOXM1, and E-cadherin play important roles intheir expression in nuclear and nonnuclear compartments.

Suppression of the growth, migration, invasion, and stemnessof pancreatic cancer cells by activation of VDR signaling

Researchers have demonstrated the antiproliferative activity ofVitamin D and VDR in several types of tumors (27–35). Indeed,both 1,25D and EB inhibited the growth of mPanc96, PANC-1,MDA-28, PA-TU-8902, BxPC-3, and FG cells in vitro in a time- anddose-dependent manner (Fig. 5A; Supplementary Fig. S4). Inter-estingly, 1,25DandEBalso suppressed themigration, invasion of,and, most importantly, spheroid formation by PDAC cells (Fig.5B–5D; Supplementary Fig. S5). Attenuationof tumor growthwasconsistent with suppression of FOXM1 expression in the tumors(Supplementary Fig. S6A and S6B). Thus, we clearly establishedfor the first time that 1,25D and EB inhibit PDAC cell stemness,invasion, and metastasis.

Impact of genetic and pharmacologic manipulation of VDRexpression and function on PDAC cell stemness andmetastasis

Finally, increased expression of VDR by using lentivirus-medi-ated gene transfer suppressed the growth of PDAC cells in ortho-topic (Fig. 6A) and ectopic (Fig. 6B)nudemousemodels of PDAC.Moreover, overexpression of VDR suppressed both experimentallung (Fig. 6C) and liver (Fig. 6D) metastases in these models.Importantly, we isolated the tumor stem cells frommPanc96, andtheir tumorigenic potential was determined (Supplementary Fig.S7A). Their tumor-spheroid formation (Supplementary Fig. S7B)and in vivo tumorigenicity were suppressed by the treatment of1,25D or EB (Supplementary Fig. S7C). Thus, activation of VDRsignaling produced significant antistemness and antitumor activ-ity in PDAC.

DiscussionIn the present study, we discovered a novel Vitamin D/VDR/

FOXM1 signaling pathway in regulation of PDAC pathogenesis.First, VDR expression was drastically reduced in PDAC cell linesand tissues, and was inversely correlated with that of FOXM1.

Figure 2.Treatment with 1,25D and EB inhibits expression of FOXM1 and itsdownstream genes. mPanc96, MDA-28, and PANC-1 cells were treated with1,25D and EB (100 nmol/L for 48 hours). A, total cell lysate proteins wereextracted for Western blot analysis using specific antibodies, and (B) mRNAwas extracted for quantitative PCR analysis. C, the FOXM1 promoter reporterwas transfected into pancreatic cancer cells, which were then treated with1,25D or EB (100 nmol/L for 48 hours). The promoter activity was assessed 48hours after treatment using a dual luciferase assay kit. Both mPanc96 andPANC-1 cells were transfected with (D) FOXM1 siRNA or control siRNA, orFOXM1 expression vector (pFOXM1) or control vector (pCTRL). Total celllysate proteins were extracted for Western blot analysis of gene expressionusing specific antibodies. GAPDH, glyceraldehyde-3-phosphatedehydrogenase; CTRL, control.

Li et al.





Reduced or lost VDR expression correlated with PDAC progres-sion. Second, activation of the Vitamin D/VDR pathway sup-pressed the proliferation, migration, stemness, tumorigenicity,

and metastasis of PDAC cells. Third, treatment with 1,25D or EBinhibited the expression of FOXM1 and its downstream targets byrepressing FOXM1 transcription and by blockade of nuclear

Figure 3.Impact of altered expression of VDRon FOXM1 expression. PANC-1 andmPanc96 cells were transfected withpVDR or pCTRL or infected with alentiviruswith control enhanced greenfluorescent protein [EGFP (L-EGFP)]or a lentivirus with VDR (L-VDR).PANC-1 and mPanc96 cells weretransfected with VDR siRNA (siVDR)or control siRNA (siCTRL). A, total celllysate proteins were extracted forWestern blot analysis of geneexpression using specific antibodies,and (B) mRNA was extracted forquantitative PCR analysis. C,Luciferase assays using mPanc96 andPANC-1 cells demonstrated thataltered VDR recruitment wasconsistent with changes in FOXM1promoter activity. D, mPanc96 cellswere transfected with VDR siRNA(siV) or control siRNA (siC) and thentreatedwith 1,25D, EB (100nmol/L), ora dissolvent for 48 hours. Total celllysate proteins were extracted forWestern blot analysis of geneexpression using specific antibodies.

Figure 4.Impact of altered VDR signaling onsubcellular localization of FOXM1.mPanc96 cells were treated with1,25D, EB, or a dissolvent (100 nmol/Lfor 48 hours; A) or transfected withcontrol vector (pCTRL) or VDR vector(pVDR; B). Cytosolic (Cyt) and nuclear(Nuc) proteins were extracted forWestern blot analysis of geneexpression using specific antibodies(respective left plot), and relativeexpression levels of proteins werequantitated (respective right plots).Protein localization in the PDACcells was determined usingimmunofluorescent staining. DAPI,40 ,6-diamidino-2-phenylindole (C).






FOXM1 expression. Fourth, treatment with 1,25D or EB and VDRtransgenics reduced the stemness of PDAC cells. These novelclinical and mechanistic findings strongly indicate that inactiva-tion of the Vitamin D/VDR pathway and consequential elevationof FOXM1 expression and function promote PDAC progression.

FOXM1expression is elevated inhumanPDAC(12, 16, 17) andis a key regulator of PDAC biology (12, 15–17). Our current studyhas shown that the levels of FOXM1 expression correlate withtumor grade anddifferentiation, further substantiating the clinicalsignificance of FOXM1 expression in PDAC pathogenesis. How-ever, we observed that VDR expressionwas pronouncedly reducedin primary PDAC cells and tissues, and correlated with advancedstage and poor pathologic grade. Those results suggest that bothFOXM1 and VDR are potentially novel biomarkers for predictingprognosis of patients with PDAC. For example, the frequentlyreduced VDR in advanced PDAC could be responsible for noobjective antitumor activity of Seocalcitol clinical trials (41).Seocalcitol and other approaches to activate VDR signaling could

have significant impact on this malignancy in early or minimaldisease states.

Besides the inverse correlation of their expressions in PDAC,FOXM1 and VDR exhibit opposite cellular functions in a varietyof contexts (43–45). In addition to its antiproliferation activity(33–35), we have demonstrated that VDR over-expression cansuppress the migration, invasion of PDAC cells, and most impor-tantly can attenuate the stemness of PDAC cells. In contrast,overexpression of FOXM1 promotes many aspects of PDAC cellbiology, including stemness (12). As regulatory molecules, VDRand FOXM1 opposingly regulate the expression and/or functionof various genes related to cell cycle, stemness, and mesenchymalcell markers (27–30), i.e., many downstream molecules of VDRare in fact those of FOXM1. Significantly, VDR suppressed theexpression of FOXM1, suggesting that the impact of VDR activa-tion on its downstream targets and consequential antitumoractivities may likely be executed through suppression ofFOXM1 expression. Evidently, VDR negatively regulates FOXM1

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Figure 5.Impact of altered VDR signaling onpancreatic cancer cell biology.Treatment with 1,25D and/or EB at 100nmol/L for 48 hours or as indicatedinhibited the (A) growth of mPanc96,PANC-1, andMDA-28 cells in vitro (MTTassay); (B) horizontal migration ofmPanc96 cells (gap-closing assay); (C)vertical migration and invasion ofmPanc96 cells (Boyden chamberassay); and (D) stemness of PANC-1and mPanc96 cells (spheroid colonyformation assay). Moreover,transfection of PANC-1 cells with a VDRexpression vector (pVDR) suppressedcell stemness more so than didtransfection with a control vector(pCTRL). � , P < 0.01 as with the propercontrol.

Li et al.





expression and function by two potentially distinct mechanisms,i.e., repressing FOXM1 gene transcription and attenuating nuclearFOXM1 expression.

Moreover, VDR activation caused concomitant changes ofexpression levels and subcellular distribution of both FOXM1and b-catenin, i.e., predominantly reduced expression levels ofnuclear FOXM1 and b-catenin. This indicates the existence of anovel VDR/FOXM1/b-catenin axis. This notion is supported byour early study, which has shown that FOXM1 interacts directlywith b-catenin and facilitates the nuclear translocation of b-cate-nin (42), and other early reports, which have shown that VDRinteracts with b-catenin and influences its nuclear content incolorectal cancer cells (18, 19). Presumably, VDR can influencethe nuclear level of b-catenin via increased binding of b-catenin tomembrane E-cadherin (44). This notion is supported by ourstudy, showing that the expression of nonnuclear E-Cadherincorrelated directly with the relatively stable levels of nonnuclearb-catenin and FOXM1. However, we clearly observed that VDRreduced the expression of both FOXM1 and b-catenin in bothnuclear and nonnuclear compartments. We believe that VDR-mediated downregulation of FOXM1 critically contributed to thereduction of nuclear b-catenin, given that FOXM1 critically reg-ulates the nuclear translocalization of b-catenin and that FOXM1is apositive transactivator ofb-catenin gene expression (42). Thus,VDR-induced downregulation of FOXM1 could result in a

decrease in overall expression and particularly nuclear accumu-lation of b-catenin protein.

Finally, the great potential of Vitamin D as a cancer chemo-preventive and therapeutic agent has spurred investigationsinto the molecular mechanisms that govern its effects on cancerand the development of Vitamin D/VDR-based strategies forprevention and treatment of cancer (17, 24, 45–47). However,the mechanisms underlying VDR underexpression in cancercells remain unclear. VDR promoter methylation and over-expression of negative regulators potentially contribute to thesilencing of VDR signaling pathway (47–49). Future study isclearly warranted to develop strategies to restore VDR expres-sion and function and translate those relevant findings to PDACinterventions.

In summary, a reduced or lost expression of VDR and itsattenuated signaling led to the overexpression of FOXM1 and itsdownstream targets, thus promoting PDAC cell proliferation,stemness, invasion, and metastasis. The clinicopathologic rele-vance and significance of this aberrant Vitamin D/VDR/FOXM1signaling inPDACpathogenesis havebeendemonstrated byusingmolecular biology, animalmodels, and human PDAC specimens.Therefore, activation and/or restoration of Vitamin D/VDR sig-naling likely produces an antitumor effect by repressing FOXM1signaling. Further investigations into molecular mechanismsunderlying dysregulation of this novel pathway would help

Figure 6.Impact of altered VDR signaling onpancreatic tumor growth andmetastasis. A, mPanc96 and PANC-1cellswere transfectedwith L-VDRor L-EGFP. The cellswere then injected intothe pancreases of nudemice. Themicewere killed 35 days after tumor-cellinjection, and their tumors wereharvested: Photographs of grosstumors (left plots) and tumor weights(right plots). The cells were injectedinto the subcutis (B), tail vein forexperimental lung metastasis (C), orileocolic vein for experimental livermetastasis (D). Affected mice (B, leftplot), mouse tumor weights (B, rightplot), experimental lung metastases(C, top plot), numbers and sizes ofexperimental lung metastases (C,bottom plots), photos and sections ofliver metastases (D, left plots), andnumbers of liver metastases (D, rightplot) were shown.






identify promising targets for designing novel preventive andtherapeutic modalities to control PDAC.

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

Authors' ContributionsConception and design: Z. Li, L. Mishra, S. Huang, K. XieDevelopment of methodology: Z. Li, Z. Jia, D. Xie, J. Cui, K. XieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Li, Z. Jia, Y. Gao, D. Xie, D. Wei, J. Cui,K. XieAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Li, Z. Jia, Y. Gao, D. Xie, D. Wei, J. Cui, S. Huang,Y. Zhang, K. XieWriting, review, and/or revision of the manuscript: Z. Li, S. Huang, K. XieAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Jia, D. Wei, L. Mishra, K. XieStudy supervision: Y. Gao, D. Wei, L. Mishra, S. Huang, Y. Zhang, K. Xie

AcknowledgmentsThe authors thank Don Norwood for editorial comments.

Grant SupportThis work was supported in part by grants R01-CA129956, R01-

CA148954, R01-CA152309, and R01-CA172233 (to K. Xie); R01-CA116528and R01-CA157933 (to S. Huang); MD Anderson Cancer Center SupportGrant CA016672 from the NIH; grant LC2013C28 (to Z. Li) from theHeilongjiang Province Foundation for Returnees of China; and grant81172265/H1617 (to Y. Zhang) from the National Natural Science Foun-dation of China.

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 24, 2014; revised November 11, 2014; accepted Decem-ber 1, 2014; published OnlineFirst October 10, 2014.

References1. Siegel R, NaishadhamD, Jemal A. Cancer statistics, 2013. CA Cancer J Clin

2013;63:11–30.2. Wolfgang CL, Herman JM, Laheru DA, Klein AP, Erdek MA, Fishman EK,

et al. Recent progress in pancreatic cancer. CA Cancer J Clin 2013;63:318–48.

3. Paulson AS, TranCaoHS, TemperoMA, Lowy AM. Therapeutic advances inpancreatic cancer. Gastroenterology 2013;144:1316–26.

4. Hartwig W, Werner J, J€ager D, Debus J, B€uchler MW. Improvement ofsurgical results for pancreatic cancer. Lancet Oncol 2013;14:e476–85.

5. Abel EV, Simeone DM. Biology and clinical applications of pancreaticcancer stem cells. Gastroenterology 2013;144:1241–8.

6. Zheng L, Xue J, Jaffee EM,Habtezion A. Role of immune cells and immune-based therapies in pancreatitis and pancreatic ductal adenocarcinoma.Gastroenterology 2013;144:1230–40.

7. Stanger BZ, Hebrok M. Control of cell identity in pancreas developmentand regeneration. Gastroenterology 2013;144:1170–9.

8. Wierstra I. The transcription factor FOXM1 (Forkhead box M1): prolifer-ation-specific expression, transcription factor function, target genes,mousemodels, and normal biological roles. Adv Cancer Res 2013;118:97–398

9. Kalin TV, Ustiyan V, Kalinichenko VV. Multiple faces of FoxM1 transcrip-tion factor: lessons from transgenic mouse models. Cell Cycle 2011;10:396–405.

10. Raychaudhuri P, Park HJ. FoxM1: a master regulator of tumor metastasis.Cancer Res 2011;71:4329–4333.

11. Halasi M, Gartel AL. FOX(M1) news–it is cancer. Mol Cancer Ther2013;12:245–54.

12. Huang C, Du J, Xie K. FOXM1 and its oncogenic signaling in pancreaticcancer pathogenesis. Biochim Biophys Acta 2014;1845:104–16.

13. Wang Z, Ahmad A, Li Y, Banerjee S, Kong D, Sarkar FH. Forkhead box M1transcription factor: a novel target for cancer therapy. Cancer Treat Rev2010;36:151–6.

14. Koo CY,Muir KW, Lam EW. FOXM1: From cancer initiation to progressionand treatment. Biochim Biophys Acta 2012;1819:28–37.

15. Wang Z, Banerjee S, Kong D, Li Y, Sarkar FH. Down-regulation of ForkheadBox M1 transcription factor leads to the inhibition of invasion andangiogenesis of pancreatic cancer cells. Cancer Res 2007;67:8293–8300.

16. Huang C, Qiu Z, Wang L, Peng Z, Jia Z, Logsdon CD, et al. A novel FoxM1-Caveolin signaling pathway promotes pancreatic cancer invasion andmetastasis. Cancer Res 2012;72:655–65.

17. Shah S, Islam MN, Dakshanamurthy S, Rizvi I, Rao M, Herrell R, et al. Themolecular basis of vitamin D receptor and b-catenin crossregulation. MolCell 2006;21:799–809.

18. Larriba MJ, Gonz�alez-Sancho JM, Barb�achano A, Niell N, Ferrer-MayorgaG,Mu~noz A. VitaminD is amultilevel repressor ofWnt/b-catenin signalingin cancer cells. Cancers (Basel) 2013;5:1242–60.

19. Beildeck ME, Islam M, Shah S, Welsh J, Byers SW. Control of TCF-4expression by VDR and vitamin D in the mouse mammary gland andcolorectal cancer cell lines. PLoS ONE 2009;4:e7872.

20. Evans PM, Chen X, Zhang W, Liu C. KLF4 interacts with b-catenin/TCF4and blocks p300/CBP recruitment by beta-catenin. Mol Cell Biol2010;30:372–81.

21. Kennedy DA, Cooley K, Skidmore B, Fritz H, Campbell T, Seely D. Vitamind: pharmacokinetics and safety when used in conjunction with the phar-maceutical drugs used in cancer patients: a systematic review. Cancers(Basel) 2013;5:255–80.

22. Lin R, Nagai Y, Sladek R, Bastien Y, Ho J, Petrecca K, et al. Expressionprofiling in squamous carcinoma cells reveals pleiotropic effects of vitaminD3 analog EB1089 signaling on cell proliferation, differentiation, andimmune system regulation. Mol Endocrinol 2002;16:1243–56.

23. Wood RJ, Tchack L, AngeloG, Pratt RE, Sonna LA.DNAmicroarray analysisof vitamin D-induced gene expression in a human colon carcinoma cellline. Physiol Genomics 2004;17:122–29.

24. Byers SW, Rowlands T, Beildeck M, Bong YS. Mechanism of action ofvitamin D and the vitamin D receptor in colorectal cancer prevention andtreatment. Rev Endocr Metab Disord 2012;13:31–8.

25. Sakai Y, Demay MB. Evaluation of keratinocyte proliferation and differ-entiation in vitamin D receptor knockout mice. Endocrinology 2000;141:2043–9.

26. Di Rosa M, Malaguarnera M, Nicoletti F, Malaguarnera L. Vitamin D3: ahelpful immuno-modulator. Immunology 2011;134:123–39.

27. Thorne J, Campbell MJ. The vitamin D receptor in cancer. Proc Nutr Soc2008;67:115–27.

28. P�almer HG, S�anchez-Carbayo M, Ord�o~nez-Mor�an P, Larriba MJ, Cord�on-Card�o C, Mu~noz A. Genetic signatures of differentiation induced by1alpha,25-dihydroxyvitamin D3 in human colon cancer cells. Cancer Res2003;63:7799–806.

29. Wang TT, Tavera-Mendoza LE, Laperriere D, Libby E, MacLeod NB, Nagai Y,et al. Large-scale in silico and microarray-based identification of direct1,25-dihydroxyvitamin D3 target genes. Mol Endocrinol 2005;19:2685–95.

30. Mullin GE, Dobs A. Vitamin d and its role in cancer and immunity: aprescription for sunlight. Nutr Clin Pract 2007;22:305–22.

31. Spina CS, Ton L, YaoM,Maehr H, Wolfe MM, Uskokovic M, et al. Selectivevitamin D receptor modulators and their effects on colorectal tumorgrowth. J Steroid Biochem Mol Biol 2007;103:757–62.

32. de Lyra EC, da Silva IA, Katayama ML, Brentani MM, Nonogaki S, G�oes JC,et al. 25(OH)D3 and 1,25(OH)2D3 629 serum concentration and breasttissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin Dreceptor in women with and without breast cancer. J Steroid BiochemMolBiol 2006;100:184–92.

33. Zugmaier G, J€ager R, Grage B, Gottardis MM, Havemann K, Knabbe C.Growth-inhibitory effects of vitaminD analogues and retinoids on humanpancreatic cancer cells. Br J Cancer 1996;73:1341–6.

34. Kanemaru M, Maehara N, Chijiiwa K. Antiproliferative effect of 1a,25-dihydroxyvitamin D3 involves upregulation of cyclin-dependent kinaseinhibitor p21 in human pancreatic cancer cells. Hepatogastroenterology2013;60:1199–205.


Li et al.




35. Kawa S, Nikaido T, Aoki Y, Zhai Y, Kumagai T, Furihata K, et al. Vitamin Danalogues up-regulate p21 and p27 during growth inhibition of pancreaticcancer cell lines. Br J Cancer 1997;76:884–9.

36. Persons KS, Eddy VJ, Chadid S, Deoliveira R, Saha AK, Ray R. Anti-growth effect of 1,25-dihydroxyvitamin D3–3-bromoacetate alone orin combination with 5-amino-imidazole-4-carboxamide-1-beta-4-ribofuranoside in pancreatic cancer cells. Anticancer Res 2010;30:1875–80.

37. Ghous Z, Akhter J, Pourgholami MH, Morris DL. Inhibition of hepatocel-lular cancer by EB1089: in vitro and in vivo study. Anticancer Res2008;28:3757–62.

38. Mouratidis PX, Dalgleish AG, Colston KW. Investigation of the mechan-isms bywhich EB1089 abrogates apoptosis inducedby 9-cis retinoic acid inpancreatic cancer cells. Pancreas 2006;32:93–100.

39. Zhang X, Jiang F, Li P, Li C, Ma Q, Nicosia SV, et al. Growth suppression ofovarian cancer xenografts in nude mice by vitamin D analogue EB1089.Clin Cancer Res 2005;11:323–8.

40. MillikenEL, ZhangX, FlaskC,Duerk JL,MacDonald PN, Keri RA. EB1089, avitamin D receptor agonist, reduces proliferation and decreases tumorgrowth rate in a mouse model of hormone-induced mammary cancer.Cancer Lett 2005;229:205–15.

41. Evans TR, Colston KW, Lofts FJ, Cunningham D, Anthoney DA, GogasH, et al. A phase II trial of the vitamin D analogue Seocalcitol (EB1089)in patients with inoperable pancreatic cancer. Br J Cancer 2002;86:680–5.

42. Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1promotes b-catenin nuclear localization and controls Wnt target-geneexpression and glioma tumorigenesis. Cancer Cell 2011;20:427–42.

43. Hendrickson WK, Flavin R, Kasperzyk JL, Fiorentino M, Fang F, Lis R, et al.Vitamin D receptor protein expression in tumor tissue and prostate cancerprogression. J Clin Oncol 2011;29:2378–85.

44. P�almer HG, Gonz�alez-Sancho JM, Espada J, BercianoMT, Puig I, Baulida J,et al. Vitamin D(3) promotes the differentiation of colon carcinoma cellsby the induction of E-cadherin and the inhibitionof beta-catenin signaling.J Cell Biol 2001;154:369–87.

45. Dalhoff K, Dancey J, Astrup L, Skovsgaard T, Hamberg KJ, Lofts FJ, et al. Aphase II study of the vitamin D analogue Seocalcitol in patients withinoperable hepatocellular carcinoma. Br J Cancer 2003;89:252–7.

46. Cho M, Peddi PF, Ding K, Chen L, Thomas D, Wang J, et al. Vitamin Ddeficiency and prognostics among patients with pancreatic adenocarcino-ma. J Transl Med 2013;11:206.

47. Marik R, Fackler M, Gabrielson E, Zeiger MA, Sukumar S, Stearns V, et al.DNAmethylation-related vitamin D receptor insensitivity in breast cancer.Cancer Biol Ther 2010;10:44–53.

48. Larriba MJ, Martín-Villar E, García JM, Pereira F, Pe~na C, de Herreros AG,et al. Snail2 cooperates with Snail1 in the repression of vitamin D receptorin colon cancer. Carcinogenesis 2009;30:1459–68.

49. Yang H, Zhang Y, Zhou Z, Jiang X, Shen A. Snail-1 regulates VDR signalingand inhibits 1,25(OH)-D(3) action in osteosarcoma. Eur J Pharmacol2011;670:341–6.






2015;21:844-853. Published OnlineFirst December 11, 2014.Clin Cancer Res Zhiwei Li, Zhiliang Jia, Yong Gao, et al. Cancer Cell StemnessExpression of Nuclear FOXM1 Protein and Suppresses Pancreatic Activation of Vitamin D Receptor Signaling Downregulates the

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