FGFR4 Promotes Stroma-Induced Epithelial-to- Mesenchymal ...Molecular and Cellular Pathobiology FGFR4 Promotes Stroma-Induced Epithelial-to-Mesenchymal Transition in Colorectal Cancer

Molecular and Cellular Pathobiology

FGFR4 Promotes Stroma-Induced Epithelial-to-Mesenchymal Transition in Colorectal Cancer

Rui Liu1, Jingyi Li3, Ke Xie4, Tao Zhang3, Yunlong Lei1, Yi Chen2, Lu Zhang1, Kai Huang1, KuiWang1, HongWu2,Min Wu5, Edouard C. Nice6, Canhua Huang1, and Yuquan Wei1

AbstractTumor cells evolve by interacting with the local microenvironment; however, the tumor–stroma

interactions that govern tumor metastasis are poorly understood. In this study, proteomic analyses revealthat coculture with tumor-associated fibroblasts (TAF) induces significant overexpression of FGFR4, but notother FGFRs, in colorectal cancer cell lines. Mechanistic study shows that FGFR4 plays crucial roles inTAF-induced epithelial-to-mesenchymal transition (EMT) in colorectal cancer cell lines. AccumulatedFGFR4 in cell membrane phosphorylates b-catenin, leading to translocation of b-catenin into the nucleus.Further, TAF-derived CCL2 and its downstream transcription factor, Ets-1, are prerequisites for TAF-induced FGFR4 upregulation. Furthermore, FGFR4-associated pathways are shown to be preferentiallyactivated in colorectal tumor samples, and direct tumor metastasis in a mouse metastasis model. Our studyshows a pivotal role of FGFR4 in tumor–stroma interactions during colorectal cancer metastasis, andsuggests novel therapeutic opportunities for the treatment of colorectal cancer. Cancer Res; 73(19); 5926–35.�2013 AACR.

IntroductionColorectal cancer is one of the most frequent causes of

cancer-related death worldwide (1). Although significantadvances have been made in the treatment of metastaticcolorectal cancers, in particular the introduction of novelchemotherapies and targeted agents, approximately 60% ofpatients receiving curative resection will undergo local recur-rence or distant metastases, and 85% of patients will relapsewithin the first 2.5 years after surgery (2). Thus, a thoroughunderstanding of the molecular mechanisms of colorectalcancer metastasis is urgently required to facilitate early diag-nosis of individuals with a high risk of metastasis.

Fibroblast growth factor (FGF) ligands bind and activatecell-surface FGF receptors (FGFR) to mediate a broad spec-trum of biological processes in both embryonic and adult

organisms (3). FGFs exert biological effects as potent growthfactors for primary epithelial cells, which makes FGF signalingsusceptible to hijacking by cancer cells. Accumulating evidencehas linked carcinogenesis in a range of tissue types with thedysregulation of FGF signaling, including control of cancer cellproliferation and motility, and support of tumor angiogenesis(4, 5).

The tumor microenvironment provides the necessary sig-nals that not only support growth and survival of the primarytumor, but also facilitate its metastatic dissemination todistant organs (6). Tumor-associated fibroblasts (TAF) haverecently been linked to a number of prometastatic capabilities,including induction of epithelial-to-mesenchymal transition(EMT) and remodeling of the extracellular matrix (ECM; ref. 7).Although TAFs are considered to be among the key determi-nants in the metastatic progression of cancer (8), mechanismsunderlying the regulatory effect of TAFs on cancer cells arestill largely unknown. In this study, we have identified a pivotalrole of FGFR4 in the TAF-mediated signaling network, whichconfers an aggressive metastatic phenotype on colorectalcancer cells.

Materials and MethodsCoculture of TAFs and cancer cells

Colorectal tumor-associated fibroblasts were isolated fromprimary colorectal tumors fromfive individual patients (TAFs),or liver metastatic foci from five individual patients (TAF-Ms).Normal colonic fibroblasts were isolated from noncancerouscolonic tissues from five patients undergoing segmental colon-ic resection for injuries. To observe the fibroblast–cancer cellcommunication, we used a noncontact coculture system, asdescribed previously (9).

Authors' Affiliations: 1The State Key Laboratory of Biotherapy andCancerCenter, and 2Department of Hepatobiliary Pancreatic Surgery, WestChina Hospital, Sichuan University; 3The School of Biomedical Sciences,Chengdu Medical College; 4Department of Oncology, Sichuan ProvincialPeople's Hospital, Chengdu, People's Republic of China; 5Department ofBiochemistry and Molecular Biology, University of North Dakota, GrandForks, North Dakota; and 6MonashUniversity, Department of Biochemistryand Molecular Biology, Clayton, Victoria, Australia

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

R. Liu, J. Li, K. Xie, and T. Zhang contributed equally to this work.

Corresponding Author: Canhua Huang, The State Key Laboratory ofBiotherapy, West China Hospital, Sichuan University, No. 1 Keyuan 4 Rd.GaopengSt.High-TechZoneChengdu, People'sRepublic of China. Phone:86-13258370346; Fax: 86-28-85164060; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4718

�2013 American Association for Cancer Research.

CancerResearch

Cancer Res; 73(19) October 1, 20135926

on December 26, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4718

http://cancerres.aacrjournals.org/

Cell linesHuman colorectal cancer cell lines RKO, HCT116, LoVo,

and SW480 were purchased from and tested by the AmericanType Culture Collection. All the cell lines were used within 6months after receipt or resuscitation. All the cell lines weremaintained in Dulbecco's Modified Eagle Medium (DMEM;Invitrogen) containing 10% fetal calf serum (Hyclone), pen-icillin (107 U/L), and streptomycin (10 mg/L) at 37�C in ahumidified chamber containing 5% CO2.Further details of experimental procedures are described in

Supplementary Materials.

ResultsFGFR4 is upregulated in response to TAF-derived signalsIn a pilot study, we analyzed the regulatory role of TAFs

on colorectal cancer cells. Organ-specific fibroblasts wereisolated from primary colorectal cancer tissues (TAFs), livermetastatic foci (TAF-Ms), and noncancerous colonic tissues(NAF). Purity of these fibroblasts was confirmed by expres-sion of vimentin and loss of pan-Cytokeratin (Supplemen-tary Fig. S1A). As expected, coculture with TAFs or TAF-Ms,but not NAFs, induced a stem cell-like phenotype (Supple-mentary Fig. S1B and 1C), enhanced both migratory andinvasive capacity (Supplementary Fig. S2A–2C), and trig-gered significant EMT in colorectal cancer cells (Supple-mentary Fig. S2D and 2E). No apparent differences werefound in the ability of TAFs and TAF-Ms to activate colo-rectal cancer cells.To explore the molecular mechanisms underlying TAF-

induced migration and invasion of colorectal cancer cells, acomparative proteomic analysis of RKO cells solo-culturedor cocultured with TAFs, was conducted (SupplementaryFig. S3A). A total of 41 proteins were identified with alteredexpression (Supplementary Table S1). Notably, bioinformat-ics analysis of these changed proteins using Batch Querysoftware, a Web-based tool, showed a dramatic alteration inthe FGF pathway (P ¼ 0.0103; Fig. 1A). Furthermore, usingSTRING software, a significant group of regulated proteinswere also found to be associated with FGFR4 (Fig. 1B), whichwas shown to be elevated more than eightfold in RKO cellscocultured with TAFs compared with the solo-cultured cells(Supplementary Fig. S3B). Overexpression of FGFR4 wasfurther validated in a series of colorectal cancer cell linesof epithelium origin by immunoblot (Fig. 1C and Supple-mentary Fig. S4A) and/or immunocytofluorescence analysis(Fig. 1D). As RKO and HCT116 cells showed most significantalteration in FGFR4 expression, both of these cell lines wereselected for further validation. TAF-induced upregulation ofFGFR4 was accompanied by increased phosphorylation ofFGFR4 (Fig. 1C and Supplementary Fig. S4B), enhancedphosphorylation of FRS2, a direct substrate of FGFR4, andErk, the major downstream effector of FGFR4 (Fig. 1C andSupplementary Fig. S4B; ref. 10), and elevated cell sensitivityto FGF19, which uniquely binds to, and signals throughFGFR4 (Fig. 1E and Supplementary Fig. S4C; ref. 11). How-ever, compared with TAFs, NAFs were unable to induceactivation of either FGFR4 or its downstream signaling

pathways in colorectal cancer cell lines (Fig. 1C–E andSupplementary Fig. S4A–4C). Aware of previous reports thatboth FRS2 and Erk can be activated by either FGFR1, FGFR2,or FGFR3 (5), we further examined whether expressionof these FGFRs was altered in response to TAFs. A slightincrease in FGFR1 and a moderate decrease in FGFR2 werefound in both RKO and HCT116 cells after 48 hours ofcoculture (Fig. 2A and Supplementary Fig. S4D). In contrast,expression of FGFR3 was constantly low in RKO (Fig. 2A),and almost undetectable in HCT116 cells.

FGFR4 mediates a TAF-induced aggressive phenotype incolorectal cancer cells

To evaluate the functional role of individual FGFRs inmediating TAF-derived signals, specific siRNAs were intro-duced to repress the expression of each FGFR, respectively.Intracellular downstream cascades of FGFRs were estimatedbymeasuring the phosphorylation status of FRS2 and Erk (12).Only a slight decrease in phosphorylation of both Erk andFRS2 was found when the cells were treated with siFGFR1,siFGFR2, or siFGFR3, compared with the siNC control. How-ever, knockdown of FGFR4 substantially reduced TAF-induced phosphorylation of Erk or FRS2 (Fig. 2B and Supple-mentary Fig. S4E), suggesting that FGFR4, but not FGFR1,FGFR2, or FGFR3, was a potential surface sensor formediatingTAF-derived signals.

To determine the role of FGFR4 in the TAF-induced aggres-sive phenotype of colorectal cancer cells, the migratory andinvasive capabilities of colorectal cancer cells were comparedin the presence or absence of FGFR4. Repression of FGFR4, byeither siFGFR4 or a dominant negative form of FGFR4(FGFR4-DN), resulted in a remarkable decrease in motilityof colorectal cancer cells cocultured with TAFs, accompaniedwith reversion to amore compact epithelium-likemorphology(Fig. 2C–E and Supplementary Fig. S4F–4I). Correspondingly,loss of FGFR4 increased expression of the epithelial marker E-cadherin and reduced the levels of mesenchymal markersvimentin and Snail in TAF-treated cells (Fig. 2F and G).Together, these results indicated that FGFR4 is required forthe TAF-induced aggressive phenotype of colorectal cancercells.

It was of particular interest to examine the contributionof FGF signaling in the TAFs themselves. As shown in Fig.2H, treatment with SU5402, a pan FGFR inhibitor, markedlyreduced the phosphorylation of both Erk and FRS2. Fur-thermore, siRNA-mediated knockdown of either FGFR1,FGFR2, FGFR3, or FGFR4 substantially decreased the phos-phorylated forms of both Erk and FRS2 (Fig. 2I), suggestingthat each FGFR was involved in FGF signal transduction inTAFs.

FGFR4 mediates TAF-induced activation of Wntsignaling pathway via b-catenin Y142 phosphorylation

Cross-talk between FGF and Wnt signaling pathways isinvolved in a range of biological processes, including carci-nogenesis (5). To explore the potential signaling cascadesdownstream of FGFR4, of our particular interest, Wnt sig-naling was examined. As results, TCF/LEF transcription

FGFR4 Promotes Stroma-Induced EMT in Colorectal Cancer

www.aacrjournals.org Cancer Res; 73(19) October 1, 2013 5927




activity and expression of Cyclin D1, a Wnt target gene, wereboth enhanced in either RKO or SW480 cells cocultured withTAFs, compared with cells solo-cultured or cocultured withNAFs (Fig. 3A and Supplementary Fig. S5A). Furthermore,repression of Wnt signaling by siRNA targeting b-cateninmarkedly abrogated TAF-induced EMT in these two celllines (Fig. 3B and Supplementary Fig. S5B), suggesting thatWnt signaling is required for TAF-induced metastasis ofcolorectal cancer cells.

Next, we investigated whether FGFR4 plays a role in TAF-induced activation of Wnt signaling. Inhibition of FGFR4 byeither specific siRNA or FGFR4-DN markedly repressed TCF/LEF transcription activity and expression of Cyclin D1 in bothRKO and SW480 cells cocultured with TAFs (Fig. 3C andSupplementary Fig. S5C).

Furthermore, we set out to explore the molecular basis ofthe regulatory effect of FGFR4 on Wnt signaling. It is knownthat disruption of the APC/Axin/GSK-3b complex stabilizesb-catenin, which is a crucial step in the activation of Wntsignaling (13). However, the observation that FGFR4 repres-sion could inhibit TAF-induced activation of Wnt signalingin SW480 cells, which harbors a truncated APC (14), sug-gested other mechanisms that are likely to be involved. Ithas been reported that some receptor tyrosine kinases,including EGFR, are capable of directly phosphorylatingb-catenin at Y142, leading to b-catenin translocation frommembrane to nucleus (15). Indeed, coimmunoprecipitationdetected an FGFR4–b-catenin complex in SW480 cells, andsuch a complex was accumulated when the cells werecocultured with TAFs (Fig. 3D and E). Furthermore,

Figure 1. Coculture with TAFs induces FGFR4 upregulation. A, the regulated proteins were analyzed by the Web-based software Batch Query. The pathwaysshowing significant changes (P < 0.05) are presented with the corresponding P value at the top of the column. The FGF signaling pathway is highlighted.B, the protein–protein interaction network of identified proteins was analyzed using String software. A significant group of regulated proteins werefound to be associated with FGFR4. C, RKO cells were cocultured with TAFs or NAFs for 24 or 48 hours. Expression of FGFR4 and phosphorylation ofFRS2 (Y436) and Erk (Thr202/Tyr204) were examined by immunoblot. Phosphorylation of FGFR4 was examined by immunoprecipitation using FGFR4antibody followed by immunoblot using an antibody specific for p-Tyrosine. D, subcellular location of FGFR4 was examined by immunocytochemistry. Scalebar, 10 mm. E, RKO cells were cocultured with TAFs for indicated time. Cells were washed with fresh serum-free DMEM medium (three times) and thentreated with 200 ng/mL FGF19 and 1 ng/mL heparin for 15 minutes. Phosphorylation of FRS2 (Y436) was examined by immunoblot. All data wererepresentative of at least three independent experiments.

Liu et al.

Cancer Res; 73(19) October 1, 2013 Cancer Research5928




overexpression of FGFR4 resulted in significant b-cateninphosphorylation at Y142 and accumulation of nuclearb-catenin in both RKO and SW480 cells, which could bereversed by treatment with SU5402 (Fig. 3F and Supple-mentary Fig. S5D; ref. 16). Furthermore, coculture with TAFsmarkedly enhanced b-catenin Y142 phosphorylation, whichcould be largely abolished on FGFR4 suppression (Fig. 3Gand Fig. S5E). These results suggested that FGFR4 mediatedTAF-induced activation of Wnt signaling pathway viab-catenin Y142 phosphorylation.

TAF-derived CCL2 is required for TAF-induced FGFR4upregulationTo explore the mechanisms responsible for TAF-induced

FGFR4 upregulation, bioinformatics analysis was employedto highlight the key regulatory factors on the basis of theproteomic profiling data. Intriguingly, a cluster of altered

proteins were found to be associated with the CCL2–CCR2axis (Fig. 4A). CCL2 is highly expressed in TAFs (17), andits downstream transcription factor, Ets-1, can initiateFGFR4 expression by direct binding to FGFR4 promoter(18, 19). Therefore, we investigated whether CCL2 wasrequired for TAF-induced FGFR4 upregulation. As shownin Fig. 4B, TAFs showed a markedly elevated expression ofCCL2, compared with NAFs. Notably, coculture with colo-rectal cancer cells further enhanced the CCL2 production inTAFs. Furthermore, addition of CCL2 in the culture induceda dramatic upregulation of FGFR4 in colorectal cancer celllines (Fig. 4C). Inhibition of CCL2 signaling via either aneutralizing antibody against CCL2 or siRNA targetingCCR2, the receptor for CCL2, markedly attenuated TAF-induced FGFR4 expression and its promoter activity (Fig.4D and Supplementary Fig. S5F), as well as the subsequentphosphorylation of b-catenin Y142 (Fig. 4E). In addition,

Figure 2. Upregulation of FGFR4 is required for TAF-induced aggressive phenotype of colorectal cancer cells. A, RKOcellswere coculturedwith TAFs for 24 or48 hours. Expression of FGFR1, FGFR2, and FGFR3 was examined by immunoblot. B, RKO cells were transfected with siFGFR1, siFGFR2, siFGFR3,siFGFR4, or siNC, respectively, and then cocultured with TAFs for 48 hours. Phosphorylation of FRS2 (Y436) and Erk (Thr202/Tyr204) were examined byimmunoblot. C, RKO cells were transfected with mock vector, FGFR4-DN, siNC, or siFGFR4, respectively, and then cocultured with TAFs for 48 hours.Cell migration was examined by wound healing assay. Scale bar, 500 mm. D, cell invasion was examined by Matrigel assay. Scale bar, 250 mm. E,representative phase-contrast images of cell morphology of RKO cells. Scale bar, 30 mm. F, expression of Snail, E-cadherin, and vimentin was examined byimmunocytochemistry. Scale bar, 20 mm. G, expression of Snail, E-cadherin, and vimentin was examined by immunoblot. H, TAFs were treated withSU5402 (10 mmol/L) for 24 hours. Phosphorylation of FRS2 (Y436) and Erk (Thr202/Tyr204) was examined by immunoblot. I, TAFs were transfected withsiFGFR1, siFGFR2, siFGFR3, siFGFR4, or siNC, respectively. Phosphorylation of FRS2 (Y436) andErk (Thr202/Tyr204)was examinedby immunoblot. All datawere representative of at least three independent experiments.






we investigated whether Ets-1 was involved in FGFR4upregulation. As shown, siRNA-mediated suppression ofEts-1 dramatically abolished TAF-induced FGFR4 over-expression (Fig. 4F and Supplementary Fig. S5F). Theseresults suggested that TAF-derived CCL2 was required forTAF-induced FGFR4 upregulation in an Ets-1-dependentmanner.

TAF-derived FGF19 is required for TAF-inducedFGFR4/Wnt activation

It has been reported that FGF19 is a specific FGFR4 acti-vator that has been implicated in colorectal cancer develop-ment (5). Therefore, we next examined the expression ofFGF19 in TAFs and NAFs. As shown in Fig. 4G, expression ofFGF19 was enhanced in TAFs compared with NAFs, revealedby both immunoblot and ELISA. Furthermore, inhibition ofFGF19 by a neutralizing antibody decreased TAF-inducedphosphorylation of both FGFR4 and b-catenin (Fig. 4H), sugg-esting that FGF19 played a role in TAF-induced FGFR4/Wntactivation.

FGFR4 governs colorectal cancer cell metastasis in vivoTo investigate whether FGFR4 accelerated colorectal can-

cer metastasis in vivo, we generated a mouse liver metastaticmodel by injecting mixed TAFs and colorectal cancer cells inspleen (20). Histopathologic analysis confirmed that the livermetastatic nodules were composed of cancer cells, but notfibroblasts (Supplementary Fig. S5G). As shown, inhibitionof FGFR4-associated pathways markedly decreased thenumber of liver metastatic nodules (TAFs þ RKO-shNC vs.TAFs þ RKO-shFGFR4, P ¼ 0.0002; TAFs þ RKO-shNC vs.TAFs þ RKO þ shb-catenin, P < 0.0001; TAFs þ RKO-shNCVS. TAFsþ RKO-shCCR2, P¼ 0.001; n¼ 8; Fig. 5A and B) andprolonged the survival time of tumor-bearing mice (TAFs þRKO-shNC vs. TAFs þ RKO-shFGFR4, P ¼ 0.0018; TAFs þRKO-shNC vs. TAFs þ RKO þ shb-catenin, P ¼ 0.0024; TAFsþ RKO-shNC VS. TAFs þ RKO-shCCR2, P ¼ 0.0062; n ¼8; Fig. 5C). These studies suggested that FGFR4 couldfunction as a crucial signaling node in tumor–stroma inter-actions by promoting a TAF-induced CCL2/FGFR4/Wntsignaling pathway.

Figure 3. FGFR4 mediates TAF-induced EMT via activating the Wnt signaling pathway. A, RKO cells were cocultured with TAFs or NAFs for 24 or 48hours. TCF/LEF transcription activity was examined by Top/Fop flash. Expression of Cyclin D1 was examined by immunoblot. B, RKO cells weretransfected with sib-catenin or siNC, respectively, and then cocultured with TAFs for 48 hours. Expression of E-cadherin and vimentin was examined byimmunoblot. C, RKO cells were transfected with mock vector, FGFR4-DN, siNC, or siFGFR4, respectively, and cocultured with TAFs for 48 hours. TCF/LEF transcription activity was examined by Top/Fop flash. Cyclin D1 expression was examined by immunoblot. D, SW480 cells were cotransfectedwith b-catenin-Flag and FGFR4-Myc. Interaction between b-catenin and FGFR4 was determined by coimmunoprecipitation. E, SW480 cells werecocultured with TAFs for 48 hours. Interaction between endogenous b-catenin and FGFR4 was determined by coimmunoprecipitation. F, RKO cellswere transfected with mock vector or a plasmid coding full-length of FGFR4, and then treated with or without 10 mmol/L SU5402. Phosphorylation ofb-catenin (Y142) was examined by immunoblot using whole-cell lysate. The level of nuclear b-catenin was examined by immunoblot by usingnuclear extracts. Histone H3 was used as internal control. H3, Histone H3. G, RKO cells were transfected with mock vector, FGFR4-DN, siNC, orsiFGFR4, respectively, and cocultured with TAFs for 48 hours. Phosphorylation of b-catenin (Y142) was examined by immunoblot. All data wererepresentative of at least three independent experiments. ��, P < 0.001; ��, P < 0.01; �, P < 0.05.

Liu et al.





FGFR4 and its associated pathways are preferentiallyactivated in colorectal cancerTo verify whether our findings with cancer cell lines

and animal models were of clinical relevance, we evaluated

the correlation between FGFR4 expression and clinicopath-ologic parameters in clinical samples. Clinicopathologicinformation for the clinical samples is summarized in Supp-lementary Table S2. FGFR4 immunoreactivity was more

Figure 4. TAF-derived CCL2 is required for TAF-induced FGFR4 up-regulation. A, the protein–protein interaction network of the regulated proteins identifiedby proteomic profiling was analyzed by String software. A significant group of proteins were found to be involved in CCL2-associated pathways. B, TAFswere cocultured with RKO cells for 48 hours (cocultured TAFs). Expression of CCL2 in untreated NAFs, untreated TAFs, and cocultured TAFs was examinedby immunoblot. The untreated NAFs, untreated TAFs, and cocultured TAFs were incubated with serum-free medium for 24 hours. The CCL2 concentrationin the supernatant was determined by ELISA. C, RKO and SW480 cells were treated with CCL2 (50 ng/mL) for 12 hours. Expression of FGFR4 wasexamined by immunoblot. D, RKO cells were transfected with siCCR2 or siNC, and then cocultured with TAFs for 48 hours in the presence or absence ofneutralizing antibodies against CCL2 (40 mg/mL). FGFR4 promoter activity was examined by luciferase assay; expression of FGFR4 was determined byimmunoblot. E, RKO cells were cocultured with TAFs for 48 hours in presence of neutralizing antibodies against CCL2 (40 mg/mL). TCF/LEF transcriptionactivity was examined by Top/Fop flash. Phosphorylation of b-catenin (Y142) was examined by immunoblot. F, RKO cells were transfected withsiEts-1 or siNC, and then cocultured with TAFs for 48 hours. FGFR4 promoter activity was examined by luciferase assay, and expression of FGFR4was determined by immunoblot. G, expression of FGF19 in TAFs and NAFs was examined by immunoblot. TAFs or NAFs were incubated with serum-freemedium for 24 hours, and the concentration of FGF19 in the supernatant was determined by ELISA. H, RKO cells were cocultured with TAFs for 48 hoursin the presence or absence of neutralizing antibodies against FGF19 (30 mg/mL). Phosphorylation of FGFR4 was examined by immunoprecipitationusing FGFR4 antibody followed by immunoblot using an antibody specific for p-Tyrosine. Phosphorylation of b-catenin (Y142) was examined by immunoblot.All data were representative of at least three independent experiments. ��, P < 0.001; ��, P < 0.01; �, P < 0.05.






intense in cancer tissues compared with noncancerousadjacent tissues (P < 0.0001; Fig. 6A). Furthermore, a highlevel of FGFR4 expression was more likely to be associatedwith lymph node metastasis (Fig. 6B) and poor outcome(Fig. 6C). In addition, we analyzed the correlation betweenthe distribution of fibroblasts and the expression of FGFR4.As shown in Fig. 6D, the tumor cells adjoining the stromalfibroblasts displayed strong FGFR4 immunoreactivity,whereas those tumor cells residing in the central part oftumor region expressed a relatively low level of FGFR4,which suggested the occurrence of a paracrine effect ofTAFs on FGFR4 expression. Furthermore, immunostainingusing consecutive sections revealed positive correlationsbetween the expression of FGFR4, CCL2, and phospho-rylated b-catenin (Y142; Fig. 6E and F). These data sug-gested that FGFR4 and its associated pathways were pref-erably activated in colorectal cancer, and were stronglyrelated to a high risk of tumor metastasis and poor patientoutcome.

DiscussionMultiple interactions between tumor cells and TAFs gov-

ern tumor progression, promoting the escape of tumor cellsfrom immune-surveillance or hormonal control, and achiev-ing the invasive phenotype required for translocation to a

distant organ (4). However, the key molecular regulator andsignaling pathways involved in the cross-talk between tumorcells and TAFs remain largely unknown. In this study, weshow that FGFR4 is a potential regulator of this process.FGFR4, but not other FGFRs, was markedly upregulated incolorectal cancer cells cocultured with TAFs. Inhibition ofFGFR4 attenuated TAF-induced intracellular signaling cas-cades, including phosphorylation of Erk and FRS2. Further-more, in both in vitro and in vivo models, TAF-inducedmigration and invasion of colorectal cancer cells could bereversed upon suppression of FGFR4. These data suggestthat exposure to TAFs prime colorectal cancer cells with aFGFR4-rich surface, which initiates intracellular signalingleading to an aggressive phenotype.

We found that the Wnt signaling pathway was required forthe TAF-induced EMT in colorectal cancer cells. More impor-tantly, we found in the SW480 cell line, which possessed adefective APC, that FGFR4 mediated TAF-induced activationof Wnt signaling pathway via b-catenin Y142 phosphorylation.Gene mutations of the APC tumor suppressor, which result indisruption of the APC/Axin/GSK-3b complex, are among themost frequent oncogenic molecular abnormalities observed incolorectal cancer (21). Therefore, the present data mightsuggest that TAF-induced and FGFR4-mediated metastasis ofcolorectal cancer cells is, at least partially, APC/Axin/GSK-3bcomplex-independent.

CCL2/CCR2 chemokine signaling has been previouslyshown to be involved in cancer metastasis (22). TAFs arean important reservoir of CCL2, which is released into thetumor microenvironment (17). Our data show that expres-sion of CCL2 was elevated in TAFs compared with NAFs.Furthermore, treating colorectal cancer cells with CCL2triggered FGFR4 expression. In contrast, inhibition of theCCL2–CCR2 axis attenuated TAF-induced FGFR4 upregula-tion and subsequent activation of Wnt signaling, as well asliver metastasis of colorectal cancer cells in a mouse model.In addition, Ets-1, a downstream transcription factor ofCCL2, seemed to be involved in this process: siRNA-medi-ated knockdown of Ets-1 markedly suppressed TAF-inducedFGFR4 upregulation. These results suggest that the CCL2signaling pathway plays crucial roles in early steps of TAF-induced colorectal cancer metastasis by mediating FGFR4upregulation.

Analysis of FGFR4 expression in colorectal tumor tissuessuggested that the mechanistic results obtained from invitro and in vivo experiments were of clinical relevance.We show that expression of FGFR4 directly correlatedwith lymph node metastasis and metastasis-free survivalrate. Furthermore, expression of FGFR4 was preferentiallyexpressed in the tumor cells adjacent to the fibroblasts, andwas positively correlated with expression of CCL2 or phos-phorylated b-catenin (Y142), respectively. These data sug-gest that FGFR4 and its associated pathways are potentialbiomarkers that could be used to predict the risk ofmetastasis and guide further diagnostic and therapeuticdecisions.

Efforts have been made to understand the inter-commu-nication between tumor and stroma cells, but few rational

Figure 5. FGFR4 governs colorectal cancer cell metastasis in vivo. A,representative images of liver metastasis. Normal RKO cells or RKO cellsthat were stably transfected with shNC, shFGFR4, shb-catenin, orshCCR2 were injected in the spleens of nude mice with or without TAFs.B, tumor cell metastasis was examined by counting the metastaticnodules in mouse liver. C, mice survival curves were plotted by theKaplan–Meier method, and the log-rank test was used to determine thesignificant difference among groups. All data were representative of atleast three independent experiments. ��, P < 0.001; ��, P < 0.01.

Liu et al.





signaling pathways have been identified thus far. The presentdata suggest that FGFR4 is a key regulator in TAF-inducedcolorectal cancer metastasis (Fig. 7). Briefly, TAF-derivedCCL2 induces expression of FGFR4 in an Ets-1-dependentmanner. Overexpression of FGFR4 phosphorylates membra-nous b-catenin at Y142, which results in its translocationinto the nucleus. Increased nuclear b-catenin initiatesexpression of Snail and subsequently represses expressionof E-cadherin, leading to induction of EMT in colorectalcancer cells.

Many other signaling pathways were also found to be alteredin colorectal cancer cells exposed to TAFs, including the plate-let-derived growth factor (PDGF) signaling pathway, as shownby our proteomic profiling and bioinformatics analysis (Fig. 1A).The FGFRs are phylogenetically closely related to the platelet-derived growth factor receptors (PDGFR). Both FGF and PDGFsignaling pathways were found to be implicated in colorectalcancer development (5, 23). Therefore, further studies will beconducted to determine other potential interactions of thesepathways in stroma-induced EMT in colorectal cancer cells.

Figure 6. FGFR4 and its associated pathway is preferably activated in colorectal cancer. A, representative images of FGFR4 immunostaining incolorectal tumor tissues and adjacent noncancerous tissues. Scale bar, 500 mm. B, expression of FGFR4 in the primary tumors without (N0) or with(N1/N2) lymph node metastasis was analyzed. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. C, metastasis-free survival curvesbased on the FGFR4 expression in primary tumors. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. D, representative images oftumor cells adjacent to TAFs. Arrows, the tumor cells adjacent to TAFs with high FGFR4 expression; asterisks, the tumor cells far from TAFs withlow FGFR4 expression. Scale bar, 75 mm. E, representative images of concurrent expression of FGFR4 with phosphorylated b-catenin (Y142)or CCL2, respectively, in consecutive sections of colorectal cancer tissues. Scale bar, 75 mm. F, linear regression analyses of immunostainingintensity between FGFR4 and CCL2 or phosphorylated b-catenin (Y142), respectively. All data were representative of at least three independentexperiments.






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

Authors' ContributionsConception and design: C. Huang, Y. WeiDevelopment of methodology: J. Li, K. Xie, T. Zhang, Y. Lei, Y. Chen, K. Huang,K. Wang, H. Wu, C. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Liu, T. Zhang, Y. Lei, Y. Chen, L. Zhang, K. Huang, K.WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Zhang, Y. Lei, L. Zhang, K. Huang, K.Wang, H.Wu,C. HuangWriting, review, and/or revision of the manuscript: R. Liu, Y. Lei, E.C. Nice,C. Huang

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Li, K. Xie, Y. Lei, H. Wu, M. WuStudy supervision: E.C. Nice, C. Huang, Y. Wei

Grant SupportThis work was financially supported by grants from the National 973 Basic

Research Program of China (2013CB911300, 2011CB910703, and 2012CB518900),the National Science and Technology Major Project (2011ZX09302-001-01 and2012ZX09501001-003), and the ChineseNSFC (81225015, 81072022, and 81172173).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 31, 2012; revised April 23, 2013; accepted July 5, 2013;published OnlineFirst August 13, 2013.

References1. Kim J, Takeuchi H, Lam ST, Turner RR, Wang H-J, Kuo C, et al.

Chemokine receptor CXCR4 expression in colorectal cancer patientsincreases the risk for recurrence and for poor survival. J Clin Oncol2005;23:2744–53.

2. Davies RJ, Miller R, Coleman N. Colorectal cancer screening: pro-spects for molecular stool analysis. Nat Rev Cancer 2005;5:199–209.

3. Neugebauer JM, Amack JD, Peterson AG, Bisgrove BW, Yost HJ. FGFsignalling during embryo development regulates cilia length in diverseepithelia. Nature 2009;458:651–4.

4. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.Cell 2011;144:646–74.

5. Turner N, Grose R. Fibroblast growth factor signalling: from develop-ment to cancer. Nat Rev Cancer 2010;10:116–29.

6. Robinson BD, Sica GL, Liu Y-F, Rohan TE, Gertler FB, Condeelis JS,et al. Tumor microenvironment of metastasis in human breast carci-noma: a potential prognostic marker linked to hematogenous dissem-ination. Clin Cancer Res 2009;15:2433–41.

7. HwangRF,Moore T, ArumugamT,Ramachandran V, AmosKD,RiveraA, et al. Cancer-associated stromal fibroblasts promote pancreatictumor progression. Cancer Res 2008;68:918–26.

8. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer 2006;6:392–401.

Figure 7. Schematic illustrating the potential role of FGFR4 in TAF-induced metastasis in colorectal cancer. TAF-derived CCL2 upregulates FGFR4 inbystander colorectal cancer cells in an Ets-1-dependent manner. Subsequently, FGFR4 phosphorylates membranous b-catenin at Y142, resulting in itsdissociation with a-catenin and translocation into nucleus. Nuclear b-catenin enhances expression of Snail and represses expression of E-cadherin, leadingto induction of EMT in colorectal cancer cells.

Liu et al.





9. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki J. Leukemia cell toendothelial cell communication via exosomal miRNAs. Oncogene2012;32:2747–55.

10. Beenken A, Mohammadi M. The FGF family: biology, pathophysiologyand therapy. Nat Rev Drug Discov 2009;8:235–53.

11. XieM-H,Holcomb I, Deuel B, DowdP,HuangA, Vagts A, et al. FGF-19,a novel fibroblast growth factor with unique specificity for FGFR4.Cytokine 1999;11:729–35.

12. Hinsby AM, Olsen JV, Bennett KL, Mann M. Signaling initiated byoverexpression of the fibroblast growth factor receptor-1 investigatedby mass spectrometry. Mol Cell Proteomics 2003;2:29–36.

13. MacDonald BT, Tamai K, He X. Wnt/b-catenin signaling: components,mechanisms, and diseases. Dev Cell 2009;17:9–26.

14. Yoshie T, Nishiumi S, Izumi Y, Sakai A, Inoue J, Azuma T, et al.Regulation of the metabolite profile by an APC gene mutation incolorectal cancer. Cancer Sci 2012;103:1010–21.

15. Krejci P, Aklian A, Kaucka M, Sevcikova E, Prochazkova J, Masek JK,et al. Receptor tyrosine kinases activate canonical WNT/b-cateninsignaling via MAP kinase/LRP6 pathway and direct b-catenin phos-phorylation. PLoS ONE 2012;7:e35826.

16. Fischer H, Taylor N, Allerstorfer S, Grusch M, Sonvilla G, HolzmannK, et al. Fibroblast growth factor receptor-mediated signals con-tribute to the malignant phenotype of non-small cell lung cancercells: therapeutic implications and synergism with epidermal

growth factor receptor inhibition. Mol Cancer Ther 2008;7:3408–19.

17. Tsuyada A, Chow A, Wu J, Somlo G, Chu P, Loera S, et al. CCL2mediates cross-talk between cancer cells and stromal fibroblasts thatregulates breast cancer stem cells. Cancer Res 2012;72:2768–79.

18. Stamatovic SM, Keep RF, Mostarica-Stojkovic M, Andjelkovic AV.CCL2 regulates angiogenesis via activation of Ets-1 transcriptionfactor. J Immunol 2006;177:2651–61.

19. Yu S, Zheng L, Asa SL, Ezzat S. Fibroblast growth factor receptor 4(FGFR4) mediates signaling to the prolactin but not the FGFR4 pro-moter. Am J Physiol Endocrinol Metab 2002;283:E490–E5.

20. Zhao L, Wang H, Liu C, Liu Y, Wang X, Wang S, et al. Promotion ofcolorectal cancer growth and metastasis by the LIM and SH3 domainprotein 1. Gut 2010;59:1226–35.

21. Roh H, Green DW, Boswell CB, Pippin JA, Drebin JA. Suppression ofb-catenin inhibits the neoplastic growth of APC-mutant colon cancercells. Cancer Res 2001;61:6563–8.

22. Qian B-Z, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, et al. CCL2recruits inflammatory monocytes to facilitate breast-tumour metasta-sis. Nature 2011;475:222–5.

23. McCarty MF, Somcio RJ, Stoeltzing O, Wey J, Fan F, Liu W, et al.Overexpression of PDGF-BB decreases colorectal and pancreaticcancer growth by increasing tumor pericyte content. J Clin Invest2007;117:2114–22.






2013;73:5926-5935. Published OnlineFirst August 13, 2013.Cancer Res Rui Liu, Jingyi Li, Ke Xie, et al. Transition in Colorectal CancerFGFR4 Promotes Stroma-Induced Epithelial-to-Mesenchymal

Updated version

10.1158/0008-5472.CAN-12-4718doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2013/08/13/0008-5472.CAN-12-4718.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/73/19/5926.full#ref-list-1

This article cites 23 articles, 9 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/73/19/5926.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/73/19/5926To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-12-4718

http://cancerres.aacrjournals.org/content/suppl/2013/08/13/0008-5472.CAN-12-4718.DC1

http://cancerres.aacrjournals.org/content/73/19/5926.full#ref-list-1

http://cancerres.aacrjournals.org/content/73/19/5926.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/73/19/5926


Documents

FGFR4 Promotes Stroma-Induced Epithelial-to- Mesenchymal ...Molecular and Cellular Pathobiology FGFR4 Promotes Stroma-Induced Epithelial-to-Mesenchymal Transition in Colorectal Cancer