zic5 drives melanoma aggressiveness by pdgfd-mediated ... · because many neural crest-related...

Molecular and Cellular Pathobiology

ZIC5 Drives Melanoma Aggressiveness byPDGFD-Mediated Activation of FAK and STAT3Reiko Satow1,2, Tomomi Nakamura1, Chiaki Kato1, Miku Endo1, Mana Tamura1,Ryosuke Batori1, Shiori Tomura1, Yumi Murayama1, and Kiyoko Fukami1,2

Abstract

Insights into mechanisms of drug resistance could extendthe efficacy of cancer therapy. To probe mechanisms in mel-anoma, we performed siRNA screening of genes that mediatethe development of neural crest cells, from which melanocytesare derived. Here, we report the identification of ZIC5 as amediator of melanoma drug resistance. ZIC5 is a transcrip-tional suppressor of E-cadherin expressed highly in humanmelanoma. ZIC5 enhanced melanoma cell proliferation, sur-vival, drug resistance, in vivo growth and metastasis. Micro-array analysis revealed that ZIC5 downstream signaling

included PDGFD and FAK activation, which contributes todrug resistance by enhancing STAT3 activation. Silencing ofZIC5 or PDGFD enhanced the apoptotic effects of BRAFinhibition and blocked survival of melanoma cells resistantto BRAF inhibitors. Furthermore, inhibition of FAK or STAT3suppressed expression of ZIC5, which was positively regulatedby PDGFD, FAK, and STAT3 in a positive feedback loop. Takentogether, our results identify ZIC5 and PDGFD as candidatetherapeutic targets to overcome drug resistance in melanoma.Cancer Res; 77(2); 366–77. �2016 AACR.

IntroductionMetastatic melanoma is a highly aggressive disease character-

ized by high mortality and poor chemotherapeutic response.Despite the recent conception of novel therapeutic approaches,such as molecular targeted therapies and immunotherapies (1),the prognosis remains very poor. Approximately 50%melanomasharbor an activating mutation in the BRAF gene, leading toaberrant MEK–ERK signaling pathway activation that promotescell growth and survival. BRAF kinase inhibitors such as vemur-afenib have improved the rates of overall and progression-freesurvival in melanoma patients with BRAF mutations (2). How-ever, tumor recurrence frequently occurs as a result of acquiredresistance to BRAF inhibitors; thus, the identification of noveldrug targets based on molecular mechanisms of malignant mel-anoma is urgently required.

The epithelial–mesenchymal transition (EMT) is a processwhereby cancer cells acquire mesenchymal phenotypes and sub-sequently lose their epithelial characteristics, resulting inenhanced malignancy, cancer stem cell-like properties, and ther-apeutic resistance. EMT is regulated by several transcription fac-tors, including SNAIL, SLUG, TWIST, and ZEB1, which repressE-cadherin expression (3). A previous microarray analysis dem-onstrated that the EMT transcriptional program is also involved in

melanoma metastasis (4). Importantly, constitutive BRAF activa-tion has been shown to promote the expression of ZEB1 andTWIST to enhance the malignant mesenchymal phenotype ofmelanoma cells (5). Moreover, low E-cadherin expression inmelanoma is associated with diminished relapse-free survival(6). These lines of evidence indicate that the EMT transcriptionalprogram is critical for melanoma progression, of which E-cad-herin suppression is an important hallmark.

Because many neural crest-related genes participate in EMTprogram in cancer (3, 7), we speculated that many unidentifiedfactors control the EMT transcriptional program in both devel-opment and tumor progression. In this study, we performedsiRNA screening of genes essential for neural crest developmentto identify the genes involved in malignant melanoma progres-sion by monitoring E-cadherin expression, leading to the iden-tification of ZIC5. We found that ZIC5 is a transcriptional sup-pressor of E-cadherin and is highly expressed in human melano-ma. Besides the E-cadherin–suppressive function, ZIC5 enhancesmelanoma proliferation, survival and drug resistance by activat-ing FAK and STAT3 through PDGFD induction.

Materials and MethodsSupplementary Materials and Methods for plasmids, siRNA,

shRNA, transfections, Western blot analysis, reagents, luciferasereporter assays, chromatin immunoprecipitation, immunocyto-chemistry, immunohistochemistry, scratch assays, cell prolifera-tion assays, cell cycle analysis, quantitative real-time PCR, migra-tion and invasion assays, animal experiments, microarray anal-ysis, apoptosis assays, and generation of vemurafenib-resistantcell lines have been provided.

Cell cultureSK-MEL-28, COLO829, HT144, and A375melanoma cell lines

were obtained from the ATCC (acquired between 2011 and2012). These cell lines were re-validated by short tandem repeat

1Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and LifeSciences, Hachioji-shi, Tokyo, Japan. 2AMED-CREST, Japan Agency for MedicalResearch and Development, Hachioji-shi, Tokyo, Japan.

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

Corresponding Author: Kiyoko Fukami, Tokyo University of Pharmacy and LifeSciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan. Phone/Fax:81-426-76-7214; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-0991

�2016 American Association for Cancer Research.

CancerResearch

Cancer Res; 77(2) January 15, 2017366

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Published OnlineFirst September 26, 2016; DOI: 10.1158/0008-5472.CAN-16-0991

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profiling in 2016 (Promega). Cells were maintained at 37�C witha 5% CO2 humidified atmosphere in RPMI-1640 medium (Invi-trogen) supplementedwith 10%FBS.Normal humanmelanocytecells were obtained from KURABO, and processed according tothe manufacturer's instructions.

siRNA library screenThe siRNA screening library consisted of 26 selected genes

essential for neural crest development (2 siRNAs per gene, Sup-plementary Table S1). These siRNAs were transfected into SK-MEL-28 and COLO829 BRAF V600E-carrying melanoma celllines, which were harvested for analysis 3 days posttransfection.Transfected cells were fixed in 4% formaldehyde at room tem-perature for 15 minutes, permeabilized with 0.1% Triton X-100,blocked with 5% bovine serum albumin (Wako), and thenincubated with Alexa Fluor 488-conjugated anti-E-cadherin anti-body (BD Biosciences) at room temperature for 2 hours. Nucleiwere counterstained with Hoechst 33342 (Dojindo). Imageswere acquired using an IN Cell Analyzer 2000 (GE Healthcare).E-Cadherin expression was quantified using In Cell AnalyzerWorkstation 3.7 software (GE Healthcare) by normalizing theE-cadherin staining intensity to the total nuclear intensity.

RNA isolation, cDNA synthesis, and quantitative real-time PCRTotal RNA isolation, cDNA synthesis, and quantitative real-

time PCR (qRT-PCR) were performed as previously described (8).The primer sequences were provided in Supplementary Materialsand Methods.

Western blot analysisWestern blotting was performed as described previously (9)

with some modifications. Representative Western blots from twoor three independent experiments are shown. Antibodies usedwere listed in Supplementary Materials and Methods.

Statistical analysisStatistical analyses were performed using the R statistical soft-

ware package (v. 3.0.1). P < 0.05 was considered statisticallysignificant. Data are represented as themean� SD (n¼ 3), unlessotherwise stated.

ResultsIdentification of ZIC5 as a suppressor of E-cadherin expressionin melanoma

The EMT program is critical for both neural crest developmentandmelanoma progression. To find novel pathways that regulatemelanoma progression, we designed a strategy to identify genesinvolved in melanoma progression by focusing on the mecha-nistic similarities between embryonic development and tumorprogression. We constructed an siRNA library consisting of 26genes (2 siRNAs per gene) with essential roles in neural crestdevelopment, but whose precise mechanisms in melanoma havenot been fully elucidated (Supplementary Table S1). SK-MEL-28and COLO829melanoma cells harboring BRAF V600E-activatingmutations were transfected with these siRNAs, and E-cadherin(CDH1) expression was analyzed by immunocytochemistry. Theknockdownof six genes,BMPER, SOX10, SULF1, SULF2, TES, andZIC5, resulted in CDH1 expression greater than 1.5-fold higher

than that observed in the negative control-transfected counter-parts in both cell lines (Fig. 1A).

ZIC5 is a member of the ZIC protein family, which consists ofZIC1 toZIC5, all ofwhich containC2H2-type zincfinger domains(10, 11); however, the precise functions of ZIC5 remainunknown. To characterize the role of ZIC5 in tumor progression,we first examined its CDH1-suppressive function in melanoma.We established an SK-MEL-28 cell line with stable ZIC5 over-expression (FLAG-ZIC5 cells) as well as control cell lines(pFLAG cells). Western blotting and qRT-PCR analysis demon-strated reduced CDH1 expression in FLAG-ZIC5 cells (Fig. 1Band C). This effect was reversed by subsequent siRNA-mediatedZIC5 knockdown, indicating that ZIC5 functions as a bona fideregulator of CDH1 expression (Fig. 1C). Knockdown of endog-enous ZIC5 using siRNA upregulated CDH1 expression in SK-MEL-28 but CDH1 expression was not detected in A375 mel-anoma cells (Fig. 1D).

Next, we performed chromatin immunoprecipation and lucif-erase assays to determine whether ZIC5modulates CDH1 expres-sion directly by binding to and regulating the CDH1 promoter.TheCDH1 promoter region was prominently detected only in theZIC5-HA immunoprecipitated sample,whereas anegative controlregion located downstreamof theCDH1 genewas not enriched inany sample (Fig. 1E). Luciferase assays using the 407 bp (–283 toþ124) CDH1 promoter (pCDH1) revealed that pCDH1 activitywas enhanced approximately 2- to 3-fold by ZIC5 knockdown(Fig. 1F). To determine the region in pCDH1 essential for regu-lation by ZIC5, the activity of pCDH1 deletion mutants wasassessed by luciferase assays (Fig. 1G). Notably, ZIC5 knockdownresulted in a significant increase in pCDH1 (–283 to þ124) andpCDH1 (–104 to þ124) activity, but not pCDH1 (–71 to þ124)andpCDH1 (–33 toþ124) activity (Fig. 1G), suggesting that ZIC5binds the pCDH1 at 71 to 104 bp upstream of the transcriptionstart site. To confirm the importance of the zinc finger domain ofZIC5, a construct lacking the zinc finger domain was generated asshown in Fig. 1H.Wild-type ZIC5protein localized to the nucleus,whereas the zinc finger domain-deleted form of ZIC5 mainlylocalized to the cytosol (Fig. 1H). In addition, ZIC5 overexpres-sion decreased pCDH1 activity, whereas zinc finger domain-deleted ZIC5 overexpression had no such effect (Fig. 1I). Theseresults suggest that ZIC5 functions as a transcription factor thatrepresses E-cadherin promoter activity via its zinc finger domains.

ZIC5 expression is elevated in melanomaRNA sequence analysis of 44 different normal human tissues in

the Human Protein Atlas online database (http://www.proteina-tlas.org) revealed that ZIC5 gene expression occurs only in thetestis and cerebral cortex. Because the expression of ZIC5 inmelanoma remains unknown, we first performed immunohis-tochemistry with human melanoma tissue arrays containing 18benign nevi, 56 melanoma, and 26 metastatic melanoma tissues.Although faint staining was detected in benign nevi, ZIC5 expres-sion was detected strongly in the melanoma tissues (Fig. 2Aand B). In addition, ZIC5 protein expression was elevated instages II–IV compared with stage I melanoma (Fig. 2B, right). Wenext examined ZIC5 protein expression in melanocytes andmelanoma cell lines harboring BRAFV600E-activatingmutations.Western blotting of A375, HT144, COLO829, and SK-MEL-28melanoma cells and normal humanmelanocytes (NHM) showedincreased ZIC5 expression in all melanoma cell lines whencompared with NHM (Fig. 2C).

ZIC5 Promotes Melanoma Malignancy via FAK/STAT3 Activation

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 367




ZIC5 promotes malignant phenotypes of melanoma cellsWe next examined the role of ZIC5 in melanoma malignant

phenotypes. Dendrites are characteristic of differentiated melano-

cytes and enable the transfer of melanosomes to other skin cells,such as keratinocytes. Notably, ZIC5-overexpressing SK-MEL-28cells (FLAG-ZIC5) showed less dendritic-like morphology than

Figure 1.

Identification of ZIC5 as an E-cadherin suppressor. A, SK-MEL-28 and COLO829 melanoma cells were transfected with siRNAs targeting 26 neural crest-relatedgenes. E-Cadherin (CDH1) expression was analyzed by immunocytochemistry after 3 days. B, FLAG-ZIC5, CDH1, and GAPDH (loading control) expression wasexamined in SK-MEL-28 cells stably expressing control vector (pFlag) or FLAG-tagged ZIC5 (Flag-ZIC5) by Western blotting. C, pFLAG and FLAG-ZIC5 cells weretransfected with negative control (siNeg) or ZIC5 siRNA (siZIC5) as indicated, and CDH1 expression was determined by qRT-PCR analysis. Data represent theexpression normalized to that of GAPDH (n ¼ 3). D, ZIC5 expression was examined in A375 cells transfected with siNeg or siZIC5 by Western blotting (left).ZIC5 andCDH1mRNAexpressionwas examined in SK-MEL-28 andA375 cells transfectedwith siNeg or siZIC5 (right). The relative expression level was normalized tothat of GAPDH as an internal control (n ¼ 3). E, ZIC5 binding to the CDH1 promoter (pCDH1) was analyzed by chromatin immunoprecipitation. HeLa cells weretransfected with HA-tagged hZIC5 (Z5HA) or pcDNA empty vector (Control) and then immunoprecipitated with anti-HA antibody (aHA) or IgG. Purified DNA wasanalyzed by PCR with primers for the CDH1 promoter (pCDH1) or the downstream region (Neg). F, pCDH1-Luc reporter activity was monitored in A375 cellstransfectedwith siNeg or siZIC5. Luciferase activity was normalized to that of an internal control (Renilla luciferase; n¼ 3).G, CDH1 promoter activity wasmonitoredin A375 cells cotransfected with siNeg or siZIC5 and full-length pCDH1 or the pCDH1 deletion mutants as indicated in the schematic drawings on the left(n¼ 3).H, Schematic drawing of hZIC5 domains and the deletionmutant. ZIC5 (green) subcellular localizationwas assessed in A375 cells transfectedwithwild-typeZIC5 (hZIC5) or the ZF domain-deleted mutant (right). Nuclei were counterstained with Hoechst (blue; scale bar, 50 mm). I, pCDH1-Luc activity wasmonitored in A375 cells transfected with FLAG-ZIC5 or the ZF deletion mutant (n ¼ 3). Statistical analysis was performed using Student t test (C, D, F, and G) orTukey multiple comparison of means test (I; �� , P < 0.001; �� , P < 0.01; � , P < 0.05).

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Cancer Res; 77(2) January 15, 2017 Cancer Research368




control cells (pFLAG; Fig. 3A). Using qRT-PCR to analyze theexpression levels of several melanocyte biomarkers, we found thatTYRP1 and OCA2 were downregulated in FLAG-ZIC5 cells (Fig.3B). In contrast, FLAG-ZIC5 cells exhibited increased expression ofMMP2 (matrix metalloproteinase 2; Fig. 3B). Knockdown ofendogenous ZIC5 upregulated TYRP1 and TYR, and downregu-lated MMP2 in SK-MEL-28 cells (Fig. 3C). ZIC5 knockdown alsoupregulated TYRP1 in A375 cells; however, TYR andMMP2 expres-sionwasnotdetected in this cell line (Fig. 3C). These results suggestthat ZIC5 promotes dedifferentiation of melanoma cells to someextent.

FLAG-ZIC5 cells showed a higher migratory capacity in scratchand Transwell migration assays than control cells (Fig. 3Dand 3E), whereas ZIC5 knockdown decreased the migratorycapacity in A375 and SK-MEL28 cells (Fig. 3F). Matrigel invasionassays revealed that ZIC5 knockdown also decreased the inva-

siveness of A375 cells (Fig. 3G). Furthermore, cell proliferationanalyses revealed that FLAG-ZIC5 cells failed to undergo con-tact-dependent growth arrest (Fig. 3H). Conversely, lower cellproliferation rates were observed in A375, SK-MEL-28,COLO829, and HT144 melanoma cell lines following ZIC5siRNA transfection (Fig. 3I and J). Cell cycle analysis revealed asignificant accumulation of sub-G1 cells in response to ZIC5knockdown (Fig. 3K), suggesting that ZIC5 contributes tomelanoma cell viability. Thus, these observations support therole of ZIC5 as a factor promoting the dedifferentiation, motil-ity, growth, and survival of melanoma cells.

ZIC5 knockdown limits melanoma growth and metastasisin vivo

To determine whether ZIC5 has a clinically relevant effect onmelanoma proliferation in vivo, A375 cells with stable ZIC5

Figure 2.

ZIC5 expression is elevated inmelanoma. A, Immunohistochemistryfor ZIC5 expression in humanmelanoma tissue arrays. Tworepresentative images of each tissuetype are shown. The enlarged figure ofZIC5 staining in melanoma is alsoshown in the inset; scale bar, 200 mm.B, ZIC5 expression in each sample wasscored as shown in A and assessedusing theWilcoxon signed-rank test.C,ZIC5 protein expression was analyzedin melanoma cell lines and in NHM byWestern blotting.






Figure 3.

ZIC5 promotes malignant phenotypes of melanoma cells. A, Morphology of FLAG-ZIC5– and pFLAG-expressing SK-MEL-28 cells. B, TYRP1, OCA2, and MMP2mRNA expression was assessed by qRT-PCR analysis in Flag-ZIC5– and pFlag-expressing cells. Data represent the expression level normalized to that ofGAPDH (n ¼ 3). (Continued on the following page.)

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knockdown by shRNA (shZIC5-1 and shZIC5-2) were established(Fig. 4A). Lower cell proliferation rateswere confirmed in shZIC5-1and shZIC-2 cells when compared with cells expressing a negativecontrol shRNA (shNeg; Fig. 4B). These cells were inoculated intotheflanksofnudemice.Notably, theaverage volumeandweightofshZIC5 xenograft tumors were significantly reduced compared to

those of their shNeg counterparts (Fig. 4C–E). In addition, analysisof lung metastasis in mice following intravenous cell injectionsrevealed the formation of multiple lung nodules of A375-shNegcells, whereas shZIC5 nodules were scarcely found (Fig. 4F andG).Lung tumor burden was quantified by analyzing the presenceof human GAPDH mRNA, suggesting that shZIC5 cells were

Figure 4.

ZIC5 knockdown prevents melanomagrowth andmetastasis in vivo.A,A375cells with stable ZIC5 shRNA (shZIC5-1or shZIC5-2) or negative controlshRNA (shNeg) expression wereestablished. ZIC5 mRNA expressionwas then assessed by qRT-PCR. Therelative expression levels of ZIC5normalized to those of GAPDH (as aninternal control) are shown (n ¼ 3).B, The cells established in A weremonitored for cell proliferation.Statistical analysis was performedusing Tukey multiple comparisonof means test (A and B; � , P < 0.05;�� , P < 0.01; �� , P < 0.001). C, A375cells with stable expression of shZIC5-1, shZIC5-2, or shNeg were inoculatedand the tumor volumewasdeterminedat indicated points. D, Tumor weightsat 34 days after inoculation are shown.Statistical significance wasdetermined by Dunnett multiplecomparison of means testing(�� , P < 0.001). C and D, Data arerepresented as the mean � SE (n ¼ 6,each). E, Representative images ofxenografts 34 days after inoculation.F, A375 cells with stable shZIC5-1,shZIC-2, or shNeg expression cellswere intravenously injected, and lungmetastasis was assessed after 2.5months. G, Significant differences inthe number of lung nodules weredetermined using the Mann–WhitneyU test. H, Human GAPDH mRNAexpression was analyzed to quantifylung tumor burden. C and H,Differences were determined usingtheMann–WhitneyU test (��,P<0.01).

(Continued.) C, TYRP1, TYR, and MMP2mRNA expression was examined by qRT-PCR in SK-MEL-28 and A375 cells transfected with siNeg or siZIC5 as in B (n ¼ 3).D, Scratch assays performed with pFLAG- and FLAG-ZIC5–expressing cells in serum-free medium for 24 hours; scale bar, 500 mm. E and F, Transwell migrationassays performed with pFLAG- and FLAG-ZIC5–expressing cells (E) or SK-MEL-28 and A375 cells transfected with siNeg or siZIC5 (F). The relative number ofmigrated cells was normalized to the total cell number (n ¼ 3). G, Matrigel invasion assays were performed with A375 cells transfected with siNeg orsiZIC5 (n ¼ 3). H, Cell proliferation was assessed in FLAG-ZIC5 or pFLAG SK-MEL-28 cells at the indicated times (n ¼ 3). I and J, Cell proliferation was assessedin melanoma cells transfected with siNeg or siZIC5 (n ¼ 3). K, Cell-cycle analysis was performed with A375 cells transfected with siNeg or siZIC5. Percentagesof sub-G1 cells are shown in the bar graph (n ¼ 3). B, C, and E–K, Statistical analysis was performed using Student t test (B, C, E, F, G, J, and K) or Tukeymultiple comparison of means test (H and I; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






significantly less prone to form lung metastases than shNeg con-trols (Fig. 4H). Collectively, these data indicate that ZIC5 con-tributes to melanoma in vivo growth and metastasis.

ZIC5 downstream factor PDGFD contributes to melanomaproliferation, migration, and FAK activation

Because ZIC5 knockdown reduced motility, proliferation, invivo growth, and metastasis of A375 melanoma cells even though

CDH1 expressionwasnot upregulated byZIC5knockdown in thiscell line, ZIC5 may induce melanoma malignancy outside of theregulation ofCDH1 expression. To further investigate the effect ofZIC5 on gene regulation, we performed microarray analysis ofA375 and SK-MEL-28 melanoma cells with or without ZIC5knockdown. ZIC5 suppression upregulated a total of 913 genes(>1.5-fold) and downregulated 302 genes (<0.5-fold) in both celllines (Fig. 5A; Supplementary Table S2A). Pathway analysis

Figure 5.

ZIC5 downstream factor PDGFD contributes to melanoma proliferation, migration, and FAK activation. A, Microarray analysis was performed with SK-MEL-28or A375 cells transfected with siNeg or siZIC5. The heat map shows 913 and 302 genes that were upregulated and downregulated, respectively, in responseto ZIC5 suppression. B, FAK phosphorylation (Tyr576/Tyr577; pFAK), pro-PDGFD, and b-actin levels were detected by Western blot analysis in A375 cellstransfected with siNeg or siZIC5. C, PDGFD expression was determined in A375 cells transfected with siNeg or siPDGFD by Western blotting (left). Levels ofphosphorylated FAK (pFAK), total FAK, and b-actinwere detected byWestern blot analysis (right).D, PDGFDmRNA expressionwas determined in A375 and HT144cells transfected with siNeg or siZIC5 by qRT-PCR analysis. Data represent the expression level normalized to that of GAPDH (n ¼ 3). E, PDGFD mRNAexpression was determined in pFLAG or FLAG-ZIC5 cells by qRT-PCR analysis as in D. F, PDGFD mRNA expression was determined in melanoma cell lines andNHM by qRT-PCR analysis as in D. G and H, Cell proliferation was analyzed in melanoma cells transfected with siNeg or siPDGFD. I, Transwell migrationassayswere performedwith A375 cells transfectedwith siNegor siPDGFD. J, Transwellmigration assayswere performedwith pFLAGor FLAG-ZIC5 cells transfectedwith siNeg or siPDGFD. K, Cell proliferation was assessed in A375 cells transfected with siZIC5 or the PDGFD expression vector or both in combination asindicated. L, Cells treated as in L were assessed by Western blot analysis with the indicated antibodies. Statistical analysis was performed using Student t test (D),Dunnett multiple comparison of means test (E, G, H, and I), or Tukey multiple comparison of means test (J and K; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).

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revealed that the altered genes were enriched for genes related toglioma, focal adhesions, and tight junctions (SupplementaryTable S2B). Because activation of FAK, which occurs at focaladhesions, has been associated with melanoma malignancy anddrug tolerance (12, 13), and because ZIC5 knockdown signifi-cantly attenuated FAK phosphorylation (Fig. 5B), we focused onfocal adhesion-related genes (Supplementary Table S2C). Amongthe differentially regulated focal adhesion-related genes, wefocused on PDGFD (platelet-derived growth factor D), becausePDGFD is a member of the PDGF family and was recentlyidentified to have tumor progressive functions. Knockdown ofPDGFD suppressed FAK phosphorylation (Fig. 5C), thus con-firming the contribution of PDGFD to FAK activation in mela-noma. Knockdown and overexpression studies clearly demon-strated that PDGFD expression was positively regulated by ZIC5(Fig. 5B, D, and E). In addition, PDGFD expression was enhancedin melanoma cell lines compared with NHM cells (Fig. 5F), butthe level was inconsistent with the ZIC5 expression level. PDGFDexpression may be regulated by multiple factors including ZIC5.Knockdown of PDGFD in melanoma cells resulted in defectiveproliferation (Fig. 5G and H) and decreased migratory capacity(Fig. 5I). To elucidate to what extent PDGFD contributed to theZIC5-induced phenotypes, we performed rescue experimentsusing cell proliferation and migration assays. Migration assaysrevealed that the enhanced migration ability of FLAG-ZIC5 cellswas completely blocked by PDGFD knockdown (Fig. 5J). Fur-thermore, PDGFD overexpression partially rescued the cell pro-liferation defect of ZIC5 knockdown cells (Fig. 5K) and partiallyrestored FAKphosphorylation (Fig. 5L). These results indicate thatPDGFD is an important factor for ZIC5-mediated proliferationand migration.

ZIC5 and PDGFD prevent apoptosis and enhance drugresistance of melanoma via STAT3 activation

Because activation of focal adhesion kinase promotes melano-ma resistance to BRAF inhibitors (13),we examinedwhether ZIC5enhances the drug resistance of melanoma cells. Apoptosis assaysusing the BRAF inhibitor vemurafenib (PLX4032) showed thatZIC5 overexpression diminished the effect of PLX4032 treatmentwith reduced apoptosis induction in SK-MEL-28 cell (Fig. 6A).Furthermore, the siRNA-mediated knockdown of endogenousZIC5 or PDGFDmarkedly enhanced PLX4032-induced apoptosisin A375 and COLO829 cells (Fig. 6B and C), leading to thereduction of drug-resistant cells (Fig. 6D). Enhancement of apo-ptosis by ZIC5 knockdown was also observed in assays usingan MEK inhibitor (UO126) or the anticancer drug oxaliplatin(Fig. 6E). Suppression of apoptosis by ZIC5 overexpression wascompletely abolished by PDGFD knockdown (Fig. 6F), suggest-ing that PDGFD is a required downstream effector in ZIC5-mediated apoptosis repression. Taken together, these resultssuggest that the ZIC5/PDGFD axis regulates antiapoptotic signal-ing, which may enhance drug resistance in melanoma cells.

Recently, feedback activation of STAT3, which can be activatedby FAK-binding Src kinase in response to RTK/MEK pathwayinhibition, has been shown to decrease drug efficacy (14–16).Therefore, we examined the role of ZIC5 and PDGFD in STAT3activation. As shown in Fig. 6G, knockdown of ZIC5 attenuatedSTAT3 Tyr705 phosphorylation in A375 cells with or withoutPLX4032 treatment, whereas PDGFD knockdown reduced STAT3phosphorylation only in PLX4032-treated cells (Fig. 6G). Asshown in Fig. 5L, PDGFD overexpression partially rescued phos-

phorylation of both FAK and STAT3 in ZIC5 knockdown cells.Furthermore, STAT3 inhibitor treatment completely blocked theantiapoptotic effect of ZIC5 (Fig. 6H). A375 cells treated with aFAK inhibitor showed reduced STAT3 Tyr705 phosphorylation(Fig. 6I), indicating that the FAK pathway could sufficientlyenhance STAT3 activation. Collectively, these findings indicatethat ZIC5 inhibits melanoma apoptosis and promotes drugresistance through PDGFD with promotion of FAK/STAT3 acti-vation (Fig. 6J), thereby suggesting that targeting of these mole-cules could improve the therapeutic efficacy of BRAF inhibitors.

ZIC5 is upregulated by FAK and STAT3-mediated signalingTo date, the results demonstrate that ZIC5 contributes to FAK

and STAT3 activation partially through PDGFD induction. Inthese experiments, we noticed that PDGFD knockdown resultsin decreased ZIC5 protein levels (Fig. 7A). Moreover, PDGFDoverexpression induced not only FAK and STAT3 activation, butalso ZIC5 expression (Fig. 7B). We next assessed whetherZIC5 expression is regulated by FAK/STAT3-mediated signaling.We found that ZIC5 protein levels were reduced by FAK inhibitortreatment (Fig. 7C) as well as by STAT3 inhibition (Fig. 7D).When STAT3 was activated by interleukin-6 (IL-6) treatment,ZIC5 expression was increased in a dose-dependent manner(Fig. 7E). Collectively, these results suggest that ZIC5, PDGFD,FAK, and STAT3 form a positive feedback loop, which maymaintain malignancy in melanoma cells.

ZIC5-mediated signaling contributes to survival and ERKactivation in vemurafenib-resistant cells

As mentioned above, melanoma recurrence frequently occursas a result of acquired resistance to BRAF inhibitors. Recently, themechanism of resistance has been well studied, and involvementof ERK re-activation in the resistant cells has been found. Becausefocal adhesion kinase could activate MEK/ERK signaling, wespeculated that ZIC5-mediated positive feedback signaling couldcontribute to ERK activation in BRAF inhibitor-resistant melano-ma cells.We establishedfive vemurafenib-resistant cell lines usingA375 cells and three vemurafenib-resistant cell lines using HT144cells (Fig. 7F). Although PLX4032 treatment significantly reducedERK phosphorylation in the A375 and HT144 parental cell lines,ERK remained activated in the resistant cell lines (Fig. 7G). Inthese resistant cell lines, elevated levels of phosphorylated FAK,ZIC5, and PDGFD during PLX4032 treatment was also observed(Fig. 7G).WhenZIC5or PDGFDexpressionwas suppressed in theresistant cell lines, the proliferation of these cell lines decreased(Fig. 7H) and, notably, apoptosis was induced (Fig. 7I). In avemurafenib-resistant cell line, ZIC5 or PDGFD knockdowncombined with PLX4032 treatment significantly reduced phos-phorylated ERK levels, while PLX4032 treatment alone did not(Fig. 7J). These results demonstrate that ZIC5-mediated signalingcontributes to ERK activation in vemurafenib-resistantmelanoma(Fig. 7K), and that targeting of ZIC5 and PDGFDmay provide aneffective therapy for BRAF inhibitor–resistant melanoma.

DiscussionMelanoma is a highly aggressive disease and the emergence of

resistance to melanoma therapy, such as BRAF-inhibitor treat-ment, limits the overall therapeutic benefits of recent molecularresearch. In this study, we identified ZIC5 as a melanoma pro-gression factor for the first time (Fig. 1–4). We also found that






ZIC5 and its downstream factor, PDGFD, promote drug resistancethrough FAK and STAT3 signaling (Fig. 5 and 6), and these factorsform a positive feedback loop (Fig. 7). Drug resistance arises from

several mechanisms that can be either intrinsic (primary) oracquired. Intrinsic factors include genetic alterations, coexistentmutations in other signaling genes, or inactivation of

Figure 6.

ZIC5/PDGFD prevents apoptosis and enhances drug resistance of melanoma via STAT3. A, FLAG-ZIC5 or pFLAG cells treated with vemurafenib (PLX4032)at 10 mmol/L for 48 hours were incubated with FITC-labeled Annexin V and the percentage of apoptotic cells was determined. B and C, A375 or COLO829 cellstransfectedwith siNeg, siZIC5, or siPDGFDwere treatedwith PLX4032 (B,0, 1, 5, 10mmol/L;C, 5mmol/L) for 48 hours, and apoptotic cellsweredetected.D,A375 cellstransfected with siNeg, siZIC5, or siPDGFD were treated with PLX4032 (5 mmol/L). After 14 days, cells were stained with Giemsa solution. E, A375 cells weretransfected with siNeg or siZIC5 and treated with UO126 (10 mmol/L) or oxaliplatin (20 mmol/L) for 48 h. F, FLAG-ZIC5 or pFLAG SK-MEL-28 cells weretransfectedwith siNeg or siPDGFD and cultured in the presence of PLX4032 (10 mmol/L) for 48 hours.G, The levels of phosphorylated STAT3 (Tyr705), total STAT3,and b-actin were determined by Western blots in A375 cells transfected with siNeg, siZIC5, or siPDGFD and treated with PLX4032 (5 mmol/L) for 24 hours.H, FLAG-ZIC5 and pFLAG cells were treated with STAT3 inhibitor (2 mmol/L) for 24 hours and subsequently cultured with PLX4032 (10 mmol/L) for 24 hours, andthe percentage of Annexin V–positive cells was determined. I, FAK and STAT3 phosphorylation was assessed by Western blot analysis in A375 cells treatedwith focal adhesion kinase inhibitor at the indicated concentrations for 24 hours. J, A schematic model of ZIC5-mediated drug resistance. Statistical analysiswas performed using Dunnett multiple comparison of means test (C), or Tukey multiple comparison of means test (A, B, E, F, and H; � , P < 0.05; �� , P < 0.01;�� , P < 0.001).

Satow et al.





Figure 7.

ZIC5 forms a positive feedback signal that contributes to survival and ERK activation in vemurafenib-resistant cells. A, A375 cells transfected with siNeg orsiPDGFDwere assessed byWestern blot analysiswith the indicated antibodies.B,A375 cells overexpressing PDGFDwere assessed byWestern blot analysiswith theindicated antibodies. C, A375 cells treated with FAK inhibitor for 24 hours (as shown in Fig. 6I) were assessed by Western blot analysis with the indicatedantibodies. D, A375 cells treated with STAT3 inhibitor for 24 h were assessed by Western blot analysis with the indicated antibodies. E, A375 cells treated with IL6in 1% serum-containing medium for 48 hours were assessed by Western blot analysis with the indicated antibodies. F, Cell proliferation was assessed in A375,HT144, and the vemurafenib-resistant derivatives (vemR) following treatment with vemurafenib (PLX4032) for 48 hours. G, A375 and HT144 cells and the relevantvemR cellswere treatedwith PLX4032 (5mmol/L) for 48 hours, and assessed byWestern blotting.H,A375 andHT144 cells and the vemurafenib-resistant derivatives(vemR) were transfected with siNeg, siZIC5, or siPDGFD, and cell number was assessed after three days. I, PLX4032-resistant vemR-3 cells established from A375were transfected with siNeg, siZIC5, or siPDGFD, and the percentage of Annexin V–positive cells was determined three days later. J, A375 vemR-3 cells weretransfected with siNeg, siZIC5, or siPDGFD. After two days, these cells were treated with PLX4032 (5 mmol/L) for 24 hours and were assessed byWestern blotting.K, A putative positive feedback model of ZIC5, PDGFD, FAK, and STAT3 signaling, which contributes to ERK activation in PLX4032-resistant cells. Statisticalanalysis was performed using Student t test (I) and Dunnett multiple comparison of means test (H; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






proapoptotic pathways. Acquired resistance emerges after treat-ment because of target gene amplification, second site muta-tion, or through bypassing signaling activation (17, 18). Toprevent tumor cells from acquiring drug resistance, strategiesthat inhibit intrinsic mechanisms that prevent tumor cell deathduring the first treatment can be effective. We demonstratedthat inhibition of the ZIC5/PDGFD axis sensitizes melanomacells to a BRAF inhibitor, thus remarkably leading to apoptosis,which consequently suppresses the emergence of drug-resistantcells (Fig. 6B-D). Moreover, inhibition of ZIC5 or PDGFDsuppressed proliferation of BRAF inhibitor-resistant melanomacell lines (Fig. 7), and ZIC5 and PDGFD contributed to ERKreactivation during BRAF inhibitor treatment (Fig. 7J). Takentogether, these results indicate that inhibition of the ZIC5/PDGFD axis is a promising target to disrupt both the intrinsicand acquired drug resistance of melanoma cells.

Recently, FAK- and STAT3-dependent signaling have beenshown to be implicated in the drug tolerance of oncogene-addicted cancer cells (13, 16, 19). For example, induction of FAKsignaling by melanoma-associated fibroblasts with high stromaldensity causes rapid reactivation of ERK/MAPK inmelanoma cellsin response to BRAF inhibition, leading to resistance against theBRAF inhibitor (13). Positive feedback activation of ZIC5,PDGFD, FAK, and STAT3 may enhance this FAK-mediated pro-motion of drug resistance in both an autocrine and paracrinemanner, leading to maintenance and spreading of drug resistancein melanoma tissues.

STAT3 also plays essential roles in resistance acquisition,because RTK/MEK pathway inhibition by drug treatment leadsto STAT3 feedback activation, which consequently promotestumor cell survival and limits the drug response (16). In lungcancer cell lines with mutant KRAS or EGFR, FGFR and IL-6/JAKsignaling promote STAT3 activation following RTK/MEK inhib-itor treatment (16). In our experiments, PDGFD knockdown inBRAF-mutantmelanoma cell lines suppressed STAT3 activation invemurafenib-treated cells, but not in untreated cells (Fig. 6G),suggesting that PDGFD mediates STAT3 activation followingvemurafenib treatment, and that the STAT3 feedback activationmechanism may differ between melanoma and lung cancer cells.PDGFD is aplatelet-derived growth factor and functions via PDGFreceptor b (PDGFRb; ref. 20). Although PDGFB can also activatePDGFRb, it has been reported that PDGFD has a higher tumor-igenic potential than PDGFB as it specifically activates the JNKsignaling cascade and matriptase (21), suggesting a PDGFD-specific mechanism of signal activation. Although the exact

molecular mechanisms underlying PDGFD-mediated STAT3 acti-vation during BRAF-inhibitor treatment require further study,PDGFD might be a specific target to block this process.

Notably, ZIC5 expression in normal human tissues is limited tothe testis and cerebral cortex (RNA sequence data from ProteinAtlas database). PDGFD knockout mice display a mild vascularphenotype and generally in goodhealth (22). Therefore, ZIC5 andPDGFD can be promising and selective targets to overcome drugresistance in melanoma.

Thus, we demonstrated a novel drug resistancemechanism andidentified promising targets to overcome drug resistance in mel-anoma. Finally, it is worth noting that this screening methodcould be useful for further discovery of molecular mechanismsand target molecules in many tumor types by exploring therelationship between development and tumor progressionmechanisms.

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

Authors' ContributionsConception and design: R. Satow, K. FukamiDevelopment of methodology: R. Satow, T. NakamuraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Satow, T. Nakamura, C. Kato, M. Endo, M. Tamura,R. Batori, S. Tomura, Y. MurayamaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):R. Satow, T.Nakamura, C. Kato,M. Endo,M. Tamura,R. Batori, S. Tomura, Y. Murayama, K. FukamiWriting, review, and/or revision of the manuscript: R. Satow, K. FukamiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Satow

Grant SupportThis work was supported by the Funding Program for Next-Generation

World-Leading Researchers and Grants-in-Aid for Scientific Research (JapanSociety for the Promotion of Science KAKENHI grant no. 26293071 toK. Fukami) and grant-in-aid for young scientists (Japan Society for the Promo-tion of Science KAKENHI grant no. 26860221 to R. Satow). This work was alsosupported by grant-in aid from the Tokyo Biochemical Research Foundationand The Uehara Memorial Foundation (R. Satow).

The costs of publication of this articlewere defrayed inpart 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 April 6, 2016; revised August 27, 2016; accepted September 12,2016; published OnlineFirst September 26, 2016.

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2017;77:366-377. Published OnlineFirst September 26, 2016.Cancer Res Reiko Satow, Tomomi Nakamura, Chiaki Kato, et al. Activation of FAK and STAT3ZIC5 Drives Melanoma Aggressiveness by PDGFD-Mediated

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