yb-1 expression and phosphorylation regulate ... · oncogenes and tumor suppressors yb-1 expression...

Oncogenes and Tumor Suppressors

YB-1 Expression and Phosphorylation RegulateTumorigenicity and Invasiveness in Melanomaby Influencing EMTCorinna Kosnopfel1, Tobias Sinnberg1, Birgit Sauer1, Christian Busch1,2,Heike Niessner1, Anja Schmitt3, Stephan Forchhammer1, Cornelia Grimmel4,Peter R. Mertens5, Stephan Hailfinger3, Sandra E. Dunn6, Claus Garbe1, andBirgit Schittek1

Abstract

Cutaneous melanoma represents one of the most aggressivehuman tumor entities possessing a high tendency to metastasize.Cancer cells frequently exploit a highly conserved developmentalprogram, the epithelial-to-mesenchymal transition (EMT), togainmigratory and invasive properties promoting theirmetastaticspread. Cytoplasmic localization of the oncogenic transcriptionand translation factor Y-box binding protein 1 (YB-1) is a pow-erful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanomadespite its neural crest origin. Here, increased expression of YB-1negatively affects patient survival in malignant melanoma andpromotes melanoma cell tumorigenicity both in vitro and in vivo.Intriguingly, this effect seems to be mainly mediated by cyto-plasmic YB-1 that does not exhibit phosphorylation at serine-102

(S102). Moreover, S102 unphosphorylated YB-1 enhances themigratory and invasive potential of human melanoma cells intwo-dimensional (2D) and three-dimensional (3D) culture sys-tems and facilitates acquisition of a mesenchymal-like invasivephenotype in the chick embryo model. Collectively, these datademonstrate that the cytoplasmic activity of YB-1 stimulatestumorigenicity and metastatic potential of melanoma cells bypromoting EMT-like properties.

Implications: This study reveals for the first time that YB-1efficiently drives tumorigenicity and invasiveness of melanomacells in its S102 unphosphorylated cytoplasmic state and thatYB-1 expression represents a negative prognostic factor in primarymelanoma patients. Mol Cancer Res; 16(7); 1149–60. �2018 AACR.

IntroductionMalignantmelanoma is an aggressive neoplasm accounting for

the majority of skin cancer–related deaths despite its comparablylow incidence (1). While localized melanomas can be effectivelytreated by surgical resection and are associated with five-yearsurvival rates of 98%, the outlook in case of metastatic diseaseis rather bleak with a current five-year survival rate of 17% (1).Unfortunately, malignant melanomas exhibit a very high pro-pensity to metastasize, which is already prevalent at stages of lowprimary tumor thickness (2) and hypothesized to be a conse-quence of its melanocytic origin (3). During embryonic develop-

ment, highly migratory neural crest cells delaminate from theneural tube andmigrate to the skin, hair follicles, or other specificlocations within the developing embryo, where they eventuallygive rise to melanocytes (4). In line with originating from exceed-ingly motile cells, melanocytes are supposed to retain latentmigratory capabilities, which could easily be reactivated in theprocess of melanoma progression (3).

The epithelial-to-mesenchymal transition (EMT) is a highlyconserved program during embryonic morphogenesis and isassociated with epithelial cells acquiring mesenchymal character-istics including an enhanced physiologic migratory capability. Inaddition to its crucial role for mesoderm formation as well asneural crest development and function, EMT is also frequentlyexploited by epithelial cancer cells during the invasive phase ofmetastasis (5).

The Y-box binding protein 1 (YB-1, YBX1) is a multifunctionalmember of the cold-shock domain (CSD) protein family, whichcan act both as a transcription factor in the nucleus and as atranslational regulator in the cytoplasm (6). The subcellularlocalization of YB-1 is governed by regulatory sequences in theC-terminal region of the protein including two noncanonicalnuclear localization signals (NLS) as well as a cytoplasmic reten-tion site (CRS; refs. 7, 8). Owing to a dominant effect of the CRSunder normal cellular conditions, YB-1 is predominantly found inthe cytoplasm (9). Upon environmental stresses and in responseto promitogenic stimuli, however, YB-1 translocates to the nucle-us (9–11). The serine/threonine kinase AKT and the p90 ribo-somal S6 kinase (RSK) are capable of phosphorylating YB-1 at a

1Division of Dermatooncology, Department of Dermatology, University ofT€ubingen, T€ubingen, Germany. 2Dermateam, Winterthur, Switzerland. 3Inter-faculty Institute of Biochemistry, University of T€ubingen, T€ubingen, Germany.4FACS Core Facility, Department of Dermatology, University of T€ubingen,T€ubingen, Germany. 5Department of Nephrology and Hypertension, Diabetesand Endocrinology, Otto-von-Guericke University, Magdeburg, Germany.6Phoenix Molecular Designs, Vancouver, British Columbia, Canada.

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

Corresponding Author: Birgit Schittek, Division of Dermatooncology, Depart-ment of Dermatology, University of T€ubingen, Liebermeisterstr. 25, T€ubingenD-72076, Germany. Phone: 4970-7129-80832; Fax: 4970-7129-5187; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-17-0528

�2018 American Association for Cancer Research.

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serine residue (S102) within the nucleic acid–binding cold-shockdomain (10, 12, 13). Intriguingly, this phosphorylation eventwasreported to impair the association of YB-1 with mRNAs in thecytoplasm and to simultaneously induce its nuclear translocationand activity as a transcription factor in breast and ovarian cancer,thuspromoting tumor cell proliferation and chemoresistance (10,12–15). Apart from its nuclear function, also increased expressionof cytoplasmic unphosphorylated YB-1 proved to mediate apotent protumorigenic effect by stimulating the translation ofmRNAs encoding EMT-regulating transcription factors in a breastcancer model (16). Indeed, both nuclear and cytoplasmic YB-1have been repeatedly associated with poor prognosis and diseaserecurrence in a variety of human malignancies (6).

On the basis of earlier work providing first evidence forincreased YB-1 expression during melanoma progression andfor a functional role of the cold-shock domain protein in thebiology of metastatic melanoma cells (17–19), we now morespecifically addressed the impact of YB-1 protein levels and itsS102 phosphorylation status on the aggressive phenotype ofmalignant melanoma reflected by patient survival as well asmelanoma cell tumorigenicity, migratory activity, and invasivecapacity.

Materials and MethodsCulture of human cells

The human metastatic melanoma cell lines MeWo and A375were purchased from ATCC (20) and authenticated via ShortTandem Repeat (STR) profiling (Leibniz Institute DSMZ);1205LU was kindly provided by the laboratory of M. Herlyn (TheWistar Institute, Philadelphia, PA; ref. 21), which routinely per-forms STR profiling using the AmpFlSTR Identifiler PCR Ampli-ficationKit (Life Technologies). The cultivation ofmelanoma cellswas conducted as described previously (22). All melanoma celllines were used no longer than 2 months upon thawing of thefrozen stock. Mycoplasma testing was regularly performed usingthe Venor GeM Classic Mycoplasma Detection Kit (MinervaBiolabs).

YBX1 geneknockout (CRISPRYBX1)was carriedout byCRISPR/Cas9–mediated genome engineering as described previously (18).1205LU CRISPR YBX1 #1 and #2 (from two separate approachesusing different sgRNA sequences targeting YBX1) represent pooledpopulations of cells with effective knockout and unaffected cells,while theA375 andMeWoCRISPR YBX1 cells constitute respectivesingle-cell clones with effective YBX1 knockout.

Lentiviral gene transfer was used for the generation of mela-noma cells with constitutive overexpression of HA-tagged wild-type YB-1 (HA-YB-1; cloned in pWPI; ref. 17), of cells withinducible overexpression of 3XFLAG-tagged wild-type YB-1(YB-1WT) or S102 phospho-site mutant YB-1 (YB-1S102D,YB-1S102A; ref. 23; subcloned into pLVX-Tight-Puro and cotrans-duced with pLVX-Tet-On Advanced, both Clontech) as well as ofmelanomacellswith inducible expressionof YB-1–specific shRNA(shYB-1, TRIPZ, clone V2THS_232997) or nonsilencing shRNA(NonSil, TRIPZ, #RHS4743; both Dharmacon/GE Healthcare).Transgene expression was induced by 2 mg/mL doxycycline(AppliChem) in the culture medium.

Lentiviral gene transferProduction of lentiviral particles and transduction of melano-

ma cells were conducted as described previously (17).

In vivo tumor growth assayTo assess melanoma growth in vivo, 5 � 105 1205LU cells with

stable HA-YB-1 overexpression or transduced with the emptyvector were subcutaneously injected into NOD/SCID mice.Tumor size was monitored up to 56 days after injection bymeasurement of tumor length and width using a caliper. Thetumor volume (V ¼ 0.4 � length � width2) was calculated as acorrelate for tumor growth.

For the in vivo tumor growth assay assessing the effect of YB-1knockdown, 1� 106 1205LU shYB-1 cells in 100 ml PBSwith 10%Matrigel were subcutaneously injected into SCID hairless outbred(SHO)mice. Themicewere randomized into two groups (n¼10).One group was continuously fed with 200 mg/mL doxycycline(AppliChem) in the drinking water for shRNA induction startingone day prior to the tumor cell injection, the other group served asan untreated control. Tumor size was monitored for two weeksand the tumor volume calculated employing the following for-mula: V ¼ 0.4 � length � width2. All animal experiments wereapproved by the responsible regional authority (Regier-ungspr€asidium T€ubingen, AZ HT2/12).

Viability assayCell viability was assessed at the indicated time points with the

4-methylumbelliferyl heptanoate (MUH) assay as described pre-viously (19).

Cell-cycle analysisPropidium iodide–based analysis of cell-cycle distribution was

performed as described previously (22).Flow cytometric determination of DNA synthesis was con-

ducted by a nucleoside analogue–based approach using anEdU (5-ethynyl-20deoxyuridine) Flow Cytometry Kit (baseclickGmbH) according to the manufacturer's instructions. Shortly,cells were labeled with 20 mmol/L EdU for 4 hours, before theywere fixed, permeabilized, and stained for EdU incorporation.Additional propidium iodide–based staining of total DNA wasperformed as described previously (22). Cells were analyzed witha LSR II flow cytometer using FACSDiva software (both BDBiosciences).

Anchorage-independent growth assayAnchorage-independent growth was examined as described

previously (24).

Wound healing assayMelanoma cells were grown until confluency in cavities of a

6-well plate, their proliferative capacity blocked by addition of0.625 mmol/L hydroxyurea (Sigma) to the cell culture medium,and after 16 hours a scratch was introduced into the cell layer. Themigratory front wasmonitored at four locations per well for up to48 hours by means of light microscopy. Pictures were taken andthe relative wound closure over time analyzed with ImageJ(Wayne Rasband, NIH, Bethesda, MD).

Migration and Matrigel invasion assaysMigration and Matrigel invasion assays were performed using

Boyden chambers containing polycarbonate filters with a poresize of 8 mm, which were either untreated (migration, controlinserts) or coated with a Matrigel basement membrane matrix(invasion, BD Biocoat Matrigel invasion chambers; both BD

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Mol Cancer Res; 16(7) July 2018 Molecular Cancer Research1150




Biosciences) as described previously (19). Depending on the celllines, different cell numbers were applied to the transwell inserts(5 � 104 1205LU, 3 � 104 MeWo). After overnight incubation at37�C, the invaded cells were fixed and stained with hematoxylinand eosin. The assayswere carried out in triplicates andperfilter atleast 5 fields were counted.

Spheroid collagen invasion assayA total of 2.5 � 103 melanoma cells were seeded under

nonadherent conditions into cavities of a 96-well plate coatedwith 1.5% agar noble (BD Difco) in PBS. After 3 days, thespheroids were reseeded into a 1 mg/mL collagen I matrix andgrown for further 4 days with normal cell culture mediumcontaining 2 mg/mL doxycycline. Then, spheroids were stainedwith Calcein-AM (1 mmol/L) for 30 minutes. Spheroids wereanalyzed with an inverse fluorescence microscope (Axiovert,Zeiss).

Chick embryo invasion assayThe chick embryo invasion assay was conducted as compre-

hensively described inBusch and colleagues (25). In short, at stage12 to 13 of the chick embryo development, according to Ham-burger and Hamilton (26), MeWo cells, which had been inducedto overexpress either YB-1S102D or YB-1S102A, were injected intothe rhombencephalic brain vesicle of the embryo (n¼ 4 or n¼ 7,respectively). After a 4-day incubation at 38�C, the embryos werefixed, embedded in paraffin, and sectioned serially. Sections werestainedwith hematoxylin and eosin aswell as antibodies targetingthe human cell–specific proliferation marker Ki67 or YB-1(Abcam) using standard methods. To quantify tumor cell inva-siveness, the invadingmelanoma cells were identified on the basisof their morphology and counted in the three sections of theembryo exhibiting the largest tumor diameter. The resultantmeanvalue was taken as a measure of the invading melanoma cells perembryo.

Tissue microarrayA tissue microarray (TMA) constructed from single but repre-

sentative punches of 270 primary melanoma cases (253 evalu-able) and 22 benign nevi was kindly provided by K. Ikenberg(University Hospital Zurich, Zurich, Switzerland) in collabora-tion with C. Busch and C. Garbe (University of T€ubingen,T€ubingen, Germany) and more detailed characteristics of theTMA are presented in Wagner and colleagues (27). Immunohis-tochemistry (IHC) staining of freshly cut 3-mm–thick sections ofthe TMA block was performed as described previously (22) usingan IHC-validated YB-1–specific mAb (F-E2G5, 1:25 dilution;ref. 28) and a hematoxylin counter staining. The YB-1 stainingintensity was evaluated and assigned a semiquantitativehisto-score ranging from "0" for completely absent staining, over"1" for weak, and "2" for moderate staining to "3" indicatingstrong expression levels. In case of heterogeneity within the tumorsample, the predominant staining intensity was determined. Thehisto-scores "0" and "1" as well as the intensities "2" and "3" werecombined to account for tissue samples with low YB-1 expression(YB-1low) and with high YB-1 levels (YB-1high), respectively. Spotsof the TMA lacking tumor tissue were not considered for analysis.Tumor analyses were performed in accordance with theDeclaration of Helsinki Principles and approved by the localmedical ethical committee (261/2009BO2).

ImmunofluorescenceImmunofluorescence staining of melanoma cells was per-

formed as described previously (22) using antibodies targetingPS102-YB-1 (rabbit, 1:100 dilution, Cell Signaling Technology),YB-1 (rabbit, 1:100 dilution, Cell Signaling Technology ormouse,1:50 dilution, Santa Cruz Biotechnology), HA (rabbit, 1:100dilution, ClonTech), or FLAG (mouse, 1:200 dilution, Sigma).The cells were fluorescently labeled by incubation with Cy5-coupled donkey anti-rabbit and Cy3-coupled donkey anti-mousesecondary antibodies (1:250 dilution, Dianova) and their nucleistained with YO-PRO-1 (Thermo Scientific). Analysis was con-ducted with a confocal laser microscope (Leica TCS SP).

Western blottingWhole-cell lysates as well as nuclear- and cytoplasmic-enriched

fractions were generated and used for Western blot analysis asdescribed previously (22). The following primary antibodies wereapplied: anti-HA (ClonTech); anti-FLAG M2 (Sigma); anti-YB-1,anti-Slug (Abcam); anti-PS102-YB-1, anti-Snail, anti-GAPDH (CellSignaling Technology) and anti-Twist, anti-Lamin B (Santa CruzBiotechnology). Immunodetection was performed as describedpreviously (22).

Statistical analysisStatistical analysiswas conductedwithGraphPadPrismversion

7.0 (GraphPad Software) unless stated otherwise. One-wayANOVA followed by Tukey multiple comparisons tests or atwo-tailed unpaired Student t test were used for P value calcula-tion and significance determination, where applicable. P values<0.05 were considered statistically significant, with �, P < 0.05;��, P < 0.01; ��, P < 0.001and ��, P < 0.0001. Analysis of patientsurvival depending on YB-1 protein levels was performed usingthe Kaplan–Meier method to generate survival curves and sub-sequent log-rank (Mantel–Cox) testing to determine significantdifferences. Kaplan–Meier survival analysis for patients from theTCGA cutaneous melanoma dataset evaluating the effect of YB-1mRNA expression was conducted using the TCGA cancer browserwebtool (29, 30).

ResultsIncreased YB-1 expression promotes melanoma celltumorigenicity

Previously, we could show an increased expression of YB-1 inmelanoma tissue compared with benignmelanocytic nevi using aTMA incorporating biopsies from 100 melanocytic lesions (17).To validate this finding on a larger scale and to allow for corre-lationwith patient survival, we nowperformed YB-1–specific IHCon a second tissue microarray comprising biopsies from 22 neviand 253 primary melanomas with matched overall survival (OS)data. In line with our previous results, YB-1 expression wassignificantly enhanced in primary melanomas as opposed tomelanocytic nevi (P < 0.0001) with the majority of samples(163) exhibiting high YB-1 protein levels and only 90 samplesshowing a low YB-1 expression level (Fig. 1A and B; Supple-mentary Fig. S1A). Intriguingly, high YB-1 protein expressioncorrelated with a shorter overall survival of melanoma patients(P ¼ 0.077; Fig. 1B and C) and closer examination of bothcytoplasmic (Supplementary Fig. S1B) and nuclear stainingpatterns (Supplementary Fig. S1C) revealed a strong linkbetween elevated cytoplasmic YB-1 and the patients' outcome

YB-1 Promotes Melanoma Cell Tumorigenicity and Invasiveness

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Figure 1.

Elevated YB-1 levels are associated with shorter patient survival and enhanced tumorigenicity of melanoma cells. A, Analysis of YB-1 protein expression in253 primary melanomas and 22 benign nevi arranged on a TMA by IHC. Histo-scores for YB-1–staining intensities were assigned: "0" absent, "1" weak (YB-1Low); "2"moderate, "3" strong (YB-1High). Statistical significance was determined using a two-tailed unpaired Student t test. B, Microphotographs of three representativeTMA samples visualizing each of the assigned histo-scores. YB-1 expression levels are shown in red (Fast Red substrate) with a hematoxylin counterstaining. Scale bars, 100 mm. C, Kaplan–Meier curves and corresponding survival rates illustrating overall survival of 253 patients diagnosed with primary melanomadepending on YB-1 expression (YB-1Low: 90 samples, YB-1High: 163 samples). Statistical differences were evaluated by a two-sided log-rank test. D, In vivotumor growth of HA-YB-1–overexpressing 1205LU or empty vector (EV) control cells after subcutaneous injection into NOD/SCIDmice. Mean tumor volumes� SEM(n ¼ 4) are depicted. E, In vivo tumor growth of 1205LU cells with inducible YB-1–specific shRNA (shYB-1) after subcutaneous injection into SHO mice. shRNAinduction was achieved by permanent administration of 200 mg/mL doxycycline in the drinking water supplied ad libitum. Mean tumor volumes (�SEM, n¼ 10) andrepresentative YB-1 protein levels of the respective tumor lysates are shown. GAPDH served as a loading control. F–H, Anchorage-independent growth assays of1205LU melanoma cells with HA-YB-1 overexpression compared with EV control cells (F), of 1205LU with inducible YB-1–specific shRNA (shYB-1) or controlshRNA (NonSil; G) or of 1205LU cells with YBX1 knockout (CRISPR YBX1 #1, #2) compared with the parental control cells (H). In case of the inducible knockdownsystem (G), cells were continuously treated with 2 mg/mL doxycycline. After 14 days, colonies were visualized with crystal violet, counted, and normalizedto the respective controls. Representative data of at least two independent experiments are shown (mean � SD, n ¼ 4). Two-tailed unpaired Student t test(F and G) or one-way ANOVA with Tukey multiple comparisons test (H) were employed to determine significant differences.

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(P ¼ 0.0027). The negative correlation of YB-1 expression andpatient survival could further be confirmed on the transcrip-tional level by analysis of 364 cutaneous melanoma patientsfrom the database of "The Cancer Genome Atlas" (TCGA;refs. 29, 30) showing a significantly reduced median overallsurvival of patients with high YB-1 mRNA expression (3.12years) as opposed to those exhibiting lower expression of YB-1mRNA (3.75 years; P ¼ 0.049; Supplementary Fig. S1D).

On the basis of the negative association of YB-1 expression andpatient survival, we next addressed the question whether modu-lation of YB-1 protein levels affects the tumorigenicity of mela-noma cells. To this end, we either overexpressed HA-tagged YB-1(Supplementary Fig. S2A) or knocked-down endogenous YB-1levels in 1205LU melanoma cells via a doxycycline-inducibleshRNA system (Supplementary Fig. S2B) and evaluated theirtumorigenicity after subcutaneous (s.c.) injection into immuno-compromised mice. While increased YB-1 levels dramaticallyaccelerated tumor formation in vivo (Fig. 1D), the reverse wasthe case upon knockdown of YB-1 protein expression (Fig. 1E). Acorresponding picture could be observed when assessing theability of cells to grow in an anchorage-independent manner, atrait which has been shown to serve as a reliable in vitro correlatefor the tumorigenicity of transformed cells in vivo (31): HA-YB-1overexpression greatly enhanced the colony-forming capacity ofthe 1205LU melanoma cells in vitro (Fig. 1F), whereas YB-1knockdown via shRNA significantly reduced the relative colonynumber in the soft agar (Fig. 1G). This could be confirmed inanother loss-of-function model system using 1205LUmelanomacells that underwent CRISPR/Cas9–based genome-editing aimingfor a YBX1 gene knockout (Fig. 1H; Supplementary Fig. S2C).Interestingly, cell viability–based growth curves and cell-cycleanalyses revealed no effects of YB-1 overexpression, knockdown,or knockout onmelanoma cell growth (Supplementary Fig. S3A–S3H). In summary, our data indicate that increased YB-1 expres-sion in melanoma patients correlates with poor overall survival,which is presumably based on an enhanced tumorigenicity of thecells and not based on elevated tumor cell proliferation.

YB-1 localization is predominantly cytoplasmic and regulatedby S102 phosphorylation in melanoma cells

Next, we wanted to identify the mechanism underlying theenhanced tumorigenicity elicited by YB-1 overexpression in mel-anoma cells. YB-1 can act as a regulator of both transcription in thenucleus and mRNA translation in the cytoplasm. Therefore, wefirst analyzed YB-1 localization using both immunofluorescenceand Western blot analysis of nuclear- and cytoplasmic-enrichedfractions. Endogenous YB-1 in control vector–transduced 1205LUmelanoma cells displayed a preponderant cytoplasmic stainingpattern (Fig. 2A and B, left). Similarly, exogenously expressedHA-tagged YB-1 mainly localized to the cytoplasm (Fig. 2A and B,right). This confirms previous observations of a predominantcytoplasmic localization of overexpressed YB-1 in another mel-anoma cell line (17) and suggests that the observed effects of YB-1on tumorigenicity of melanoma cells could critically rely on itscytoplasmic activities.

Phosphorylation of YB-1 at serine-102 has been reported toenhance its nuclear shuttling and to mediate a switch fromtranslational regulation to transcriptional activity in breast andovarian cancer models (10, 13–15). However, the functionaleffect of YB-1 S102 phosphorylation in melanoma cells hashitherto not been analyzed. To this end, we generated melanoma

cell lines with an inducible expression of FLAG-tagged YB-1mutants containing either a disrupted S102 phosphorylation site(YB-1S102A) or an amino acid exchange mimicking constitutivephosphorylation (YB-1S102D; Supplementary Fig. S4A). Immuno-fluorescence as well as Western blot analyses using nuclear- andcytoplasmic-enriched fractions of the transduced 1205LU mela-noma cells showed a predominant cytoplasmic localization ofYB-1S102A, comparable with the overexpressed wild-type YB-1(YB-1WT), whereas a higher tendency of nuclear translocation wasdetectable for YB-1S102D (Fig. 2C and D). A similar picture couldbe seen for A375 and MeWo melanoma cells with inducibleectopic expression of the YB-1 wild-type or the phospho-sitevariants (Supplementary Fig. S4B). These data clearly indicatethat comparable with the situation in breast and ovarian cancer,the preponderant cytoplasmic localization of YB-1 in melanomacells is mainly, but not exclusively, governed by its S102 phos-phorylation status.

YB-1mediates its stimulatory effect on anchorage-independentgrowth in the absence of S102 phosphorylation

With the help of the above described YB-1 phospho-sitemutants, we proceeded to evaluate whether S102 phosphoryla-tion of YB-1 affects the tumorigenicity ofmelanoma cells. Intrigu-ingly, overexpression of YB-1S102A, which resulted in a mainlycytoplasmic localization of YB-1 (Fig. 2C and D), stronglyenhanced the ability of 1205LU melanoma cells to grow anchor-age-independently in an in vitro tumorigenicity assay, phenoco-pying the effect of the likewise predominantly cytoplasmic occur-ring wild-type YB-1 (YB-1WT) protein (Fig. 3). Conversely, over-expression of the phospho-mimic mutant YB-1S102D, exhibitingan increased tendency of nuclear occurrence (Fig. 2C and D), didnot influence anchorage-independent growth compared with thecorresponding cells harboring the empty vector (EV) control. Thissuggests that it is cytoplasmic, S102 unphosphorylated YB-1,which mediates the stimulating effect of YB-1 overexpression onmelanoma cell tumorigenicity and anchorage-independentgrowth. Similar results were obtained with two additional mel-anoma cell lines (Fig. 3): Intriguingly, while the nonphosphor-ylatable YB-1S102A profoundly enhanced the tumorigenicity in allcell lines tested,wild-type YB-1 did not exert a stimulatory effect inMeWo melanoma cells, which went along with an increasedtendency of nuclear localization and phosphorylation of theoverexpressed YB-1WT in this cell line (Supplementary Fig.S4B–S4D). Comparable with overexpression of wild-type YB-1and knockdown of endogenous YB-1 levels in 1205LU (Supple-mentary Fig. S3A–S3H), ectopic expression of the different S102phospho-site mutants did not significantly affect melanoma cellgrowth, pointing to a specific positive effect of S102 unpho-sphorylated YB-1 on the anchorage-independent growth of mel-anoma cells (Supplementary Fig. S5A and S5B).

S102 unphosphorylated YB-1 enhances themigratory potentialand invasiveness of melanoma cells

As a next step, we assessed a potential implication of YB-1 inmelanoma cell invasion and migration depending on its S102phosphorylation status. Downregulation of YB-1 levels by meansof RNA interference or YBX1 gene knockout drastically decreasedthe migratory potential of melanoma cells, as could be observedin a wound closure assay (Fig. 4A). Most importantly, overexpres-sion of YB-1S102A robustly accelerated wound closure by allanalyzed melanoma cell lines, whereas YB-1S102D did not






significantly affect cell motility (Fig. 4B). Both the negative effectof YB-1 downregulation and the stimulating effect observed forthe overexpression of S102 unphosphorylated YB-1 could beconfirmed in a Boyden chamber–based cell migration assay (Fig.4C and D). Matrigel invasion assays furthermore showed that YB-1 expression levels positively correlate with the invasive capacityof melanoma cells (Fig. 4E) and that overexpression of YB-1S102A

significantly enhances melanoma cell invasion (Fig. 4F).To further evaluate the functional effects of the S102 phospho-

site mutants in a more physiologic setting, we conducted a three-dimensional spheroid-based collagen invasion assay. Supportingthe results of the Boyden chamber invasion assay, overexpressionof YB-1S102A, mimicking S102 nonphosphorylated YB-1, stronglyenhanced melanoma cell invasion into the collagen matrix,whereas the phospho-mimic YB-1S102D–overexpressing cellsinvaded only marginally (Fig. 5A).

Altogether, these data strongly indicate a critical functionalrole of S102 unphosphorylated YB-1 not only in anchorage-independent growth, but also in both motility and invasivenessof melanoma cells in two-dimensional and three-dimensionalmodel systems.

Overexpression of S102 nonphosphorylatable YB-1 facilitatesacquisition of an EMT-like capacity in melanoma cells

In an epithelial breast cancer model, cytoplasmic S102nonphosphorylated YB-1 has been shown to translationallyregulate genes involved in epithelial-to-mesenchymal transi-tion and, thereby, to greatly promote the migratory and inva-sive capability of the tumor cells (16). Indeed, Western blotanalysis revealed an enhanced expression of EMT-promotingtranscription factors such as Twist (TWIST1), Slug (SNAI2), orSnail (SNAI1) upon overexpression of S102 unphosphorylated

Figure 2.

The localization of YB-1 in melanoma cells is shaped by S102 phosphorylation. A, Confocal immunofluorescence analysis of control vector–transduced (EV)or HA-YB-1–overexpressing 1205LU cells with antibodies specific for YB-1 (Cy3-labeled, red) and the HA-tag (Cy5-labeled, blue). Nuclei were stained withYO-PRO-1 (green). An overview is depicted in the top and the indicated selection further magnified in the bottom. Scale bars, 25 mm. B, Immunoblot analysis ofHA-YB-1–overexpressing 1205LU or control vector–transduced cells using equivalent volumes of the respective cytoplasmic- and nuclear-enriched fractions. GAPDHand Lamin B served as the respective subcellular markers. C, Confocal immunofluorescence analysis for endogenous YB-1 and ectopically expressed FLAG-taggedYB-1 species (YB-1S102D, YB-1S102A, YB-1WT) using YB-1–specific (Cy5-labeled, blue) and FLAG-specific (Cy3-labeled, red) antibodies. 1205LU melanoma cellsharboring the respective overexpression system or an empty vector control (EV) were induced for 5 days with 2 mg/mL doxycycline prior to analysis. Nuclei werestained with YO-PRO-1 (green). Scale bars, 25 mm. D, Left, immunoblot analysis of FLAG-tagged YB-1 phospho-site variants in cytoplasmic- and nuclear-enriched fractions obtained from 1205LU cells with inducible overexpression of YB-1S102D, YB-1S102A, or YB-1WT or from empty vector control cells. Cells werepreinduced with 2 mg/mL doxycycline for 5 days. Fifteen micrograms or 5 mg protein were loaded of the cytoplasmic- and nuclear-enriched fractions, respectively.GAPDH and Lamin B served as the corresponding subcellular markers. Right, semiquantitative densitometric analysis was performed. Relative cytoplasmicoccurrence of the different YB-1 species was calculated as the ratio of cytoplasmic and nuclear values, representing the FLAG-specific signal intensities normalizedto the respective loading control (mean � SD; n ¼ 3). Significance was determined with one-way ANOVA and subsequent Tukey multiple comparison test.

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YB-1 in melanoma cell lines (Fig. 5B), whereas their expressionwas strongly reduced upon downregulation of endogenous YB-1 levels as well as upon YBX1 gene knockout (SupplementaryFig. S6A and S6B). To further assess the functional impact of theYB-1 phospho-site variants on acquisition of an EMT-likephenotype in malignant melanoma, MeWo cells with ectopicexpression of either YB-1S102A or YB-1S102D were injected intothe rhombencephalic brain vesicle of early chick embryos. Afterfour days, distribution of melanoma cells was analyzed, reveal-ing tumor nodules in the dorsal midline of the rhombence-phalic roof plate for both MeWo YB-1S102A and YB-1S102D (Fig.5C, I; Supplementary Fig. S6C). Similar staining patterns couldbe observed for the human cell–specific proliferation markerKi67 (Fig. 5C, II) indicating that melanoma cell proliferationwas not significantly affected by the YB-1 phospho-site mutantsin this experimental setting. However, while MeWo YB-1S102D

cells formed compact tumor lesions with tight cell–cell adhe-sion reminiscent of epithelial cells, YB-1S102A–overexpressingtumors exhibited reduced cell–cell contacts reflected in a scat-tered distribution of cells with a rather mesenchymal-likemorphology (Fig. 5C, I–III; Supplementary Fig. S6C). Intrigu-ingly, the difference in the structural architecture of the tumornodules went along with a readily detectable pigment produc-tion in the tumor lesions expressing the phospho-mimic YB-1variant YB-1S102D, which was absent from YB-1S102A tumors,further suggesting a differentiation switch in response to theembryonic setting facilitated by the nonphosphorylated YB-1mutant (Fig. 5C, II and III). In line with the precedent assaysaddressing melanoma cell invasiveness, YB-1S102A–overexpres-sing melanoma cells strongly invaded into the surroundingmesenchymal host tissue, whereas this was not the case forMeWo cells expressing YB-1S102D (Fig. 5C and D). Collectively,these data strongly suggest that S102 unphosphorylated YB-1effectively promotes EMT-like processes in malignant melano-ma cells.

DiscussionThe Y-box binding protein 1 is not only critically involved in

inflammatory diseases but has further been reported to be over-expressed and to serve as a negative prognostic factor in a numberof humanmalignancies (6, 32). Indeed, increasedYB-1 expressionhas been associated with a shorter disease-free and/or overallsurvival in patients with sarcomas, gliomas, breast, gastric, blad-der, and non–small cell lung cancer, as well as with diffuse large

B-cell and non-Hodgkin lymphomas (15, 28, 33–39). Although,we had already shown in previous work that YB-1 expressionlevels correlate with melanoma progression (17, 19), a possibleprognostic role of YB-1 in malignant melanoma has not beenaddressed so far. Using a TMA-based analysis, we could now showthat elevated YB-1 protein expression negatively correlates withoverall survival of patients suffering from primary melanomas.This could be confirmed on the level ofmRNA expression exploit-ing the TCGA cancer genome datasets for cutaneous melanomas,suggesting a potential useof YB-1 as anegative prognostic factor inmalignant melanoma.

In line with the enhanced YB-1 expression observed inmelanoma lesions compared with benign melanocytic nevi,we could further reveal a clear association between YB-1 proteinlevels and melanoma cell tumorigenicity in different experi-mental models. This is in agreement with previous studiesreporting an increased tumorigenicity of breast and kidneyepithelial cells following YB-1 overexpression (40, 41) andstrongly suggests an important role of YB-1 in melanomadevelopment and progression.

YB-1 is a versatile protein that can exert various functionaleffects by regulating gene expression both on a transcriptional andon a translational level (6). Under normal cellular conditions,YB-1 has been described to primarily localize to the cytoplasmowing to a dominant effect of the cytoplasmic retention sitelocalized in its C-terminal domain (7–9). Indeed, YB-1 serves asan important packaging protein for mRNAs in the cytoplasm,enhancing their stability and regulating their translational activity(42–44). Correspondingly, a predominant cytoplasmic occur-rence of YB-1 has been observed in various cancer entities includ-ing sarcomas, breast, gastric, bladder, and lung cancer as well asnon-Hodgkin lymphomas (15, 28, 34–36, 38, 39, 44). However,the validity of these results is controversial, being limited by thedifferential sensitivity of the applied antibodies, which is deter-mined by the accessibility of the respective YB-1 epitopes andsuspected to give rise to distinct nucleo-cytoplasmic stainingpatterns in IHC analyses (28, 45). Consequently, here, immuno-fluorescence analysis evaluating the subcellular localization ofoverexpressed YB-1 was conducted with the help of antibodiestargeting both YB-1 itself and its associated protein tag andwas additionally validated by immunoblot analyses usingcytoplasmic- and nuclear-enriched fractions. In agreement withprevious data in malignant melanoma (17), we could alsoobserve in this study a predominant cytoplasmic localization ofboth endogenous and especially ectopically expressed YB-1.

Figure 3.

Overexpression of S102 unphosphorylated YB-1promotes anchorage-independent growth.Anchorage-independent growth assays of 1205LU,A375, and MeWo melanoma cells with inducibleectopic expression of YB-1S102D, YB-1S102A, or YB-1WT

or transduced with the empty vector control (EV).After 14 days, colonies were visualized with crystalviolet and counted. Colony numbers obtained aftercontinued induction with 2 mg/mL doxycycline werenormalized to the respective uninduced controls anddisplayed relative to EV. Representative data of twoindependent experiments are shown (mean � SD,n ¼ 4). One-way ANOVA with Tukey multiplecomparisons test was used to assess significantdifferences.






Kosnopfel et al.





Together with the negative prognostic value of elevated cyto-plasmic YB-1 in patients with primary melanomas, this indicatesa critical role of YB-1 activity as a translation factor in melanomacell tumorigenicity.

S102 phosphorylation has been shown to govern YB-1 func-tional activity in epithelial cancers with phosphorylated YB-1detaching frommRNAs in the cytoplasm, shuttling to the nucleusand binding to the gene promoters of YB-1 transcriptional targets(10, 12–15). S102 unphosphorylated YB-1, in contrast, has beenreported to primarily localize to the cytoplasm, promoting cap-independent mRNA translation (16). Although phosphorylationof YB-1 at S102 seems to influence its subcellular distribution inbreast cancer cells, this posttranslational modification is not nec-essary for nuclear shuttling andpresumably rather directly controlsYB-1 functional activity reflected amongothers by itsDNA-bindingcapacity (13, 15).Wecan showhere that inagreementwith thedataobtained in epithelial cancer, also in malignant melanoma, S102phosphorylation has a moderate effect on the intracellular local-ization of YB-1, with the phospho-mimic YB-1S102D possessing ahigher tendency to shuttle to the nucleus as opposed to thepredominantly cytoplasmic YB-1S102A. These data, on the onehand, strongly suggest a universal mechanism of YB-1 functionalregulation by phosphorylation at the serine-102 residuewithin thecold-shock domain, but on the other hand also indicate theinvolvement of additional mechanisms fine-tuning the nucleo-cytoplasmic distribution of YB-1 in malignant melanoma.

In line with the predominant cytoplasmic localizationobserved for both wild-type YB-1 and YB-1S102A, overexpressionof either of the two YB-1 variants in 1205LU and A375 stronglyinduced melanoma cell tumorigenicity as opposed to the phos-pho-mimic YB-1S102D and the empty vector–transduced controls.These findings further argue for a crucial role of YB-1 cytoplasmicactivity in mediating the observed oncogenic effects of YB-1overexpression. In the third cell line (MeWo), however, only theconstitutively unphosphorylated YB-1 species YB-1S102A couldpromote melanoma cell tumorigenicity, whereas this was not thecase for YB-1WT in this cell line, which exhibited not only astronger S102 phosphorylation but also an increased tendencyof nuclear localization. On the one hand, these results corrob-orate the stimulating effect associated with expression of S102unphosphorylated YB-1. On the other hand, this indicates thatthe phosphorylation and, therefore, activity status of wild-typeYB-1 strongly depends on the cellular context, and by thatprofoundly influences the functional effects associated with theYB-1 overexpression.

Interestingly, overexpression of S102 unphosphorylated YB-1not only proved to promote the tumorigenicity of melanomacells, but also strongly enhanced their migratory and invasivecapability indifferent two- and three-dimensionalmodel systems.In contrast, modulation of YB-1 levels did not affect tumor cellproliferation similar topreviousfindings characterizing YB-1 as animportant driver of sarcoma cell invasion and metastasis inde-pendent of their proliferative capacity (39, 44). In earlier work, wecould provide first evidence for an important role of YB-1 expres-sion in melanoma cell motility and invasiveness (19). Here, wecould extend thesefindings and show that theobserved functionaleffect relies on YB-1, which is not posttranslationally modifiedat S102.

In a mammary cell model, cytoplasmic S102 unphosphory-lated YB-1 has been shown to act as a translational activator ofEMT-inducing transcription factors such as Snail, Twist, andZeb2 (ZEB2) and to thereby potently stimulate the migratoryand invasive capacity of the cells (16). The epithelial-to-mes-enchymal transition is an important developmental programthat is regularly hijacked by epithelial cancer cells during themetastatic process allowing them to break away from theprimary tumor and to invade into the adjacent tissue (5).Despite its neuroectodermal origin, processes similar to anEMT have also been observed in cutaneous melanoma andshown to correlate with the metastatic capacity of primarytumors based on the acquisition of a highlymigratory and invasivephenotype (46, 47). The chicken embryo represents a valuablemodel to study physiologic epithelial-to-mesenchymal transitionincluding the biology of the neural crest. On the basis of theancestral relationship of melanoma cells with the highly motileneural crest cells, the chick embryo further provides a suitablemodel to analyze their invasive capability (48). Indeed, melanomacells have been shown to readily respond to exposure to theembryonic neural crest microenvironment by acquiring a geneexpression pattern highly reminiscent of their motile ancestors aswell as by spontaneously resuming neural crest cell migration (3,49–51). Here, we could show that overexpression of S102 unpho-sphorylated YB-1 clearly facilitates the acquisition of a mesenchy-mal-like, highly invasive phenotype in malignant melanoma cellsin response to the embryonic environment. This went along notonly with a significantly enhanced invasion of the melanoma cellsinto the surrounding host tissue, but also with a dedifferentiatedcellular state reflectedbya lackofpigmentproductionaswell asbyacell morphology reminiscent of mesenchymal cells. In line withthese results, we could observe increased protein levels of EMT-

Figure 4.Expression of S102 unphosphorylated YB-1 correlates with melanoma cell motility and invasiveness. A, Wound closure assay of 1205LU melanoma cells withinducible YB-1 knockdown (shYB-1) as well as of A375 single-cell clones with YBX1 knockout compared with the respective control cells. In case of theinducible knockdown system, cells were preinduced for 5 days with 2 mg/mL doxycycline. Sixteen hours before the scratch was introduced, hydroxyurea wasadded to the culturemedium to block proliferation. Left, cell migration into thewoundwas assessed for 48 hours and quantificationwas performed at four regions ofthe migrating front (mean� SD, n¼ 3). Right, representative pictures are shown after 0, 24, and 48 hours. B,Wound closure assay of melanoma cells with inducibleoverexpression of YB-1S102D, YB-1S102A, or YB-1WT and the appropriate control cells (EV) after a 5-day preinduction with 2 mg/mL doxycycline and a 16-hourpretreatmentwith hydroxyurea toblock cell proliferation. Cellmigration into thewoundwasmeasured after 24hours, quantification carried out for four regions of themigrating front and significant differences were evaluated by one-way ANOVA with subsequent Tukey multiple comparisons test (mean � SD, n ¼ 3; top).Representative pictures are shown (bottom). C–F, Boyden chamber–based cell migration (C and D) and cell invasion (E and F) assays analyzing the effectof YB-1 downregulation (C and E) and overexpression of YB-1 phospho-site variants (D and F) in 1205LU and MeWo cells. In case of the inducible expressionsystems, cells were preinduced with 2 mg/mL doxycycline for 5 days. The relative number of migrated (C and D) and invaded (E and F) cells was calculated onthe basis of the evaluation of 5 optical fields and normalized to the respective control cell line (mean � SD, n ¼ 3). Significance was determined with atwo-tailed unpaired Student t test or by one-way ANOVA with subsequent Tukey multiple comparisons test, where applicable.






promoting transcription factors upon overexpression of S102unphosphorylated YB-1, further indicating its implication inEMT-like processes in malignant melanoma cells.

Intriguingly, the acquisition of a mesenchymal-like phenotypeis one popular strategy exploited by tumor cells to enable anchor-age-independent growth, which in turn represents an importantprerequisite for efficient metastases formation (52). Indeed, wecould report a significantly increased colony formation in softagar upon overexpression of S102 nonphosphorylated YB-1.These results, consequently, further argue for an involvement ofS102 unphosphorylated cytoplasmic YB-1 in propagating a mes-enchymal-like state with a high propensity to metastasize inmalignant melanoma cells.

On the basis of the effects of intracellular YB-1 levels on cellmigration, invasion, and anchorage-independent growth, ourdata indicate that S102 unphosphorylated YB-1 is able to promoteall key steps of tumor metastasis in malignant melanoma. Futureinvestigations should now focus on a more detailed functional

evaluation of cytoplasmic YB-1 including the identificationand characterization of potential translational targets to fullyelucidate the molecular mechanism behind the observed effectsof unphosphorylated YB-1 in melanoma cells. Moreover, theidentification of further posttranslational modifications of theYB-1 protein apart from the S102 phosphorylation mightprovide further insight into the regulation of YB-1 localizationand functional activity.

In conclusion, we can show here for the first time that YB-1 playsan important role in melanoma cell tumorigenicity and the acqui-sition of a highly invasive phenotype reminiscent of an epithelial-to-mesenchymal transition. This effect seems to be mediated byS102 unphosphorylated cytoplasmic YB-1 and proposes a prog-nostic role of the Y-box binding protein 1 in malignant melanomaas a valid marker reflecting tumor cell aggressiveness.

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

Figure 5.

YB-1S102A promotes the invasiveness of melanoma cells in three-dimensional in vitro and in vivo model systems. A, Collagen-based invasion assays usingspheroids generated from 1205LU and MeWo melanoma cells with inducible expression of either YB-1S102D or YB-1S102A. The spheroids were implemented into acollagen type I matrix and ectopic YB-1 expression was induced by addition of 2 mg/mL doxycycline. After 4 days, spheroids were stained with Calcein-AM.Immunofluorescentmicrophotographs of two representative spheroids are depicted and the initial spheroid size before cellular invasion indicated (white circle, left).Invasion into the collagen matrix was quantified with ImageJ by evaluation of the area covered after invasion of the cells and its normalization to the initialspheroid core (mean � SD, n ¼ 10 or n ¼ 5; right). Significance was determined with a two-tailed unpaired Student t test. B,Western blot analyses evaluating theexpression of EMT-promoting transcription factors using whole-cell lysates from 1205LU, A375, and MeWo melanoma cells with inducible overexpressionof the YB-1 phospho-site variants (YB-1S102D, YB-1S102A) or harboring the empty control vector (EV). Cells were induced with 2 mg/mL doxycycline for 5 days prior toanalysis. GAPDH was detected as a loading control. C and D, Chick embryo invasion assay of MeWo cells overexpressing either YB-1S102D or YB-1S102A. Tumorformation andmelanoma cell invasion was assessed 96 hours after injection into the rhombencephalic brain vesicle. Sections of the chick embryo were stained withhematoxylin-eosin (HE; I) or Ki67-specific antibodies including a hematoxylin counter staining (II, III). Orange arrows show invading melanoma cells, blackarrows point to areas with pigment accumulation. Scale bars, 200 mm (C). Invading cells were quantified by morphologic analysis of the three sections representingthe largest tumor diameter for each embryo (mean � SD, n ¼ 4 or n ¼ 7). Significance was determined using two-tailed unpaired Student t tests (D).

Kosnopfel et al.





Authors' ContributionsConception and design: C. Kosnopfel, T. Sinnberg, C. Garbe, B. SchittekDevelopment of methodology: C. Kosnopfel, T. Sinnberg, C. Busch,H. Niessner, A. Schmitt, S.E. Dunn, B. SchittekAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Kosnopfel, T. Sinnberg, B. Sauer, C. Busch,H. Niessner, A. Schmitt, S. Forchhammer, C. Grimmel, B. SchittekAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Kosnopfel, T. Sinnberg, S. Forchhammer,P. Mertens, C. Garbe, B. SchittekWriting, review, and/or revision of the manuscript: C. Kosnopfel, C. Busch,A. Schmitt, S. Forchhammer, P. Mertens, C. Garbe, B. SchittekAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Schmitt, C. Grimmel, S. Hailfinger, C. GarbeStudy supervision: C. Kosnopfel, T. Sinnberg, B. Schittek

AcknowledgmentsThis work was supported by the Melanoma Research Network of the

Deutsche Krebshilfe e.V. (German Cancer Aid) and the German ResearchFoundation (GRK1302) to B. Schittek and by the German Research Foundation(Me1365/7-2; SFB854; to P.R. Mertens).

The authors thank Kristian Ikenberg for kindly providing the primarymelanoma tissue microarray.

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 22, 2017; revised December 20, 2017; accepted January16, 2018; published first May 9, 2018.

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Kosnopfel et al.




2018;16:1149-1160. Published OnlineFirst May 9, 2018.Mol Cancer Res Corinna Kosnopfel, Tobias Sinnberg, Birgit Sauer, et al. Invasiveness in Melanoma by Influencing EMTYB-1 Expression and Phosphorylation Regulate Tumorigenicity and

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yb-1 expression and phosphorylation regulate ... · oncogenes and tumor suppressors yb-1 expression...

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