The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary

Published OnlineFirst June 5, 2020; DOI: 10.1158/0008-5472.CAN-19-2373

CANCER RESEARCH | MOLECULAR CELL BIOLOGY

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype Niels Vandamme1,2,3, Geertrui Denecker1,2, Kenneth Bruneel1,2, Gillian Blancke1,2, Ozden€ Akay1,2,3,4,5, Joachim Taminau1,2, Jordy De Coninck1,2, Eva De Smedt1,2, Nicolas Skrypek1,2, Wouter Van Loocke2,6, Jasper Wouters7, David Nittner4,5, Corinna Kohler€ 4,5, Douglas S. Darling8, Phil F. Cheng9, Marieke I.G. Raaijmakers9, Mitchell P. Levesque9, Udupi Girish Mallya10,11, Mairin Rafferty11, Balazs Balint11, William M. Gallagher10,11, Lieve Brochez12, Danny Huylebroeck13,14, Jody J. Haigh15,16, Vanessa Andries2, Florian Rambow4,5, Pieter Van Vlierberghe2,6, Steven Goossens1,2,6, Joost J. van den Oord7, Jean-Christophe Marine4,5, and Geert Berx1,2

ABSTRACT ◥ Epithelial-to-mesenchymal transition (EMT)-inducing tran- Graphical Abstract: http://cancerres.aacrjournals.org/content/ scription factors (TF) are well known for their ability to induce canres/80/14/2983/F1.large.jpg. mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 Promotes ZEB1 Drives ZEB2 Promotes ZEB2 in human primary melanoma has been shown to correlate primary tumor growth melanoma metastatic outgrowth cell invasion and survival with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary

sites. Ablation of Zeb2 hampered outgrowth of primary melanomas Micro- in vivo, whereas ectopic expression enhanced proliferation and environment growth at both primary and secondary sites. Gain of Zeb2 expres- MITFlow sion in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear ZEB2high ZEB1high ZEB2high low low low that melanoma cells undergo a conversion in state where ZEB2 ZEB1 ZEB2 ZEB1 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These ﬁndings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination.

Signiﬁcance: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and con- versely inhibiting invasiveness, which could be of future clinical ZEB1 and ZEB2 levels impact melanoma population dynamics. relevance.

Introduction melanocytes is often associated with constitutive MAPK signaling, Cutaneous melanoma is the most aggressive form of skin cancers mostly driven by activating mutations in BRAF or NRAS, and inac- and remains a major clinical challenge. Malignant transformation of tivation of tumor suppressors such as PTEN, CDKN2A,orTP53.

1Molecular and Cellular Oncology Laboratory, Department of Biomedical Molec- Medical Center, Rotterdam, the Netherlands. 14Department of Development and ular Biology, Ghent University, Ghent, Belgium. 2Cancer Research Institute Ghent Regeneration, KU Leuven, Leuven, Belgium. 15Department of Pharmacology and (CRIG), Ghent, Belgium. 3VIB-UGent Center for Inflammation Research, Ghent, Therapeutics, Rady Faculty of Health Sciences, University of Manitoba, Winni- Belgium. 4Laboratory for Molecular Cancer Biology, VIB Center for Cancer peg, Manitoba, Canada. 16Research Institute in Oncology and Hematology, Biology, Leuven, Belgium. 5Laboratory for Molecular Cancer Biology, Depart- CancerCare Manitoba, Winnipeg, Manitoba, Canada. ment of Oncology, KULeuven, Leuven, Belgium. 6Center for Medical Genetics, Note: Supplementary data for this article are available at Cancer Research Department of Biomolecular Medicine, Ghent University and University Hospital, Online (http://cancerres.aacrjournals.org/). Ghent, Belgium. 7Laboratory of Translational Cell and Tissue Research, Depart- ment of Pathology, KULeuven and UZ Leuven, Leuven, Belgium. 8Department of N. Vandamme and G. Denecker contributed equally to this article. Oral Immunology and Infectious Diseases, and Center for Genetics and Molecular Corresponding Author: Geert Berx, CRIG and Ghent University, Technologie- Medicine, University of Louisville, Louisville, Kentucky. 9Department of Derma- park-Zwijnaarde 71, Ghent 9052, Belgium. Phone: 32-9-3313650; Fax: 32-9- tology, University of Zurich, University of Zurich Hospital, Zurich, Switzerland. 3313609; E-mail: [email protected] 10Cancer Biology and Therapeutics Laboratory, UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College, Dublin, Ireland. Cancer Res 2020;80:2983–95 11OncoMark Limited, Nova UCD, Belfield Innovation Park, University College doi: 10.1158/0008-5472.CAN-19-2373 Dublin, Belfield, Dublin, Ireland. 12Department of Head and Skin, Ghent University Hospital, Ghent, Belgium. 13Department of Cell Biology, Erasmus University 2020 American Association for Cancer Research.

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Melanoma is a highly heterogeneous tumor, composed of phenotyp- informed consent was approved by the local institutional review ically distinct subpopulations (1). While the mechanisms that con- board (IRB; EK647 and EK800). Melanoma cells were brought into tribute to intratumor heterogeneity remain poorly understood, it is culture as described previously (9). SK-MEL28 and 501MEL were without a doubt a major contributor to both intrinsic and acquired obtained from ATCC. TGFb treatment of cell lines M000921 therapy resistance and metastatic dissemination. Identification of and M010817 was performed with recombinant hTGFb, 5 ng/mL mediators of intratumor heterogeneity may therefore lead to new (PeproTech, 100-21). All cell lines were screened periodically for therapeutic avenues that exhibit longer-lasting antitumor responses, Mycoplasma contamination. prevent metastatic dissemination and identification of prognostic markers for therapy stratification. Mice Melanoma progression and therapy resistance does not rely solely Mice were kept in accordance with the institutional guidelines regard- on mutation-driven mechanisms. There is increasing evidence that ing the care and use of laboratory animals and all procedures and nongenetic mechanisms that modulate the epigenetic landscape and/ experiments were approved by the institutional review ethics committee. or transcriptional and metabolic state, also contribute to progression Mice with the following alleles have been described elsewhere: conditional fl fl and drug resistance (2). It is increasingly recognized that phenotypic Zeb2 / , Rosa-YFP, Rosa-Zeb2TG/TG-IRES-GFP, Tyr::CreERT2 and heterogeneity stems, at least partly, from the ability of melanoma cells Tyr:NRAS p53 (10–13). Conditionally, Rosa-Zeb2TG/TG-IRES-GFP– to switch back and forth between a proliferative cell state and a overexpressing mice were generated using G4 hybrid ES cells and the mesenchymal-like, invasive state. Both cell-intrinsic and -extrinsic Gateway-compatible Rosa26 locus targeting vector, as described previ- (i.e., microenvironmental) factors such as oxygen, cytokines, and ously (14). For TyrCreERT2 induction, the dorsal hair of anesthetized growth factors modulate this reversible phenotype switch. Over recent mice was removed with a (50:50 w/w) mix of beeswax and gum rosin years, the reported reversible mechanisms of melanoma invasion have (Sigma-Aldrich, 243221 and 60895-250G). 4-hydroxytamoxifen (4-OHT; supported this model (3–7). Phenotype switching is reminiscent to 70% Z-isomer, Sigma Sigma-Aldrich, H-6278) was dissolved at 50 mg/mL epithelial-to-mesenchymal transition (EMT). This phenotype switch- in DMSO at 37C under gentle agitation. 4-OHT was further diluted at ing event was also shown to promote therapy resistance and is a key 5 mg/mL in ethanol and 5 mL of the 4-OHT solution was applied using driver of metastatic spreading. In epithelial tissues, EMT-inducing a pipette on the depilated area directly. All Tyr::CreERT2 animals transcription factors, such as SNAIL and ZEB family members, in including control animals received 4-OHT treatment. Mice were exam- addition to promote invasion and metastasis may also contribute to de ined at a regular basis and sacrificed at an endpoint defined by adverse novo carcinogenesis by bypassing cellular senescence and expanding clinical symptoms, such as weight loss (>15%), a hunched posture or the cancer stem cell pool. The functional role of EMT drivers has thus multiple skin tumors (diameter > 5 mm). Quantification of the percentage been expanded beyond their ability to induce a mesenchymal tran- of mice bearing macro-metastases in each organ was visually examined scriptional program. The regulation and role of EMT-inducing tran- under the binocular at necropsy. scription factors in nonepithelial contexts such as melanocytes and melanoma may even be more diverse and pleiotropic. We have, for Histology and IHC instance, shown that ZEB2 is an important driver of normal mela- Tumors, organs, and skin were isolated and fixed overnight in 4% nocyte development and differentiation and established correlations paraformaldehyde solution, dehydrated, embedded in paraffin and cut between nuclear ZEB2 expression in the primary melanoma tumor into 5-mm sections. For histology, samples were stained with hema- and recurrence-free survival (8). We also showed that ZEB2 positively toxylin and eosin. For IHC staining, antigen retrieval was done in regulate MITF levels and/or activity during melanocyte homeostasis. citrate buffer and endogenous peroxidases were blocked with 3% H2O2 These data raised the possibility that ZEB2 may contribute to mela- in methanol. The sections were incubated with primary antibodies and noma plasticity and, as such, modulate melanoma growth and pro- stained with biotin-conjugated secondary antibodies followed by gression and metastatic dissemination in a manner that is unconven- Streptavidin-HRP based development (substrate development with tional for a EMT-TF. To test this possibility, we examined the DAB). When necessary, the signal was amplified using the Tyramide contribution of ZEB2 to melanoma development and metastatic Signal Amplification (TSA) kit (Perkin Elmer). TMA microarray slides spreading. Our data establish a critical role for ZEB2 as a key modulator containing 178 primary melanoma samples were digitally scanned on of melanoma heterogeneity and cell population dynamics in vivo. an Aperio ScanScope scanner and analyzed using Aperio algorithms. Correlation between the IHC data was compared with clinical para- meters using the two-tailed Fisher exact test and the x2 test. Immu- Materials and Methods nostaining against ZEB1 and ZEB2 on mouse sections was performed Human tissues using in-house generated rabbit and mouse mAb, respectively, GFP Tissue samples were obtained from the Department of Dermatol- was detected using a rabbit monoclonal antibody (clone D5.1 XP ogy, Universitair Ziekenhuis Gent (Ghent, Belgium), from the Depart- Rabbit mAb #2956, Cell Signaling Technology). Rabbit anti-ZEB2 ment of Pathology, University Hospital Leuven, KU Leuven, Belgium (HPA003456, Sigma) and rabbit anti-ZEB1 (Clone H-102, sc-25388, and from archival paraffin-embedded patient samples from St. Vin- Santa Cruz Biotechnology) was used for human sections. cent's University Hospital (Dublin, Ireland). All patient specimens were used in accordance with institutional and national policies at the Lentiviral transductions respective locations, with appropriate approval provided by the rel- For generation of melanoma cells overexpressing ZEB1, virus evant Ethics committees at the respective institutions. All patient- production was performed in HEK293T cells using calcium phosphate related information was anonymized. transfection with pMD2.G (envelope plasmid), psPAX2 (packaging plasmid), and pSIN-TRE-GW-3xHA ZEB1. Transduced cells were Cell lines selected by puromycin (2 mg/mL for all cell lines except 501MEL and M-series primary melanoma cell cultures have been established SK-MEL28: 1 mg/mL). The shRNA vectors are pLKO1.5-based from from surplus material from cutaneous melanoma metastases. Written Sigma, Mission shRNA series.

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siRNA and plasmid transfections centrifugal filters (Millipore, UFC510096) in two consecutive diluting For RNAi, we used siRNA pools (Dharmacon) and a scrambled washes. cDNA synthesis was performed with iScript advanced cDNA siRNA pool (Dharmacon) as a control. Transfections were performed synthesis kit (Bio-Rad, 172-5038) following the manufacturer's with HiPerfect (Qiagen) according to the manufacturer's instructions instructions. Quantitative PCR was done using the SYBR Master Mix (siZeb1: Dharmacon, M-051513-01-0005; siZeb2: M-059671-01-0005; Kit SensiFast SYBR No-Rox Kit (Bioline, CSA-01190) for the genes of siZEB1: M-006564-02-0005; siZEB2: M-006914-00-0005). Plasmids interest and reference genes. Plates were run on the LightCycler 480 carrying myc-tagged ZEB2, HA-tagged MITF, or empty cDNA expres- (Roche). The average threshold cycle of triplicate reactions was used sion cassettes were transiently transfected. 501MEL cells were trans- for all subsequent calculations using the DCt method, calculated in fected using FugeneHD (Promega E-231): cells were seeded in a 6-well qBasePLUS (BioGazelle). The following reference genes were used for to reach 75% confluency prior to the transfection in 3 mL growth a first read-out: Calm2, Matr3, Hmbs, Rpl13a, Oaz1, Gapdh, Ywhaz, medium with a 4:1 ratio DNA:Lipofectant. Subsequently, for each Tbp, Cox4i1,andEef1a for mouse, EEF1A1, DCAF6, OAZZ1, well, 3.3 mg total DNA was diluted in 160 mL OptiMEM, 13 mL CALM2, MATR3, COX4I1, RPL37A, FTL, SH3KBP1, TPT1 for Fugene HD reagent was added, and vortexed briefly, and the human. Subsequently, the most stable reference (housekeeping) solution was incubated at room temperature during 5 minutes, genes from this set were determined via qBaseþ (geNorm) prior and 150 mL of the mixture was added dropwise on the cells (in 3 mL to repeating the experiment. growth medium). MITF-VP16 chimera, a transcriptionally more active MITF-derivative, and MITF cDNA expression constructs RNA sequencing were kindly provided by Prof. Vachtenheim (Department of Tran- Melanoma cell cultures were established from patient biopsies. scription and Cell Signaling, Charles University, Prague, Czech Patients gave their informed consent and was approved by the local Republic). IRB (EK647 and EK800). RNA from the melanoma cell cultures were isolated with the QIAGEN RNeasy Kit. RNA capture was performed Electric cell surface impedance sensing with TruSeq RNA Library Prep Kit v2 (Illumina) and sequenced on a 8W10E array plates (Applied Biophysics) were washed and treated HiSeq4000. RNA counts were quantified from single-end reads using with 10 mmol/L cysteine for 30 minutes at room temperature. Next, for STAR aligner (15). Normalization of RNA counts was performed with HUVEC assays, array plates were coated with laminin before cell edgeR (16). RNA sequencing (RNA-seq) data of TGFb treatment seeding. HUVECs (Lonza) were seeded and challenged with melano- human melanoma cell lines available at GEO database (GSE148767). ma cells once stable impedance (and visible confluency of the HUVECs) was achieved. As a control, heat-inactivated cells (10 min- Single-cell RNA-seq analyses utes at 55C) in conditioned medium were included. Next, arrays were ZEB1 and ZEB2 expression values (TPMs) were inferred from mounted onto the array holder connected to the electric cell surface single-cell RNA-sequencing analyses (scRNA-seq) data (GSE115978) impedance sensing (ECIS) module and placed in a standard incubator from human malignant melanoma cells and log-normalized expres- ¼ at 37 C and 5% CO2 throughout the experiment. All impedance sion values represented for n 1,951 cells. measurements were performed in the multifrequency measurement Seurat R package (version 3.1.3) was used to log normalize the data, mode. perform dimensionality reduction analysis, clustering of the cells and performing differential gene expression. We extracted the melanoma RNA isolation cells that were annotated by Jerby-Arnon and colleagues (17). How- þ þ RNA was extracted from cell cultures by using RNeasy extraction ever, we deleted a small cluster of contaminating CD3E CD45 T columns (Qiagen) or TRIsure lysis buffer (BioLine) according to the cells that were initially annotated as melanoma cells in the original manufacturer's guidelines. For tumors, after collecting, the tissue is dataset. 526 of 1,951 cells were double negative for ZEB1 and ZEB2. subsequently disinfected in a povidone–iodine solution (iso-Betadine) Remaining cells came from 23 patients. 1,373 cells showed ZEB2 and washed twice in 70% ethanol and three times in PBS. Skin and 238 cells ZEB1 expression. ZEB1 and ZEB2 expression per single preparations were carefully dissected, followed by removal of fat tissue cells are significantly anticorrelated, as shown by Spearman correlation using a scalpel. Tissue pieces were incubated in RNAlater (Ambion, (r ¼0.28; P < 2.2E-16). AM7021; BioLine, BIO-38032) at 4C overnight. Subsequently, the pieces were put in a petridish in 1 mL TRISure on ice and cut into small SRB proliferation assay pieces. Subsequently, the pieces were homogenized using a mechanical The proliferative capacity of melanoma cells was analyzed by mixer followed by passing the sample five times through a 21-gauge performing the Sulforhodamine B (SRB) proliferation assay. Briefly, needle. To reduce the carryover of RNases during further processing, cells were seeded at 3,000 to 7,000 cells per 96-well and at day 0 and 4, the homogenized sample is centrifuged for 15 minutes, 20,000 g,4C the cells were fixed with 30% (wt/vol) trichloroacetic acid for 1 hour, to eliminate debris. The supernatant is transferred to a new RNAse- and stained for 30 minutes at room temperature with 0.4% SRB in 1% free Eppendorf tube and incubated for 5 minutes at room temperature. acetic acid. Excessive dye was washed off with 1% acetic acid and After phase separation by adding 200 mL chloroform and vigorous dissolved in 10 mmol/L Tris buffer (pH 10.5). The plates were shaking, 250 mL from the aqueous upper phase was added to 500 mL measured with a microplate reader (Bio-Rad, Eke, Belgium) at 570 nm. cold RNase-free ethanol. Samples were snap-frozen in liquid nitrogen Day 0 was used as a reference sample. and stored at 80C. Western blot analysis qRT-PCR Tissues were mechanically separated from the dermis and ground RNA was treated with 1 U of RNAse-free DNase RQ1 (Promega) into small pieces prior to lysis, culture cells were immediately lysed per mg RNA for 30 minutes at 37C in appropriate buffer. DNAse was in Laemmli-lysis buffer (50 mmol/LTris-HCl pH 6.8, 10% glycerol, inactivated by incubation in Promega stop solution for 10 minutes at 2% SDS) using standard practices. After sonicating and centrifuging þ 65C. Bulk Mg2 was removed by using Amicon ultra 0.5-mL the samples, 20 mg of protein was separated on a acrylamide gel

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and transferred to a polyvinylidene difluoride membrane. Membranes this possibility, we engineered a conditional ZEB2 allelic series onto the were incubated with primary antibodies and appropriate HRP-labeled NRASQ61K-driven mouse melanoma background (Fig. 1C). The secondary antibodies (GE Healthcare). Primary antibodies were dilut- Tyr::NRASQ61K;TyrCreERT2;Trp53lox/lox mice (henceforth NRASQ61K ed in 3% milk. Detection was performed with the Western Lightning p53 / mice) were intercrossed with mice carrying a Zeb2 condi- Chemiluminescence Reagent Plus Kit (PerkinElmer) or the Immobi- tional transgenic allele, Rosa26-(loxP-STOP-loxP)-Zeb2. Note that lion Western HRP Substrate (Millipore). the Zeb2 open reading frame is followed by an IRES-EGFP reporter sequence, GFP expression can therefore be used to fate map trans- Colony formation in soft agar genic cells. Mice carrying two copies of this allele are thereafter Petri dishes (60-mm diameter) were coated with 0.5% Bacto agar referred to as Rosa-Zeb2. In addition, the Tyr::NRASQ61K;Tyr- (Difco) in growth medium. A suspension of 1,104 cells in growth CreERT2;Trp53lox/lox mice have also been intercrossed with mice fl fl medium containing 0.35% Noble agar (Difco) was seeded on top of the carrying a conditional Zeb2 knockout allele (Zeb2 / ;refs.10,14).To agar coating and then covered with growth medium. Fresh medium modulate ZEB2 expression the compound mice were exposed to was applied weekly. Cell colonies were allowed to form over a time topical application of 4-hydroxytamoxifen (4-OHT) onto their back period of 55 days, after which, they were stained for 1 hour with 0.005% skin. Nevus formation and melanoma development was monitored crystal violet (Sigma) in PBS and photographed. weekly. Strikingly, Zeb2 ablation dramatically attenuated nevus and mel- Invasion and migration assay anoma outgrowth (Fig. 2A and B). Moreover, targeting Zeb2 in a An QCM ECMatrix 24-well (8 mm) Fluorimetric Cell Invasion NRASQ61K p53 / background delayed melanoma initiation (Fig. 2C). Assay kit (Chemicon; Sigma-Aldrich) was used according to the Interestingly, all analyzed pigmented tumors that arose in NRASQ61K fl fl manufacturer's protocol. An insert polycarbonate membrane with a p53 / Zeb2 / mice contained only Zeb2 wild-type melanoma cells, pore size of 8 mm) was coated with a thin layer of ECMatrix. Cells were indicating that these lesions originated from melanoma cells that seeded in the insert (top chamber) at a density of 25 103 cells/well in escaped Zeb2 ablation (Supplementary Fig. S1A and S1B). As such, serum-free RPMI. RPMI medium supplemented with 10% FCS was no genuine Zeb2-knockout melanoma tumors are formed. These added to the bottom chamber. Following 24-hour incubation, invading results indicate that ZEB2 is required for the growth and expansion cell numbers were determined via fluorescent read-out. For migration of primary NRAS-driven melanoma. In contrast, ectopic ZEB2 over- assays, similar inserts were not coated with ECMatrix. expression accelerated melanoma initiation, decreased the latency and increased melanoma penetrance. ZEB2 expression did not specifically Statistical analyses affect metastatic spread on a p53-null–mutated background. Results were evaluated using the statistical tests and indicated with P values in the figure legends. P < 0.05 was considered statistically Zeb2 drives a melanoma “proliferative” transcriptional significant. Survival curves and metastasis-free survival were analyzed program by Kaplan–Meier analysis, which were compared by log-rank To assess whether ZEB2 can promote melanomagenesis in the (Mantel–Cox–Kaplan Meyer Plot). GraphPad Prism 7 was used to presence of functional p53, we conditionally overexpressed ZEB2 in perform log-rank (Mantel–Cox), Student t test, two-way ANOVA a Tyr::NRASQ61K (p53 wild-type) background. Ectopic expression of followed by post-hoc Tukey HSD (SRB assays). R (http://www.cran.r- transgenic ZEB2 and GFP was confirmed via immunoblot analysis project.org) was used to perform Fisher exact test and the x2 test (for (Supplementary Fig. S2A). Forced expression of ZEB2 in Tyr:: TMA staining), Benjamini–Hochberg Multiple Testing Correction NRASQ61K Rosa26-Zeb2 mice (NRASQ61K Rosa-Zeb2) did not signif- (adjusted) P value (for bulk RNA-seq data). Differential gene expres- icantly affect tumor latency or incidence (Supplementary Fig. S2B). sion during single-cell analysis was performed via a nonparameteric Ectopic ZEB2 expression is therefore not sufficient to transform Wilcoxon rank sum test in Seurat. NRASQ61K–expressing melanocytes. Molecular analysis of NRASQ61K and NRASQ61K Rosa-Zeb2 tumor cells showed an increased expression in genes that belong to the previously described melanoma “prolifer- Results ative” gene signature including Mitf, Mc1r, and Sox10 (19, 20). This Zeb2 is required for melanoma growth and development increase occurred concomitantly to a decrease in expression of Zeb1 ZEB2 expression in human melanoma is heterogenous (Fig. 1A). expression (Fig. 3A). RNAi-mediated Zeb2 knockdown in melanoma Such a heterogeneous pattern was also observed in lesions from the cell lines Mel3 and Mel6 derived from the Rosa-Zeb2 melanoma mouse previously established NRASQ61K-driven mouse model of melanoma model attenuated and reversed this gene signature, as illustrated by a (Fig. 1B). Note that NRAS is consistently altered in 20% of melanomas decrease in Mitf levels (Fig. 3B). This was accompanied by an and is the second most common oncogenic driver mutation (18). increase in Zeb1 levels. As soon as cells regained Zeb2 expression Constitutive melanocyte-specific expression of one copy of the acti- (6 days after transfection of the siRNA molecules), the “proliferative” vated form of human NRAS (NRASQ61K) triggers melanoma devel- gene signature reemerged, highlighting the reversibility and dynamic opment in a small fraction of Tyr::NRASQ61K mice. The mouse skin is nature of the ZEB2-driven transcriptional program (Fig. 3C). Impor- hyperpigmented and reminiscent of patients with a giant congenital tantly, Zeb2 depletion led to a decrease in the proliferation potential of nevus (GCN). These nevi are large pigmented lesions that frequently primary melanoma cell lines derived from 5 different NRASQ61K Rosa- progress to melanoma. On average 15%–20% of Tyr::NRASQ61K mice Zeb2 mice and the B16 melanoma cell line (Supplementary Fig. S3A) develop melanomas within 1 year in the absence of secondary genetic and induces a melanoma “invasive/mesenchymal-like” gene expres- alterations (11). Concomitant loss of p53 or p16INK4A functionally sion in B16 cells (Supplementary Fig. S3B; refs. 19, 20). Zeb2 depletion, increases the incidence to nearly 100% and shortens the latency Mitf depletion or ectopic Zeb1 expression in B16 cells promoted an significantly (11). The biological significance of the varying ZEB2 invasive and migratory phenotype in vitro (Supplementary Fig. S3C levels remains unclear. We hypothesized that graded levels of ZEB2 and S3D). A transendothelial migration assay as described by Tar- may associate with distinct melanoma phenotypic properties. To test antola and colleagues (21) and Keese and colleagues (22) showed how

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A Human melanoma B Mouse melanoma Tyr::NRASQ61K

Tyr::NRASQ61K p53 fl/fl 50 µm 200 µm

50 µm 50 µm C Zeb2 Group level Full genotype Tyr::NRASQ61K;p53fl/fl ;Rosa-Zeb2 Tyr::NRASQ61K;TyrCreERT2;Trp53fl/fl ;Rosa26mZeb2/Zeb2 Tyr::NRASQ61K;p53fl/fl Tyr::NRASQ61K;TyrCreERT2;Trp53fl/fl ;Zeb2+/+ Tyr::NRASQ61K;p53fl/fl ;Zeb2fl/fl Tyr::NRASQ61K;TyrCreERT2;Trp53fl/fl ;Zeb2fl/fl

Figure 1. ZEB2 is heterogeneously expressed in melanoma. A, ZEB2-stained human melanoma samples exhibit heterogeneity in ZEB2 expression. Top and bottom selected rectangles depict areas with respectively strong and weak nuclear ZEB2 expression. Micrograph images were taken with 4/0.1 and 20/0.4 objective. B, ZEB2-stained sections from Tyr::NRASQ61K and Tyr::NRASQ61K p53fl/fl mouse melanoma models. C, Overview of mouse models with graded Zeb2 levels on a Tyr::NRASQ61K p53fl/fl-targeted background.

ZEB1 promotes melanoma cells to attach to and cause retraction of the levels was observed in a fraction of the melanoma cells thus recapit- endothelial monolayer (Supplementary Fig. S3E). We validated our ulating the naturally occurring cellular heterogeneity observed in the conclusions in a panel of human melanoma cell lines. Lu1205 mel- NRASQ61K wild-type Zeb2 melanoma lesions. Because, the Zeb2 trans- anoma cells show increased or attenuated invasive capacity upon gene does not contain untranslated regions, we concluded that silenc- respectively enhanced or reduced ZEB1 expression (Supplementary ing of its expression is likely due to a (post)translational regulatory Fig. S3F). Upon overexpression of ZEB1 in the human 501MEL event. Interestingly, tumor areas that had lost Zeb2-transgenic expres- melanoma cell line, we readily observed a dramatic growth attenuation sion became strongly positive for Zeb1. This was in keeping with the (Supplementary Fig. S3G). Three days following doxycycline admin- above in vitro and with our previous observation that Zeb2-deﬁcient istration, the 501MEL iZEB1 cell line altered predominantly genes melanocytes acquire a dedifferentiated stem cell phenotype exhibiting associated with the proliferative phenotype, whereas the invasive gene elevated Zeb1 levels (8). repertoire is not induced yet to the fullest. ZEB1 induction shifts the cells toward an invasive gene signature after 6 days, similar to ZEB2 Zeb2 promotes the growth from micro- to macrometastasis depletion (Supplementary Fig. S3G). ZEB1-mediated depletion of Complete necropsy of NRASQ61K and NRASQ61K Rosa26-Zeb2 MITF protein levels and increased migratory capacities were con- tumor-bearing mice revealed the presence of large pigmented pulmo- ﬁrmed in the differentiated melanoma cell lines 501MEL and nary metastases that had colonized the lung parenchyma, the liver, and SK-MEL28 (Supplementary Fig. S3H and S3I). muscle tissue in a large fraction of Zeb2 transgenic mice (n ¼ 26), We next assessed whether the ectopically induced Zeb2 transcrip- whereas no macrometastases could be observed in the control animals tional program could be spontaneously reversed in the in vivo setting. (n ¼ 19; Fig. 4A). In the lung parenchyma of NRASQ61K control We took advantage of the GFP-reporter gene expression to fate map animals, we did observe small clusters of melanoma cells, only visible the Zeb2-transgenic cells in primary NRASQ61K Rosa-Zeb2 melanoma through microscopic analysis ofhematoxylinandeosin(H&E) (Fig. 3D). Remarkably, an attenuation of ZEB2 transgenic protein sections. Although such pulmonary micrometastases were detected

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A Tyr::NRASQ61K p53 fl/fl Figure 2. ZEB2 is required for melanoma develop- Zeb2fl/fl Zeb2+/+ Rosa-Zeb2 ment and growth on a p53-null background. A, Morphologic appearance of skins of mice with 32 weeks of age. B, H&E staining of skin section at defined time points posttreatment. C, Kaplan– Meier tumor-free survival curves for Tyr::NRASQ61K p53fl/fl Rosa26-Zeb2 (n ¼ 13) or Tyr::NRASQ61K p53fl/fl Zeb2fl/fl mice (n ¼ 15) and their wild-type controls (respectively, n ¼ 17 and n ¼ 21). Tumor-free percentages were compared using the log-rank (Mantel–Cox) test.

B Tyr::NRASQ61K p53 fl/fl Zeb2fl/fl Zeb2+/+ Rosa-Zeb2

12 weeks

22 weeks

32 weeks

C Tyr::NRASQ61K p53 fl/fl

Zeb2+/+ (n = 21) Rosa+/+ (n = 17) Zeb2fl/fl (n = 30) Rosa-Zeb2 (n = 13) 100 Log-rank 100 Log-rank P = 0.01 P = 0.01

50 50

0 0

Percentage tumor-free mice 0204060

Percentage tumor-free mice 0204060 Weeks Weeks

in 88% of the control animals, none of them was able to succesfully pulmonary macrometastases (>50 lesions), in the remaining grow to a visible metastatic lesion seen with the naked eye (Fig. 4B). 54%, only micrometastases were detected through the analyses of Such micrometastases were not observed in other organs. From H&E sections (Fig. 4C). Collectively, these ﬁndings indicated that the NRASQ61K Rosa26-Zeb2 mice,46%ofthemicedidhavemany ectopic Zeb2 expression facilitates the outgrowth of “dormant”

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Figure 3. A GFP Zeb2 Zeb1 ZEB2 promotes reversible expression of differ- P = 0.001 P = 0.03 P = 0.05 10 3 entiation markers. A, Relative mRNA expres- 8 sion levels for Zeb2, GFP, Zeb1, Sox10, Mitf,and 8 Mc1r in melanoma samples. Data were com- 6 pared by using unpaired Student t test and are 2 6 presented as means 95% SD. B, Relative 4 mRNA expression of phenotype-specific genes 4 96 hours after siRNA treatment against Zeb2 in 1 two cell lines generated from two different 2 2 Tyr::NRASQ61K Rosa26-Zeb2 mouse melano- Q61K Relative expression (mRNA) mas. C, Relative mRNA expression of pheno- Relative expression (mRNA) Relative expression (mRNA) Tyr::NRAS ; 0 00 type-specific genes in cell line Mel6 after siRNA Rosa26+/+ withdrawal and a Zeb2 recovery phase of Q61K 6 days after knockdown. Data were compared Mitf Sox10 Mc1r Tyr::NRAS ; by using Student t test and are presented as P = 0.01 P = 0.0005 P = 0.001 Rosa26-Zeb2 30 6 80 averages þ SD. D, IHC analysis for GFP, ZEB2, and ZEB1 in primary and metastatic melanoma samples from Tyr::NRASQ61K Rosa26-Zeb2 mice 20 4 60 to evaluate heterogeneity of endogenous and exogenous Zeb2 expression. , P < 0.05; , P < 10 2 40 0.005; , P < 0.0005; ns, nonsignificant.

0 0 20

Relative expression (mRNA) −10 Relative expression (mRNA) −2 Relative expression (mRNA) 0 B Mel3 cell line C Mel6 cell line (Tyr::NRASQ61K; Rosa26-Zeb2) (Tyr::NRASQ61K; Rosa26-Zeb2)

5 5 ** siCon siCon * * 4 siZeb2 4 siZeb2 Recovery Zeb2 **

3 3 ns ns ns ns ** ** ** *

2 2

1 1

** ***

Relative expression (mRNA) *** Relative expression (mRNA) *** *** 0 0

Mitf Mitf GFP Zeb1 GFP Zeb1 Sox10 Wnt5a total Zeb2flag-Zeb2 total Zeb2total Zeb2 D Tyr::NRASQ61K; Rosa26-Zeb2 Primary melanoma Metastasis

GFP

Zeb2

Zeb1

40 µm

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disseminated melanoma cells and promote the formation of suc- ZEB1high cells have a clear loss of proliferation/differentation markers cessful metastases. such as MITF, TYR, PMEL, and SOX10 and upregulation of invasion markers such as COL1A2, TPM2, ALDH1A3, and TCF4 (Fig. 6E). ZEB2 modulates the proliferative–invasive melanoma phenotypic switching TGFb promotes ZEB switching RNA-seq analysis of a large cohort of human primary melanoma TGFb1 has been shown to promote melanoma switching from a cultures revealed a strong inverse correlation between expression levels proliferative to an invasive state (23, 24). Accordingly, exposure of two of ZEB2, MITF, and the MITF-target gene TYR, and an inverse early-passage melanoma cultures (M010817 and M000921), expres- correlation between MITF and ZEB1 expression. ZEB1 expression sing a high ZEB2/ZEB1 ratio to TGFb1 increased ZEB1 and fibronectin was correlated with AXL, WNT5A, TCF4, CDH2, and FN1 expression expression, while decreasing MITF levels, both at protein and RNA (Fig. 5A). These correlations were confirmed via qRT-PCR on a levels (Fig. 7A and B). This TGFb1 treatment shifted the gene selection of cultures and a panel of human melanoma cell lines expression profiles of these cells toward an invasive/mesenchymal- (Fig. 5B). ZEB2-positive melanomas exhibit a differentiated and like phenotype, as illustrated by the upregulation of genes such as AXL proliferative gene signature, whereas ZEB1 melanomas express the and WNT5A (Fig. 7A; Supplementary Fig. S5). Interestingly, while invasive gene signature, indicating that melanoma switch between a ZEB2 mRNA levels were not affected by this treatment in the M010817 proliferative MITFhighZEB2highZEB1low state and an invasive and cells 48 hours after TGFb1 exposure, a clear drop in ZEB2 protein metastatic ZEB1highZEB2lowMITFlow state. Upon RNAi-mediated levels was observed, suggesting the involvement of a posttranslational silencing of ZEB2, using two independent shRNAs (sh1 and sh2 mechanism in ZEB2 downregulation. Withdrawal of TGFb1 after ZEB2), SK-MEL28 melanoma cells and 501MEL cells loose MITF 96 hours, in combination with inhibitor SB431542 (inhibiting kinase expression and exhibit, a severe growth retardation, while gaining activity of the TGFb1 receptors ALK4, -5 and -7) to block all positive expression of invasive markers including ZEB1 (Fig. 5C and D; autocrine feedback loops, was able to revert this phenotype after 7 days. Supplementary Fig. S4A and S4B). ZEB2 depletion also hampers the These data show that well-established phenotype switching inducer, ability of these melanoma cells to form colonies in vitro (Fig. 5E; TGFb1, is sufficient to revert the ZEB2/ZEB1 ratio, an event that is Supplementary Fig. S4C). Upon restoration of Zeb2 expression fol- likely to directly contribute to phenotype switching. lowing doxycycline withdrawal, the cells recovered a proliferative phenotype, further confirming the reversibility of the phenotype switch (Fig. 5E). RNAi-mediated depletion of ZEB2 or MITF in Discussion 501MEL cells, results in a strong proliferation defect (Supplementary We previously showed that ZEB2 is a gatekeeper of melanocyte Fig. S4D). The decreased proliferation in siZEB2-treated 501MEL cells, development and differentiation (8). Here, we provide evidence that but not siMITF-treated cells, can partially be rescued by siZEB1 ZEB2 promotes the growth of both primary and metastatic melanoma treatment (Supplementary Fig. S4D and S4E). Reexpression of the lesions, while suppressing ZEB1 expression and thereby an invasive/ MITF-VP16 chimera, a transcriptionally more active MITF-derivative mesenchymal-like transcriptional program. Genetic ablation of Zeb2 is sufficient to restore growth in ZEB2/MITF-depleted cells. In con- indeed hampered outgrowth of primary melanoma in mice, whereas trast, ZEB2 reexpression is not able to rescue growth inhibition upon ectopic expression enhanced melanoma proliferation and the growth MITF depletion. This supports the model that ZEB2 is required for of melanoma at both primary and secondary sites. Therefore, Zeb2 MITF functionality and suppression of the alternative ZEB1 state but is cannot be regarded as a genuine tumor suppressor, as suggested unable to compensate for MITF loss, while ZEB1 may act downstream previously (25). In support of our findings, ZEB2 was instead recently of the ZEB2/MITF interplay and contributes to the phenotype switch identified as an “Achilles heel” of melanoma growth in a genome-wide when ZEB2/MITF functionality is compromised. Taken together, our RNAi-based loss-of-function screen aiming at establishing a “cancer data identified ZEB2 as a key modulator of the proliferative to invasive dependency map” (26). Here, we show how strong nuclear ZEB2 phenotype switch both in mouse and human melanoma cells. To expression in human primary melanoma was associated with lower further substantiate these findings, we examined the correlation Breslow index and increased metastasis-free survival. An increased between ZEB2 expression and Breslow Depth of Invasion index. This metastatic burden in mice ectopically expressing Zeb2 has, at first, been is used as a standard prognostic factor in melanoma pathology difficult to reconcile with these observations. However, we noticed that measuring how deep the tumor cells have invaded the dermis and although presence of increased Zeb2 mRNA levels, ZEB2 protein levels the surrounding tissue in human melanoma biopsies. A robust asso- were not elevated in all GFP-tagged transgenic cells, indicating that ciation between intense nuclear ZEB2 immunoreactivity and lower specific microenvironmental cues were able to destabilize ZEB2 pro- Breslow score was identified, thus confirming that loss of ZEB2 in tein expression in a fraction of the cells within the primary tumor. primary melanoma is associated with increased invasiveness in human We hypothesize that the transgenic cells that lose ZEB2 expression patients (Fig. 6A and B). Moreover, IHC expression analyses on are the cells that leave the primary lesions and eventually contribute to human melanoma samples made clear that ZEB2 is attenuated and the seeding of metastasis-initiating cells in various distant organs. ZEB1 nuclear expression is enhanced in the invasive front at the Interestingly, in control Tyr::NRASQ61K mice, only microscopic clus- deepest margins of the tumor, strongly associating ZEB1 with the ters of tumor cells were observed at distant sites. In contrast, multiple invasive behavior of human primary melanoma cells (Fig. 6C). To macroscopic colonies could be detected in Zeb2 transgenic mice. We assess whether the ZEB1- and ZEB2-expressing cells are distinct therefore propose that increased Zeb2 expression facilitates the awak- subpopulations in human melanomas, we analyzed single-cell expres- ening of otherwise dormant ZEB2-negative cells. As such, the observed sion data of human melanoma (GSE115978; ref. 17). This analysis oscillation of both endogenous and transgenic ZEB2 levels within the made clear that the majority of the melanoma cells (1,951 cells coming primary tumor suggests that the increased metastatic burden may be from 33 patients analysed by scRNA-seq) are ZEB2high ZEB1low and due to enhanced outgrowth at distant sites, rather than the promotion that ZEB2 and ZEB1 are clearly anticorrelated as shown by Spearman of primary tumor invasion and metastasis. The ZEB2-mediated correlation (r ¼0.28, P < 2.2E-16; Fig. 6D). Interestingly, ZEB2low growth effect at distant sites is reminiscent of the enhancement of

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ZEB2 Driver of Melanoma Metastasis

A B Tyr::NRASQ61K; Tyr::NRASQ61K; Rosa26+/+ Rosa26-Zeb2 Frequency of metastasis (n = 19) (n = 26) Lung 100 80 Metastasis 60 40 no metastases 20 detected Lung 0 Percentage of mice Control Rosa- Zeb2

Liver 100 80 60 Metastasis 40 No metastases 20 detected Liver 0 Percentage of mice Control Rosa- Zeb2

Muscle 100 80 60 Metastasis 40 No metastases 20 detected Muscle 0 Percentage of mice Control Rosa- Zeb2

Tyr::NRASQ61K; Rosa26+/+ (n = 19)

Tyr::NRAS Q61K; Rosa26-Zeb2 (n = 26)

Figure 4. Forced Zeb2 expression enhances metastatic outgrowth on a Tyr::NRASQ61K–targeted background. A, Frequency of metastases (one or more) detected in Tyr:: NRASQ61K p53fl/fl Zeb2fl/fl mice and their wild-type controls. B, Photographs of liver, lung, and muscle tissue of control mice and metastasis-bearing Tyr::NRASQ61K p53fl/fl Zeb2fl/fl mice. C, Micrograph of lungs of Tyr::NRASQ61K p53fl/fl Zeb2fl/fl mice and their wild-type controls (left) and H&E sections (right).

melanoma cell growth at primary cutaneous sites. When melanoma entiated and proliferative phenotype, forced expression of Zeb2 did not cells reach growth-permissive microenvironments, Zeb2 expression is necessarily eliminate phenotype-switching abilities in our mouse reinstated to enable expansion. Melanoma cells that acquire elevated model of melanoma. Indeed, to achieve local invasion, tumor cells Zeb2 expression have a growth advantage outcompete Zeb2-negative may temporarily abolish ZEB2 protein levels, which results in an cells and contribute to the development of macrometastases, an event oscillation from ZEB2 toward ZEB1, even in tumors with transgenic that is generally considered as one of the severe rate-limiting steps ZEB2 expression. Phenotype switching involving a dual role of ZEB2 in during metastasis and that is likely more efﬁciently bypassed via the melanoma development shows striking parallels with the need for ZEB2 transgenic expression (27). Although ZEB2 reinforces a differ- reversibility of EMT in metastasis of carcinomas (28, 29). Metastases of

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Figure 5.

ZEB2 affects the phenotypical switch between proliferation and invasion in human melanoma A, Log2 (fold change) expression values determined via RNA-seq analysis of a large cohort of human primary melanoma cultures. B, Log2 (fold change) expression values of phenotype-specific genes in short-term melanoma cultures (passage < 5; left) or melanoma cell lines (right). Differential expression determined via qRT-PCR and normalized to the median of the expression levels and represented in a heatmap. C, Immunoblot analysis of ZEB2, MITF, and ZEB1 in SK-MEL28 cells transduced with lentiviral vectors expressing doxycycline-inducible shRNA directed against ZEB2. D, Relative proliferation after ZEB2 knockdown in SK-MEL28 melanoma cells measured via SRB assay. Data are represented as mean þ SEM and compared using two-way ANOVA, post hoc Tukey HSD. E, Left, images of colony formation in doxycycline-induced SK-MEL28 cells. Cells were untreated (DOX) or pretreated (þDOX) with doxycycline and grown in soft agar in the absence (DOX) or presence (þDOX) of doxycycline. After 15 days, cell colonies were imaged using an inverted microscope. Right, quantification of colony formation of cells pretreated with doxycycline for 7 or 14 days, grown in soft agar in the presence (on DOX) of absence (withdrawal) of doxycycline in the culture medium. Data were compared by using unpaired Student t test and are presented as means 95% SD. , P < 0.05; , P < 0.005; , P < 0.0005; ns, not significant.

the most common human carcinomas often exhibit a redifferentiation. regards to this, it is generally accepted that organ colonization by Reexpression of differentiation markers such as E-cadherin, induced circulating tumor cells is the most complex and rate-limiting step in by microenvironmental signals, confers a selective advantage and the metastatic cascade. Our data also establish antagonistic roles for enhances communication with the neighboring parenchyme (30). In ZEB2 and ZEB1, two members of one EMT TF family. We provide

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A B Primary melanoma TMA (n = 178)

High ZEB2 expression level (above avg) Low ZEB2 expression level (below avg) Melanoma patients Classification Breslow’s Classification Breslow’s depth of invasion <0.75 mm depth of invasion >4 mm

C ZEB2 ZEB1 Skin surface

*: Deepest margin * of the tumor * Subcutaneous adipose

DE− ZEB2high ZEB1high ρ = −0.28 ZEB2+ ZEB1 P > 2.2E-16 ZEB2+ ZEB1+ ZEB2− ZEB1+ ZEB2 expression

650 0 ZEB1 expression cells 0

1,800 cells

Figure 6. High ZEB2 levels are associated with a recurrence-free survival and lower Breslow index of invasion in human melanoma. A, ZEB2-stained tissue microarray slides containing 178 primary melanoma samples were digitally scanned on an Aperio ScanScope scanner. Digital slides were analysed using Aperio algorithms. ZEB2 protein expression values were determined using an automated image analysis approach (IHC-MARK, Oncomark) designed to quantify IHC stained slides. The correlation between the IHC data (ZEB2 expression below or above average expression) was compared with the Breslow category using the two-tailed Fisher exact test and the x2 test. B, Representative ZEB2-stained human melanoma samples. Micrograph images were taken with a 10/0.25 objective. C, ZEB2- and ZEB1-stained human melanoma samples with histologic assessment of the invasive regions and the deepest margin of the tumor. D, ZEB1 and ZEB2 expression values (TPMs) were inferred from scRNA-seq data (GSE115978) from human malignant melanoma cells and log-normalized expression values are represented for n ¼ 1,951 cells. Five- hundred and twenty-six of 1,951 cells were double negative for ZEB1 and ZEB2. Remaining cells came from 23 patients. The 1,373 cells showed ZEB2 expression and 238 cells showed ZEB1 expression. ZEB1 and ZEB2 expression per single cells were significantly anticorrelated, as shown by Spearman correlation (r ¼0.28), P < 2.2E-16. The histogram next to the axis reflects the number of cells in specific ranges of expression levels for ZEB2 (left) or ZEB1 (bottom). E, Heatmap representing top 100 differentially expressed genes between Zeb1high and Zeb2high cells, performed via a nonparametric Wilcoxon rank sum test.

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Figure 7.

TGFb treatment mediates the switch from ZEB2 to ZEB1 and induces an invasive gene signature. A, Relationship of log2 (fold change) RNA-seq expression (base mean) and statistical signiﬁcance [Benjamini–Hochberg multiple testing correction (adjusted) P value] of differently expressed genes after 96-hour TGFb treatment with three biological replicates of short-term melanoma cultures from two independent cell lines (M010817 and M000921), represented in volcano plots. B, Immunoblot analysis of several markers involved in ZEB-switching in M010817 and M000921 cells after addition of TGFb1 to the culture medium and reversion of the phenotype by administration of the TGFb inhibitor SB431542, inhibiting kinase activity of TGFb receptors ALK4, -5, and -7.

evidence that the ZEB2/ZEB1 ratio is a critical determinant of the TGFb activates both EMT inducers in epithelium-derived malig- melanoma phenotypic state. A high ratio is associated with prolifer- nancies. However, because the role of ZEB2 in carcinoma progres- ation, whereas a low ratio favors invasion and migration. The strict sion is unrelated to the functions of ZEB2 in neural crest–derived dichotomy between proliferation and invasion can be nuanced, as it malignancies, the regulation and interplay with TGFb is also likely has been reported that proliferation and invasion are not always to be different. From a therapeutic standpoint, our data indicate mutually exclusive states in melanoma and that several intermediate that approaches aiming at targeting ZEB2 expression/function may states exist (31, 32). Also, our single-cell analyses indicate that be beneficial to limit the growth of metastatic lesions. However, ZEB1/ZEB2 are coexpressed in a subset of melanoma cells. As such an approach is expected, in the same time, to promote the elevated ZEB1 levels have been associated with drug resistance of transition towards an invasive stateandassuch,favormetastatic many cancer types and increased survival of circulating tumor cells spreading. Such an approach should therefore only be considered after extravasation (33), it is tempting to speculate that coexpression very carefully, in meticulously selected cases, or in combination of ZEB2 may favor the growth of drug-resistant ZEB1high melanoma with drugs that compromise the viability of invasive melanoma cells cells or the survival of ZEB1high-invasive circulating tumor cells. as well. Our data also call for the careful (re-)examination of the While we hypothesize that dormant, disseminated melanoma cells individual biological and pathologic functions of the EMT TFs as need to restore ZEB2 expression to a certain minimal level to regain the repertoire of their activities keeps expanding beyond what was proliferation potential, survival of circulating tumor cells prior to previously anticipated. extravasation might also be enhanced by ZEB2 levels, contributing to the successful organ colonization of ZEB2-positive melanoma Disclosure of Potential Conflicts of Interest cells. While the role of oncogene signaling to direct the reversible W.M. Gallagher has a paid consulting position and ownership interest with reprogramming of EMT-inducing transcription factors in melano- OncoMark Limited, and also has received commercial research funding and has an ma was characterized thoroughly by Caramel and colleagues, the advisory relationship with Carrick Therapeutics. No potential conflicts of interest microenvironmental signals that contribute to this ZEB1/ZEB2 were disclosed by the other authors. switch in melanoma are largely unknown (25). Changes in the Authors’ Contributions microenvironment are presumed to direct melanocyte plasticity and Conception and design: N. Vandamme, G. Denecker, G. Berx melanoma phenotype switching (2). In line with this, phenotype Development of methodology: N. Vandamme, G. Denecker, G. Blancke, O.€ Akay, switching can initially be triggered by genetic instability that G. Berx b sensitizes the cells toward microenvironmental cues. TGF ,which Acquisition of data (provided animals, acquired and managed patients, provided is abundant in the tumor microenvironment, is known to regulate facilities, etc.): N. Vandamme, G. Denecker, K. Bruneel, G. Blancke, O.€ Akay, phenotype switching (24). The extensive interplay between ZEB J. Taminau, E. De Smedt, N. Skrypek, W. Van Loocke, J. Wouters, D. Nittner, transcription factors and members of the TGFb family in the C. Kohler,€ P.F. Cheng, M.I.G. Raaijmakers, M.P. Levesque, F. Rambow, V. Andries, context of epithelial cells is well established, but has been poorly B. Balint, W.M. Gallagher, J.J. Haigh, P. Van Vlierberghe, S. Goossens, J.J. van examined in melanocytes and melanoma (34, 35). Our findings den Oord, J.C. Marine, G. Berx b Analysis and interpretation of data (e.g. statistical analysis, biostatistics, indicate that TGF may affect melanoma phenotype switching, at computational analysis): N. Vandamme, J. De Coninck, F. Rambow, G. Berx least by partly modulating the ZEB2/ZEB1 ratio. This antagonstic Writing, review, and/or revision of the manuscript: N. Vandamme, J.C. Marine, effect on ZEB1/ZEB2 levels is in contrast with the classic view that G. Berx

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Administrative, technical, or material support (i.e. reporting or organizing data, Research (SBO; S008518N), the Geconcerteerde Onderzoeksacties Ghent University constructing databases): U. Girish Mallya, M. Rafferty, B. Balint, W.M. Gallagher, (GOA-01GB1013W), Vlaamse Liga tegen Kanker (365U8914U), and the Stichting D.S. Darling, L. Brochez, D. Huylebroeck, J.J. van den Oord tegen Kanker (FAF-F/2016/814). Study supervision: G. Berx The costs of publication of this article were defrayed in part by the payment of page Acknowledgments charges. This article must therefore be hereby marked advertisement in accordance N. Vandamme was supported by a personal PhD fellowship at the Institute for the with 18 U.S.C. Section 1734 solely to indicate this fact. Promotion of Innovation by Science and Technology in Flanders (IWT) (2012-2016) and a personal PhD fellowship from Kom op tegen Kanker (2016-2017). K. Bruneel and E. de Smedt are predoctoral fellows with the FWO. G. Blancke's laboratory is Received August 1, 2019; revised March 2, 2020; accepted May 18, 2020; supported by the Fonds Wetenschappelijk Onderzoek (3G050217W), Strategic Basic published ﬁrst June 5, 2020.

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AACRJournals.org Cancer Res; 80(14) July 15, 2020 2995

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype

Niels Vandamme, Geertrui Denecker, Kenneth Bruneel, et al.

Cancer Res 2020;80:2983-2995. Published OnlineFirst June 5, 2020.

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