Cross-Talk Between Receptor Tyrosine Kinases AXL and ERBB3

Published OnlineFirst March 26, 2019; DOI: 10.1158/0008-5472.CAN-18-2316 Cancer Tumor Biology and Immunology Research

Cross-Talk between Receptor Tyrosine Kinases AXL and ERBB3 Regulates Invadopodia Formation in Melanoma Cells Or-Yam Revach, Oded Sandler, Yardena Samuels, and Benjamin Geiger

Abstract

The invasive phenotype of metastatic cancer cells is accompa- invadopodia regulators and enhance invadopodia function. nied by the formation of actin-rich invadopodia, which adhere to Bioinformatic analysis of multiple melanoma cell lines points the extracellular matrix and degrade it. In this study, we explored to an inverse expression pattern of AXL and ERBB3. High the role of the tyrosine kinome in the formation of invadopodia expression of AXL in melanoma cells is associated with high in metastatic melanoma cells. Using a microscopy-based siRNA expression of invadopodia components and an invasive pheno- screen, we identified a series of regulators, the knockdown of type. These results provide new insights into the complexity of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, metastasis-promoting mechanisms and suggest that targeting of ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia multiple invadopodia signaling networks may serve as a poten- formation and function. Notably, the receptor tyrosine kinase tial anti-invasion therapy in melanoma. AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. Significance: These findings uncover a unique interplay This apparent contradiction was attributed to the capacity of AXL between AXL and ERBB3 in invadopodia regulation that to directly stimulate invadopodia, yet its suppression upregulates points to the need for combined therapy in order to prevent the ERBB3 signaling pathway, which can also activate core invadopodia-mediated metastasis in melanoma.

Introduction development of physical protrusive force, generated by actin polymerization,thatpushestheplasmamembraneoutward(4,6). The morbidity and mortality rates caused by melanoma tumors Their proteolytic activity, exerted by secreted and membrane- are primarily associated with the high capacity of these tumors to bound metalloproteinases (MMP), and serine proteinases (7) metastasize, resulting in a 10-year survival rate of 12.5%–26% (1). distinguishes invadopodia from other migration-promoting The initial steps of the metastatic process include local invasion of membrane protrusions (e.g., lamellipodia, filopodia) or ECM the melanoma cells into the surrounding connective tissue, driven adhesions (e.g., focal adhesions, podosomes; ref. 5). by signaling pathways that stimulate cell migration across tissue The temporal sequence of events that drives invadopodia barriers, turnover of cell-matrix and cell–cell adhesions, and formation and function is not fully understood, yet it has been penetration of the cancerous cells into the vascular and lymphatic proposed that this process is initiated by ligand-induced activa- systems (2). The migratory and invasive processes are commonly tion of receptor tyrosine kinases (RTK; ref. 8) that activate Src driven by reorganization of the actin cytoskeleton, and the for- family kinases (9), and the PI3K–AKT pathway (10). Consequent- mation of protrusive cell structures such as lamellipodia, filopo- ly, TKS5 (5, 11) and cortactin (12) are phosphorylated, and dia, ruffles, and invadopodia (3). participate in the early stages of actin machinery assembly in Invadopodia are actin-rich protrusions of the plasma mem- invadopodia (5, 6). A few RTKs, such as members of the EGFR brane, which attach to the extracellular matrix (ECM) via integrin family (13), the DDR family kinases (14), and Tie2 (15), were receptors, and degrade it (4). These structures are commonly shown to affect invadopodia formation and function, suggesting found in metastatic cancer cells, and are believed to promote the that tyrosine phosphorylation is a major regulator of invadopodia dissemination of metastases in vivo (5). The local invasion-pro- formation and function in different cells. That said, an under- moting activity of invadopodia is achieved by coordination of standing of the specific roles of individual kinases in the regula- local adhesions, enzymatic degradation of the ECM, and the tion of invadopodia formation and action remains limited. We addressed the issue in this study by conducting a microscopy-based siRNA screen, targeting each of the 85 human tyrosine Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, kinases in A375 metastatic melanoma cells, and tested the effect of Israel. their knockdown (KD) on invadopodia formation and on ECM Note: Supplementary data for this article are available at Cancer Research degradation. The screen revealed 7 kinases (TYK2, IGFR1, ERBB3, Online (http://cancerres.aacrjournals.org/). TYRO3, FES, ALK, PTK7), whose suppression decreased invado- Corresponding Author: Benjamin Geiger, Weizmann Institute of Science, PO podia function, and 3 kinases (ABL2, AXL, CSK) whose KD Box 26, Rehovot 76100, Israel. Phone: 972-8934-3910; Fax: 972-8946-5607; increased invadopodia function. Testing the involvement of these E-mail: [email protected] genes in the regulation of invadopodia formation in another doi: 10.1158/0008-5472.CAN-18-2316 metastatic melanoma cell line, 63T, confirmed some, but not all, 2019 American Association for Cancer Research. of the regulators identified in the original screen, which may be

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attributed to a different driver mutation in the cells (a BRAF/NRAS control, with siTOX as the transfection efficiency reporter. Cells mutation in A375/63T cells, respectively) and cell-specific varia- were incubated for 48 hours before replating for an experi- tions in the tyrosine kinome. ment. RNA sequences are listed in Supplementary Material S1. In-depth characterization of the role of the AXL RTK in invadopodia formation indicated that this kinase exerts a dual role as an AXL knockdown by shRNA. TRC AXL shRNA was purchased from invadopodia regulator, with an intrinsic capacity to stimulate inva- GE Healthcare Dharmacon. pCMV-VSV-G (helper plasmid) and dopodia, yet its suppression upregulates an alternative signaling pHRCMV-8.2DR (packaging plasmid) were both kindly provided pathway for invadopodia formation: the ERBB3 signaling pathway, by Prof. Yardena Samuels (Weizmann Institute of Science, Reho- which can activate core invadopodia regulators, and greatly enhance vot, Israel). Mission pLKO.1-puro (Sigma-Aldrich) was used as a invadopodia formation and function. We discuss the mechanism negative control vector. HEK293 cells were transfected with the underlying the apparent cross-talk between AXL and ERBB3, and its target plasmid, together with the helper and the packaging plas- potential relevance to metastatic melanoma therapy. mid, using Lipofectamine 2000 (Invitrogen). Conditioned medium was collected from the cells 48 hours posttransfection, and placed on the target cells (A375) for 24 hours. Cells were treated Materials and Methods with 3 mg/mL puromycin for selection. Antibodies and reagents R428 inhibitor and Gas6 were purchased from R&D Systems. Cell cultures R428 was used at concentrations of 1–5 mmol/L, depending A375 metastatic melanoma cells, HEK293T, 4T1, and on the cell type. Gas6 was used at a range of concentrations MDA-231 metastatic breast carcinoma cells were obtained (200 ng–1,000 ng/mL) depending on the cell type. Effective from the ATCC. WM793 and A2508 cells were a kind gift from concentrations of Gas6 varied between experiments. Neuregulin Prof. Meenhard Herlyn (Wistar Institute, Philadelphia, PA). All was purchased from PeproTech and was used in concentrations of human cell lines, except 293T cells, were authenticated using short 100 ng/mL. Puromycin was used for selection of infected cells at tandem repeat (STR) profiling in the last year. concentrations of 3 mg/mL (Sigma). A subset of cell lines (104T, 63T, and 76T) used in the study was The following antibodies were used in this study: mouse derived from a panel of pathology-confirmed metastatic mela- monoclonal antiphosphorylated tyrosine is a self-prepared soup. noma tumor resections collected from patients who consented by Rabbit polyclonal anti-p-AXL(Y779) was purchased from written approval to enroll to an immunotherapy study. The trails R&D Systems. Rabbit monoclonal antibody anti-p-ERBB3 were approved by the Institutional Review Board and conducted (Y1289) # 4791; rabbit polyclonal antibody anti-Cortactin # by the US Common Rule. Clinical trials (#03-C-0277) were 3503; and rabbit polyclonal antibody p-SRC (Y416) # 2101 were conducted at the Surgery Branch of the National Cancer Institute ordered from Cell Signaling Technology. Mouse monoclonal anti- (Bethesda, MD). Pathology-con firmed melanoma cell lines were ERBB3, SC-7390; rabbit polyclonal anti-TKS5; goat polyclonal derived from mechanically or enzymatically dispersed tumor anti-AXL, SC-1096; rabbit polyclonal anti-SRC, SC-19; rabbit cells. All patient-derived cell lines were authenticated using STR polyclonal anti-AKT, SC-8312, and rabbit polyclonal anti-p-AKT profiling. Cells were cultured in DMEM or RPMI medium sup- (s437), SC-514032 were supplied by Santa Cruz Biotechnology. plemented with 10% FCS (Gibco), 2 mmol/L glutamine, and Mouse monoclonal anti-GAPDH, #AM4300, came from Thermo 100 U/mL penicillin–streptomycin. Cultures were maintained in fi Mycoplasma Fisher Scientific. F-Actin was fluorescently labeled with TRITC- a humidi ed atmosphere of 5% CO2 in air, at 37 C. phalloidin from Sigma-Aldrich. Nuclei were labeled with DAPI tests were carried out routinely using a Mycoplasma PCR Detec- staining (Sigma). tion Kit (BI CT) or by Mycoplasma Fast Kit (Biotool). Last check The following secondary antibodies were used in this study: for A375 cells was on September 27, 2018. goat anti-mouse IgG conjugated to Cy5 (Jackson ImmunoRe- search Laboratories); goat anti-rabbit IgG H&L (HRP) #ab97080; Quantitative real-time PCR and goat anti-mouse IgG H&L (HRP) #ab97040 (Abcam). Total RNA was isolated using an RNeasy Mini Kit (Qiagen), according to the manufacturer's protocol. A 2 mg aliquot of total Plasmids, siRNA, and transfections RNA was reverse-transcribed, using a High-Capacity cDNA IRES-GFP empty vector, AXL-WT-IRES-GFP, and AXL-KD-IRES Reverse Transcription Kit (Applied Biosystems). Quantitative GFP were a kind gift from Dr. Aaron S. Meyer (Koch Institute for real-time PCR (qRT-PCR) was performed with a OneStep Instru- Integrative Cancer Research at MIT, Cambridge, MA). ment (Applied Biosystems), using Fast SYBR Green Master Mix (Applied Biosystems). Gene values were normalized to a GAPDH DNA transfection. Cells were transfected using Lipofecta- housekeeping gene. Primer sequences can be found in Supple- mine2000 (Invitrogen) in antibiotic-free media, according to the mentary Material S1. manufacturer's instructions. Media were replaced with full media after 6 hours. After 48 hours, cells were replated onto cross-linked Gelatin coating gelatin-coated plates (for more details, see "Gelatin coating" Gelatin gel. Ninety-six–well glass-bottomed plates (Nunc; section) for 3–6 hours, depending on the experiment, then either Thermo Fisher Scientific) or plastic tissue culture plates were fixed or lysed for protein. coated with 50 mg/mL poly-L-lysine solution (Sigma, catalog no. P-7405) in DPBS, and incubated for 20 minutes at room tem- RNA transfection. Transfection of the siRNA siGENOME perature. The plates were then gently washed three times with PBS. SMARTpool (GE Healthcare Dharmacon) was performed using Porcine skin gelatin, 0.2 mg (Sigma, catalog no. G2500) was DharmaFect1 (Dharmacon) at a 50 mmol/L concentration. In dissolved at 37 C in 100 mL ddH2O. Gelatin was cross-linked every transfection, siNON-TARGETING POOL #2 was used as a with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC,

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AXL and ERBB3 Regulates Invadopodia in Melanoma Cells

Sigma, catalog no. 03450) and N-hydroxysuccinimide (NHS, was conducted under 1% serum conditions (only throughout the Sigma, catalog no. 130672), each prepared as 10% solutions in duration of the experiment). ddH2O. The 96-well glass-bottomed plates were coated with 50 mL of gelatin and cross-linker mixture, and the plastic plates Immunoblotting were coated with various volumes, according to their size. The Whole-cell lysates were prepared using SDS lysis buffer (2.5% ratios of gelatin to cross-linkers were 82.5:12.5:5 (gelatin: NHS: SDS, 25% glycerol, 125 mmol/L Tris-HCL pH ¼ 6.8, 0.01% EDC). bromophenol blue, and 4% freshly added b-mercaptoethanol). Surfaces were incubated for 1 hour at room temperature, Samples were resolved on 8% SDS-PAGE gels, and transferred into washed three times in PBS, and sterilized by 30 minutes of UV polyvinylidene difluoride/nitrocellulose membranes. Blots were radiation. probed with antibodies to AXL (1:1,000), p-ERBB3 (1:1,000), ERBB3 (1:500), pCortactin (1:1,000), cortactin (1:1,000), pSRC (1:1,000), Src (1:1,000), pAKT (1:1,000), AKT (1:1,000), and Immunofluorescence staining, immunofluorescence GAPDH (1:1,000; for details, see Antibodies and reagents sec- microscopy, and image analysis tion), and developed using SuperSignal ECL reagents (Thermo For immunostaining, cells were plated at approximately 70% Fisher Scientific). confluence on gelatin-coated glass-bottomed plates (see above) For R428 inhibitor experiments, cells were plated on gelatin- for varying time periods. Cells were then fixed for 3 minutes in coated plates with the indicated concentrations of the inhibitor warm 3% PFA (Merck), 0.5% Triton X-100 (Fluka-Chemie AG), (1–2 mmol/L, depending on the cell type) or DMSO, and lysed for followed by 3% PFA alone for an additional 30 minutes. Post- immunoblotting after 3 hours. For prolonged R428 treatment, fixation, cells were washed three times with PBS and incubated cells were cultured with the indicated concentrations of the with primary antibody for 1 hour, washed three times in PBS, and inhibitor (1–2 mmol/L, depending on the cell type) or DMSO incubated for an additional 30 minutes with the secondary anti- for 2 days, then replated on gelatin-coated plates with the same body, washed again, and kept in PBS for imaging. Images were inhibitor concentrations, and lysed for immunoblotting after acquired using the DeltaVision microtiter system (Applied Preci- 3 hours. sion, Inc.), using a 40/0.75 air objective, or a 60/1.42 oil objective (Olympus). Image analysis was performed using ImageJ Cell invasion assay software (rsbweb.nih.gov/ij). A375 cells (50,000 cells) were cultured in serum-free media in the top chamber of a BioCoat Matrigel Invasion Chamber Gelatin degradation assay #354480 (Corning), in duplicates. Full medium containing Gelatin (0.2 mg; catalog no. G2500, Sigma) was labeled by 10% FBS was placed in the bottom chamber. Cells were allowed Alexa Fluor 488 Protein Labeling Kit (Molecular Probes, Thermo to invade into the Matrigel for 24 hours, then fixed with methanol, Fisher Scientific) according to the manufacturer's instructions. and stained with Giemsa. Cells that remained in the top part of the Glass-bottomed 96-well plates were coated as mentioned above chamber were removed with a cotton bud, and the inserts were (gelatin coating), with ratios of 1:10 labeled gelatin: nonlabeled washed three times, before imaging the inserts. Four quarters were gelatin. imaged for each filter using a 10 lens, and cells were counted For degradation assays, cells were plated on the gelatin matrix, manually, using an ImageJ cell counter. Cell counts from the two and cultured for varying lengths of time (on average, 5–6 hours). duplicates were averaged. Each experiment was normalized to its Cells were fixed and stained for actin and DAPI, and degradation control, as a percentage of invading cells. area was assessed by ImageJ software. In every well, 36–64 fields of view were imaged in an automated Screen targeting tyrosine kinome using siRNA fashion by the DeltaVision microtiter system, using a 40 objec- A screen for invadopodia regulators among tyrosine kinase tive. Cells in every field of view were counted using DAPI staining, family members was performed, using a tyrosine kinase library and the degradation area was calculated by the fluorescently (siGENOME SMARTpool, Dharmacon) for A375 and 63T meta- labeled gelatin channel, using the Analyze Particles plug-in in static melanoma cells (for more details regarding cell lines, see ImageJ software. The value for each well assessing invadopodia Cell cultures section). function was total degradation area/cell (mm2). To compare The siRNA library targets 85 genes of the tyrosine kinome (see results between experiments, control values were normalized to Supplementary Material S2). Cells were cultured in a 96-well plate 1, and all the samples were normalized accordingly. format in antibiotic-free media, 1 day prior to transfection. The For R428 inhibitor experiments, cells were plated on gelatin- next day, the siRNA library was transfected into the cells, using a coated plates with the indicated concentrations of the inhibitor DharmaFect1 protocol calibrated to achieve the highest transfec- (1–5 mmol/L, depending on the cell type) or DMSO, and fixed tion ef ficiency in those cells. Cells were transfected with 50 mmol/L after 5 hours. For prolonged R428 treatment, cells were cultured siRNA that targets tyrosine kinases, using nontargeting siRNA as a with the indicated concentrations of the inhibitor (1–2 mmol/L, control (Supplementary Fig. S1A). In each experiment, siTOX was depending on the cell type) or DMSO for 2 days, then replated on used to test transfection efficiency in a qualitative fashion. gelatin-coated plates with the same inhibitor concentrations, and To test transfection efficiency in a quantitative fashion, a few fixed (after 5 hours) for the invadopodia assay. wells in every experiment were transfected with siCON (nontar- For Gas6 stimulation, A375 cells were cultured with 200– geting control) or with siSRC, and Src levels were analyzed using 1,000 ng/mL of Gas6 for 5 hours. Cells were fixed and stained real-time PCR. Transfection efficiency was 80%–90%. The next for actin and DAPI, invadopodia function was assessed as men- day, the medium was replaced by full media, and cells were tioned above, and the results compared with control cells. For 63T incubated for another 48 hours. Three days after transfection, cells, Gas6 stimulation in concentrations of 100–1,000 ng/mL cells were replated in fluorescently labeled gelatin on 96-well

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Table 1. Invadopodia regulators in A375 cells broadinstitute.org/ccle) containing Affymetrix microarray expres- Gene sion data. Hierarchical clustering by Euclidian distance was knock- Degradation/cell applied on the Z-score values, and four groups of cell lines were down (normalized) P Phenotype manually defined. P values were calculated for the differences TYK2 0.19 4.03E-04 Less invadopodia IGF1R 0.38 9.24E-05 Less invadopodia between ERBB3 and AXL levels, and the other 4 genes within each TYRO3 0.21 1.54E-03 Less invadopodia group (see Supplementary Material S3, tab 1). Invadopodia- FES 0.17 4.59E-03 Less invadopodia related genes and tyrosine kinases were compared only between ALK 0.26 3.88E-11 Nonmature invadopodia two groups of cell lines with alternating patterns of AXL and ERBB3 0.32 9.79E-05 Less structures, less TKS5 ERBB3 (Clusters 1 and 2; see Supplementary Material S3, tab 2 PTK7 0.34 1.92E-03 Less invadopodia and 3). Significance level was determined by a Mann–Whitney Control 1.00 two-tailed test, following multiple hypothesis correction. ABL2 2.27 2.31E-10 More invadopodia AXL 2.84 5.60E-03 More invadopodia CSK 8.31 6.77E-03 More invadopodia Human phosphotyrosine-RTK array A Human Phosphotyrosine-RTK array (R&D Systems, no. NOTE: Normalized degradation/cell is presented for each gene KD in A375 cells. P value for each gene is presented. Three repeats; n > 100 for each sample. The ARY001) was performed, according to the manufacturer's instruc- phenotype caused by KD of each of the genes, visualized by actin and TKS5 tions. Briefly, cells were lysed on ice in lysis buffer with protease staining, and evaluated in a qualitative fashion is described under the Phenotype inhibitors (Sigma) for 15 minutes. Five-hundred micrograms of heading. lysates were incubated with blocked array membranes overnight. Detection was performed using an enhanced Chemiluminescent glass-bottomed plates (for details, see Gelatin coating section). SuperSignal West Femto substrate (Thermo Fisher Scientific). After 6 hours, cells were fixed and stained for actin, TKS5 and DAPI. Plates were then imaged in an automated fashion with a Statistical analysis DeltaVision microtiter system, using a 40 objective. In each well, Quantitative data for statistical analysis were expressed as 16–36 fields of the gelatin, actin, TKS5, and DAPI were imaged mean SEM (shown as an error bar) from at least three inde- automatically by the microscope; each field was then analyzed pendent experiments, unless stated otherwise. The significance using ImageJ software for degradation/cell (mm2), and all the of the difference between samples was calculated using a two- fields were quantified into average degradation rates per cell tailed Student t test. [n.s (not significant), P > 0.05); , P 0.05; (Supplementary Fig. S1A). , P 0.01; , P 0.001; , P 0.0001]. Quantities and shapes of invadopodia were analyzed in a quantitative and qualitative fashion, using actin and TKS5 staining (Table 1, Phenotype; Supplementary Material S2; and Sup- Results plementary Fig. S1B and S1C). Primary screen for novel invadopodia regulators in melanoma In Supplementary Material S2, data from the 3 screens in the cells A375 cell line, and from the 2 screens in the 63T cell line, are The search for invadopodia regulators was conducted by siRNA presented. In every tab, control values are presented first, followed screening, whereby each of the human tyrosine kinases was by all the genes that were analyzed. Degradation values were knocked down from the metastatic human melanoma cell line normalized to control, that was set as 1, to compare different A375 (See Supplementary Material S2). These cells form prom- experiments. A P value was calculated for each gene, according inent invadopodia in 30%–40% of the cells (16), displaying a to the degradation/cell values in all the fields, relative to the moderate capacity to degrade the underlying gelatin matrix, and control values. In the first screen, only KD of genes that showed enabling the quantification of both suppression and enhance- any effect on matrix degradation (52 of 85 tyrosine kinases) ment of matrix degradation. Immunolabeling of these cells for were analyzed. Genes whose KD caused a statistically significant phosphotyrosine, together with actin, as a marker for invadopo- difference (P 0.05) and led to a 3-fold reduction or a 1.5-fold dia, revealed extensive localization of tyrosine-phosphorylated elevation or more in gelatin degradation, were chosen for further residues both within invadopodia cores, and throughout the validation (Supplementary Material S2, Screen 2). Altogether, adhesion rings surrounding them (Fig. 1A; invadopodia are the primary screen was performed three times (see results in denoted by white arrows). A comparable level of labeling was Supplementary Material S2). also detected in focal adhesions located mainly at the cell periph- In 63T cells, the 10 invadopodia regulators that were found in ery (Fig. 1A). A375 cells were tested two additional times (Supplementary The siRNA "SMARTpool" library was transfected into A375 cells Material S2). in a 96-well format, and transfection efficiency was found to be 80%–90%, based on the ability of siTOX to kill the cells (Fig. 1B), Invadopodia quantification or quantitative real-time PCR analysis of Src in Src KD cells Invadopodia quantification was determined as a percentage of (Fig. 1C). To assess the effect of tyrosine kinase KD on invado- invadopodia-forming cells in 16–36 fields, based on their actin podia function, the treated cells were replated on fluorescently and TKS5 labeling. Cell counts were performed by DAPI, as labeled gelatin in 96-well glass-bottomed plates for 6 hours, then described above. fixed and stained for actin and TKS5, as invadopodia markers, and DAPI, for cell number quantification and assessment of cell Gene expression analysis toxicity (Supplementary Fig. S1A). Plates were then imaged auto- Gene expression profiles of AXL, ERBB3, MITF, and three of the matically (for details, see Materials and Methods), and gelatin MITF targets (PMEL, TYRP1, and MELANA), were extracted from degradation area/ per cell (mm2) was quantified (Supplementary 62 melanoma cell lines from the CCLE dataset (https://portals. Fig. S1A; Supplementary Material S2). To compare the results of

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AXL and ERBB3 Regulates Invadopodia in Melanoma Cells

Figure 1. Microscopy-based siRNA screen for tyrosine kinases invadopodia regulators in A375 cells. A, A375 cells were cultured on gelatin gel for 3 hours and stained for phosphorylated tyrosine and actin. White arrows, invadopodia structures. Scale bar, 20 mm. B, Representative images from the siCON versus siTOX samples of the screen, used to evaluate transfection efficiency in A375. C, Real-time analysis of Src gene expression following transfection under screening conditions in A375; n ¼ 3. D and E, Quantification of normalized degradation/cell for the final list of genes of the screen (invadopodia assay) in A375 cells. D, Genes that caused a reduction in invadopodia function upon KD. E, Genes that caused an elevation of invadopodia function upon KD. Three repeats; n > 100 in each sample. F, Representative images of A375 cells from the screen. Cells were cultured for 6 hours on fluorescently labeled gelatin and stained with DAPI. Scale bar, 20 mm.

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the different experiments, gelatin degradation values were nor- Given the potential functional redundancy between tyrosine malized to the control, which was set as 1. kinases, which may vary in different cell types, we further tested Invadopodia formation was evaluated qualitatively, based the effectiveness of the regulators that were found in A375 on on staining for actin and TKS5 (Table 1, phenotype). For the another metastatic, patient-derived melanoma cell line, 63T, final list of regulators, quantitative analysis of the percentage of which differs from A375 cells in its genetic background (a invadopodia-forming cells was performed (Supplementary BRAF/NRAS mutant in A375/63T cells, respectively; ref. 22). Fig. S1B and S1C). Only genes whose KD resulted in a statis- 63T cells displayed conspicuous invadopodia with extensive tically significant difference (P < 0.05) and led to a >3-fold matrix degradation in 20%–30% of the cells, (Fig. 2A), and are reduction or a >1.5-fold elevation in gelatin degradation, suitable for siRNA transfection (Fig. 2B and C). Testing the compared with control, were selected for further characteriza- effects of siRNA KD of the A375 invadopodia regulators on 63T tion (Supplementary Material S2, Screen 2). Altogether, the cells (see Supplementary Material S2) indicated that only KD of primary screen was performed three times (see full results in FES, one of the 7 positive regulators obtained with A375 cells, Supplementary Material S2). also induced a reduction in invadopodia function in 63T cells. Of the 85 human tyrosine kinases tested, KD of 7 genes Conversely, the 3 regulators whose KD enhanced invadopodia reproducibly reduced gelatin degradation, while 3 enhanced it in A375 cells (CSK, AXL, and ABL2), displayed a similar effect (Table 1; Fig. 1D–F). Notably, all the genes KD that affected in 63T cells (Supplementary Table 1; Fig. 2D; Supplementary gelatin degradation also induced comparable changes in invado- Fig. S1D). podia formation (Table 1, Phenotype; Supplementary Fig. S1B AXL is a receptor tyrosine kinase, associated with an invasive and S1C). KD of each of the three genes causing an elevation in phenotype in multiple cancers, including melanoma (23, 24). In invadopodia function (CSK, AXL, and ABL2) resulted in an addition, AXL was shown to be involved in drug resistance increase in the percentage of cells forming invadopodia with a mechanisms in melanoma and other cancers (25). high content of TKS5 in their cores (Table 1, Phenotype; Supple- Intrigued by the unexpected enhancement of invadopodia mentary Fig. S1B and S1C). In contrast, KD of most genes that following the KD of AXL, and its novel association with invado- caused reduction in gelatin degradation resulted in a decrease in podia regulation, we chose to focus our study on the mechanism invadopodia-forming cells (Table 1, Phenotype; Supplementary underlying the effect of this kinase on invadopodia formation and Fig. S1B and S1C). function. Notably, some of the invadopodia-regulating genes identified in this screen were previously shown to affect invadopodia The AXL RTK regulates invadopodia in melanoma cells formation or function, including CSK (17), which inhibits the To validate the specificity of the augmentation of invadopodia activity of Src family kinases (18) and, upon KD, causes a prominence and matrix degradation upon AXL KD, we tested each dramatic elevation in invadopodia function. PTK7 and ERBB3 of the four duplexes present in the siRNA "SMARTpool" in both were shown to be positive invadopodia regulators in other A375 and 63T cell lines, and confirmed that 3 of 4 siRNA duplexes cellular systems (19, 20) and ABL2 was found in our screen of display an elevated degradation effect similar to that seen in the melanoma cell lines to be a negative regulator, while in breast siAXL siRNA pool (Supplementary Fig. S2A and S2B). Further cancer cells, it was shown to positively regulate invadopo- quantification of the effect indicated that upon KD of AXL, dia (13, 21). normalized gelatin degradation levels (degraded area/cell)

Figure 2. Microscopy-based siRNA screen for tyrosine kinases invadopodia regulators in 63T cells. A, 63T cells cultured for 3 hours on fluorescently labeled gelatin and stained for actin show that they degrade the matrix (right) and form actin dots (left). White arrows, invadopodia structures. Scale bar, 10 mm. B, Representative images from the siCON versus siTOX samples of the screen, used to evaluate transfection efficiency in 63T cells. C, Real-time analysis of Src gene expression following transfection under screening conditions in 63T cells; n ¼ 3. D, Representative images of 63T cells from the screen. Cells were cultured for 6 hours on fluorescently labeled gelatin and stained with DAPI. Scale bar, 20 mm.

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AXL and ERBB3 Regulates Invadopodia in Melanoma Cells

Figure 3. AXL is localized to invadopodia and regulates their function. A and B, Invadopodia assay of siCON versus siAXL in A375 and 63T cells, respectively. Cells were cultured for 6 hours. Ten repeats for each cell; n > 100 in each sample. C, Representative image of A375 siCON and siAXL cells. Scale bar, 20 mm. D, Western blot analysis of AXL protein in A375 and 63T cells upon AXL KD. GAPDH was used as a loading control. E and F, Quantification of invadopodia-forming cells (expressed as a percentage) in A375 and 63T cells, respectively. Cells were cultured on gelatin gel for 6 hours. Three repeats; n > 100 in each sample. A375 (G)or 63T (H) cells were cultured on fluorescently labeled gelatin for 1.5, 3, and 6 hours, and stained for actin and phosphorylated AXL. AXL localized to invadopodia cores from the early stages of their formation (1.5 hours) through the mature stage (6 hours). AXL also localized to focal adhesions. White arrows, invadopodia structures. Scale bar, 20 mm. increased by a factor of 7 (in A375 cells) or of 4 (63T cells; pointed to a strong association of AXL with invadopodia cores Fig. 3A–C). AXL suppression efficiency was up to 90%, as deter- at various time points following cell culturing and invadopodia mined by Western blot analysis (Fig. 3D) and real-time PCR formation (Fig. 3G and H). Taken together, these results suggest (Supplementary Fig. S2C and 2D). Percentage of invadopodia- that AXL plays an important role in the regulation of invadopodia forming cells in both cell lines was elevated comparably with the in melanoma cells. increased gelatin degradation (Fig. 3E and F, respectively). A similar effect involving elevated invadopodia function was AXL is a dual-function regulator of invadopodia, with both obtained when cells were stably infected with shAXL (Supple- suppressive and stimulatory activity mentary Fig. S2E and S2F), and upon siRNA transfection in two Further characterization of the molecular mechanisms under- other melanoma cell lines, WM793 and A2508, that also form lying the role of AXL in invadopodia formation and function invadopodia (Supplementary Fig. S2G–S2I). indicated that AXL might have a dual regulatory effect, both The role of invadopodia in elevated matrix degradation negative and positive. First, we tested the effect of AXL overexpres- induced by AXL suppression was confirmed by double KD of sion on invadopodia function (e.g., gelatin degradation) in A375 both AXL and TKS5 (an invadopodia-specific marker), which cells (Fig. 4A and B) or in 63T cells (Fig. 4C). The overexpression of eliminated the elevation in matrix degradation (Supplementary wild-type AXL, but not of its kinase-dead (DK) derivative, or empty Fig. S2J and S2K). vector, caused a significant elevation in invadopodia function, Finally, localization of phosphorylated AXL in A375 and 63T suggesting that kinase activity is required for the positive regulation melanoma cells, as seen by immunofluorescence microscopy, of invadopodia (Fig. 4A–C). The transfection resulted in a marked

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Figure 4. AXL is a positive regulator of invadopodia in melanoma cells. Invadopodia assay of A375 cells (A and B) or 63T cells (C) overexpressing AXL-WT, AXL kinase- dead (DK), or control plasmid, cultured on fluorescently labeled gelatin for 6 hours, and stained for DAPI. Representative images in A; quantification in B and C. Three repeats for each cell type; n > 100 in each sample. Scale bar, 20 mm. D and E, Invadopodia assay of control or 200 ng/mL Gas6-treated cells cultured on fluorescently labeled gelatin for 6 hours. Gas6 effective concentrations were varied between experiments and ranged between 200 and 1,000 ng/mL. Four repeats; n > 100 in each sample. Scale bar, 20 mm. F and G, Invadopodia assay of A375 cultured on fluorescently labeled gelatin for 6 hours with DMSO or the AXL inhibitor R428 (in concentrations of 5 mmol/L, 2 mmol/L, or 1 mmol/L). Three repeats for each cell type; n > 100. Cells are stained for actin. Scale bar, 20 mm. H and I, Invadopodia assay of 63T cultured on fluorescently labeled gelatin for 6 hours with DMSO or the AXL inhibitor R428 (in concentrations of 5 mmol/L, 2 mmol/L, or 1 mmol/L). Three repeats for each cell type; n > 100. Cells were stained for actin. Scale bar, 20 mm.

elevation in total AXL levels (Supplementary Fig. S3A). The ele- S3B and S3C). The localization of both the WT and DK mutant of vation in gelatin degradation also correlated with the ability of the AXL to the membrane was conﬁrmed by immunolabeling of cells to invade through a transwell ﬁlter in vitro (Supplementary Fig. nonpermeabilized 293T cells (that do not express endogenous

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AXL and ERBB3 Regulates Invadopodia in Melanoma Cells

AXL), following transfection with the WT and mutant AXL, com- When we analyzed, in A375 cells, the levels of ERBB3 activation pared with an empty vector (Supplementary Fig. S3D). under conditions of AXL KD, we found an increase in total ERBB3 Additional support for the positive role of AXL in invadopodia levels, as well as in ERBB3 phosphorylation (Fig. 5C). RNA levels formation and function in A375 or 63T melanoma cells was of ERBB3 were only slightly elevated following AXL inhibition obtained through elevated levels of invadopodia, following treat- (Supplementary Fig. S4E), suggesting that the upregulation of ment with the AXL ligand Gas6 (Fig. 4D and E; Supplementary ERBB3 is primarily posttranscriptional. It is noteworthy that the Fig. S3E; ref. 26) that did not cause elevation in AXL KD cells interplay between AXL and ERBB3 was not symmetrical, as ERBB3 compared with the stimulated control (Supplementary Fig. S3F). KD did not affect AXL levels and phosphorylation on Y779 In addition, there was suppression of invadopodia in both cell (Supplementary Fig. S4F; quantifications in Supplementary types by treatment with the AXL inhibitor R428 for 6 hours [Fig. 4F Fig. S4G). In line with that, Gas6 stimulation did not rescue and G (A375) and 4H and I (63T)]. Invadopodia inhibition by the invadopodia function in ERBB3 KD cells (Supplementary AXL inhibitor was also observed in two additional, patient- Fig. S4H). Moreover, Src, Cortactin, and AKT-activating phos- derived melanoma cells lines, 76T and 104T, as well as in 4T1 phorylation levels were also elevated under those same conditions murine metastatic breast cancer cells that form invadopodia (Fig. 5D–F, respectively, quantifications to all Western blots (Supplementary Fig. S3G and S3H). Elevated levels of invadopo- shown in Fig. 5G). Activation of these components is an indica- dia function upon AXL KD was specific to melanoma cells, as tion of more active invadopodia, and higher levels of actin when AXL was KD in MDA-231 breast cancer cells, invadopodia polymerization (4). Indeed, the SRC and PI3K–AKT pathways function was decreased by factor of two (Supplementary Fig. S3I can potentially be activated directly by ERBB3 (31), and cortactin and S3J), corroborating our observation that AXL acts as a general is a downstream target of Src in invadopodia formation (5). These positive invadopodia regulator. results suggest that AXL inhibition activates the ERBB3 pathway in melanoma cells, and this activation may lead to an increase in ERRB3 activation is upregulated upon AXL suppression, invadopodia formation. leading to enhanced invadopodia function Consistently, invadopodia function was also elevated follow- Given existing evidence that AXL promotes invasion and migra- ing long-term (2 days) inhibition of AXL by the R428 inhibitor, tion in a variety of cancer cells (23, 24), together with our current comparable with that observed following AXL siRNA-mediated findings, we hypothesized that an alternative invasion-promoting KD (Fig. 5H), and in contrast with short-term (6 hours) exposure pathway may be activated following AXL inhibition by siRNA, to the inhibitor, which blocks invadopodia formation (Fig. 4F–I). leading to an elevation in invadopodia formation. Furthermore, Moreover, prolonged AXL inhibition using R428 results in higher this alternative pathway is not activated following short-term levels of total and phosphorylated ERBB3 (Fig. 5I, two right inhibition of AXL by R428 (Fig. 4F–I). lanes). Notably, no such elevation was observed following Surprisingly, the levels of pAXL (phosphorylated on Y779) short-term (6 hours) treatment with the inhibitor (Fig. 5I, two were not decreased, and even slightly elevated, in AXL KD cells, left lanes). Cortactin phosphorylation was also elevated under compared with control (Fig. 5A; quantifications in 5B), although prolonged AXL inhibition using R428 (Fig. 5J, two right lanes), total AXL levels were decreased by about 90% (Fig. 5A). However, and not under short-term treatment (Fig. 5J, two left lanes). RTK array, total phosphotyrosine analysis, revealed a approxi- Quantifications of ERBB3 and cortactin upon inhibitor treatment mately 50% reduction in AXL phosphorylation on other tyrosine are shown in Fig. 5K. residues (Supplementary Fig. S4A and S4B). Further staining of The notion that the elevation in invadopodia function in AXL AXL KD cells showed strong localization of pAXL (Y779), in KD cells depends on ERBB3 activation was further tested by invadopodia (Supplementary Fig. S4C). Moreover, short-term double-KD experiments. In accordance with the siRNA screen, inhibition (up to 6 hours) of AXL KD cells by R428, caused a KD of ERBB3 alone led to suppression of invadopodia activity, reduction of AXL phosphorylation on Y779, demonstrated while AXL KD increased it (Fig. 5L and M). Double KD of AXL and by Western blot analysis (Fig. 5A and B) and cell staining ERBB3 indicated that invadopodia elevation by AXL KD does not (Supplementary Fig. S4C). Reduced invadopodia function was take place in the absence of ERBB3 (Fig. 5L and M). Specifically, detected following this treatment (Supplementary Fig. S4D). the difference between invadopodia function following ERBB3 These results indicate that the residual AXL (10%–15%) in AXL KD and AXLþERBB3 double KD was found to be highly signif- KD cells is hyperphosphorylated on Y779 and strongly localized icant (Fig. 5L and M). to invadopodia structures, suggesting that this phosphorylation Finally, Neuregulin, an ERBB3 ligand (32), stimulation ele- may be important for invadopodia function. These findings vated invadopodia function in both A375 and 63T cells led us to hypothesize that another tyrosine kinase is driving (Fig. 5N and O), and resulted in slightly higher invadopodia AXL hyperphosphorylation, thereby enhancing invadopodia activity in AXL KD cells, compared with nontreated AXL KD formation. cells (Supplementary Fig. S4I). Cross-talk between AXL and EGFR family members was previ- To further test whether AXL KD affects other RTKs, we analyzed ously proposed by studies in several cancer systems as a potential the phosphorylation of all 49 RTKs, using an RTK array (Supple- mechanism for drug resistance, usually in the form of AXL mentary Fig. S4A and S4B). As shown, ALK and ERBB3 phosphor- activation following EGFR inhibition (27–29). Moreover, in ylations were slightly elevated, while VEGFR2 and AXL phosphor- breast cancer cells, the opposite process was found, when pro- ylations were reduced upon AXL KD (Supplementary Fig. S4A and longed inhibition of AXL leads to an increase in ERBB3 expression S4B). No further alterations in the phosphorylation of RTKs were and phosphorylation (30). This phenotype was not linked to detected upon AXL KD. invasion or invadopodia. Moreover, to our knowledge, no such Our findings suggest that when AXL is suppressed or inhibited interplay between AXL and ERBB3 was documented in melanoma for prolonged periods, the ERBB3 pathway, an alternative, highly cells. potent signaling pathway for invadopodia formation, is activated,

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A BG siCON siAXL siA+R 1.8 7.5 pAXL 1.6 J (Y779) 1.4 1.2 2.5 DMSO R428 D-2d R-2d 1

0.8 2 Cortactin AXL 0.6 0.4 (Y421) 1.5 0.2 Tubulin 0

siCON siAXL siA+R 1 Cortactin pAXL Relative expression level expression Relative pAXL

0.5 Relative expression level expression Relative 0 C D siCON siAXL Control AXL/ pERRB3/ ERBB3/ pAKT/ pCORT/ pSRC/ siCON siAXL GAP GAP GAP AKT CORT SRC K 2.50 Cortactin pERBB3 (Y421) 2.00 (Y1289) Cortactin 1.50 ERBB3 DMSO R428 D-2d R-2d E I 1.00 pAKT pERBB3 0.50 AXL (S437) (Y1289) AKT 0.00

GAPDH ERBB3

Relative expression level expression Relative

R428

DMSO DMSO

F pSRC DMSO

R4282d

DMSO2d DMSO2d (Y416) GAPDH DMSO2d pERBB3/GAP ERBB3/GAP pCortactin/GAP H Src

DMSO R428 2d Degradation/cell (fold change) (fold Degradation/cell M L n.s 4 3.5 3 2.5 2 1.5 1 0.5 0 Degradation/cell (fold change) (fold Degradation/cell siCON siAXL siERBB3 siA+siE O A375 63T N

5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

Control 100 ng/mL Control 100 ng/mL Degradation/cell (fold change) (fold Degradation/cell A375 63T

Figure 5. The ERBB3 pathway is activated when AXL is inhibited for prolonged periods. A, Western blot analysis of A375 cells showing AXL phosphorylation (Y779) and total AXL levels of control (siCON), AXL KD (siAXL), or AXL KD with R428 AXL inhibitor treatment (siAþR). Tubulin served as loading control. Quantifications in B; n ¼ 3. C, Western blot analysis of A375 cells, showing ERBB3 phosphorylation (Y1289) and total ERBB3 protein in siAXL versus siCON cells. AXL levels are presented. GAPDH served as a loading control. Quantification in G; n ¼ 4. D–F, Western blot analysis of A375 cells, showing Src phosphorylation (Y416) and total Src, cortactin phosphorylation (Y421), and total cortactin and AKT (S437) phosphorylation, and total AKT protein levels in siAXL versus siCON cells, respectively. Quantification in G; n ¼ 3 or more for each protein. G, Quantifications of Western blots in C–F. H, Invadopodia assay of A375 cells incubated for 2 days with 1 mmol/L R428, AXL inhibitor, or DMSO. After 48 hours, cells were replated on fluorescently labeled gelatin for 6 hours for the invadopodia assay. White arrows, invadopodia. Four repeats; n > 100 in each sample. I, pSRC/SRC graph is truncated due to a wide range of differences in phosphorylation elevation between the phosphoproteins. Western blot analysis of A375 cells, showing ERBB3 phosphorylation (Y1289) and total protein in DMSO (D) versus R428 short-term treatment (1 mmol/L for 6 hours) and DMSO (D-2d) versus R428-2d prolonged treatment (1 mmol/L for 2 days). GAPDH served as a loading control. Quantifications in K; n ¼ 3. J, Western blot analysis of A375 cells, showing cortactin phosphorylation (Y421) and total protein in DMSO (D) versus R428 short-term treatment (1 mmol/L for 6 hours) and DMSO (D-2d) versus R428-2d prolonged treatment (1 mmol/L for 2 days). Quantifications in K; n ¼ 3. L and M, Invadopodia assay of A375 cells with KD of AXL, ERBB3, AXLþERBB3, or siCON cultured on fluorescently labeled gelatin for 6 hours. Six repeats; n > 100 in each sample. N and O, Invadopodia assay of A375 or 63T cells (two repeats or three repeats, respectively; n > 100 in each sample) cultured on fluorescently labeled gelatin for 6 hours with 100 ng/mL neuregulin or vehicle.

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AXL and ERBB3 Regulates Invadopodia in Melanoma Cells

leading to increased invadopodia formation and function. This Discussion increase in invadopodia function can be directly mediated by ERBB3 or indirectly, through overactivation of AXL by phosphor- In this study, we describe the molecular cross-talk between ylation on tyrosine 779 (Fig. 5A; see further activation mechanism different tyrosine kinases involved in the regulation of invado- in the Discussion). podia formation and function in melanoma cells. Using a comprehensive siRNA screen, we identified several novel positive AXL expression exhibits an expression pattern inverse to that of invadopodia regulators in A375 metastatic melanoma cells, ERBB3 and MITF in melanoma cells including TYK2, IGF1R, TYRO3, FES, ALK, ERBB3, and PTK7 To test whether the interplay between AXL and ERBB3 consti- (Fig. 1D and F; Table 1). In addition, we found three kinases tutes a common feature of melanoma cells, we analyzed the (CSK, ABL2, and AXL) whose KD upregulated invadopodia for- expression levels of AXL and ERBB3 in 62 melanoma cell lines mation and function (Fig. 1E and F; Table 1). A few of these from the CCLE database (https://portals.broadinstitute.org/ccle). kinases (CSK, ABL2, ERBB3, and PTK7) were reported to regulate MITF and three of its downstream targets were added to the invadopodia in other cellular systems (5, 13, 18, 20, 36); CSK, for analysis, due to the known inverse correlation of AXL and example, is a negative regulator of invadopodia, due to its inhib- MITF expression levels in melanoma, and the relevance of itory effect on SRC family kinases (SKF; refs. 5, 18), which are the MITF/AXL ratio to melanoma state, progression, and drug known to play an essential role in invadopodia formation by resistance (33). phosphorylating and activating key structural components of On the CCLE database–derived expression Z-score values, we invadopodia, including TKS5 and cortactin (37). applied hierarchical clustering, and four groups of cell lines It is noteworthy that while KD of CSK, which phosphorylates were manually defined(Fig.6A).Twooftheclusters(cluster1 multiple SFKs, elevates invadopodia formation (5), KD of indi- and cluster 2; a total of 31 cell lines) encompassing 50% of the vidual SFKs (including Src) had a limited effect on invadopodia dataset, displayed a distinct, inverse expression pattern between formation (Supplementary Material S2). This phenotype may be AXL and ERBB3 (P <0.0006; see Supplementary Material S3). attributed to the compensatory activity of other SFKs, which are Moreover, ERBB3 expression levels were positively correlated prominently expressed in melanoma tumors (38). with the levels of MITF and its downstream targets (Fig. 6A, Similarly, functional redundancy between tyrosine kinases that clusters 1 and 2). Clusters 3 and 4 showed different expression regulate invadopodia, was also implied by marked differences fi patterns: either all three genes (AXL, ERBB3, and MITF) were between the pro les of invadopodia modulators in A375 and expressed at high levels (Fig. 6A, cluster 3); or both ERBB3 and 63T cells (Fig. 1D and E; Supplementary Fig. S1D; Table 1; fi AXL were highly expressed, but MITF was low (Fig. 6A, cluster Supplementary Table S1). Speci cally, only 4 genes out of 10 4). This analysis demonstrates that in many cases of melanoma caused similar effects on invadopodia in 63T cells (see Table 1; (50%, in our dataset), the expression patterns of AXL and Supplementary Table S1). These differences may also be attrib- ERBB3 are mutually exclusive, suggesting that they act on uted to the different driver mutations in the cells, as A375 is a fi alternative oncogenic pathways, as we see in the context of BRAF mutant, while 63T is an NRAS mutant. Clari cation of invadopodia regulation (Fig. 5). these cellular variations is required for a deeper understanding of Next, we classified the cells as proliferative or invasive, based invadopodia formation, and for assessing their roles in melanoma on their melanoma phenotype–specific expression (MPSE) metastasis. signature (34, 35). MPSE predicts whether the cell phenotype In this comprehensive analysis, we found that the RTK AXL acts will be proliferative or invasive, based on the expression of a set as a dual-function regulator of invadopodia in both the A375 and of genes. Cluster 1 (AXL low/ERBB3 high) is enriched with 63T melanoma cell lines. AXL is known to be associated with an proliferative cell lines (Fig. 6B, bottom bar), whereas cluster 2 invasive phenotype in multiple cancers, including melano- (AXL high/ERRB3 low) is dominated by an invasive phenotype ma (39). In addition, AXL was shown to be involved in drug (Fig. 6B, bottom bar). resistance mechanisms in melanoma and other cancers (25). Our fi Consistently, cluster 2 exhibits a significantly higher expression novel nding that AXL is involved in the regulation of invado- of invadopodia components [e.g., TKS5 (SH3PXD2A), Fascin, podia formation and function (Fig. 3 and 4) adds another MMP2, ABL1, and vinculin; see Fig. 6C; Supplementary Material dimension to our understanding of how AXL contributes to the S3 for P values]. development of these aggressive phenotypes. To obtain an overview of the contribution of the tyrosine The dual role of AXL is manifested by increased invadopodia kinome to melanoma invasion and invadopodia formation, we formation and function upon AXL KD in 4 different melanoma – analyzed the expression patterns of all tyrosine kinases, and cells lines (A375, 63T, WM793, and A2058; Fig. 3A F; Supple- – their distribution among the 4 clusters (Supplementary Fig. mentary Fig. S2G S2I). On the other hand, we demonstrate the S5). A group of 13 tyrosine kinases were coexpressed with high positive role of AXL in invadopodia formation and function, AXL/low ERRB3 levels (cluster 2; see Supplementary Material based on a series of experiments involving two melanoma cell fi S3); among these, PDGFRA and PDGFRB, EGFR, and FGFR1. In lines (A375 and 63T). AXL localizes speci cally to invadopodia contrast, another group of 11 tyrosine kinases were coexpressed structures at various stages of their formation (Fig. 3G and H) and with high ERRB3/low AXL (cluster 1; see Supplementary causes an increase in invadopodia activity following overexpres- – Material S3); among them, two other members of the TAM sion of WT-AXL, but not its kinase-dead mutant (Fig. 4A C). receptor family, MER and TYRO3, as well as Syk and CSK Consequently, the capacity of these cells to invade in a Matrigel (Supplementary Fig. S5). Other tyrosine kinases were not transwell invasion assay was increased as well (Supplementary differentially expressed among the clusters. This analysis fur- Fig. S3B and S3C). Activation of AXL by its ligand, Gas6, increased ther supports the notion of two alternative pathways that invadopodia function in both cell lines (Fig. 4D and E; Supple- regulate cell invasion in melanoma. mentary Fig. S3E), while short-term (6 hours) treatment of cells

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Figure 6. AXL negatively correlates with ERBB3 in 50% of melanomas. A, Hierarchical clustering by Euclidian distance was applied to the Z-score values of AXL, ERBB3, MITF, and three MITF targets: PMEL, TYRP1, and MELANA, from 62 melanoma cell lines of the CCLE Cell Line dataset. Four clusters were selected manually. AXL and ERBB3 are signiﬁcantly negatively correlated (for P values, see Supplementary Material S3). B, Prediction of cell phenotype based on the MPSE (melanoma phenotype-speciﬁc expression) signature (34, 35). C, Analysis of Z-score values of 14 core invadopodia proteins among the four clusters described in A. P values for the differences of the proteins in clusters 1 and 2 are presented in Supplementary Material S3. D, Proposed model for AXL-ERBB3 interplay in invadopodia regulation. AXL and ERBB3 can both positively regulate invadopodia, depending on their initial balance in the cell. Each of them is activated by its ligand (AXL by Gas6; ERBB3 by neuregulin). A one-directional interplay exists between the two receptors. Once AXL is subjected to short-term inhibition (6 hours) bythe inhibitor, invadopodia function is inhibited; while AXL is subjected to prolonged inhibition (2 days) by siRNA or inhibitor treatment, ERBB3 can be activated, by destabilization of its E3 ligase complex mediated by an AKT negative feedback loop (see Discussion). The elevation in ERBB3 expression and phosphorylation can activate invadopodia function directly by activation of PI3K, or mediate the phosphorylation of AXL on Y779 together with another RTK, and lead to invadopodia formation. Both AXL and ERBB3 can activate PI3K and SFK, leading to phosphorylation of TKS5 and cortactin, which integrate into the invadopodia core and initiate their formation. Further actin polymerization and actin bundling by fascin occurs, eventually resulting in the secretion of MMPs and matrix remodeling.

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with the AXL inhibitor R428 was found to block invadopodia will predominantly express, at steady state, only one of them, at function in 4 melanoma cell lines (Fig. 4F–I; Supplementary high levels. Fig. S3G and S3H), as well as in 4T1 breast cancer cells (Supple- One possible mechanism underlying posttranscriptional mentary Fig. S3G and S3H). Finally, KD of AXL in the metastatic ERBB3 regulation is an AKT negative feedback loop, mediated breast cancer cell line MDA-231 caused a significant reduction in through the E3 ligase Nrdp1 and the Nrdp1 regulator USP8, which invadopodia function (Supplementary Fig. S3I and S3J). Further target ERBB3 to proteasomal degradation (32). USP8 can be experiments clarified the dual role of AXL in the regulation of phosphorylated and stabilized by AKT, which is activated by invadopodia. Upon AXL KD, there was no reduction in AXL ERBB3, thus creating a negative feedback loop for ERBB3 activa- phosphorylation on tyrosine Y779 (Fig. 5A and B; Supplementary tion (32). In line with that, ERBB3 was shown to display increased Fig. S4C), although total AXL phosphorylation levels were membrane localization and activity that compensate for the loss reduced (Supplementary Fig. S4A and S4B). Under those condi- of ERBB family kinases upon tyrosine kinase inhibitor treatment. tions, both the expression and phosphorylation levels of ERBB3 This compensation was also mediated by AKT negative feedback increased (Fig. 5C and G), suggesting a compensatory mechanism signaling (42). for the loss of AXL by ERBB3, potentially leading to an increase in Inhibition of the PI3K–AKT pathway can occur upon AXL invadopodia function (Figs. 3A, B, and 5H). This phosphorylation perturbation (23, 43, 44). In the absence of AKT, there is less state was accompanied by enhanced phosphorylation of key ERBB3 ubiquitination for degradation by Nrdp1, a situation that invadopodia regulators, including SRC, AKT, and cortactin can result in increased ERBB3 protein levels and phosphoryla- (Fig. 5D–G). tion (32). ERRB3 overexpression and activation, together with A role for ERBB3 in invadopodia function in melanoma is also activation of another RTK (28, 30) can result in increased AXL supported by the fact that ERBB3 was determined to be one of the Y779 phosphorylation, activated PI3K pathway, and invadopodia invadopodia regulators in the A375 screen described above formation (10). Both EGFR and HER2 were shown to directly (Fig. 1D and F; Table 1), and by increased invadopodia formation phosphorylate AXL on Y779 (45, 46). Y779 phosphorylation was upon treatment of both A375 and 63T cells with the ERBB3 ligand, shown to be one of three autophosphorylation sites in the protein neuregulin (Fig. 5N and O). c-terminus, and constitute a docking site for a variety of signaling Is ERBB3 unique in its capacity to compensate for AXL loss in proteins, including PI3K, Grb2, PLCg, c-src, and lck (23), which invadopodia regulation? We cannot exclude the possibility that can lead to enhanced invadopodia formation (4). other tyrosine kinases can compensate for the loss of AXL; yet To conclude, the findings reported here suggest two possible double KD of both AXL and ERBB3 is sufficient to abrogate the modes of action for invadopodia regulation in melanoma increase in invadopodia formation (Fig. 5L and M), and other (Fig. 6D, Proposed Model). One is governed by AXL, which is RTKs did not show dramatic changes in their phosphorylation activated by Gas6 (39) and can promote cell invasion and migra- levels upon AXL KD (Supplementary Fig. S4A and S4B). tion, cytoskeletal remodeling (47), and invadopodia formation Examination of AXL/ERBB3 expression levels in 62 melanoma and function (our results; Fig. 4). This can be achieved by acti- cell lines included in the CCLE database shows significant inverse vation of invadopodia-associated proteins (e.g., TKS5 and cortac- expression patterns in approximately 50% of the cells (Fig. 6A, tin) by Src family kinases and the PI3K pathway (see also Fig. 6D, clusters 1 and 2). Interestingly, ERBB3 levels positively correlate proposed model; refs. 10, 48). Indeed, high expression levels of with MITF expression levels, and with three of its main down- core invadopodia components such as TKS5, MMP2, ABL1, fascin, stream targets (Fig. 6A, clusters 1 and 2). MITF has been shown to and vinculin (4) correlate with high AXL levels (Fig. 6C, cluster 2), negatively correlate with AXL in melanoma cells (40, 41). MITF as well as an invasive phenotype (Fig. 6A and B, cluster 2). levels are also known to affect melanoma progression, and fine- The second invadopodia-stimulating pathway is induced by tuning of its expression leads to various physiologic and patho- ERRB3, which predominantly correlates with a proliferative phe- logic manifestations, including cell differentiation, proliferation, notype (Fig. 6B). Yet upon suppression of AXL, the negative apoptosis, and invasion (33, 41). Furthermore, the balance feedback loop mediated by AKT is broken, and ERBB3 is less between MITF and AXL can change cell fate, invasiveness, and degraded by its E3 ligase, Nrdp1 (32, 49). Under those conditions, drug resistance in melanoma (33, 41). ERBB3 protein can be elevated and phosphorylated, and either The fact that only prolonged inhibition of AXL ( 2days), promote invadopodia function on its own, or mediate residual leads to elevated ERBB3 levels (Fig. 5I) and consequently, to AXL phosphorylation with another active member of the ERBB increased invadopodia formation (Fig. 5H), suggests that the family (50), leading to increased invadopodia formation (Figs. 5 compensatory mechanism is regulated either at the transcrip- and 6D, proposed model). tional level, or during a slow, posttranslational process that The process of cell invasion includes differential regulation of affects ERBB3 stability. Notably, similar reciprocal cross-talk the migratory and invasive capacities of the cells (2). A finely- between AXL and ERBB3 was reported in breast cancer cells, tuned balance between the invadopodia regulators AXL and where prolonged (1–2 days) inhibition of AXL by inhibitor or ERBB3 is necessary for the first steps of invasion, that require siRNA treatment led to an elevation in ERBB3 phosphorylation immediate remodeling of the extracellular matrix. Further cellular and activity (30). dissemination will require the integration of the migratory Note that the CCLE database is based on RNA expression levels machinery, which can also be regulated by AXL (24, 26). in the various cells lines, and demonstrates heterogeneity in the Our new insights into the unique abilities of AXL and ERBB3 expression patterns of AXL and ERBB3 (Fig. 6A). Our data show an to regulate invadopodia function, together with a deeper under- interplay between them mostly on the protein level (Fig. 5C), in standing of the molecular cross-talk within the kinome network, cells that express both high AXL and high ERBB3 (A375, cluster 4). and their downstream targets in invadopodia, will facilitate Under AXL perturbation, changes in ERBB3 expression and phos- the design of an effective combinatorial therapy targeting the phorylation are seen. Other cells (e.g., those in clusters 1 and 2) metastatic process.

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Disclosure of Potential Conflicts of Interest expression plasmids. We thank Meenhard Herlyn for his kind gift of WM793 No potential conflicts of interest were disclosed. and A2508 cells (Wistar Institute, Philadelphia, PA). We thank the Genomics Center of Biomedical Core Facility (Technion, Israel) for cell authentication Authors' Contributions tests. We thank Itay Tirosh (Weizmann Institute of Science, Rehovot, Israel), Conception and design: O.-Y. Revach, B. Geiger Daniel Peeper and Oscar Krijgsman (Netherlands Cancer Institute, Amsterdam, Development of methodology: O.-Y. Revach the Netherlands), and Tamar Geiger (Tel-Aviv University, Israel) for insightful fi Acquisition of data (provided animals, acquired and managed patients, discussions and scienti c advice regarding AXL/ERBB3 cross-talk in melanoma. provided facilities, etc.): O.-Y. Revach This work was supported by Israel Ministry of Science (IMOS) French Program Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Grant no. 3-13024 and an Israel Science Foundation Grant no. 2749/17. computational analysis): O.-Y. Revach, O. Sandler B. Geiger is the incumbent of the Erwin Neter Professorial Chair in Cell and Writing, review, and/or revision of the manuscript: O.-Y. Revach, B. Geiger Tumor Biology. Y. Samuels is supported by the Israel Science Foundation grant Administrative, technical, or material support (i.e., reporting or organizing number 696/17. This project has received funding from the European Research ’ data, constructing databases): O.-Y. Revach Council (ERC) under the European Union s Horizon 2020 research and Study supervision: B. Geiger innovation program (grant agreement No. 770854). Other (assisted in analyzing the gene expression patterns): O. Sandler Other (provided the melanoma cell lines and relevant reagents (see Materials The costs of publication of this article were defrayed in part by the fi and Methods), as well as scienti c contributions regarding melanoma payment of page charges. This article must therefore be hereby marked biology and gene expression analysis): Y. Samuels advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments We thank Yosef Yarden (Weizmann Institute of Science, Rehovot, Israel) for providing reagents for ERRB3 detection and scientific advice regarding ERBB3. Received July 27, 2018; revised January 16, 2019; accepted March 22, 2019; We thank Aaron Meyer (MIT, Boston, MA) for his generous gift of AXL published first March 26, 2019.

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www.aacrjournals.org Cancer Res; 79(10) May 15, 2019 OF15

Cross-Talk between Receptor Tyrosine Kinases AXL and ERBB3 Regulates Invadopodia Formation in Melanoma Cells

Or-Yam Revach, Oded Sandler, Yardena Samuels, et al.

Cancer Res Published OnlineFirst March 26, 2019.

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