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 micros- copy-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, Ó2019 American Association for Cancer Research. of the regulators identified in the original screen, which may be www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 26, 2019; DOI: 10.1158/0008-5472.CAN-18-2316 Revach et al. 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 invado- podia 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 medi- um 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