Tumor Biology and Immunology miR-519d Promotes Melanoma Progression by Downregulating EphA4 Kuo-Tai Hua 1 , Jin-Bong Hong 2 , Yi-Shuan Sheen 2 , Hsin-Yi Huang 3 , Yi-Ling Huang 2 , Jau-Shiuh Chen 2 , and Yi-Hua Liao 2 Abstract Increasing evidence suggests that there is a unique cell sub- population in melanoma that can form nonadherent melano- spheres in serum-free stem cell medium, mimicking aggressive malignancy. Using melanospheres as a model to investigate progression mechanisms, we found that miR-519d overexpres- sion was sufficient to promote cell proliferation, migration, invasion, and adhesion in vitro and lung metastatic capability in vivo. The cell adhesion receptor EphA4 was determined to be a direct target of miR-519d. Forced expression of EphA4 reversed the effects of miR-519d overexpression, whereas silencing of EphA4 phenocopied the effect of miR-519d. Malignant pro- gression phenotypes were also affected at the level of epithelial- to-mesenchymal transition and the ERK1/2 signaling pathway inversely affected by miR-519d or EphA4 expression. In clinical specimens of metastatic melanoma, we observed significant upregulation of miR-519d and downregulation of EphA4, in the latter case correlated inversely with overall survival. Taken together, our results suggest a significant functional role for miR-519d in determining EphA4 expression and melanoma progression. Significance: These results suggest a significant role for miR- 519d in determining expression of a pivotal cell adhesion mol- ecule that may impact risks of malignant progression in many cancers. Cancer Res; 78(1); 216–29. Ó2017 AACR. Introduction Malignant melanoma, which is associated with high morbidity and mortality, is a tumor resulting from transformed melanocytes or nevocytes that are derived from the neural crest. Melanoma cells and neural crest stem cells were also found to exploit common molecular mechanisms (1). Historically, neural stem cells are typically cultured in vitro under nonadherent conditions as neuro- spheres (2). Sphere-forming assays are also widely used in diverse cancer tissues and the cancer stem cell research (3, 4). It has been found that a subpopulation of cells in human melanomas can form nonadherent melanospheres in serum-free stem cell medi- um (5). In comparison with the adherent melanoma cells, these spheroid cells were demonstrated to have the stem-like properties of self-renewing and multipotency to differentiate under appro- priate conditions into multiple cell lineages, such as melanocytes, adipocytes, osteocytes, and chondrocytes (5). The spheroid cells were highly tumorigenic that intradermal injections in SCID mice of as few as 100 cells generated tumors that maintained tumor- igenic potential into subsequent recipients (6–8). Although recent evidence has challenged the notion of only a selected subpopu- lation of cancer cells having the ability to form a melanoma tumor (9), melanospheres still represent an enrichment of heteroge- neous cell groups with enhanced clonogenic potential (6). More- over, melanospheres presented a more aggressive phenotype with higher invasiveness and increased antitumor immune escape (10, 11). Taking advantage of this in vitro spheroid culture model, we exploited this system to further characterize the mechanism of melanoma progression and aggressiveness. miRNAs are a class of single-stranded, small noncoding RNAs (22 nucleotides) that act through complementary interaction with the target mRNAs, resulting in translational repression or cleavage of the target mRNAs (12, 13). Studies have shown that dysregulation of miRNAs are implicated in melanoma formation and progression. As melanospheres represent a cell model of stemness as well as aggressiveness, we searched the literature for a possible miRNA candidate and found miR-519d, one of the members in chromosome 19 miRNA cluster (C19MC), which is the largest primate-specific miRNA cluster (14) preferentially expressed in stem cells (15–17), was shown to be differentially expressed between primary melanomas and metastases (18). In this study, we are interested in investigating whether miR-519d contributed to melanoma progression by studying three-dimen- sional spheroid system. EphA4 belongs to the large Eph (erythropoietin-producing hepatocellular carcinoma) family of receptor tyrosine kinases and has been demonstrated to have different, even paradoxical, effects in various human tumor context. For example, upregu- lation of EphA4 correlated with liver metastasis in colorectal cancer (19), and overexpression of EphA4 could also enhance cell adhesion to facilitate metastasis of hepatocellular carcino- ma (20). Through the EphA4–FGFR1 signaling pathway, EphA4 promoted cell proliferation and migration in the human glio- ma U251 cell line (21). In contrast, EphA4 overexpression in 1 Graduate Institute of Toxicology, College of Medicine, National Taiwan Univer- sity, Taipei, Taiwan. 2 Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan. 3 Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Yi-Hua Liao, Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, 7 Chung-Shan South Road, Taipei 10002, Taiwan. Phone: 886-2-2356-2141; Fax: 886-2-2393-4177; E-mail: yihualiao@ntu.edu.tw doi: 10.1158/0008-5472.CAN-17-1933 Ó2017 American Association for Cancer Research. Cancer Research Cancer Res; 78(1) January 1, 2018 216 on May 31, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-1933