www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 14 Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma Aparna Jayachandran1,2, Matthew Anaka1,2, Prashanth Prithviraj1,2, Christopher Hudson1, Sonja J McKeown3, Pu-Han Lo1, Laura J Vella1,2, Colin R Goding4, Jonathan Cebon1,2, Andreas Behren1,2 1 Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Cancer Immunobiology Laboratory, Heidelberg, VIC 3084, Australia 2 Department of Medicine, University of Melbourne, Victoria, 3010, Australia 3 Department of Anatomy and Neuroscience, University of Melbourne, Victoria, 3010, Australia 4 Ludwig Institute for Cancer Research, University of Oxford, Oxford, OX3 7DQ, UK Correspondence to: Andreas Behren, e-mail: [email protected] Key words: Thrombospondin 1, melanoma, epithelial-to-mesenchymal transition, chick embryo, invasion, drug resistance Received: April 29, 2014 Accepted: June 23, 2014 Published: July 08, 2014 ABSTRACT Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. INTRODUCTION new treatments targeting specific melanoma driver mutations such as BRAF V600E have a significant Melanoma is a frequently fatal malignancy of the impact, the benefit of treatment is often short lived neural crest-derived melanocytes, the pigment producing and the disease becomes rapidly fatal once resistance cells in skin, uveal tract and mucosal membranes. The develops [2, 3]. Accumulating evidence indicates that cause of death from melanoma is metastasis [1]. While the acquisition of invasive and metastatic characteristics www.impactjournals.com/oncotarget 5782 Oncotarget by melanoma cells involves the reactivation of a These changes in EMT markers are often associated with developmental epithelial-to-mesenchymal transition functional change toward an invasive phenotype [21]. We (EMT)-like program [4-6]. The complex mechanisms evaluated the expression of classical EMT genes, E- and involved in this process in melanoma remain largely N-cadherins using quantitative real-time RT-PCR (qRT- unknown. PCR) in a panel of 54 human melanoma cell lines that EMT describes a reprogramming of epithelial were derived from resected melanoma metastases [22]. cells that leads to the loss of epithelial characteristics, Expression patterns of these two molecules in the cell notably polarity and cell adhesion, and the acquisition of a lines varied from high N-cadherin with no E-cadherin mesenchymal phenotype with increased invasive abilities. expression, high E-cadherin with no or low N-cadherin, It occurs during normal development as part of processes to intermediate levels of both (Figure 1A). such as gastrulation and neural crest cell migration [7]. We divided the lines into those expressing During cancer progression, this phenotype is associated E-cadherin and those lacking E-cadherin expression, with tumor invasion, metastatic dissemination, and and their gene expression patterns were compared acquisition of resistance to drug treatment [8, 9]. Cadherin using previously generated whole genome microarray switching is a hallmark of EMT, leading to the down- expression profiling data [22]. 634 probes representing regulation and replacement of the cell surface adhesion 552 genes were differentially expressed between the molecule E-cadherin by N-cadherin, enabling motility two classes of cell lines (Supplementary Table S1). [10-12]. As expected E-cadherin expression was higher in the In various cancers, EMT leads to the generation lines identified as E-cadherin expressing by qRT-PCR of cancer cells possessing stem cell attributes of (13.5 fold), and N-cadherin expression was higher in tumor- initiation and resistance to chemotherapy [8]. the lines lacking E-cadherin expression by qRT-PCR. In melanoma, a sub-population of slow-cycling cells A principal components analysis based on the differential (defined by label-retention) which exhibit efficient expression gene list largely segregated the two classes tumor-initiating capacity, have properties associated with of cell lines, although with some overlap between lines stemness, and are resistant to various anticancer drugs has with intermediate levels of E- and N-cadherin, perhaps been identified [13]. representing a mixed phenotype (Supplementary Thrombospondins comprise a family of homologous Figure S1). A gene-set enrichment analysis (GSEA) of proteins that regulate cellular phenotype and extracellular the cell lines revealed gene sets associated with TGF-beta structure during tissue genesis and remodelling. signaling [23], cell migration [24, 25], ECM modulation Thrombospondin 1 (THBS1) was the first member to [26] and EMT [27-29]. Significantly enriched gene sets be identified, and has been shown to modulate tumor can be found in Supplementary Table S2. Based on the progression and metastasis [14, 15]. Whereas the role of GSEA results, and the evidence of opposing E- and THBS1 in angiogenesis in melanoma is well documented, N-cadherin expression, we therefore labelled the cell lines its role in tumor metastasis is only just emerging [16, mesenchymal- and epithelial-like. 17]. THBS1 has been identified as a major physiological We chose to focus on Thrombospondin 1 activator of transforming growth factor (TGF)-beta, a (THBS1), which showed 19 fold higher expression in the potent elicitor of EMT [18, 19]. However, a role of THBS1 mesenchymal-like cells. qRT-PCR confirmed THBS1 in mediating EMT in melanoma remains to be elucidated. mRNA expression levels were higher in mesenchymal- This study therefore aimed to reveal the functional roles of like cells (Figure 1B). A subset of high and low THBS1 THBS1 during melanoma progression by assessing THBS1 expressing cell lines, as determined by qRT-PCR, was expression and its effects on the functional characteristics subjected to solid-phase ELISA. This detected little or no of melanoma cells, particularly those associated with THBS1 secretion in conditioned medium from epithelial- mesenchymal transformation. like cells, whereas mesenchymal-like cells secreted significant amounts of THBS1 into the medium (Figure 1C). As THBS1 is a known activator of TGF-beta [18] RESULTS and TGF-beta has a pivotal function in the progression of EMT [30, 31], we examined the level of TGF-beta Melanoma cells exhibiting a mesenchymal secretion in a subset of high and low THBS1 secreting phenotype express high levels of Thrombospondin melanoma cell lines. THBS1 high cell lines secreted 1 associated with TGF-beta signaling high TGF-beta1 in contrast to THBS1 low cell lines that secreted no TGF-beta1 into the medium (Supplementary At the molecular level, EMT in melanoma cells is Figure S2A). To extend these findings, we analyzed characterized by a series of coordinated changes including a cutaneous melanoma dataset available from The down-regulation of the adherens junction molecule Cancer Genome Atlas (TCGA) (http://www.cbioportal E-cadherin and upregulation of N-cadherin [5, 20]. .org) [32, 33]. Mutual exclusivity data from 376 melanoma www.impactjournals.com/oncotarget 5783 Oncotarget Figure 1: Classification of a panel of melanoma cells lines based on gene expression.qRT-PCR analysis revealed expression of (A) E- and N-cadherins and (B) THBS1 in a panel of 54 melanoma cell lines. (C) Media from 4 mesenchymal- and 4 epithelial-like melanoma cells were collected and subjected to THBS1 ELISA. ANOVA analysis of the two sets of cell lines was significant ( p<0.005). patients revealed that both THBS1 and TGF-beta1 are MelanA (MLANA) (fold change: 37), Tyrosinase (TYR) co-expressed (Odds ratio =3.4, p=0.036, Fisher’s exact (fold change: 27), and MITF (fold change: 10) were test). TGF-beta1 treatment in two epithelial-like melanoma up-regulated in the epithelial-like cells examined. qRT-PCR cell lines induced THBS1 expression in a time-dependent