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Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Background The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell’s phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype. Methods The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2’-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells. Results Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells. Conclusions Our data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2185-5) contains supplementary material, which is available to authorized users.

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

8542 Background: The acquisition of new mutations can explain resistance to targeted therapies; however resistance also develops without demonstrable new mutations. To better understand potential mechanisms for treatment failure we studied subpopulations of chemo-resistant cells that can be identified in early passage cell lines and the cellular plasticity that yields these. Methods: Melanoma subpopulations were identified and sorted on the basis of functional assays. Gene expression was evaluated with Illumina HT12 arrays and qPCR to identify potential mechanisms and targets. Sorted cells were evaluated in vitro for the induction of phenotype switching and effects of target inhibition. Results: A subset of slow proliferating label-retaining cells (LRC) was identified using a membrane dye. Direct isolation of LRC from a pt showed these were not an in vitro artefact. LRC isolated from multiple cell lines comprised the majority of cells that resisted cytotoxic drugs and could invade through an artificial extracellular membrane. Gene expression profiling identified a network of over-expressed genes related to epithelial-to-mesenchymal transition (EMT) notable for the expression of Thrombospondin-1 (TSP-1), Transforming-Growth Factor, Beta Induced, (TGFBI) and other extracellular matrix molecules. Initial studies show that LRC shared gene expression characteristics with PLX4032-resistant cells. Although interrogating LRC by qPCR showed up-regulation of the putative melanoma stem cell marker ABCB5 and the lysine specific demethylase Jarid1B, cell sorting and serial re-labelling experiments revealed a dynamic and interchangeable phenotype for these cells, challenging the hierarchical stem cell model for melanoma. The observed slow-cycling and chemo-resistant phenotype could be induced in vitro through TGF beta-mediated phenotype switching and cellular invasion was blocked using an antibody against TSP-1. Conclusions: Cytokine-induced plasticity in melanoma yields invasive, therapy-resistant cells which express EMT-related genes and pathways. We hypothesize that these cells are a source of treatment resistance in vivo and blocking their emergence may prevent treatment failure.

// Aparna Jayachandran 1 , 2 , Matthew Anaka 1 , 2 , Prashanth Prithviraj 1 , 2 , Christopher Hudson 1 , Sonja J McKeown 3 , Pu-Han Lo 1 , Laura J Vella 1 , 2 , Colin R Goding 4 , Jonathan Cebon 1 , 2 , Andreas Behren 1 , 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: andreas.behren@ludwig.edu.au Keywords: 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.