528 MicroRNA-130a (miR-130a) regulates self-renewal capacity and tumor growth in cutaneous squamous cell carcinoma W Lohcharoenkal, K Das Mahapatra, N Xu Lande ´n, E Sonkoly and A Pivarcsi Department of Medicine, Unit of Dermatology and Venereology, Karolinska Institutet, Stockholm, Sweden Cutaneous Squamous Cell Carcinoma (cSCC) is a malignant neoplasm of epidermal kerati- nocytes resulting from the accumulation of somatic mutations in oncogenes and tumor suppressor genes due to solar radiation. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein-coding genes at the posttranscriptional level. Previ- ously, we performed a global analysis of miRNA expression in cSCC and identified miR-130a to be downregulated in tumors; However, its role in cSCC has been unknown. In this study, we investigated the function of miR-130a in cSCC using in vitro and in vivo models of cancer. qPCR results demonstrated the decreased expression of miR-130a in cSCC compared to healthy skin, which was also confirmed by in situ hybridization. The ectopic overexpression of miR-130a in two different SCC cell lines (A431 and UT-SCC-7) resulted in the suppression of their self-renewal capacity as determined by colony-forming assays. Moreover, miR-130a inhibited the migratory capacity of SCC cells as determined by scratch- and Transwell migration assays and their capacity to invade through Matrigel in Transwell invasion assays. To address the role of miR-130a in vivo, we engineered a stable miR-130a-overexpressing SCC cell line via lentiviral transduction and performed tumor xenograft experiments by injecting miR-130a-overexpressing cells as well as control cells into immunodeficient mice subcutaneously. Measurement of the tumor volume revealed that miR-130a suppressed cancer growth on mice from 4 weeks after injection and onwards significantly. The tumors were harvested at week 6 and we observed that tumors formed by miR-130a-overexpressing SCC cells weighed significantly less compared with controls. Taken together, miR-130a suppresses the self-renewal capacity and invasiveness in vitro and tumor growth in vivo in cSCC. Our results will contribute to the understanding of the molecular processes leading to the neoplastic transformation of keratinocytes. 529 Microphthalmia-associated transcription factor regulates dynamic melanoma heterogeneity NK Haass 1,2 , L Spoerri 1 , CA Tonnessen 1 , KA Beaumont 2 , DS Hill 2 , RJ Jurek 2 , SM Daignault 1 ,F Ahmed 1 , AG Smith 1 and W Weninger 2 1 University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia and 2 Centenary Institute, Sydney, NSW, Australia Dynamic heterogeneity, is a prime source for drug resistance. As distinct proliferative and invasive capabilities reflect variable drug sensitivities, identifying these different responses is crucial to design effective therapies. We utilize real-time cell cycle imaging (FUCCI) in 3D in vitro and in vivo to study melanoma heterogeneity. Mouse xenograft tumors generated from cell lines with high microphthalmia-associated transcription factor (MITF) level displayed a homogeneous distribution of cycling cells throughout. In contrast, tumors generated from cell lines with low MITF levels were composed of clusters of cycling cells and clusters of G1- arrested cells. The proliferating areas were in close proximity to blood vessels, presumably characterized by oxygen/nutrient availability. Indeed, knock-down of MITF in MITF-high melanoma cells resulted in the same clustered phenotype presented in xenografts generated from MITF-low melanoma cells. Melanoma spheroids recapitulated the in vivo cycling behavior, considering that here oxygen and nutrients are supplied by diffusion. MITF was undetectable within the hypoxic G1-arrested spheroid core, indicating hypoxia-induced MITF downregulation. Modulation of MITF expression impacted spheroid architecture and size, with overexpression giving rise to less compacted structures and vice versa. We show that MITF protects from cell cycle arrest induced by oxygen/nutrient deprivation. High MITF levels prevent cell cycle arrest by reducing the cell-intrinsic propensity to arrest in response to low oxygen/nutrient and concurrently by allowing sufficient supply of oxygen/nutrients to cells. The latter is achieved through decreased cell-cell adhesion resulting in the generation of looser, ‘spongier’ tumors that may allow more efficient oxygen/nutrient diffusion. Taken together, MITF is a potent regulator of dynamic heterogeneity, which in turn impacts on drug sensitivity. 530 Deciphering tumour heterogeneity towards optimised characterisation of malignant melanoma J Griss 1 , R Mayer 2 , C Wagner 1 , C Gerner 2 and S Wagner 1 1 Dermatology, Medical University of Vienna, Vienna, Austria and 2 Analytical Chemistry, University of Vienna, Vienna, Austria Targeted therapies have become a mainstay of melanoma therapies. However, resistance or heterogeneous responses to these therapies is diminishing their clinical success. One explanation for this phenomenon is that tumours contain diverse clones driven by different driver mechanisms causing varying responses to therapy. This tumour heterogeneity is an unsolved problem for the selection of appropriate therapies. In this first phase of our project we aimed to estimate whether and how intra-tumour heterogeneity can be mapped by in depth whole proteome analysis. Two metastases from two patients were split in four and five parts respectively. Each of these parts was subsequently analysed using MS/MS based label- free shotgun proteomics with one technical replicate. Spectra were identified using the search engine MSGF+ and clustered using our novel spectra-cluster algorithm. The clustering results were used to improve the accuracy of the label-free quantitation. On average, 2.500 proteins were identified per metastasis piece. Using the clustering results significantly reduced the observed technical variability. As expected, the predominant factor of variability was the two different patients with 698 proteins differentially expressed between them. A pathway anal- ysis revealed that while one patient had a predominantly innate immune response, the other patient showed a strong adaptive immune response (i.e. changes in MHC 1 and 2 complex, IFN and NFkB related proteins). Additionally, one patient showed a strong up-regulation of proteins responsible for extracellular matrix degradation and adherens junctions. These ex- periments show that we are able to identify central processes to cancer biology using pro- teomics technologies. Additionally, through our in-depth analysis we are able to pinpoint regional changes of these altered pathways within a single metastasis and thereby assess the influence of intra-tumour heterogeneity on future projects. 531 Galectin-9 induces apoptosis of tumor cells through caspase pathways in cutaneous T-cell lymphoma R Nakajima, T Miyagaki, H Kamijo, T Oka, N Takahashi, H Suga, M Sugaya and S Sato University of Tokyo Graduate School of Medicine, Bunkyo-ku, Japan Galectin-9 is a member of the tandem-repeat galectin family, which is widely distributed in human tissues. Galectin-9 has the capacity to regulate multiple physiological and patho- logical processes including antitumor immunity. T cell immunoglobulin and mucin domain 3 (Tim-3), one of the receptors of galectin-9, is expressed on IFN-g-producing Th1 cells and CD8 + T cells, and galectin-9 induces apoptosis of these cells. Actually, anti-Tim-3 antibody promotes IFN-g-mediated antitumor immunity. On the other hand, galectin-9 is known to induce apoptosis of malignant cells, such as chronic myeloid leukemia cells and malignant melanoma cells. Thus, galectin-9 has the capacity to both suppress antitumor immunity and promote apoptosis of tumor cells. To elucidate galectin-9 involvement in cutaneous T-cell lymphoma (CTCL), we first investigated the direct effect of galectin-9 on CTCL cell lines. We revealed that galectin-9 induced apoptosis in CTCL cell lines (HUT78, MyLa, SeAx, HH, and MJ cells). This effect was independent of Tim-3 expression, as anti-Tim-3 antibody failed to suppress galectin-9-induced apoptosis of CTCL cell lines, although these CTCL cell lines expressed Tim-3 on the cell surface. In addition, inhibitors of caspase-3, caspase-8, or cas- pase-9 suppressed galectin-9 induced apoptosis in CTCL cell lines. We next examined galectin-9 expression in blood and lesional skin of patients with CTCL. Serum galectin-9 levels were elevated in advanced CTCL patients, and significantly correlated with serum levels of lactate dehydrogenase and soluble IL-2 receptor. Furthermore, Galectin-9 mRNA expression levels are increased in lesional skin of CTCL, especially that of advanced CTCL. As previously reported in other cancers, patients with advanced CTCL expressing a high level of galectin-9 mRNA showed a more favorable survival prognosis than those with low galectin-9 mRNA levels. These results suggest that administration of galectin-9 can be a new therapeutic strategy for CTCL. 532 Variable but distinct metabolic signature in malignant melanoma R Lang 1 , RG Feichtinger 5 , R Geilberger 5 , F Rathje 5 , JA Mayr 2 , W Sperl 2 , JW Bauer 1 , C Hauser- Kronberger 3 , M Emberger 4 and B Kofler 5 1 Department of Dermatology, Paracelsus Medical University Salzburg, Salzburg, Austria, 2 Department of Pediatrics, Paracelsus Medical University Salzburg, Salzburg, Austria, 3 Institute of Pathology, Paracelsus Medical Uni- versity Salzburg, Salzburg, Austria, 4 Laboratory for Pathology Weger/Emberger, Salzburg, Austria and 5 Department of Pediatrics, Research Program for Receptor Biochemistry and Tumor Metabolism, Paracelsus Medical University Salzburg, Salzburg, Austria The Warburg theory of cancer postulates that an important driver of tumorigenesis is insuf- ficient respiration caused by mitochondrial defects, and simultaneous boost of lactate gen- eration due to exaggerated aerobic glycolysis. Here we provide direct evidence that two types of malignant melanomas can be distinguished by their bioenergetic characteristics. We analyzed 48 melanoma samples by immunohistochemistry and found that 38% of mela- nomas are characterized by areas of isolated or combined deficiencies of complexes of the oxidative phosphorylation (OXPHOS). The incidence of OXPHOS deficient areas is associ- ated with an increased Breslow index suggesting pathological significance. 62% of mela- nomas showed high levels of all OXPHOS complexes. Mitochondrial mass was high in all melanomas. Carbonic anhydrase IX protein levels were low, indicating that melanomas generally are well-oxygenated tumors. Expression of hypoxia-inducible factor-1a (HIF-1a) and monocarboxylate trasporter- 4 (MCT4) was high, which might be explained by the sig- nificant increase of lactate dehydrogenase A and presumable elevated levels of lactate in melanomas. Thus, our data indicate that there are two types of malignant melanoma: one that features a classic Warburg signature (high glycolysis, low OXPHOS), whereas the other one, despite being glycolytic, maintains a relatively high level of OXPHOS complexes. 533 PDE4D is a therapeutic target in melanoma N Dumaz 1,2 , D Julie 1,2,3 , S Becherirat 1,2 , J Andre 1,2 and C Lebbe 1,2,3 1 INSERM U976, Paris, France, 2 UMRS 976, University Paris Diderot, Paris, France and 3 Dermatology Depart- ment, Saint Louis Hospital, Paris, France The cAMP pathway is both important for normal melanocyte biology and implicated in melanoma development. We have shown that transformation of melanocytes by oncogenic BRAF, NRAS or KIT inhibits cAMP signalling. This inhibition involves the phosphodiesterase PDE4D, enzyme that physiologically degrades cAMP. PDE4D expression, and in particular the expression of the PDE4D5 isoform, is increased in patients with advanced melanoma. We have previously shown that PDE4D promoted melanoma invasion by interacting with focal adhesion kinase (FAK) through the scaffolding protein RACK1. We are now showing that inhibiting PDE4D by chemical inhibitor or RNA interference significantly reduces clone formation in BRAF, NRAS and KIT mutated melanoma cell lines. Inhibition of PDE4D also reduces the growth of melanoma cells in 3D as spheroids. Moreover, we demonstrate that PDE4 inhibitors inhibit the proliferation of melanoma cell lines, which were resistant to the BRAF inhibitors used to treat BRAF-mutated melanoma. PDE4 isoforms may therefore serve as a new therapeutic target for the treatment of aggressive melanoma and may prevent the emergence of melanoma clones with enhanced metastatic capabilities. Indeed, specific PDE4 inhibitors are available in the clinics and could be used as a complementary therapeutic approach. Melanoma and Other Skin Cancers | ABSTRACTS www.jidonline.org S283