Purpose The presence of epithelial cells is a bad prognostic factor in uveal melanoma (UM) and epithelial tumors are characterized by high expression of E‐cadherin. However, CDH1, the gene encoding for E‐cadherin, is located at chromosome 16q which is lost in UM with a bad prognosis. In order to solve this paradox we studied the underlying epigenetic mechanism of the spindle to epithelial transition and the accompanying E‐cadherin expression. Methods UM cell lines were in vitro treated with methylation and deacetylation inhibitors, or were cultured under hypoxic conditions to mimic in vivo UM conditions to induce epigenetic reprogramming. RNA and DNA was isolated for gene expression, DNA methylation, and chromosome structure analysis, while cell lysates were used for E‐cadherin analysis. Results Treatment with methylation and deacetylation inhibitors resulted in a phenotypic change from a spindle to more epithelial cell type. Furthermore, inhibition of methylation and hypoxia treatment both upregulated E‐cadherin expression in vitro and induced euchromatin formation at 16q22, where CDH1 is located. Conclusion By revealing epigenetic regulation of CDH1 in UM we resolve a step in the dynamic process of spindle to epitheloid celltype switch in UM. Since this switch could be induced by hypoxia we establish a link between environmental stress and progression. With these data we provide evidence for epigenetic regulation of UM progression as well as an option for future treatment.
Purpose Highly malignant uveal melanoma contain increased numbers of lymphocytes and macrophages. We wondered whether hypoxia plays a role in the development of this inflammation. We analysed whether hypoxia induces uveal melanoma cells to release pro‐inflammatory cytokines, and whether tumor supernatant (TSN) affects monocyte migration and differentiation. Methods The expression of pro‐inflammatory genes in freshly cultured uveal melanoma was studied in an in vitro 24 hour hypoxic culture system using qPCR. Cell lines were cultured under normoxic and hypoxic conditions. Chemotaxis was tested using a transwell system with purified monocytes and TSN. Differentiation was tested by adding TSN to a monocyte‐DC culture. CCL2, IL‐6 and PGE2 levels in TSN were determined by ELISA. Results Exposure of freshly‐cultured uveal melanoma cells to hypoxia led to an increased expression of the pro‐inflammatory cytokines PLGF, TGFß, END1, ICAM1 and a lower expression of AIMP1 (EMAP2), CCL2 (MCP‐1), and IL1b. TSN from melanoma cells lines, cultured in normoxic as well as hypoxic conditions, was able to attract monocytes. Migration was independent of tumor‐produced CCL2. Uveal melanoma supernatant inhibits monocyte differentiation. Conclusion Under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. TSN from uveal melanoma cell lines is capable of affecting the chemotactic response of monocytes in vitro, irrespectively of the hypoxic or normoxic conditions. Our data suggest that the induction of immune cells is not dependent on intratumoral oxygen level, and the uveal melanoma TSN does not skew macrophage polarization phenotype.
Purpose Uveal melanoma (UM) is the most common intra‐ocular tumor in adults that often leads to metastases for which no effective treatment is available. The aim of this study was to analyze molecular mechanisms in UM in order to reveal treatment targets. Methods Efficacy of kinase inhibitors and molecular mechanisms are analyzed in preclinical models of UM. Focus in this study was on the kinases and pathways that are associated with metastasis. Results Based on c‐Met expression in UM with a bad prognosis (monosomy 3) and activation of c‐Met in all metastasis cell lines c‐Met was chosen as treatment target and Crizotinib was chosen as candidate drug. Crizotinib is a dual kinase inhibitor that inhibits both ALK and c‐Met. Molecular analysis revealed that Crizotinib was able to inhibit c‐Met in UM effectively at nano‐molar range. Proliferation was however not affected by Crizotinib treatment when cells were grown attached to culture dishes. To achieve growth inhibition with Crizotinib cells had to be grown non‐attached. Moreover, only tumor cells that contained activated c‐Met were affected by Crizotinib while c‐Met negative cells continued to grow in the presence of the drug. Molecular analysis furthermore revealed that focal adhesion kinase (FAK) is a downstream target that was also inactivated in response to Crizotinib treatment. FAK is involved in tumor cell motility and tumor cell extravasation that are both crucial processes in metastasis. Conclusion We propose Crizotinib as a possible treatment option for metastasizing UM. As c‐Met expression is highly correlated with monosomy 3, both c‐Met and monosomy 3 may be evaluated as biomarker for Crizotinib treatment.
Purpose Uveal melanoma patients that present a tumor with monosomy 3 have a high propensity to develop metastases. Based on the two‐hit hypothesis, one might expect that the remaining copy of chromosome 3 contains a genetically‐modified gene and that loss of this gene is responsible for malignant progression. Identification of the gene and the genetic or epigenetic mechanisms that influence genes on chromosome 3 will enlighten the pathway(s) that determine uveal melanoma progression. In addition, studying epigenetics may result in useful molecular markers: whereas analysis of monosomy 3 is useful in primary tumors, it is useless in the detection of spreading tumor cells in the blood. We propose that a molecular marker in the form of a (tumor specific) genetic or epigenetic modification will be more helpful in this respect. Methods We have analyzed genes on chromosome 3 for epigenetic regulation that may suffice in non‐invasive testing for spreading tumor cells. Results We have identified RASSF1a methylation as a predictor of metastasis in uveal melanoma based on analysis of uveal melanoma tissues. In order to detect disseminating tumor cells in the bloodstream of uveal melanoma patients we have developed a very sensitive assay for methylation of RASSF1a. The detection limit of RASSF1a methylation exceeds 1/10,000 and in combination with isolation cell‐free DNA we are able to detect tumor DNA in the background of a vast excess of normal blood cells. With this assay we will test patient blood and validate the prognostic and diagnostic value of this marker. Conclusion With this assay we will test patient blood and validate the prognostic and diagnostic value of this marker.
Purpose Chromosomal aberrations and the inflammatory phenotype have been identified as predictive factors for survival of uveal melanoma (UM). The mechanism by which these factors are linked remains obscure. We took the human leukocyte antigen (HLA), located on chromosome 6p as marker for inflammation and studied whether aberrations of chromosome 6p influenced the HLA expression in UM. Methods SNP‐array copy number analysis and gene expression profiling were performed on 28 uveal melanomas of patients who underwent enucleation between 1999 and 2004 at the Leiden University Medical Center, Leiden, in The Netherlands. UM protein expression of HLA‐A, ‐B and –DR was measured with immunohistochemistry. The status of chromosome 3, 6p, and gene expression of HLA and HLA regulators were analyzed, as well as protein expression of HLA. Results Gain of 6p was present in 8 cases (29%) and not related with survival. An increased gene expression was seen in the group who died due to metastatic UM for HLA‐A and –B, and B2M. Gene expression correlated with protein expression for HLA‐A, HLA‐B, but not with HLA‐DR. Tumors did not differ in level of HLA protein expression, HLA gene expression, and HLA regulator gene expression with regard to chromosome 6p status. Conclusion High HLA gene‐ and protein expression in UM are not influenced by gain of 6p. Yet for the first time we demonstrated that in UM an increased expression of HLA class I genes correlated with the elevated protein expression of HLA class I.
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