ABSTRACT
PURPOSE: Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors. METHODS: Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice. RESULTS: Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice. CONCLUSIONS: Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , MART-1 Antigen/genetics , Melanoma/genetics , RNA, Neoplasm/genetics , Uveal Neoplasms/genetics , Animals , Blotting, Western , Cell Count , Cell Line, Tumor , Female , Humans , Immunohistochemistry , MART-1 Antigen/biosynthesis , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Nude , Microscopy, Phase-Contrast , Neoplasms, Experimental , Polymerase Chain Reaction , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathologyABSTRACT
PURPOSE: Uveal melanoma (UM) has been the subject of intense interest due to its distinctive metastatic pattern, which involves hematogenous dissemination of cancerous cells toward the liver in 50% of patients. To search for new UM prognostic markers, the Suppressive Subtractive Hybridization (SSH) technique was used to isolate genes that are differentially expressed between UM primary tumors and normal uveal melanocytes (UVM). METHODS: A subtracted cDNA library was prepared using cDNA from uncultured UM primary tumors and UVM. The expression level of selected genes was further validated by cDNA microarray, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence analyses. RESULTS: One hundred-fifteen genes were identified using the SSH technique. Microarray analyses comparing the gene expression profiles of UM primary tumors to UVM validated a significant differential expression for 48% of these genes. The expression pattern of selected genes was then analyzed by semi-quantitative RT-PCR and was found to be consistent with the SSH and cDNA microarray findings. A down-regulation of genes associated with melanocyte differentiation was confirmed in UM primary tumors. Presence of undifferentiated cells in the UM was demonstrated by the expression of stem cell markers ATP-binding cassette sub-family G member 2 (ABCG2) and octamer-binding protein 4 (OCT4). CONCLUSIONS: We demonstrated that the SSH technique is efficient to detect differentially expressed genes between UM and UVM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor. In addition, deregulation of the melanocyte differentiation pathway revealed the presence of UM cells exhibiting a stem cell-like phenotype.
Subject(s)
Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Melanocytes/metabolism , Melanoma/genetics , Neoplasm Proteins/genetics , Uvea/metabolism , Uveal Neoplasms/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Adult , Aged , Biomarkers, Tumor/genetics , Cell Differentiation/genetics , Comparative Genomic Hybridization/methods , DNA, Complementary , Female , Gene Library , Humans , Liver Neoplasms/mortality , Liver Neoplasms/secondary , Male , Melanocytes/pathology , Melanoma/mortality , Melanoma/pathology , Middle Aged , Octamer Transcription Factor-3/genetics , Survival Rate , Tumor Cells, Cultured , Uvea/pathology , Uveal Neoplasms/mortality , Uveal Neoplasms/pathologyABSTRACT
Cancer aggressiveness is related to the ability of cancer cells to escape the anchorage dependency toward the extracellular matrix, a process regulated by the integrin α5ß1 and its ligand fibronectin. Here, we characterized the expression of the α5 gene in human uveal melanoma cell lines with distinct tumorigenic properties and investigated some of the mechanisms underlying the variations of their malignancy. Strong and weak expression of α5 was observed in cells with no (T108/T115) and high (T97/T98) tumorigenic properties, respectively. Expression and DNA binding of the transcription factors Sp1, activator protein 1 (AP-1) (both acting as activators), and nuclear factor I (NFI) (a strong repressor) to the α5 promoter were demonstrated in all cell lines. A reduced expression of AP-1 combined with a dramatic increase in NFI correlated with the suppression of α5 expression in T97 and T98 cells. Restoring α5 expression in T97 cells entirely abolished their tumorigenicity in immunodeficient mice. These uveal melanoma cell lines might therefore prove particularly useful as cellular models to investigate α5ß1 function in the pathogenesis of invasive uveal melanoma.
