ABSTRACT
Metastatic tumor cells originating from cancers of a variety of tissues such as breast, skin, and prostate may remain dormant for long periods of time. In the case of uveal melanoma, the principal malignancy of the eye, complete removal of the primary tumor by enucleation can nonetheless be followed by metastatic tumor growth in distant organs months, years, or even decades later. This suggests that tumor cells have already spread to secondary sites at the time of treatment and remain dormant as micrometastases. Identifying factors that govern long-lived survival of metastatic tumor cells is therefore key to decreasing mortality associated with this and other diseases. While investigating factors differentially expressed in melanoma cells and normal melanocytes, we identified the receptor tyrosine kinase Axl and found up-regulation of Axl in uveal melanomas and melanoma cell lines by RNase protection, Western analysis, and immunohistochemistry. Axl has been shown to mediate cell growth and survival through its ligand Gas6 in non-transformed cells. To test whether stimulation of Axl can enhance survival of uveal melanoma cells, we assessed the degree of mitogenesis and cell survival by bromodeoxyuridine incorporation and trypan blue exclusion, respectively, upon stimulation of Mel 290 uveal melanoma cells with Gas6 in vitro. We show that Gas6 mediates mitogenesis and cell survival in Mel 290 cells. We further demonstrate that these effects occur specifically through the Axl receptor by modulating the expression of Axl with an antisense construct. cDNA microarray analysis of 12,687 genes then revealed that Gas6 stimulation of Axl in Mel 290 cells results primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data show that the Axl pathway mediates increased survival of uveal melanoma cells, potentially advantageous during cancer dormancy, and that Axl may function in part through regulation of Cyr61.
Subject(s)
Cell Survival/physiology , Melanoma/enzymology , Melanoma/pathology , Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Uveal Neoplasms/enzymology , Uveal Neoplasms/pathology , Base Sequence , Cell Division/physiology , Cells, Cultured , DNA Primers , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/genetics , Melanocytes/cytology , Polymerase Chain Reaction/methods , Proto-Oncogene Proteins , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured , Axl Receptor Tyrosine KinaseABSTRACT
OBJECTIVE: To study the expression of angiogenic factors Cyr61 and tissue factor (TF) in uveal melanoma and its correlation with blood vessel density. METHODS: Suppression subtractive hybridization was used to identify genes that are differentially expressed between cell lines of uveal melanoma and normal uveal melanocytes. Expression of these genes was subsequently verified in primary uveal melanomas and correlated with the number of blood vessels in archival specimens by immunohistochemical analysis. RESULTS: Cyr61 and TF are expressed at elevated levels in cell lines of uveal melanoma compared with normal uveal melanocytes. Duplication of a region of chromosome arm 1p, encompassing the genes encoding Cyr61 and TF, was observed in the melanoma cell line used in the initial subtractive hybridization. Both genes are also expressed in primary uveal melanomas, and a correlation was found between expression of TF and the number of blood vessels in archival specimens. CONCLUSIONS: Cyr61 and TF may contribute to the angiogenic phenotype associated with uveal melanoma. A region of chromosome arm 1p also may contain oncogenes or tumor suppressor genes pertinent to the origins of this type of ocular tumor. CLINICAL RELEVANCE: New immunotherapies have been devised for the treatment of cancer based on the expression of TF. Similar approaches may be effective in treating uveal melanoma.
