Malignant mesothelioma : An experimental study with emphasis on proteoglycans in mesothelial cell growth and differentiation

Sammanfattning: Malignant mesothelioma is a highly aggressive tumor with median survival ranging from 4 to 12 months and, despite intense therapeutical efforts, it is invariably fatal. Mesothelioma cells are unique in the sense that they possess a biphasic growth potential and can be stimulated by serum growth factors to differentiate into stable epithelial or fibrous phenotypes. The prognosis of this tumor varies greatly depending on the differences in growth pattern, the most important predictor of poor prognosis being a fibrous phenotype. To study the molecular basis of mesothelial differentiation, we used benign and malignant mesothelial cells in various stages of phenotypic differentiation. In order to evaluate the impact of proteoglycans (PG) on this process, a series of PGs were analyzed by semi quantitative reversed transcriptase polymerase chain reaction. The cells with epithelial phenotype showed increased expression of syndecan-2, syndecan-4 and hyaluronan synthase, and fibroblast-like cells expressed more matrix PGs: versican, decorin and biglycan. The PG profile may serve as a "fingerprint", and reflect the maturation of mesothelial cells. The functional importance of syndecans in mesothelial differentiation was further shown by antisense targeting; down-regulation of each particular syndecan caused a loss of epithelial morphology, syndecans - 1 and -4 being also essential for cell adhesion. The differentiation of mesothelioma cells was influenced by treatment with various growth factors (TGF-beta2, EGF, FGF-2, IGF-I and PDGF-BB). These factors affected the proliferation and morphology of mesothelioma cells to various extents, and the PG profile changed, in parallel, with an induced epithelial-mesenchymal transition. Exposure to EG17 and IGF-I caused a fibroblast-like morphology simultaneously with a reduction in the syndecan expression levels. At the same time, the levels of shed syndecan-1 increased in the culture medium. The involvement of other regulatory molecules in mesothelioma differentiation was assessed by subtractive hybridization, which has revealed a limited number of genes being differentially expressed between cells of epithelial or fibrous phenotypes. Most of these genes were recovered from the epithelial cells, which may indicate a more mature phenotype. The expression level of thioredoxin reductase, a small redox-active protein involved in drug resistance, was extremely high in both cell sub-lines, and may reflect the generic insensitivity of mesotheliomas to chemotherapy. Although syndecans play a major role in regulating cell morphology, little is known about their subcellular distribution. Using confocal laser microscopy we found a substantial proportion of syndecans at intracellular locations, and syndecan-1 accumulated in the nucleus in a time-dependent manner. There was a close spatial relation of syndecans to tubulin in both interphase and mitotic cells. Vinblastine treatment interfered with the nuclear transport, and syndecan-1 and tubulin co- polymerize in paracrystalline occlusion bodies, in parallel with impaired nuclear transport. These findings suggest a tubulin-mediated transport mechanism. TGF-beta2 reduced the proliferation rate of mesothelioma cells, concomitantly with a delay in nuclear transport of syndecan-1. These data show that all syndecans are involved in maintaining the epithelial morphology, and that various amounts and translocation of syndecans may participate in molecular switches that regulate cell differentiation and proliferation. The above mechanisms may represent crucial steps, and possible future targets for therapy, that can be used to improve the management of patients with malignant mesothelioma.