Imatinib mesylate alters the expression of genes related to disease progression in an animal model of uveal melanoma

Imatinib mesylate (IM) is a compound that inhibits both BCR-ABL tyrosine kinase and c-kit receptors. Tyrosine kinases are important in cellular signaling and mediate major cellular processes such as proliferation, differentiation, apoptosis, attachment, and migration. Twenty-six albino rabbits were injected with 1 x 10(6) human uveal melanoma (UM) cells (92.1) into the suprachoroidal space. Animals were immunosuppressed (cyclosporin A) over the course of the 12-week experiment and divided into two groups (n = 13). the experimental group received IM once daily by gavage while the control group received a placebo. One animal per group was sacrificed every week after the 2nd week. Upon necropsy, organs were harvested for histopathological examination. Cells from the primary tumors were recultured and tested in proliferation and invasion assays. A PCR array was used to investigate the differences in expression of 84 genes related to tumor metastasis. in the treated group, 4 rabbits developed intraocular tumors, with an average largest tumor dimension (LTD) of 2.5 mm and 5 animals reported metastatic disease. Whereas 6 rabbits in the control group developed intraocular tumors, with an average LTD of 5.8 mm and 6 animals reported metastatic disease. the recultured cells from the treated group demonstrated lower proliferation rates and were less invasive (p < 0.001). the PCR array showed differences in expression of genes related to metastasis. Notably, there was 290-fold increase in SERPINB5, a tumor suppressor gene, and a 10-fold higher expression of KISS I, a metastasis suppressor gene, in the treated group. Proangiogenic genes such as VEGFA, PDGFA and PDGFB were downregulated in the treated group. To the best of our knowledge, this is the first report detailing the altered expression of specific genes in UM cells after treatment with IM.McGill Univ, Ctr Hlth, Dept Ophthalmol & Pathol, Montreal, PQ, CanadaHenry C Witelson Ocular Pathol Lab, Montreal, PQ, CanadaUniversidade Federal de São Paulo UNIFESP EPM, Dept Ophthalmol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP EPM, Dept Ophthalmol, São Paulo, BrazilWeb of Scienc

Lysyl Oxidase: A potential modulator of early events in the Uveal Melanoma Metastatic Cascade

LOX is a marker of poor prognosis in several malignancies and is hypothesized to promote a migratory phenotype in hypoxic breast carcinomas. This thesis aims to characterize the expression of LOX and LOXL2 in human UM and to evaluate the transcriptional control of LOX under simulated hypoxic conditions. DPA and BAPN, both LOX catalytic inhibitors, were used to evaluate the effect of inhibiting this enzyme on cellular proliferation and invasion. Eighty-one percent of FFPE samples stained positive for LOX. High LOX expression correlated with the aggressive epithelioid cell type and was associated with shorter metastasis-free survival. Simulated hypoxia resulted in a significant increase in LOX mRNA expression likely mediated by HIF-1 alpha transcriptional promotion. Inhibiting LOX's catalytic activity significant reduced cellular invasion but had no effect on cell proliferation. These findings suggest that LOX may facilitate the escape of malignant cells from the eye following the development of tissue hypoxia.LOX est un indicateur d'un moins bon pronostic pour certaines tumeurs malignes et, hypothétiquement, il facilite le phénotype migrateur dans les carcinomes hypoxiques du sein. Les objectifs de cette thèse sont de caractériser l'expression de LOX et LOXL2 dans le mélanome de l'uvée humaine et d'évaluer le contrôle transcriptionel de LOX sous des conditions hypoxiques simulées. DPA et BAPN, deux inhibiteurs catalytiques de LOX, ont été utilisés pour bloquer cette enzyme et évaluer les effets sur la prolifération et l'invasion cellulaires. Quatre vingt et un pour cent des échantillons FFPE sont positifs pour LOX. Une corrélation étroite existe entre une forte expression de LOX et le type agressif de cellules épithélioïdes qui est associé avec une survie sans métastase de courte durée. L'hypoxie simulée a causé une grande augmentation de l'expression de LOX mRNA, probablement facilitée par l'activation de la transcription HIF-1 alpha. En bloquant l'activité catalytique de LOX, l'invasion cellulaire est considérablement réduite mais, par contre, la prolifération cellulaire n'est pas affectée. Ces résultats indiquent que LOX aide l'échappement des cellules cancéreuses de l'oeil après le développement de l'hypoxie des tissus

Expression profiling of formalin-fixed paraffin embedded primary human uveal melanomas using DASL matrices

Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue analyzed by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two uveal melanoma (UM) clinical-histopathological parameters (metastasis, cell type).A total of 43 FFPE UM were used. the expression of RPL13a, a ribosomal protein gene, for each sample was used to evaluate the quality of RNA extracted from FFPE tissue. Gene expression values generated from the array were analyzed using the GeneSpring GX software (Agilent). Immunohistochemistry was used in order to validate transcriptional findings at the protein level.A total of 106 genes were identified with (P < 0.05, Welch ANOVA test) a difference in transcript abundance for the metastasis clinical parameter. Furthermore, we identified 64 genes with a statistically significant (P < 0.05) difference in transcript abundance between the spindle and epithelioid cell types. Each individual sample for both groups (metastasis, cell type) exhibited distinct transcriptional profiles that were separated on a PCA. Positive nuclear immunostaining for LIG4-metastasis, ErbB3-cell type was found to be associated with better patient prognosis and outcome.To the best of our knowledge, this is the first time that a successful retrospective analysis has been done with UM FFPE RNA. This data may lead to future customized therapeutic targets, which may improve the now unchanged mortality rate of this particular malignancy.Cedars Cancer InstituteHellen Keller FoundationMcGill Univ, Ctr Hlth, Henry C Witelson Ophthalm Pathol Lab, Montreal, PQ H3A 2B4, CanadaMcGill Univ, Ctr Hlth, Henry C Witelson Ophthalm Pathol Registry, Montreal, PQ H3A 2B4, CanadaNatl Res Council Canada, Biotechnol Res Inst, Montreal, PQ H4P 2R2, CanadaNCI, NIH, Bethesda, MD 20892 USAUniversidade Federal de São Paulo, UNIFESP EPM, Dept Ophthalmol, São Paulo, BrazilUniv Valladolid, Dept Ophthalmol, Ocular Pathol Lab, Valladolid, SpainUniversidade Federal de São Paulo, UNIFESP EPM, Dept Ophthalmol, São Paulo, BrazilWeb of Scienc