A Comparative Transcriptomic Analysis of Uveal Melanoma and Normal Uveal Melanocyte

Abstract

BackgroundUveal melanoma is the most common primary intraocular tumor in adults in western countries. It is associated with very severe visual morbidity and may lead to distant metastases even after successful treatment of the primary tumor. In order to gain better insight into molecular mechanisms related to tumorigenesis and metastasis of uveal melanoma, we used next-generation sequencing technology (SOLiD, Life Technologies) to acquire global transcriptome alteration between posterior uveal melanoma cells and normal uveal melanocyte.ResultsFrom mRNAs of the cultured uveal melanoma cells and normal uveal melanocytes, we annotated more than 3.7×107 and 2.7×107 sequencing tags based on human Ensembl databases, respectively. For detailed analysis, we chose 5155 well-annotated genes mainly involved in the MAPK signaling pathway, cell cycle, cell adhesion junction, apoptosis, and P53 signaling pathways as well as melanogenesis. In an effort to confirm the authenticity of our sequencing results, we validated twenty-one identically differentially expressed genes by using quantitative real time PCR from cultured cell lines of other posterior uveal melanoma cells and normal uveal melanocytes.ConclusionWe have identified a large number of potentially interesting genes for biological investigation of uveal melanoma. The expression profiling also provides useful resources for other functional genomic and transcriptome studies. These 21 potential genes could discriminate between uveal melanoma cells and normal uveal melanocyte, which may be indicative of tumorigenesis process. Our results further suggest that high-throughput sequencing technology provides a powerful tool to study mechanisms of tumogenesis in the molecular level.

Molecular Pathology of Uveal Melanoma

“…Recent analysis of gene expression data of UM would suggest that de-differentiation indeed does occur during UM development, but this requires further investigation. 11,39 It is also unclear whether the naevus stage is a prerequisite in UM development: it has been estimated that o1 in 8000 naevi undergo malignant transformation to form UM. 62 Histologically, it is exceptionally rare for a residual naevus to be evident adjacent to or within a choroidal melanoma, supporting this observation.…”

“…These events include: mutation or amplification of proto-oncogenes; inactivating mutations or deletions of tumour (and metastasis) suppressor genes; and chromosomal aberrations (Tables 1, 2 and 3). 11 Some of these genetic alterations are considered to occur in the early stages of UM development; others, at later stages (ie, prior to or at haematogeneous dissemination). It has been proposed that the genetic developmental pathway of UM bifurcates at an early stage to result in two very distinct genetic signatures: (1) disomy 3 with chromosome 6p gain and class 1 gene expression profile, and (2) monosomy 3 with class 2 molecular signature and a high metastatic propensity.…”

“…79 This has been observed in longitudinal studies using next-generation sequencing in paired primary and metastatic carcinomas, 89 and warrants further investigation using such techniques in UM. The retinoblastoma tumour suppressor pathway is disrupted in most UM either through: hyperphosphorylation of Rb; elevated expression of cyclin D1; or through methylation and inactivation of the INK4A gene [11][12][13][14][15][16] 2. Self-sufficiency in growth signals Gain of cell cycle stimulator-activation of pathways The PI3K-AKT prosurvival pathway is constitutively activated in UM.…”

“…Self-sufficiency in growth signals Gain of cell cycle stimulator-activation of pathways The PI3K-AKT prosurvival pathway is constitutively activated in UM. LOH of the PTEN locus occurs in 76% of UM [17][18][19][20][21] The RAF/MEK/ERK pathway is constitutively activated: activating mutations in GNAQ or GNA11 occur in 480% of UM and can activate the RAF/ MEK/ERK pathway 11,[22][23][24][25][26][27][28] …”

“…The p53 pathway is functionally blocked by its inhibitor MDM2 [13][14][15]29,30 BCL-2 Defects in the Bcl2 pathway in UM contribute to apoptosis resistance 11,14,29 PTEN downregulation The PI3K-AKT prosurvival pathway is constitutively activated in UM to avoid apoptosis 19,20 Produce insulin-like growth factor (IGF- …”