Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas

Abstract

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Figure 1

Divergence of gene expression patterns between class 1 and class 2 uveal melanomas reflects a shift from neural crest to epithelial-like differentiation. A, unsupervised principal component analysis showing clustering of class 1 and class 2 tumors. Spheres, individual tumors. B, mean mRNA expression of melanocyte, Wnt pathway, neural crest, and epithelial genes in 14 class 1 melanomas and 11 class 2 melanomas (Affymetrix GeneChip expression units). Bars, SE. Graphs are depicted on log₁₀ scales. C, representative tissue sections from class 1 and class 2 uveal melanomas stained with H&E, immunostained using antibodies against E-cadherin, β-catenin, or Id2 (blue, positive staining),or stained with PAS. PAS staining is displayed using grayscale images to show extracellular matrix patterns (arrows). Bars, 20 μm.

Figure 2

Id2 down-regulation in primary uveal melanomas is associated with epithelioid cytology and metastatic death. A, scatter plot of Id2 mRNA expression (Affymetrix GeneChip expression units) versus the proportion of epithelioid cells in human uveal melanomas, measured by rank score from 1 to 25. Significance level was calculated using Pearson correlation coefficient. B, Kaplan-Meier analysis comparing metastatic death in patients with “low Id2” versus “high Id2” tumors. Using receiver operating characteristic curves to discretize tumors for maximal sensitivity and specificity to predict metastasis based on Id2 expression, a threshold of 1,714 GeneChip units assigned 7 tumors to the low Id2 group and 16 tumors to the high Id2 group.

Figure 3

Id2 inhibits epithelial differentiation in uveal melanoma cells by negatively regulating E-cadherin expression. A, Mel202 class 1 melanoma cells transfected with an empty control vector or an Id2 antisense vector and viewed by phase-contrast microscopy. Arrows, an epithelial-like cluster of melanoma cells. Bars,10 μm. B, Mel202 and Mel290 class 1 uveal melanoma cells cotransfected with Id2 antisense and CD20 expression vectors and immunostained for E-cadherin and CD20 (a marker for transfected cells). Id2-deficient MM80 class 2 uveal melanoma cells were transfected with an Id2 expression vector and immunostained for E-cadherin and Id2. Bars,10 μm. C, E-cadherin mRNA levels measured by quantitative real-time PCR (fold change compared with glyceraldehyde-3-phosphate dehydrogenase) in Mel202 cells at the indicated time points after transfection with an Id2 antisense vector or the empty control vector. Bars, SE. D, Luciferase transcription assays in Mel290 and Mel202 class 1 uveal melanoma cells cotransfected with an E-cadherin promoter-luciferase reporter along with an Id2 antisense vector or an empty control vector. Luciferase activity was measured 48 hours after transfection. Epal mut, mutation of E-pal palindromic element; Ebox2 mut, mutation of the E-box2 element; Ebox-null, mutation of all E-boxes. Bars, SE. Cartoon depicts current hypothesis to explain effect of Id2 on E-pal and E-box2 elements (see text for details).

Figure 4

Depletion of Id2 leads to up-regulation of E-cadherin and anchorage-independent growth in uveal melanoma cells. A, anchorage-independent colony formation of Mel202 melanoma cells in soft agar, measured as the number of colonies per ×20 field following transfection with an Id2 antisense vector or an empty control vector. DECMA1 indicates that soft agar was incubated with neutralizing anti-E-cadherin antibodies. Columns, mean of triplicate experiments; bars, SE. B, representative bright-field photomicrographs from soft agar assays, showing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide–stained viable tumor cells. Large multicellular spheroids were seen only in cells transfected with the Id2 antisense vector (middle). Bars, 150 μm.

Figure 5

Deletion of Id2 recapitulates the class 2 phenotype in a mouse model of uveal melanoma. A, low-magnification images of representative ocular tumors from 15-week-old mice with the indicated Id2 genotypes stained with H&E. Arrow, location of a typical small,flat ocular tumor observed in Id2^+/+ mice. Bars,100 Am. Bottom, corresponding graph summarizing the vertical growth phase cross-sectional tumor areas for all eyes with that genotype at age 6,9, and 15 weeks. B, representative high-magnification images of ocular tumors with the Id2^+/+ and Id2^−/− genotypes stained with H&E (arrow indicates mitotic figure),immunostained using antibodies against E-cadherin or β-catenin (blue, positive staining),or stained with PAS. Note the low-grade fusiform cells in the Id2^+/+ tumor compared with the large,round epithelioid cells in the Id2^−/− tumor. PAS staining is displayed using grayscale images to show extracellular matrix patterns (arrows). Bars,20 Am. C, proportion of epithelioid cells in TyrTAg ocular tumors with the three Id2 genotypes,measured as epithelioid score (mean nuclear circularity).