Gene expression profiling in melanoma identifies novel downstream effectors of p14ARF

Abstract

p14ARF is inactivated by deletions/mutations in many cancer types and can suppress cell growth by both p53-dependent and p53-independent mechanisms. To identify novel downstream effectors of p14ARF, we used gene expression profiling as a primary screening tool to select candidates for follow up validation studies using in vitro cell-based assays. Gene expression profiles of a panel of 35 melanoma cell lines with either wild-type (n 5 12) or mutant (n 5 23) p14ARF were compared to identify genes associated with inactivation of p14ARF. Analysis of the microarray data identified 1,316 probe sets that were significantly (p < 0.01) differentially expressed between the p14ARF wild-type and mutant cell lines. Pathway analysis of these genes showed an overrepresentation of many receptor-mediated signal transduction pathways, e.g. TGFb, EGF, HGF, PDGF, MAPK, Wnt and integrin pathways. A number of components of these pathways, including FLRT3, RUNX2, MIG-6 and SMURF2 were confirmed as downstream targets of p14ARF using p14ARF-inducible cell lines and RNAi. We propose that regulation of these genes may contribute to melanoma development when p14ARF function is lost. ' 2007 Wiley-Liss, Inc.

Cross-Platform Array Screening Identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as Genes Frequently Silenced by Methylation in Melanoma

“…Integrating the degree of methylation and the level of mRNA expression, as assessed by previous Affymetrix microarray analysis [19] gave high correlation coefficients (Spearman coefficients = −0.75/−0.82/−0.6/−0.52 respectively, p<0.005) for four genes: COL1A2 , THBS1 , TNFRSF10D and UCHL1 .…”

“…These new data were then integrated with previous data-sets of global mRNA expression [18], [19] and expression post-demethylation treatment [14] in order to focus on identifying additional candidate TSGs down-regulated through promoter methylation. Genes were further filtered to identify those in which ≥60% methylation correlated with a 4-fold decrease in mRNA levels in at least 2 samples, together with an average post-demethylation re-expression fold-change of >4 across the panel of 11 melanoma lines ( Figure 1 ).…”

“…All cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum as described previously [19], [61]. Primary human melanocytes were obtained from neonatal foreskins and cultured in 10% heat-inactivated fetal calf serum (CSL, Melbourne, Australia) in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 3 mM HEPES with the addition of 6 ng/ml cholera toxin and 16.2 nM phorbol 12-myristate 13-acetate (PMA) (Sigma Chemical Co., St. Louis, MO) as previously described [62].…”

“…In order to provide a more complete picture of methylation in melanoma, these data were integrated with our previous constitutive mRNA expression [18], [19] and post-demethylation treatment expression data [14] to identify a list of potential genes for assessment by comprehensive promoter methylation analysis using the Epityper system in an extended panel of melanoma cell lines and tumors.…”

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion

“…In addition, the tumor suppressor p14ARF was reported to repress RUNX2 expression in melanoma cell lines. In this study, the authors speculated that increased RUNX2 resulting from p14ARF mutation might contribute to melanoma development [22]. As a first approach to studying the role of RUNX2 in melanoma development, we determined that RUNX2 was overexpressed in melanoma cell lines as compared with primary cultures of melanocytes or an immortalized melanocyte cell line.…”