Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer

Abstract

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays 1 and array-based comparative genomic hybridization 2,3 for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.Inactivation of tumor-suppressor genes (TSGs) in cancer is often a two-step process 4 involving mutation of the target gene and loss of the wild-type allele. Mapping of chromosomal deletions and losses of heterozygosity in cancer cells has been widely applied to guide the identification of TSGs. On its own, however, this approach is slow, labor-intensive and complicated by genomic instability, which often leads to numerous candidate regions for further study. In an alternative approach, the nonsense-mediated decay (NMD) mechanism, which normally targets transcripts with nonsense mutations for rapid degradation 5,6 , is blocked to cause the differential stabilization of genes that contain truncating mutations. This approach, coupled with microarrays to measure transcript levels after NMD inhibition, has been proposed for the genome-wide identification of mutated genes in cell lines 1 .Here we combined results from NMD microarray experiments highlighting putative nonsense mutations with high-resolution data on deleted genomic regions in cancer cell lines obtained with arraybased comparative genomic hybridization (CGH) 2,3 . We applied this integrated approach, which focuses on biallelic gene inactivation events, to the identification of candidate TSGs in prostate cancer.We pretreated the DU 145, PC-3 and LNCaP prostate cancer cell lines with emetine (which inhibits the NMD pathway) and then exposed them to actinomycin D to block new mRNA synthesis and to distinguish post-transcriptional shifts in mRNA stability, which indicate the presence of a nonsense mutation. We used cDNA microarrays to measure changes in transcript levels in cells treated with emetine versus untreated cells. We also carried out corresponding analyses with nonmalignant control cells to distinguish drug-induced gene expression changes from mutation-induced transcript stabilization events. We used known nonsense mutations, including the C39X...

Differential gene expression profiling of large and small retinal ganglion cells

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Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells

“…Using cDNA microarrays, Noensie and Dietz (2001) have demonstrated that blocking translation with emetine, for example, results in an elevated mRNA content for genes known to contain bi-allelic inactivating mutations. Although several genes mutated in colon and prostate cancer cell lines have already been identified (Huusko et al, 2004;Ionov et al, 2004b;Rossi et al, 2005), the efficiency of identifying mutations using the GINI strategy is still low, because too many genes that do not contain mutations also show mRNA increases following inhibition of NMD as a result of a stress response. This complication often makes it difficult to select candidate genes for sequencing analysis.…”

Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells

“…EPHB2 encodes an ephrin receptor that mediates developmental processes (neurogenesis) and regulates cell adhesion and migration (Cutforth and Harrison, 2002). EPHB2 has been identified as a potential prostate tumor suppressor that is genetically inactivated in DU-145 cells, in which one EPHB2 allele is deleted, whereas the other carries a truncating mutation (Huusko et al, 2004). Thus, it is not surprising that EPHB2-siRNA had no effect on cell survival in DU-145 cells.…”