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...
Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the application of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATPdependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A reexpression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer.
S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer. ' 2008 Wiley-Liss, Inc.Key words: prostate cancer; S100P; xenograft Cancer of the prostate is the second leading cause of cancer deaths amongst men in the United States. 1 Initially, the disease responds to androgen blockade therapy, however, resistance to this hormone therapy often develops during tumor progression. Hormone-refractory tumors are also often metastatic and resistant to chemotherapy and thus present a significant clinical problem. Predictive biomarkers of prostate tumor progression, as well as new therapeutic interventions for advanced stages of prostate cancer are urgently needed. The application of microarray technologies by our group, as well as others, to interrogate the prostate cancer genome has proven to be a powerful approach for identifying genes associated with prostate cancer progression and resistance to therapy. In our analysis, using a xenograft model of hormone refractory tumors, S100P was identified as a gene whose increased expression is associated with androgen independent growth of xenografts. Subsequent analysis of over 500 prostate specimens validated S100P as a protein whose overexpression is tightly associated with clinical prostate cancer progression suggesting that it may play a causal role in progression and...
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