Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.

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...

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.