Prostate cancer, a leading cause of cancer death, displays a broad range of clinical behavior from relatively indolent to aggressive metastatic disease. To explore potential molecular variation underlying this clinical heterogeneity, we profiled gene expression in 62 primary prostate tumors, as well as 41 normal prostate specimens and nine lymph node metastases, using cDNA microarrays containing Ϸ26,000 genes. Unsupervised hierarchical clustering readily distinguished tumors from normal samples, and further identified three subclasses of prostate tumors based on distinct patterns of gene expression. High-grade and advanced stage tumors, as well as tumors associated with recurrence, were disproportionately represented among two of the three subtypes, one of which also included most lymph node metastases. To further characterize the clinical relevance of tumor subtypes, we evaluated as surrogate markers two genes differentially expressed among tumor subgroups by using immunohistochemistry on tissue microarrays representing an independent set of 225 prostate tumors. Positive staining for MUC1, a gene highly expressed in the subgroups with ''aggressive'' clinicopathological features, was associated with an elevated risk of recurrence (P ‫؍‬ 0.003), whereas strong staining for AZGP1, a gene highly expressed in the other subgroup, was associated with a decreased risk of recurrence (P ‫؍‬ 0.0008). In multivariate analysis, MUC1 and AZGP1 staining were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative prostate-specific antigen levels. Our results suggest that prostate tumors can be usefully classified according to their gene expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification.

Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease. [Cancer Res 2007;67(18):8504-10]

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Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.

Androgen receptor (AR) signaling reprograms cellular metabolism to support prostate cancer (PCa) growth and survival. Another key regulator of cellular metabolism is mTOR, a kinase found in diverse protein complexes and cellular localizations, including the nucleus. However, whether nuclear mTOR plays a role in PCa progression and participates in direct transcriptional cross-talk with the AR is unknown. Here, via the intersection of gene expression, genomic, and metabolic studies, we reveal the existence of a nuclear mTOR-AR transcriptional axis integral to the metabolic rewiring of PCa cells. Androgens reprogram mTOR-chromatin associations in an AR-dependent manner in which activation of mTOR-dependent metabolic gene networks is essential for androgeninduced aerobic glycolysis and mitochondrial respiration. In models of castration-resistant PCa cells, mTOR was capable of transcriptionally regulating metabolic gene programs in the absence of androgens, highlighting a potential novel castration resistance mechanism to sustain cell metabolism even without a functional AR. Remarkably, we demonstrate that increased mTOR nuclear localization is indicative of poor prognosis in patients, with the highest levels detected in castration-resistant PCa tumors and metastases. Identification of a functional mTOR targeted multigene signature robustly discriminates between normal prostate tissues, primary tumors, and hormone refractory metastatic samples but is also predictive of cancer recurrence. This study thus underscores a paradigm shift from AR to nuclear mTOR as being the master transcriptional regulator of metabolism in PCa.

Prostate cancer is the most common cancer among men in the United States, and aberrant DNA methylation is known to be an early molecular event in its development. Here, we have used expression profiling to identify novel hypermethylated genes whose expression is induced by treatment of prostate cancer cell lines with the DNA methyltransferase inhibitor 5-Aza-2V-deoxycytidine (5-aza-dC). Of the 271 genes that were induced by 5-aza-dC treatment, 25 also displayed reduced expression in primary prostate tumors compared with normal prostate tissue, and the decreased expression of only one gene, aldehyde dehydrogenase 1 family, member A2 (ALDH1a2), was also associated with shorter recurrence-free survival. ALDH1a2 encodes an enzyme responsible for synthesis of retinoic acid (RA), a compound with prodifferentiation properties. By immunohistochemistry, we observed that ALDH1a2 was expressed in epithelia from normal prostate but not prostate cancer. Using bisulfite sequencing, we determined that the ALDH1a2 promoter region was significantly hypermethylated in primary prostate tumors compared with normal prostate specimens (P = 0.01). Finally, transfection-mediated reexpression of wild-type ALDH1a2 (but not a presumptive catalytically dead mutant) in the prostate cancer cell line DU145 resulted in decreased colony growth (P < 0.0001), comparable with treatment with either 5-aza-dC or RA. Taken together, our findings implicate ALDH1a2 as a candidate tumor suppressor gene in prostate cancer and further support a role of retinoids in the prevention or treatment of prostate cancer. (Cancer Res 2005; 65(18): 8118-24)

Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

Prostate cancer is the most commonly diagnosed cancer among men in the United States. Recently, fusion of TMPRSS2 with ETS family oncogenic transcription factors has been identified as a common molecular alteration in prostate cancer, where most often the rearrangement places ERG under the androgen-regulated transcriptional control of TMPRSS2. Here, we carried out rapid amplification of cDNA ends (RACE) on a prostate cancer specimen carrying an atypical aberration discovered by array-based comparative genomic hybridization (array CGH), suggesting an alternative fusion partner of ERG. We identified novel transcribed sequences fused to ERG, mapping 4 kb upstream of the TMPRSS2 start site. The sequences derive from an apparent second TMPRSS2 isoform, which we found also expressed in some prostate tumors, suggesting similar androgenregulated control. In a reverse transcription-polymerase chain reaction (RT-PCR)-based survey of 63 prostate tumor specimens (54 primary and nine lymph node metastases), 44 (70%) cases expressed either the known or novel variant TMPRSS2-ERG fusion, 28 (44%) expressed both, 10 (16%) expressed only the known, and notably six (10%) expressed only the variant isoform fusion. In this specimen set, the presence of a TMPRSS2-ERG fusion showed no statistical association with tumor stage, Gleason grade or recurrence-free survival. Nonetheless, the discovery of a novel variant TMPRSS2 isoform-ERG fusion adds to the characterization of ETS-family rearrangements in prostate cancer, and has important implications for the accurate molecular diagnosis of TMPRSS2-ETS fusions. Recently, by examining outlier values of gene expression in human cancers, Tomlins et al 5 discovered elevated expression of the ETS family members ERG (v-ets erythroblastosis virus E26 oncogene like) and ETV1 (Ets variant gene 1) to be a common feature of prostate cancer. Elevated expression resulted from chromosomal rearrangement fusing TMPRSS2 (transmembrane protease, serine 2) (chr 21q22.3) to ERG (chr 21q22.2), an apparent intra-chromosomal deletion of B3 Mb on chromosome 21, or less frequently to ETV1, an interchromosomal rearrangement between chr 21q22.3 and chr 7p21.2.