Associations of prostate cancer risk variants with disease aggressiveness: results of the NCI-SPORE Genetics Working Group analysis of 18,343 cases.

Genetic studies have identified single nucleotide polymorphisms (SNPs) associated with the risk of prostate cancer (PC). It remains unclear whether such genetic variants are associated with disease aggressiveness. The NCI-SPORE Genetics Working Group retrospectively collected clinicopathologic information and genotype data for 36 SNPs which at the time had been validated to be associated with PC risk from 25,674 cases with PC. Cases were grouped according to race, Gleason score (Gleason ≤ 6, 7, ≥ 8) and aggressiveness (non-aggressive, intermediate, and aggressive disease). Statistical analyses were used to compare the frequency of the SNPs between different disease cohorts. After adjusting for multiple testing, only PC-risk SNP rs2735839 (G) was significantly and inversely associated with aggressive (OR = 0.77; 95 % CI 0.69-0.87) and high-grade disease (OR = 0.77; 95 % CI 0.68-0.86) in European men. Similar associations with aggressive (OR = 0.72; 95 % CI 0.58-0.89) and high-grade disease (OR = 0.69; 95 % CI 0.54-0.87) were documented in African-American subjects. The G allele of rs2735839 was associated with disease aggressiveness even at low PSA levels (<4.0 ng/mL) in both European and African-American men. Our results provide further support that a PC-risk SNP rs2735839 near the KLK3 gene on chromosome 19q13 may be associated with aggressive and high-grade PC. Future prospectively designed, case-case GWAS are needed to identify additional SNPs associated with PC aggressiveness.

Expression differences between African American and Caucasian prostate cancer tissue reveals that stroma is the site of aggressive changes.

In prostate cancer, race/ethnicity is the highest risk factor after adjusting for age. African Americans have more aggressive tumors at every clinical stage of the disease, resulting in poorer prognosis and increased mortality. A major barrier to identifying crucial gene activity differences is heterogeneity, including tissue composition variation intrinsic to the histology of prostate cancer. We hypothesized that differences in gene expression in specific tissue types would reveal mechanisms involved in the racial disparities of prostate cancer. We examined 17 pairs of arrays for AAs and Caucasians that were formed by closely matching the samples based on the known tissue type composition of the tumors. Using pair-wise t-test we found significantly altered gene expression between AAs and CAs. Independently, we performed multiple linear regression analyses to associate gene expression with race considering variation in percent tumor and stroma tissue. The majority of differentially expressed genes were associated with tumor-adjacent stroma rather than tumor tissue. Extracellular matrix, integrin family and signaling mediators of the epithelial-to-mesenchymal transition (EMT) pathways were all downregulated in stroma of AAs. Using MetaCore (GeneGo) analysis, we observed that 35% of significant (p < 10(-3)) pathways identified EMT and 25% identified immune response pathways especially for interleukins-2, -4, -5, -6, -7, -10, -13, -15 and -22 as the major changes. Our studies reveal that altered immune and EMT processes in tumor-adjacent stroma may be responsible for the aggressive nature of prostate cancer in AAs.

PTEN regulation by Akt-EGR1-ARF-PTEN axis.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.

From mRNA to tumor suppressor.

The combination of inhibition of RNA degradation and comparative genomic scanning is a powerful new method for detecting gene disruptions. The utility of the method is well-illustrated by a series of observations linking the ephrin receptor EPHB2 to prostate cancer.

Spatial Profiling of the Prostate Cancer Tumor Microenvironment Reveals Multiple Differences in Gene Expression and Correlation with Recurrence Risk.

The tumor microenvironment plays a crucial role in both the development and progression of prostate cancer. Furthermore, identifying protein and gene expression differences between different regions is valuable for treatment development. We applied Digital Spatial Profiling multiplex analysis to formalin-fixed paraffin embedded prostatectomy tissue blocks to investigate protein and transcriptome differences between tumor, tumor-adjacent stroma (TAS), CD45+ tumor, and CD45+ TAS tissue. Differential expression of an immunology/oncology protein panel (n = 58) was measured. OX40L and CTLA4 were expressed at higher levels while 22 other proteins, including CD11c, were expressed at lower levels (FDR < 0.2 and p-value < 0.05) in TAS as compared to tumor epithelia. A tissue microarray analysis of 97 patients with 1547 cores found positive correlations between high expression of CD11c and increased time to recurrence in tumor and TAS, and inverse relationships for CTLA4 and OX40L, where higher expression in tumor correlated with lower time to recurrence, but higher time to recurrence in TAS. Spatial transcriptomic analysis using a Cancer Transcriptome Atlas panel (n = 1825 genes) identified 162 genes downregulated and 69 upregulated in TAS versus tumor, 26 downregulated and 6 upregulated in CD45+ TAS versus CD45+ tumor. We utilized CIBERSORTx to estimate the relative immune cell fractions using CD45+ gene expression and found higher average fractions for memory B, naïve B, and T cells in TAS. In summary, the combination of protein expression differences, immune cell fractions, and correlations of protein expression with time to recurrence suggest that closely examining the tumor microenvironment provides valuable data that can improve prognostication and treatment techniques.

RNA expression differences in prostate tumors and tumor-adjacent stroma between Black and White Americans.

Prostate cancer (PCa) in Black Americans (BA) is diagnosed at an earlier median age and a more advanced stage than PCa in White Americans (WA). Tumor-adjacent stroma (TAS) plays a critical role in tumorigenesis of prostate cancer. We examined RNA expression in both tumor and TAS of BA compared to WA. After evaluating the geographical ancestry of each sample, preliminary analysis of our own RNA-seq data of 7 BA and 7 WA TAS revealed 1706 downregulated and 1844 upregulated genes in BA relative to WA PCa patients (p adj < 0.05). An assessment of published RNA-seq data of clinically matched tumor-enriched tissues from 15 BA and 30 WA patients revealed 932 upregulated and 476 downregulated genes in BA relative to WA (p adj < 0.05). When TAS and tumor epithelial cohorts were compared for the top 2500 statistically significant genes, immune responses were downregulated in BA vs WA TAS, while T cell-exhaustion pathways and the immune checkpoint gene CTLA4 were upregulated in BA vs WA tumors. We found fewer activated dendritic cells in tumor and more CD8 T-cells in TAS of BA versus WA PCa patients. Further characterization of these differences in the immune response of PCa patients of distinct geographical ancestry could help to improve diagnostics, prognostics, and therapy.

Expression of Endogenous Retroviral RNA in Prostate Tumors has Prognostic Value and Shows Differences among Americans of African Versus European/Middle Eastern Ancestry.

Endogenous retroviruses (ERVs) are abundant, repetitive elements dispersed across the human genome and are implicated in various diseases. We investigated two potential roles for ERVs in prostate cancer (PCa). First, the PCa of Black Americans (BA) is diagnosed at an earlier median age and at a more advanced stage than the PCa of White Americans (WA). We used publicly available RNA-seq data from tumor-enriched samples of 27 BA and 65 WA PCa patients in order to identify 12 differentially expressed ERVs (padj < 0.1) and used a tissue microarray of the PCa cores from an independent set of BA and WA patients to validate the differential protein expression of one of these ERVs, ERV3-1 (p = 2.829 × 10-7). Second, we used 57 PCa tumors from patients of all ancestries from one hospital as a training set to identify the ERVs associated with time to biochemical relapse. A 29-ERV prognostic panel was then tested and validated on 35 separate PCa tumors from patients obtained in two different hospitals with a dramatic increase in prognostic power relative to clinical parameters alone (p = 7.4 × 10-11). In summary, ERV RNA expression differences in the prostate tumors of patients of different ancestries may be associated with dissimilarities in the mechanism of cancer progression. In addition, the correlation of expression of certain ERVs in prostate tumors with the risk of biochemical relapse indicates a possible role for ERV expression in cancer progression.

The wisdom of the commons: ensemble tree classifiers for prostate cancer prognosis.

MOTIVATION: Classification and regression trees have long been used for cancer diagnosis and prognosis. Nevertheless, instability and variable selection bias, as well as overfitting, are well-known problems of tree-based methods. In this article, we investigate whether ensemble tree classifiers can ameliorate these difficulties, using data from two recent studies of radical prostatectomy in prostate cancer. RESULTS: Using time to progression following prostatectomy as the relevant clinical endpoint, we found that ensemble tree classifiers robustly and reproducibly identified three subgroups of patients in the two clinical datasets: non-progressors, early progressors and late progressors. Moreover, the consensus classifications were independent predictors of time to progression compared to known clinical prognostic factors.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Transcriptome Analysis of Ovarian and Uterine Clear Cell Malignancies.

PURPOSE: Ovarian and uterine clear cell carcinomas (CCCs) are rare but associated with poor prognosis. This study explored RNA transcription patterns characteristic of these tumors. EXPERIMENTAL DESIGN: RNA sequencing (RNA-seq) of 11 ovarian CCCs and five uterine CCCs was performed and compared to publicly available data from high grade serous ovarian cancers (HGSOCs). Ingenuity Pathway Analyses were performed. CIBERSORT analyses estimated relative fractions of 22 immune cell types in each RNA-seq sample. Sequencing data was correlated with PD-L1 immunohistochemical expression. RESULTS: RNA-seq revealed 1,613 downregulated and 1,212 upregulated genes (corrected p < 0.05, |FC |≥10) in ovarian CCC versus HGSOC. Two subgroups were identified in the ovarian CCC, characterized by ethnicity and expression differences in ARID1A. There were 3,252 differentially expressed genes between PD-L1+/- ovarian CCCs, revealing immune response, cell death, and DNA repair networks, negatively correlated with PD-L1 expression, whereas cellular proliferation networks positively correlated with expression. In clear cell ovarian versus clear cell uterine cancer, 1,607 genes were significantly upregulated, and 109 genes were significantly downregulated (corrected p < 0.05, |FC|≥10). Comparative pathway analysis of late and early stage ovarian CCCs revealed unique metabolic and PTEN pathways, whereas uterine CCCs had unique Wnt/Ca+, estrogen receptor, and CCR5 signaling. CIBERSORT analysis revealed that activated mast cells and regulatory T cell populations were relatively enriched in uterine CCCs. The PD-L1+ ovarian CCCs had enriched resting NK cells and memory B cell populations, while PD-L1- had enriched CD8 T-cells, monocytes, eosinophils, and activated dendritic cells. CONCLUSIONS: Unique transcriptional expression profiles distinguish clear cell uterine and ovarian cancers from each other and from other more common histologic subtypes. These insights may aid in devising novel therapeutics.

Bcl-B expression in human epithelial and nonepithelial malignancies.

PURPOSE: Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported. EXPERIMENTAL DESIGN: We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival. RESULTS: Bcl-B protein was strongly expressed in all normal plasma cells but found in only 18% of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n = 48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non-small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001). CONCLUSIONS: Tumor-specific alterations in Bcl-B expression may define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors.

Expression profile of human gingival fibroblasts induced by interleukin-1beta reveals central role of nuclear factor-kappa B in stabilizing human gingival fibroblasts during inflammation.

BACKGROUND: Interleukin (IL)-1beta is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1beta on the gene expression using expression arrays containing approximately 40,000 genes, and tested the role of nuclear factor-kappa B (NF-kappaB) in maintaining an activated HGF population. METHODS: Total RNA was isolated from IL-1beta-induced and mock-induced control cells. Gene expression analyses were performed using expression arrays and confirmed by quantitative real-time polymerase chain reaction. Western blot analysis to show inhibitor of kappa B-alpha (IkappaBalpha) phosphorylation and immunostaining of cells for NF-kappaB nuclear translocation were performed. Apoptosis was confirmed by assay of poly ADP-ribose polymerase (PARP) cleavage. RESULTS: A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1beta-induced cells (P <0.001). A total of 215 genes were upregulated, and 39 genes were downregulated. Most notable NF-kappaB pathway members (NFkappaB1, NFkappaB2, IkappaBalpha, IkappaBepsilon, IkappaBzeta, REL, RELB, and TA-NFKBH) were upregulated. IkappaBalpha was phosphorylated, and NF-kappaB accumulated in the nucleus. An IL-1beta-induced set of 27 genes was downregulated by an NF-kappaB inhibitor, leading to a decreased number of viable cells and suggesting an antiapoptotic role for NF-kappaB. CONCLUSIONS: IL-1beta leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and especially NF-kappaB-dependent antiapoptotic genes. NF-kappaB activation blocks apoptosis, thereby stabilizing the HGF population in inflammation.

Transcriptional Profiling of Age-Associated Gene Expression Changes in Human Circulatory CD1c+ Myeloid Dendritic Cell Subset.

Immune dysfunction is a hallmark of aging and is thought to be responsible for the age-associated diseases. Dendritic cells (DCs) of the immune system function as initiators and regulators of the immune responses. Recent studies have highlighted the division of labor between various DC subsets. CD1c+ DC subset has emerged as a major inducer of CD4 T cell response. There is a scarcity of information regarding the age-associated changes in the functions of DC subsets in the elderly. Here, we investigated the changes in transcriptional profile of CD1c+ DC subset from healthy aged and young individuals using RNA sequencing. Our results suggest that majority of the genes in DCs are upregulated with age. Glucose transport, GPCR, and potassium channel genes are all upregulated in DCs from aged as compared to young indicating an enhanced activation state of DCs from aged individuals. The expression of histones, small nucleolar RNA H/ACA box (SNORA) and small nucleolar RNA C/D/box (SNORD), and long non-coding RNA (lncRNA) is also substantially upregulated in the DCs from aged. In contrast, the antigen-presenting and energy generating pathways are downregulated. In summary, DCs from aged subjects display an activated state coupled with reduced antigen presentation which may be responsible for age-associate immune dysfunction.

Messenger RNAs under differential translational control in Ki-ras-transformed cells.

Microarrays have been used extensively to identify differential gene expression at the level of transcriptional control in oncogenesis. However, increasing evidence indicates that changes in translational control are critical to oncogenic transformation. This study identifies mRNA transcripts that are differentially regulated, primarily at the level of translation, in the immortalized human embryonic prostate epithelial cell line 267B1 and the v-Ki-ras-transformed counterpart by comparing total mRNA to polysome-bound mRNA by using Affymetrix oligonucleotide microarrays. Among the transcripts that were identified were those encoding proteins involved in DNA replication, cell cycle control, cell-to-cell interactions, electron transport, G protein signaling, and translation. Many of these proteins are known to contribute to oncogenesis or have the potential to contribute to oncogenesis. Differential expression of RNA-binding proteins and the presence of highly conserved motifs in the 5' and 3' untranslated regions of the mRNAs are consistent with multiple pathways and mechanisms governing the changes in translational control. Although Alu sequences were found to be associated with increased translation in transformed cells, an evolutionarily conserved motif was identified in the 3' untranslated regions of ephrinB1, calreticulin, integrin alpha3, and mucin3B that was associated with decreased polysome association in 267B1/Ki-ras.

Early growth response 1 acts as a tumor suppressor in vivo and in vitro via regulation of p53.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

Assessing Researcher Needs for a Virtual Biobank.

INTRODUCTION: Biosamples and associated clinical data accelerate translational and clinical research discoveries. A lack of high quality biosamples both stalls projects and limits research advances. In this study, we targeted a wide audience of University of California (UC) biobanking stakeholders who were either involved with the collection or the utilization of biosamples to assess the scope of their biobanking activities and their interest in virtual biobanking or cooperating in the formation of the UC-wide biorepository. MATERIALS AND METHODS: Each institutional review board from the five UC medical campuses' provided a dataset of potential researchers involved with biobanking. Once identified, a brief six item web-based questionnaire was administered electronically to these researchers. RESULTS: Most survey participants (80%) responded "yes" (n = 348) that they were actively collecting biosamples for research, and 68% of participants indicated they would either definitely (30%, n = 131) or maybe (38%, n = 166) request biosample materials now or within the next year. An equal proportion of participants responded yes (42% or n = 184) and maybe (42% or n = 182) when asked if they would voluntarily contribute specimens to a UC-wide virtual biobank. DISCUSSION: The results presented above show high levels of support among UC biobanking stakeholders for both requesting material from and contributing material to a UC-wide virtual biobank. In addition, a considerable number of individual researchers on our five UC medical campuses are conducting biospecimen research (i.e., well over n = 435 respondents).

Inhibition of Egr-1 expression reverses transformation of prostate cancer cells in vitro and in vivo.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-beta1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 54+/-3%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P=0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

Survey of differentially methylated promoters in prostate cancer cell lines.

DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

"Promoter array" studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells.

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.

C-Jun NH(2)-terminal kinase mediates proliferation and tumor growth of human prostate carcinoma.

PURPOSE: C-Jun NH(2)-terminal kinase (JNK) has been implicated in numerous functions including stress responses, apoptosis,and transformation. The role in transformation is based largely on studies of isolated cell types with little indication of whether JNK plays a general role in a specific human tumor type or whether this occurs in vivo. EXPERIMENTAL DESIGN: We examined 9 human prostate carcinoma cell lines in vitro and a representative line in vivo. RESULTS: For all of the cell lines proliferation is highly correlated with serum-supported JNK activity (r(Pearson) = 0.91; P = 0.004), whereas no relationship was observed for 10 human breast cancer cell lines (r(Pearson) = -0.32). Treatment with characterized antisense oligonucleotides complementary to sequences common to either the JNK1 or JNK2 family of isoforms showed that, whereas antisense JNK1 inhibited growth by a maximum of 57%, antisense JNK2 inhibited proliferation up to 80%. Sense and scrambled control oligonucleotides had little effect (average 3.7 +/- 1.5%). Moreover, systemic treatment of mice bearing established xenografts of PC3 prostate carcinoma cells with antisense JNK1 and JNK2 led to inhibition tumor growth by 57% (P < 0.002) and 80% (P < 0.001), respectively. The difference is significant (P < 0.012). Combined antisense treatment led to a significant increase in frequency of tumor regression (P = 0.022). CONCLUSION: These results indicate that JNK is required for growth of prostate carcinoma cells in vitro and in vivo, and additionally indicate that JNK2 plays a dominant role. The JNK pathway is a novel target in the treatment of prostate carcinoma.