Loss of FOXA1 Drives Sexually Dimorphic Changes in Urothelial Differentiation and Is an Independent Predictor of Poor Prognosis in Bladder Cancer.

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14-positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression.

A Murine Model of K-RAS and ß-Catenin Induced Renal Tumors Expresses High Levels of E2F1 and Resembles Human Wilms Tumor.

PURPOSE: Wilms tumor is the most common renal neoplasm of childhood. We previously found that restricted activation of the WNT/ß-catenin pathway in renal epithelium late in kidney development is sufficient to induce small primitive neoplasms with features of epithelial Wilms tumor. Metastatic disease progression required simultaneous addition of an activating mutation of the oncogene K-RAS. We sought to define the molecular pathways activated in this process and their relationship to human renal malignancies. MATERIALS AND METHODS: Affymetrix® expression microarray data from murine kidneys with activation of K-ras and/or Ctnnb1 (ß-catenin) restricted to renal epithelium were analyzed and compared to publicly available expression data on normal and neoplastic human renal tissue. Target genes were verified by immunoblot and immunohistochemistry. RESULTS: Mouse kidney tumors with activation of K-ras and Ctnnb1, and human renal malignancies had similar mRNA expression signatures and were associated with activation of networks centered on ß-catenin and TP53. Up-regulation of WNT/ß-catenin targets (MYC, Survivin, FOXA2, Axin2 and Cyclin D1) was confirmed by immunoblot. K-RAS/ß-catenin murine kidney tumors were more similar to human Wilms tumor than to other renal malignancies and demonstrated activation of a TP53 dependent network of genes, including the transcription factor E2F1. Up-regulation of E2F1 was confirmed in murine and human Wilms tumor samples. CONCLUSIONS: Simultaneous activation of K-RAS and ß-catenin in embryonic renal epithelium leads to neoplasms similar to human Wilms tumor and associated with activation of TP53 and up-regulation of E2F1. Further studies are warranted to evaluate the role of TP53 and E2F1 in human Wilms tumor.

Evaluation of public cancer datasets and signatures identifies TP53 mutant signatures with robust prognostic and predictive value.

BACKGROUND: Systematic analysis of cancer gene-expression patterns using high-throughput transcriptional profiling technologies has led to the discovery and publication of hundreds of gene-expression signatures. However, few public signature values have been cross-validated over multiple studies for the prediction of cancer prognosis and chemosensitivity in the neoadjuvant setting. METHODS: To analyze the prognostic and predictive values of publicly available signatures, we have implemented a systematic method for high-throughput and efficient validation of a large number of datasets and gene-expression signatures. Using this method, we performed a meta-analysis including 351 publicly available signatures, 37,000 random signatures, and 31 breast cancer datasets. Survival analyses and pathologic responses were used to assess prediction of prognosis, chemoresponsiveness, and chemo-drug sensitivity. RESULTS: Among 31 breast cancer datasets and 351 public signatures, we identified 22 validation datasets, two robust prognostic signatures (BRmet50 and PMID18271932Sig33) in breast cancer and one signature (PMID20813035Sig137) specific for prognosis prediction in patients with ER-negative tumors. The 22 validation datasets demonstrated enhanced ability to distinguish cancer gene profiles from random gene profiles. Both prognostic signatures are composed of genes associated with TP53 mutations and were able to stratify the good and poor prognostic groups successfully in 82%and 68% of the 22 validation datasets, respectively. We then assessed the abilities of the two signatures to predict treatment responses of breast cancer patients treated with commonly used chemotherapeutic regimens. Both BRmet50 and PMID18271932Sig33 retrospectively identified those patients with an insensitive response to neoadjuvant chemotherapy (mean positive predictive values 85%-88%). Among those patients predicted to be treatment sensitive, distant relapse-free survival (DRFS) was improved (negative predictive values 87%-88%). BRmet50 was further shown to prospectively predict taxane-anthracycline sensitivity in patients with HER2-negative (HER2-) breast cancer. CONCLUSIONS: We have developed and applied a high-throughput screening method for public cancer signature validation. Using this method, we identified appropriate datasets for cross-validation and two robust signatures that differentiate TP53 mutation status and have prognostic and predictive value for breast cancer patients.

FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype.

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

Signal transduction pathway analysis in desmoid-type fibromatosis: transforming growth factor-ß, COX2 and sex steroid receptors.

Despite reports of sex steroid receptor and COX2 expression in desmoid-type fibromatosis, responses to single agent therapy with anti-estrogens and non-steroidal anti-inflammatory drugs are unpredictable. Perhaps combination pharmacotherapy might be more effective in desmoid tumors that co-express these targets. Clearly, further understanding of the signaling pathways deregulated in desmoid tumors is essential for the development of targeted molecular therapy. Transforming growth factor-ß (TGFß) and bone morphogenetic proteins (BMP) are important regulators of fibroblast proliferation and matrix deposition, but little is known about the TGFß superfamily in fibromatosis. A tissue microarray representing 27 desmoid tumors was constructed; 14 samples of healing scar and six samples of normal fibrous tissue were included for comparison. Expression of selected receptors and activated downstream transcription factors of TGFß family signaling pathways, ß-catenin, sex steroid hormone receptors and COX2 were assessed using immunohistochemistry; patterns of co-expression were explored via correlational statistical analyses. In addition to ß-catenin, immunoreactivity for phosphorylated SMAD2/3 (indicative of active TGFß signaling) and COX2 was significantly increased in desmoid tumors compared with healing scar and quiescent fibrous tissue. Low levels of phosphorylated SMAD1/5/8 were detected in only a minority of cases. Transforming growth factor-ß receptor type 1 and androgen receptor were expressed in both desmoid tumors and scar, but not in fibrous tissue. Estrogen receptor-ß was present in all cases studied. Transforming growth factor-ß signaling appears to be activated in desmoid-type fibromatosis and phosphorylated SMAD2/3 and COX2 immunoreactivity might be of diagnostic utility in these tumors. Given the frequency of androgen receptor, estrogen receptor-ß and COX2 co-expression in desmoid tumors, further assessment of the efficacy of combination pharmacotherapy using hormonal agonists/antagonists together with COX2 inhibitors should be considered.

Functional remodeling of benign human prostatic tissues in vivo by spontaneously immortalized progenitor and intermediate cells.

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133(high)/CD44(high)/OCT4(high)/PTEN(high)) was characterized as a putative progenitor cell, and BHPrE1 (p63(high)/p53(high)/p21(WAF1)(high)/RB(high)) was characterized as a putative epithelial intermediate cell. Genomic analysis demonstrated an abnormal karyotype with genomic rearrangements including PTEN amplification in NHPrE1 and CTNNB1 (beta-catenin) amplification in BHPrE1 cells. Embedded three-dimensional culture of NHPrE1 showed greater branching than BHPrE1. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme, were grafted to the renal capsule of severe combined immunodeficient mice. Both cell lines were able to regenerate benign secretory ductal-acinar architecture in vivo, containing intact basal and luminal epithelial layers confirmed by the expression of appropriate CK profiles. Prostate-specific antigen, 15-lipoxygenase-2, androgen receptor, and NKX3.1 proteins were appropriately expressed in the regenerated epithelia. Regeneration of benign prostatic glandular structures could be achieved using as few as 10 NHPrE1 cells, whereas 200,000 BHPrE1 cells were required to achieve prostatic architecture. This suggests a greater proportion of progenitor/stem cells in NHPrE1 than in BHPrE1. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis, and carcinogenesis.

Human lupus T cells resist inactivation and escape death by upregulating COX-2.

Autoimmune T-helper cells drive pathogenic autoantibody production in systemic lupus erythematosus (SLE), but the mechanisms maintaining those T cells are unknown. Autoreactive T cells are normally eliminated by functional inactivation (anergy) and activation-induced cell death (AICD) or apoptosis through death receptor (Fas) signaling. However, mutations in the genes encoding Fas and its ligand (FasL) are rare in classical SLE. By gene microarray profiling, validated by functional and biochemical studies, we establish here that activated T cells of lupus patients resist anergy and apoptosis by markedly upregulating and sustaining cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 caused apoptosis of the anergy-resistant lupus T cells by augmenting Fas signaling and markedly decreasing the survival molecule c-FLIP (cellular homolog of viral FLICE inhibitory protein). Studies with COX-2 inhibitors and Cox-2-deficient mice confirmed that this COX-2/FLIP antiapoptosis program is used selectively by anergy-resistant lupus T cells, and not by cancer cells or other autoimmune T cells. Notably, the gene encoding COX-2 is located in a lupus-susceptibility region on chromosome 1. We also found that only some COX-2 inhibitors were able to suppress the production of pathogenic autoantibodies to DNA by causing autoimmune T-cell apoptosis, an effect that was independent of prostaglandin E(2) (PGE(2)). These findings could be useful in the design of lupus therapies.

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Quantitative analysis of the secretome of TGF-beta signaling-deficient mammary fibroblasts.

Transforming growth factor beta (TGF-beta) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-beta type II receptor (TbetaRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TbetaRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TbetaRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TbetaRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-beta signaling pathway in fibroblasts because systemic inhibition of TGF-beta signaling pathways is being considered as a potential cancer therapy.

Transcriptional remodeling of rapidly stimulated HL-1 atrial myocytes exhibits concordance with human atrial fibrillation.

During atrial fibrillation (AF), rapid stimulation causes atrial remodeling that increases arrhythmia susceptibility. Using an established atrial (HL-1) myocyte model, we investigated the transcriptional profile associated with early atrial myocyte remodeling. Spontaneously contracting HL-1 cells were cultured in the absence and presence of rapid stimulation for 24 h and RNA harvested for microarray analysis. We identified 758 genes that were significantly altered with rapid stimulation (626 up- and 132 down-regulated). Results were confirmed using real-time quantitative RT-PCR for selected genes based on physiological relevance in human AF and/or experimental atrial tachycardia (AT), and regulation in the microarray results. In some cases, transcriptional changes were rapid, occurring within 3 h. For a selected group of genes, results were validated for the expressed protein, with findings that correlated with observed transcriptional changes. Significantly regulated genes were classified using the Gene Ontology Database to permit direct comparison of our findings with previously published myocardial transcriptional profiles. For broad functional categories, there was strong concordance between rapidly stimulated HL-1 myocytes and human AF, but not for other remodeling paradigms (cardiomyopathy and exercise). Many individual gene changes were conserved with AF/AT, with marked up-regulation of genes encoding brain and atrial natriuretic peptide precursors, and heat shock proteins. For the conserved genes, both a cellular stress and survival response was evident. Our results demonstrate similarities with human AF/experimental AT with respect to large-scale patterns of transcriptional remodeling, as well as regulation of specific individual genes. Importantly, we identified novel pathways and molecules that were concordantly regulated in vivo.

Web-based interrogation of gene expression signatures using EXALT.

BACKGROUND: Widespread use of high-throughput techniques such as microarrays to monitor gene expression levels has resulted in an explosive growth of data sets in public domains. Integration and exploration of these complex and heterogeneous data have become a major challenge. RESULTS: The EXALT (EXpression signature AnaLysis Tool) online program enables meta-analysis of gene expression profiles derived from publically accessible sources. Searches can be executed online against two large databases currently containing more than 28,000 gene expression signatures derived from GEO (Gene Expression Omnibus) and published expression profiles of human cancer. Comparisons among gene expression signatures can be performed with homology analysis and co-expression analysis. Results can be visualized instantly in a plot or a heat map. Three typical use cases are illustrated. CONCLUSIONS: The EXALT online program is uniquely suited for discovering relationships among transcriptional profiles and searching gene expression patterns derived from diverse physiological and pathological settings. The EXALT online program is freely available for non-commercial users from http://seq.mc.vanderbilt.edu/exalt/.

Integrated genomic and transcriptional profiling identifies chromosomal loci with altered gene expression in cervical cancer.

For a better understanding of the consequences of recurrent chromosomal alterations in cervical carcinomas, we integrated genome-wide chromosomal and transcriptional profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal controls. Previous genomic profiling showed that gains at chromosome arms 1q, 3q, and 20q as well as losses at 8q, 10q, 11q, and 13q were common in cervical carcinomas. Altered regions spanned multiple megabases, and the extent to which expression of genes located there is affected remains unclear. Expression analysis of these previously chromosomally profiled carcinomas yielded 83 genes with significantly differential expression between carcinomas and normal epithelium. Application of differential gene locus mapping (DIGMAP) analysis and the array CGH expression integration tool (ACE-it) identified hotspots within large chromosomal alterations in which gene expression was altered as well. Chromosomal gains of the long arms of chromosome 1, 3, and 20 resulted in increased expression of genes located at 1q32.1-32.2, 3q13.32-23, 3q26.32-27.3, and 20q11.21-13.33, whereas a chromosomal loss of 11q22.3-25 was related to decreased expression of genes located in this region. Overexpression of DTX3L, PIK3R4, ATP2C1, and SLC25A36, all located at 3q21.1-23 and identified by DIGMAP, ACE-it or both, was confirmed in an independent validation sample set consisting of 12 SCCs and 13 normal ectocervical samples. In conclusion, integrated chromosomal and transcriptional profiling identified chromosomal hotspots at 1q, 3q, 11q, and 20q with altered gene expression within large commonly altered chromosomal regions in cervical cancer.

An Evaluation of the Accuracy of a Flash Glucose Monitoring System in Children with Diabetes in comparison with Venous Blood Glucose.

AIMS: To evaluate the performance of a factory-calibrated flash glucose monitoring system in children with diabetes compared to venous blood glucose (BG). METHODS: A total of 13 hospitalized participants newly diagnosed with type 1 diabetes, aged 1~14 years old, were involved in the study. Sensor glucose measurements on days 2, 3, 6, 7, 12, and 13 of wear were compared with venous BG. During these days, the venous BG results were obtained either 4 or 7 times per day. RESULTS: The accuracy was evaluated against venous BG, with 469 of 469 (100.0%) sensor and venous BG pairs within consensus error grid zones A and B, including 94.7% in zone A. The overall mean absolute relative difference (MARD) was 11.67%. The MARD of blood glucose lower than 4.0 mmol/L (MARD = 16.89%) was higher than blood glucose between 4 and 10 mmol/L (MARD = 11.58%) and blood glucose higher than 10 mmol/L (MARD = 7.79%). Compared to venous BG, the MARDs of wear days 2, 3, 6, 7, 12, and 13 were 11.53%, 9.66%, 11.79%, 10.89%, 13.18%, and 13.92%, respectively, with no statistically significant difference (P = 0.25). The median ARD was highest when the glucose decreased >0.11 mmol/L/min (20.27%) and lower than 10.00% when the glucose changed between 0.06 and 0.11 mmol/L/min, changed <0.06 mmol/L/min, and increased >0.11 mmol/L/min. CONCLUSIONS: The accuracy of the system is good and remains stable over 14 days of wear; however, the accuracy depends on the glucose level and rates of glucose concentration changes.

Identification of a gene-expression predictor for diagnosis and personalized stratification of lupus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a wide spectrum of clinical manifestations and degrees of severity. Few genomic biomarkers for SLE have been validated and employed to inform clinical classifications and decisions. To discover and assess the gene-expression based SLE predictors in published studies, we performed a meta-analysis using our established signature database and a data similarity-driven strategy. From 13 training data sets on SLE gene-expression studies, we identified a SLE meta-signature (SLEmetaSig100) containing 100 concordant genes that are involved in DNA sensors and the IFN signaling pathway. We rigorously examined SLEmetaSig100 with both retrospective and prospective validation in two independent data sets. Using unsupervised clustering, we retrospectively elucidated that SLEmetaSig100 could classify clinical samples into two groups that correlated with SLE disease status and disease activities. More importantly, SLEmetaSig100 enabled personalized stratification demonstrating its ability to prospectively predict SLE disease at the individual patient level. To evaluate the performance of SLEmetaSig100 in predicting SLE, we predicted 1,171 testing samples to be either non-SLE or SLE with positive predictive value (97-99%), specificity (85%-84%), and sensitivity (60-84%). Our study suggests that SLEmetaSig100 has enhanced predictive value to facilitate current SLE clinical classification and provides personalized disease activity monitoring.

Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC). Between 15% and 35% of HNSCCs harbor HPV DNA. Demographic and exposure differences between HPV-positive (HPV+) and negative (HPV-) HNSCCs suggest that HPV+ tumors may constitute a subclass with different biology, whereas clinical differences have also been observed. Gene expression profiles of HPV+ and HPV- tumors were compared with further exploration of the biological effect of HPV in HNSCC. Thirty-six HNSCC tumors were analyzed using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV by PCR and real-time PCR. Eight of 36 (22%) tumors were positive for HPV subtype 16. Statistical analysis using Significance Analysis of Microarrays based on HPV status as a supervising variable resulted in a list of 91 genes that were differentially expressed with statistical significance. Results for a subset of these genes were verified by real-time PCR. Genes highly expressed in HPV+ samples included cell cycle regulators (p16(INK4A), p18, and CDC7) and transcription factors (TAF7L, RFC4, RPA2, and TFDP2). The microarray data were also investigated by mapping genes by chromosomal location (DIGMAP). A large number of genes on chromosome 3q24-qter had high levels of expression in HPV+ tumors. Further investigation of differentially expressed genes may reveal the unique pathways in HPV+ tumors that may explain the different natural history and biological properties of these tumors. These properties may be exploited as a target of novel therapeutic agents in HNSCC treatment.

Molecular alterations in primary prostate cancer after androgen ablation therapy.

PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

NF-κB gene signature predicts prostate cancer progression.

In many patients with prostate cancer, the cancer will be recurrent and eventually progress to lethal metastatic disease after primary treatment, such as surgery or radiation therapy. Therefore, it would be beneficial to better predict which patients with early-stage prostate cancer would progress or recur after primary definitive treatment. In addition, many studies indicate that activation of NF-κB signaling correlates with prostate cancer progression; however, the precise underlying mechanism is not fully understood. Our studies show that activation of NF-κB signaling via deletion of one allele of its inhibitor, IκBα, did not induce prostatic tumorigenesis in our mouse model. However, activation of NF-κB signaling did increase the rate of tumor progression in the Hi-Myc mouse prostate cancer model when compared with Hi-Myc alone. Using the nonmalignant NF-κB-activated androgen-depleted mouse prostate, a NF-κB-activated recurrence predictor 21 (NARP21) gene signature was generated. The NARP21 signature successfully predicted disease-specific survival and distant metastases-free survival in patients with prostate cancer. This transgenic mouse model-derived gene signature provides a useful and unique molecular profile for human prostate cancer prognosis, which could be used on a prostatic biopsy to predict indolent versus aggressive behavior of the cancer after surgery.

LIM domain only-2 (LMO2) induces T-cell leukemia by two distinct pathways.

The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.

A data similarity-based strategy for meta-analysis of transcriptional profiles in cancer.

BACKGROUND: Robust transcriptional signatures in cancer can be identified by data similarity-driven meta-analysis of gene expression profiles. An unbiased data integration and interrogation strategy has not previously been available. METHODS AND FINDINGS: We implemented and performed a large meta-analysis of breast cancer gene expression profiles from 223 datasets containing 10,581 human breast cancer samples using a novel data similarity-based approach (iterative EXALT). Cancer gene expression signatures extracted from individual datasets were clustered by data similarity and consolidated into a meta-signature with a recurrent and concordant gene expression pattern. A retrospective survival analysis was performed to evaluate the predictive power of a novel meta-signature deduced from transcriptional profiling studies of human breast cancer. Validation cohorts consisting of 6,011 breast cancer patients from 21 different breast cancer datasets and 1,110 patients with other malignancies (lung and prostate cancer) were used to test the robustness of our findings. During the iterative EXALT analysis, 633 signatures were grouped by their data similarity and formed 121 signature clusters. From the 121 signature clusters, we identified a unique meta-signature (BRmet50) based on a cluster of 11 signatures sharing a phenotype related to highly aggressive breast cancer. In patients with breast cancer, there was a significant association between BRmet50 and disease outcome, and the prognostic power of BRmet50 was independent of common clinical and pathologic covariates. Furthermore, the prognostic value of BRmet50 was not specific to breast cancer, as it also predicted survival in prostate and lung cancers. CONCLUSIONS: We have established and implemented a novel data similarity-driven meta-analysis strategy. Using this approach, we identified a transcriptional meta-signature (BRmet50) in breast cancer, and the prognostic performance of BRmet50 was robust and applicable across a wide range of cancer-patient populations.

SPARCL1 suppresses metastasis in prostate cancer.

PURPOSE: Metastasis, the main cause of death from cancer, remains poorly understood at the molecular level. EXPERIMENTAL DESIGN: Based on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo. RESULTS: SPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models. CONCLUSIONS: We present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer.