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Increased expression of NUSAP1 has been identified as a robust prognostic biomarker in prostate cancer and other malignancies. We have previously shown that NUSAP1 is positively regulated by E2F1 and promotes cancer invasion and metastasis. To further understand the biological function of NUSAP1, we used affinity purification and mass spectrometry proteomic analysis to identify NUSAP1 interactors. We identified 85 unique proteins in the NUSAP1 interactome, including ILF2, DHX9, and other RNA-binding proteins. Using proteomic approaches, we uncovered a function for NUSAP1 in maintaining R-loops and in DNA damage response through its interaction with ILF2. Co-immunoprecipitation and colocalization using confocal microscopy verified the interactions of NUSAP1 with ILF2 and DHX9, and RNA/DNA hybrids. We showed that the microtubule and charged helical domains of NUSAP1 were necessary for the protein-protein interactions. Depletion of ILF2 alone further increased camptothecin-induced R-loop accumulation and DNA damage, and NUSAP1 depletion abolished this effect. In human prostate adenocarcinoma, NUSAP1 and ILF2 mRNA expression levels are positively correlated, elevated, and associated with poor clinical outcomes. Our study identifies a novel role for NUSAP1 in regulating R-loop formation and accumulation in response to DNA damage through its interactions with ILF2 and hence provides a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Androgen receptor (AR) is expressed in normal murine and human kidneys of both genders, but its physiologic role is uncertain. Several studies showed loss of AR in renal cell carcinoma (RCC) in conjunction with increasing clinical stage and pathological grade, but others found that higher AR expression correlated with worse outcomes. Limited functional studies with renal cell lines suggested tumor-promoting activity of AR. In this study, we queried transcriptomic, proteomic, epigenetic and survival data from The Cancer Genome Atlas (TCGA) to evaluate AR expression and its association with overall survival in three subtypes of RCC (clear cell [ccRCC], papillary [pRCC], and chromophobe [chRCC]). We found that although there was no significant difference in AR mRNA expression in ccRCC of males vs. females, AR protein expression in ccRCC was significantly higher in male compared to female patients. More importantly, higher expression of AR at both transcript and protein levels was associated with improved overall survival in both genders with ccRCC, but did not predict survival of either gender with pRCC or chRCC. Genes whose transcript levels were associated with AR mRNA levels significantly overlapped between ccRCC and pRCC, but not with chRCC, suggesting a similar transcriptional program mediated by AR in ccRCC and pRCC. Ingenuity pathway analysis also identified overlapping pathways and upstream regulators enriched in AR-associated genes in ccRCC and pRCC. Hypermethylation of CpG sites located in the promoter and first exon of AR was associated with loss of AR expression and poor overall survival. Our findings support a tumor suppressor role for AR in both genders that might be exploited to decrease the incidence or progression of ccRCC. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. [ABSTRACT FROM AUTHOR]

Purpose: We determined the effects of selenomethionine, the major organic selenium containing compound found in the diet and the form of selenium being used in the Selenium and Vitamin E Cancer Prevention Trial, on prostate cancer cells. Materials and Methods: We assessed global transcript profiles of selenomethionine treated LNCaP using cDNA microarrays and compared them to those of cells treated with methylselenic acid, a direct precursor of methylselenol, which is the active form of selenium in vivo. Results: After treatment with selenomethionine 2,336 unique genes showed expression changes of at least 1.5-fold in at least 3 time points during 48 hours and 366 unique transcripts differed significantly between selenomethionine and methylselenic acid treated LNCaP. Approximately half of the 76 cell cycle regulated genes affected by selenomethionine were down-regulated and enriched for genes associated with the G2/M phase. Flow cytometry analysis showed that selenomethionine induced G2/M arrest in LNCaP at low concentrations. Selenomethionine also affected expression levels of 35 known androgen responsive genes and 18 of these transcripts showed changes that were the inverse of those seen after androgen stimulation. At high concentrations selenomethionine decreased prostate specific antigen promoter driven luciferase expression. Conclusions: Selenomethionine modulates transcript levels of genes involved in a number of biological processes, including cell cycle/apoptosis androgen signaling, signal transduction and transcriptional regulation. Although the pathways affected paralleled in many ways those that are modulated by methylselenic acid, distinct differences in transcript patterns and effects on cell cycle regulation suggest that different selenium compounds could exert unique effects in prostate cells. [Copyright &y& Elsevier]

To obtain a comprehensive view of the transcriptional programs in prostatic stromal cells of different histological/pathological origin, we profiled 18 adult human stromal cell cultures from normal transition zone (TZ), normal peripheral zone (PZ), benign prostatic hyperplasia (BPH), and prostate cancer (CA) using cDNA microarrays. A hierarchical clustering analysis of 714 named unique genes whose expression varied at least threefold from the overall mean abundance in at least three samples in all 18 samples demonstrated that cells of different origin displayed distinct gene expression profiles. Many of the differentially expressed genes are involved in biological processes known to be important in the development of prostatic diseases including cell proliferation and apoptosis, cell adhesion, and immune response. Significance Analysis of Microarrays (SAM) analysis identified genes that showed differential expression with statistical significance including 24 genes between cells from TZ versus BPH, 34 between BPH versus CA, and 101 between PZ versus CA. S100A4 and SULF1, the most up- and downregulated genes in BPH versus TZ, respectively, showed expression at the protein level consistent with microarray analysis. In addition, sulfatase assay showed that BPH cells have lower SULF1 activity compared to TZ cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed differential expression of ENPP2/autotoxin and six other genes between PZ versus CA, as well as differential expression of six genes between BPH versus CA. Our results support the hypothesis that prostatic stromal cells of different origin have unique transcriptional programs and point towards genes involved in actions of stromal cells in BPH and CA. J. Cell. Physiol. 210: 111–121, 2007. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Highlights • Tumor-derived exosomes as a novel liquid biopsy biomarker for cancer diagnosis. • MUC-1 is over-expressed on the surface of adenocarcinomas, in particular MCF-7 cells. • MUC-1 is also highly expressed on the surface of exosomes secreted by MCF-7 cells. • MUC-1 aptasensor of “on-off”-type specific detection of tumor exosomes in the blood. Abstract Numerous studies have demonstrated tumor-derived exosomes (TEX) as an ideal biomarker for cancer. However, most present exosome detection methods were cumbersome and expensive, simple and specific detection of TEX in the blood still remained a challenging task. To solve the above problem, we found firstly that MUC1 was highly expressed on the surface of exosomes secreted by MCF-7 cells compared to other cells. Here, we designed an “on-off”-type aptasensor based on MUC1 activation on TEX. In the absence of TEX, the aptasensor was “off” state. Once in the presence of TEX, the aptasensor recognized the MUC1 on TEX and then turned “on” state, resulting in emitting a fluorescence signal. We used the aptasensor to detect exosomes derived from MCF-7, MCF-7/ADR, A549, MGC-803 and Hs578Bst cells and blood serum from breast cancer patients and healthy donors. The fluorescence signal value of exosomes from serum in breast cancer patients was markedly higher than healthy donors, which were a significant difference between the two groups (P < 0.05). Consequently, the method based on exosomes with high sensitivity, specificity and small amount sample were very likely to be used for breast cancer diagnosis. More importantly, this method can be universally applicable for other tumor diagnosis by changing the aptamer. [ABSTRACT FROM AUTHOR]