Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes.

BACKGROUND: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. METHODS: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). RESULTS: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. CONCLUSIONS: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.

Acute response in circulating microRNAs following a single bout of short-sprint and heavy strength training in well-trained cyclists.

Background: Heavy strength (HS) and short-sprint (SS) are commonly used training methods for competitive road cyclists, with the aim to improve the anaerobic power and short time cycling performance. Knowledge of how such training methods affects biochemical as well as molecular factors, are particularly important for determining individual recovery and long-term adaptations. The primary aim of the current study was to investigate the expression levels of small non-coding RNAs in response to HS and SS training in elite cyclists as potential biomarkers for individual optimal restitution time. Methods: Eleven well trained cyclists performed one session of HS training and one session of SS training on separate days. Blood samples were taken at baseline and 5 min, 1 h and 21 h post training. Along with physiological measurements and biochemical factors (serum creatine kinase, myoglobin, human growth hormone and plasma lactate), real-time quantitative PCR was used to explore whether HS and/or SS training influenced the abundance of 24 circulating miRNAs, in serum, associated with muscle development, angiogenesis, and/or inflammation. Results: Based on complete miRNA profiles from nine cyclists, the miRNAs showing most altered expression after both training sessions included the three striated muscle-specific miRNAs (myomiRs) miR-1-3p, 133a-3p and 133b-3p. While all three miRNAs showed significantly highest expression at 1 h post HS session, the acute effect of the SS session included a significantly higher level of miR-1-3p alone, at 5 min (highest), as well as at 1 h and 21 h post session. Correlation (negative) with biochemical markers was only shown for miR-133a-3p and CK (r = -0.786, p = 0.041) and between miR-133b-3p and [La-] (r = -0.711, p = .032), at 21 h post SS session. Conclusion: Our findings support that unique myomiRs are regulated by HS and SS training. Such knowledge may be important for individually adjusted restitution times.

MiR-182 and miR-203 induce mesenchymal to epithelial transition and self-sufficiency of growth signals via repressing SNAI2 in prostate cells.

MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR-182 and miR-203 in our previously described prostate cell model. MiR-182 and miR-203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re-expression of miR-182 or miR-203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR-182 and miR-203 provided EPT1 cells with the ability to self-sufficiency of growth signals, a well-recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR-182 and miR-203. SNAI2 was identified as a common target of miR-182 and miR-203. Knock-down of SNAI2 in EPT1 cells phenocopied re-expression of either miR-182 or miR-203 regarding both MET and self-sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re-expression of miR-182/203 and knock-down of SNAI2. Finally, P-cadherin was identified as a direct target of SNAI2. We conclude that miR-182 and miR-203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR-182/203 in cancer related processes.

Increased expression of SIM2-s protein is a novel marker of aggressive prostate cancer.

PURPOSE: The human SIM2 gene is located within the Down's syndrome critical region of chromosome 21 and encodes transcription factors involved in brain development and neuronal differentiation. SIM2 has been assigned a possible role in the pathogenesis of solid tumors, and the SIM2-short isoform (SIM2-s) was recently proposed as a molecular target for cancer therapy. We previously reported SIM2 among the highly up-regulated genes in 29 prostate cancers, and the purpose of our present study was to examine the expression status of SIM2 at the transcriptional and protein level as related to outcome in prostate cancer. EXPERIMENTAL DESIGN: By quantitative PCR, mRNA in situ hybridization, and immunohistochemistry, we evaluated the expression and significance of SIM2 isoforms in 39 patients with clinically localized prostate cancer and validated the expression of SIM2-s protein in an independent cohort of 103 radical prostatectomies from patients with long and complete follow-up. RESULTS: The SIM2 isoforms (SIM2-s and SIM2-l) were significantly coexpressed and increased in prostate cancer. Tumor cell expression of SIM2-s protein was associated with adverse clinicopathologic factors like increased preoperative serum prostate-specific antigen, high histologic grade, invasive tumor growth with extra-prostatic extension, and increased tumor cell proliferation by Ki-67 expression. SIM2-s protein expression was significantly associated with reduced cancer-specific survival in multivariate analyses. CONCLUSIONS: These novel findings indicate for the first time that SIM2 expression might be important for clinical progress of human cancer and support the recent proposal of SIM2-s as a candidate for targeted therapy in prostate cancer.

Power Production and Biochemical Markers of Metabolic Stress and Muscle Damage Following a Single Bout of Short-Sprint and Heavy Strength Exercise in Well-Trained Cyclists.

Purpose: Although strength and sprint training are widely used methods in competitive cycling, no previous studies have compared the acute responses and recovery rates following such sessions among highly trained cyclists. The primary aim of the current study was to compare power production and biochemical markers of metabolic stress and muscle damage following a session of heavy strength (HS) and short-sprint training (SS). Methods: Eleven well-trained male cyclists (18 ± 2 years with maximal oxygen uptake of 67.2 ± 5.0 mL·kg-1·min-1) completed one HS session and one SS session in a randomized order, separated by 48 h. Power production and biochemical variables were measured at baseline and at different time points during the first 45 h post exercise. Results: Lactate and human growth hormone were higher 5 min, 30 min and 1 h post the SS compared to the HS session (all p ≤ 0.019). Myoglobin was higher following the HS than the SS session 5 min, 30 min and 1 h post exercise (all p ≤ 0.005), while creatine kinase (CK) was higher following the HS session 21 and 45 h post exercise (p ≤ 0.038). Counter movement jump and power production during 4 sec sprint returned to baseline levels at 23 and 47 h with no difference between the HS and SS session, whereas the delayed muscle soreness score was higher 45 h following the HS compared to the SS session (p = 0.010). Conclusion: Our findings indicate that SS training provides greater metabolic stress than HS training, whereas HS training leads to more muscle damage compared to that caused by SS training. The ability to produce power remained back to baseline already 23 h after both training sessions, indicating maintained performance levels although higher CK level and muscle soreness were present 45 h post the HS training session.

ERG upregulation and related ETS transcription factors in prostate cancer.

The aim of this study was to identify and validate differentially expressed genes in matched pairs of benign and malignant prostate tissue. Samples included 29 histologically verified primary tumors and 23 benign controls. Microarray analysis was initially performed using a sequence verified set of 40,000 human cDNA clones. Among the genes most consistently and highly upregulated in prostate cancer was the ETS family transcription factor ERG (ETS related gene). This finding was validated in an expanded patient series (37 tumors and 38 benign samples) using DNA oligonucleotide microarray and real-time quantitative PCR assays. ERG was 20- to more than 100-fold overexpressed in prostate cancer compared with benign prostate tissue in more than 50% of patients according to quantitative PCR. Surprisingly, ERG mRNA levels were found to be significantly higher in the endothelial cell line, HUVEC, than in the prostate cell lines PC3, DU145 and LNCaP. In situ hybridization of prostate cancer tissue revealed that ERG was abundantly expressed in both prostate cancer cells and associated endothelial cells. The consistency and magnitude of ERG overexpression in prostate cancer appeared unique, but several related ETS transcription factors were also overexpressed in matched pairs of tumor and benign samples, whereas ETS2 was significantly underexpressed. Our findings support the hypothesis that ERG overexpression and related ETS transcription factors are important for early prostate carcinogenesis.

Gene expression profiles in prostate cancer: association with patient subgroups and tumour differentiation.

Prostate carcinoma is the most common cancer of western men and is a markedly heterogeneous disease. The aim of this study was to identify signatures of differentially expressed genes in prostate cancer using DNA microarray technology, evaluating expression profiles in matched pairs of benign and malignant tissue. Samples were collected from 33 radical prostatectomies, and 52 specimens were included, representing 29 histologically verified primary tumours, 19 paired samples of malignant and benign tissue, and 4 non-paired benign tissue samples. Microarray analysis was performed using an expanded sequence verified set of 40,000 human cDNA clones, revealing several genes with significant differences between malignant and benign tissue, including recently reported genes like alpha-methylacyl-CoA racemase (AMACR) and hepsin, as well as genes relevant for tumour development and progression. Leave out cross validation (LOCV) test correctly predicted tumour or benign tissue in 47 (90.3%) out of 52 cases, significantly better than cross validation tests using randomly permuted tissue labels. Unsupervised clustering analysis revealed 3 distinct patient clusters significantly associated with Gleason score, and high grade tumours (Gleason score >/=7) accumulated in cluster 1 (C1). Gene expression profiles correctly predicted 100% of tumour samples segregating to C1, as also validated by LOCV. Gene expression profiles were analysed in filtered and floored datasets with similar results, and a pair-wise design was also tested. Gene expression profiles provided tumour clusters linked to differentiation, and revealed novel markers relevant for molecular classification, grading and therapy of prostate cancer.

p63 attenuates epithelial to mesenchymal potential in an experimental prostate cell model.

The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis.

The transcription factor ERG is highly upregulated in the majority of prostate cancers due to chromosomal fusion of the androgen responsive promoter of TMPRSS2 to the ERG reading frame. Our aim was to identify this gene fusion in urine samples from prostate cancer patients prior to radical treatment and to compare fusion status with clinicopathological variables. Urine fractions from 55 patients (with and without prior prostatic massage) were analyzed for the presence of TMPRSS2:ERG isoforms using real-time qPCR. Sixty-nine percent of urine samples following prostatic massage were positive for TMPRSS2:ERG isoforms a or b, five out of which were positive for both, vs 24% of samples obtained without prior massage. Isoform a seems to be most prevalent and some patients may be positive for more than one fusion variant, reflecting the multifocality of prostate cancer. Prostatic massage prior to sampling, analysis of pelleted urine material and detection of cDNA provided the highest sensitivity. Positive statistical correlations were identified between TMPRSS2:ERG fusion and high s-PSA, pathological stage and Gleason score. Our findings contribute to the increasing elucidation of the role of TMPRSS2:ERG in the development of prostate cancer.

Genome-wide profiling of histone h3 lysine 4 and lysine 27 trimethylation reveals an epigenetic signature in prostate carcinogenesis.

BACKGROUND: Increasing evidence implicates the critical roles of epigenetic regulation in cancer. Very recent reports indicate that global gene silencing in cancer is associated with specific epigenetic modifications. However, the relationship between epigenetic switches and more dynamic patterns of gene activation and repression has remained largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide profiling of the trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) was performed using chromatin immunoprecipitation coupled with whole genome promoter microarray (ChIP-chip) techniques. Comparison of the ChIP-chip data and microarray gene expression data revealed that loss and/or gain of H3K4me3 and/or H3K27me3 were strongly associated with differential gene expression, including microRNA expression, between prostate cancer and primary cells. The most common switches were gain or loss of H3K27me3 coupled with low effect on gene expression. The least prevalent switches were between H3K4me3 and H3K27me3 coupled with much higher fractions of activated and silenced genes. Promoter patterns of H3K4me3 and H3K27me3 corresponded strongly with coordinated expression changes of regulatory gene modules, such as HOX and microRNA genes, and structural gene modules, such as desmosome and gap junction genes. A number of epigenetically switched oncogenes and tumor suppressor genes were found overexpressed and underexpressed accordingly in prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This work offers a dynamic picture of epigenetic switches in carcinogenesis and contributes to an overall understanding of coordinated regulation of gene expression in cancer. Our data indicate an H3K4me3/H3K27me3 epigenetic signature of prostate carcinogenesis.

Epithelial to mesenchymal transition of a primary prostate cell line with switches of cell adhesion modules but without malignant transformation.

BACKGROUND: Epithelial to mesenchymal transition (EMT) has been connected with cancer progression in vivo and the generation of more aggressive cancer cell lines in vitro. EMT has been induced in prostate cancer cell lines, but has previously not been shown in primary prostate cells. The role of EMT in malignant transformation has not been clarified. METHODOLOGY/PRINCIPAL FINDINGS: In a transformation experiment when selecting for cells with loss of contact inhibition, the immortalized prostate primary epithelial cell line, EP156T, was observed to undergo EMT accompanied by loss of contact inhibition after about 12 weeks in continuous culture. The changed new cells were named EPT1. EMT of EPT1 was characterized by striking morphological changes and increased invasion and migration compared with the original EP156T cells. Gene expression profiling showed extensively decreased epithelial markers and increased mesenchymal markers in EPT1 cells, as well as pronounced switches of gene expression modules involved in cell adhesion and attachment. Transformation assays showed that EPT1 cells were sensitive to serum or growth factor withdrawal. Most importantly, EPT1 cells were not able to grow in an anchorage-independent way in soft agar, which is considered a critical feature of malignant transformation. CONCLUSIONS/SIGNIFICANCE: This work for the first time established an EMT model from primary prostate cells. The results show that EMT can be activated as a coordinated gene expression program in association with early steps of transformation. The model allows a clearer identification of the molecular mechanisms of EMT and its potential role in malignant transformation.

Comparison of nucleic acid targets prepared from total RNA or poly(A) RNA for DNA oligonucleotide microarray hybridization.

The aim of this work was to compare DNA microarray results using either total RNA or affinity-purified poly(A) RNA from the same biological sample for target preparation. The high-density oligonucleotide microarrays of both Agilent Technologies (based on two-color detection) and Applied Biosystems (based on single-color detection) were evaluated. Real-time quantitative PCR was used to quantify messenger RNA (mRNA) and ribosomal RNA (rRNA) at different stages of target preparations. Poly(A) RNA versus total RNA target hybridizations exhibited slightly lower correlation coefficients than did self versus self hybridizations (i.e., poly(A) RNA targets vs. poly(A) RNA targets or total RNA targets vs. total RNA targets). Only a small fraction of all transcripts appeared to be significantly over- or underrepresented when total RNA targets or poly(A) RNA targets from the same biological sample were compared. Therefore, the conclusion is that poly(A) affinity purification from total RNA can be omitted during target preparation for routine mRNA expression analysis using high-density oligonucleotide microarrays. Among consistently overrepresented transcripts in total RNA targets were histone mRNAs known to lack poly(A) tails. Therefore, structurally exceptional RNA species can be identified by comparing targets derived from either poly(A) RNA or total RNA using microarray hybridization.