Training and validation of a novel 4-miRNA ratio model (MiCaP) for prediction of postoperative outcome in prostate cancer patients.

Background: New molecular biomarkers for prostate cancer (PC) prognosis are urgently needed. Ratio-based models are attractive, as they require no additional normalization. Here, we train and independently validate a novel 4-miRNA prognostic ratio model for PC. Patients and methods: By genome-wide miRNA expression profiling of PC tissue samples from 123 men who underwent radical prostatectomy (RP) (PCA123, training cohort), we identified six top candidate prognostic miRNAs and systematically tested their ability to predict postoperative biochemical recurrence (BCR). The best miRNA-based prognostic ratio model (MiCaP) was validated in two independent cohorts (PCA352 and PCA476) including >800 RP patients in total. Clinical end points were BCR and prostate cancer-specific survival (CSS). The prognostic potential of MiCaP was assessed by univariate and multivariate Cox-regression analyses and Kaplan-Meier analyses. Results: We identified a 4-miRNA ratio model, MiCaP (miR-23a-3p×miR-10b-5p)/(miR-133a×miR-374b-5p), that predicted time to BCR independently of routine clinicopathologic variables in the training cohort (PCA123) and was successfully validated in two independent RP cohorts. In addition, MiCaP was a significant predictor of CSS in univariate analysis [HR 3.35 (95% CI 1.34 - 8.35), P = 0.0096] and in multivariate analysis [HR 2.43 (95% CI 1.45-4.07), P = 0.0210]. As proof-of-principle, we also analyzed MiCaP in plasma samples from 111 RP patients. A high MiCaP score in plasma was significantly associated with BCR (P = 0.0036, Kaplan-Meier analysis). Limitations include low mortality rates (CSS: 5.4%). Conclusions: We identified a novel 4-miRNA ratio model (MiCaP) with significant independent prognostic value in three RP cohorts, indicating promising potential to improve PC risk stratification.

Mutations in the TLR3 signaling pathway and beyond in adult patients with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon production downstream of Toll-like receptor (TLR)3. In the present study, we used whole-exome sequencing to investigate the genetic profile of 16 adult patients with a history of HSE. We identified novel mutations in IRF3, TYK2 and MAVS, molecules involved in generating innate antiviral immune responses, which have not previously been associated with HSE. Moreover, data revealed mutations in TLR3, TRIF, TBK1 and STAT1 known to be associated with HSE in children but not previously described in adults. All discovered mutations were heterozygous missense mutations, the majority of which were associated with significantly decreased antiviral responses to HSV-1 infection and/or the TLR3 agonist poly(I:C) in patient peripheral blood mononuclear cells compared with controls. Altogether, this study demonstrates novel mutations in the TLR3 signaling pathway in molecules previously identified in children, suggesting that impaired innate immunity to HSV-1 may also increase susceptibility to HSE in adults. Importantly, the identification of mutations in innate signaling molecules not directly involved in TLR3 signaling suggests the existence of innate immunodeficiencies predisposing to HSE beyond the TLR3 pathway.

Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients.

Childhood acute lymphoblastic leukemia survival approaches 90%. New strategies are needed to identify the 10-15% who evade cure. We applied targeted, sequencing-based genotyping of 25 000 to 34 000 preselected potentially clinically relevant single-nucleotide polymorphisms (SNPs) to identify host genome profiles associated with relapse risk in 352 patients from the Nordic ALL92/2000 protocols and 426 patients from the German Berlin-Frankfurt-Munster (BFM) ALL2000 protocol. Patients were enrolled between 1992 and 2008 (median follow-up: 7.6 years). Eleven cross-validated SNPs were significantly associated with risk of relapse across protocols. SNP and biologic pathway level analyses associated relapse risk with leukemia aggressiveness, glucocorticosteroid pharmacology/response and drug transport/metabolism pathways. Classification and regression tree analysis identified three distinct risk groups defined by end of induction residual leukemia, white blood cell count and variants in myeloperoxidase (MPO), estrogen receptor 1 (ESR1), lamin B1 (LMNB1) and matrix metalloproteinase-7 (MMP7) genes, ATP-binding cassette transporters and glucocorticosteroid transcription regulation pathways. Relapse rates ranged from 4% (95% confidence interval (CI): 1.6-6.3%) for the best group (72% of patients) to 76% (95% CI: 41-90%) for the worst group (5% of patients, P<0.001). Validation of these findings and similar approaches to identify SNPs associated with toxicities may allow future individualized relapse and toxicity risk-based treatments adaptation.

Next-generation sequencing identifies germline MRE11A variants as markers of radiotherapy outcomes in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) can be cured by radical radiotherapy (RT). We previously found tumour MRE11 expression to be predictive of survival following RT in MIBC, and this was independently validated in a separate institute. Here, we investigated germline MRE11A variants as possible predictors of RT outcomes in MIBC, using next-generation sequencing (NGS). PATIENTS AND METHODS: The MRE11A gene was amplified in germline DNA from 186 prospectively recruited MIBC patients treated with RT and sequenced using bar-coded multiplexed NGS. Germline variants were analysed for associations with cancer-specific survival (CSS). For validation as a prognostic or predictive marker, rs1805363 was then genotyped in a cystectomy-treated MIBC cohort of 256 individuals. MRE11A mRNA isoform expression was measured in bladder cancer cell lines and primary tumour samples. RESULTS: Carriage of at least one of six (five novel) rare variants was associated with the worse RT outcome (hazard ratio [HR] 4.04, 95% confidence interval [95% CI] 1.42-11.51, P = 0.009). The single-nucleotide polymorphism (SNP), rs1805363 (minor allele frequency 11%), was also associated with worse CSS (per-allele HR 2.10, 95% CI 1.34-3.28, Ptrend = 0.001) following RT in MIBC, with a gene-dosage effect observed, but no effect seen on CSS in the cystectomy cohort (Ptrend = 0.89). Furthermore, rs1805363 influenced relative MRE11A isoform expression, with increased isoform 2 expression with carriage of the rs1805363 minor A allele. CONCLUSIONS: Germline MRE11A SNP rs1805363 was predictive of RT, but not of cystectomy outcome in MIBC. If successfully validated in an independent RT-treated cohort, this SNP could be a useful clinical tool for selecting patients for bladder-conserving treatment.

Genome-wide study of association and interaction with maternal cytomegalovirus infection suggests new schizophrenia loci.

Genetic and environmental components as well as their interaction contribute to the risk of schizophrenia, making it highly relevant to include environmental factors in genetic studies of schizophrenia. This study comprises genome-wide association (GWA) and follow-up analyses of all individuals born in Denmark since 1981 and diagnosed with schizophrenia as well as controls from the same birth cohort. Furthermore, we present the first genome-wide interaction survey of single nucleotide polymorphisms (SNPs) and maternal cytomegalovirus (CMV) infection. The GWA analysis included 888 cases and 882 controls, and the follow-up investigation of the top GWA results was performed in independent Danish (1396 cases and 1803 controls) and German-Dutch (1169 cases, 3714 controls) samples. The SNPs most strongly associated in the single-marker analysis of the combined Danish samples were rs4757144 in ARNTL (P=3.78 × 10(-6)) and rs8057927 in CDH13 (P=1.39 × 10(-5)). Both genes have previously been linked to schizophrenia or other psychiatric disorders. The strongest associated SNP in the combined analysis, including Danish and German-Dutch samples, was rs12922317 in RUNDC2A (P=9.04 × 10(-7)). A region-based analysis summarizing independent signals in segments of 100 kb identified a new region-based genome-wide significant locus overlapping the gene ZEB1 (P=7.0 × 10(-7)). This signal was replicated in the follow-up analysis (P=2.3 × 10(-2)). Significant interaction with maternal CMV infection was found for rs7902091 (P(SNP × CMV)=7.3 × 10(-7)) in CTNNA3, a gene not previously implicated in schizophrenia, stressing the importance of including environmental factors in genetic studies.

Common variant at 16p11.2 conferring risk of psychosis.

Epidemiological and genetic data support the notion that schizophrenia and bipolar disorder share genetic risk factors. In our previous genome-wide association study, meta-analysis and follow-up (totaling as many as 18 206 cases and 42 536 controls), we identified four loci showing genome-wide significant association with schizophrenia. Here we consider a mixed schizophrenia and bipolar disorder (psychosis) phenotype (addition of 7469 bipolar disorder cases, 1535 schizophrenia cases, 333 other psychosis cases, 808 unaffected family members and 46 160 controls). Combined analysis reveals a novel variant at 16p11.2 showing genome-wide significant association (rs4583255[T]; odds ratio=1.08; P=6.6 × 10(-11)). The new variant is located within a 593-kb region that substantially increases risk of psychosis when duplicated. In line with the association of the duplication with reduced body mass index (BMI), rs4583255[T] is also associated with lower BMI (P=0.0039 in the public GIANT consortium data set; P=0.00047 in 22 651 additional Icelanders).

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer.

BACKGROUND: Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC. METHODS: Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays representing 267 radical prostatectomy (RP) and 111 conservatively treated (CT) PC patients. Clinical end points were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. The ANPEP promoter methylation levels were determined by bisulphite sequencing or MethyLight analysis in 278 nonmalignant and PC tissue samples, and in cell lines. RESULTS: The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2'-deoxycytidine induced ANPEP expression in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis marker (vascular endothelial growth factor or microvessel density) improved risk prediction significantly in both cohorts. CONCLUSION: Our results suggest negative APN immunoreactivity as a new independent adverse prognostic factor for patients with clinically localised PC and, furthermore, that epigenetic mechanisms are involved in silencing of ANPEP in PC.

A high resolution genomic portrait of bladder cancer: correlation between genomic aberrations and the DNA damage response.

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho-epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (γH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n=319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (P<0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6-3.8) and higher T stage (P<0.05). Univariate analysis confirmed this association (P=0.006) in an independent cohort (n=241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; technology transfer from microarrays to PCR.

BACKGROUND: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. METHODS: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. RESULTS: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P<0.001) when adjusting for stage, grade and treatment. Independent validation of the 12-gene panel and the determined cut-off values is needed and ongoing. CONCLUSION: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patient's disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer.

Expression of MAGE-A3, NY-ESO-1, LAGE-1 and PRAME in urothelial carcinoma.

BACKGROUND: The potential for cancer-testis (CT) antigens as targets for immunotherapy in cancer patients has been heavily investigated, and currently cancer vaccine trials based on the CT antigens, MAGE-A3 and NY-ESO-1, are being carried out. METHODS: We used specific q-RT-PCR assays to analyse the expression of the CT genes MAGE-A3, NY-ESO-1 (CTAG1B), LAGE-1 (CTAG2) and PRAME in a panel of bladder tumours from 350 patients with long-term follow-up and detailed treatment information. RESULTS: Overall, 43% of the tumours expressed MAGE-A3, 35% expressed NY-ESO-1, 27% expressed LAGE-1 and 20% expressed PRAME. In all, 56% of the tumours expressed at least one of the CT genes analysed. Univariate Cox regression analysis of CT gene expression in non-muscle-invasive tumours showed that expression of MAGE-A3 (P=0.026), LAGE-1 (P=0.001) and NY-ESO-1 (P=0.040) was significantly associated with a shorter progression-free survival. In addition, we found that patients with tumours expressing PRAME responded poorly to chemotherapy (P=0.02, χ(2)-test). CONCLUSION: Cancer-testis genes are frequently expressed in bladder cancer and especially in tumours of high stage and grade. In addition, the CT gene expression may have both prognostic and predictive value. Development of specific immunotherapy against the CT antigens in bladder cancer may ultimately increase patient survival.

Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes.

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP-chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes.

The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer.

BACKGROUND: Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood. METHODS: We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1-T4 tumours using expression microarrays and qRT-PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays. RESULTS: We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3'UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict. CONCLUSION: This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.

Association between genetic variation in a region on chromosome 11 and schizophrenia in large samples from Europe.

Recent molecular studies have implicated common alleles of small to moderate effect and rare alleles with larger effect sizes in the genetic architecture of schizophrenia (SCZ). It is expected that the reliable detection of risk variants with very small effect sizes can only be achieved through the recruitment of very large samples of patients and controls (that is tens of thousands), or large, potentially more homogeneous samples that have been recruited from confined geographical areas using identical diagnostic criteria. Applying the latter strategy, we performed a genome-wide association study (GWAS) of 1169 clinically well characterized and ethnically homogeneous SCZ patients from a confined area of Western Europe (464 from Germany, 705 from The Netherlands) and 3714 ethnically matched controls (1272 and 2442, respectively). In a subsequent follow-up study of our top GWAS results, we included an additional 2569 SCZ patients and 4088 controls (from Germany, The Netherlands and Denmark). Genetic variation in a region on chromosome 11 that contains the candidate genes AMBRA1, DGKZ, CHRM4 and MDK was significantly associated with SCZ in the combined sample (n=11 540; P=3.89 × 10(-9), odds ratio (OR)=1.25). This finding was replicated in 23 206 independent samples of European ancestry (P=0.0029, OR=1.11). In a subsequent imaging genetics study, healthy carriers of the risk allele exhibited altered activation in the cingulate cortex during a cognitive control task. The area of interest is a critical interface between emotion regulation and cognition that is structurally and functionally abnormal in SCZ and bipolar disorder.

Pancreatic ß-cells activate a JunB/ATF3-dependent survival pathway during inflammation.

Destruction of insulin-producing pancreatic ß-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent ß-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified ß-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for ß-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary ß-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of ß-cells under inflammatory stress.

Low ANXA10 expression is associated with disease aggressiveness in bladder cancer.

BACKGROUND: Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level. METHODS: We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis. RESULTS: Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells. CONCLUSION: We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.

Repression of KIAA1199 attenuates Wnt-signalling and decreases the proliferation of colon cancer cells.

BACKGROUND: The KIAA1199 transcript is upregulated in colon adenomas and downregulated upon ß-catenin knockdown. METHODS: Transcript profiling was performed on >500 colon biopsies, methylation profiling data were compared with transcript data. Immunohistochemistry assessed KIAA1199 protein expression in 270 stage II/III tumours (>3 years follow-up). The effects of stable KIAA1199 knockdown in SW480 cells (three different constructs) were studied using transcriptional profiling, proliferation and protein analysis. RESULTS: The KIAA1199 transcript was strongly upregulated in 95% of adenocarcinomas. Absent expression in normal mucosa correlated with KIAA1199 promotor methylation. Nuclear and cytoplasmic KIAA1199 protein expression was identified in colon adenocarcinomas and other types of cancers. A subpopulation of patients with tumours strongly expressing KIAA1199 in the nucleus showed a better outcome with regard to recurrence as lung or liver metastases. The KIAA1199 knockdown affected the cell cycle and the Wnt-signalling pathway. Reduced cellular proliferation and decreased KI67, phosphorylated retinoblastoma, ß-catenin and ASCL2 protein expression supported these findings. Eighteen Wnt-signalling genes differentially expressed upon KIAA1199 knockdown correlated with the KIAA1199 expression profile in clinical specimens. CONCLUSION: The KIAA1199 knockdown attenuates the effects of the Wnt/ß-catenin signalling and it may thus be regarded as a regulatory part of this pathway.

miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors.

Downregulation of miR-145 in a variety of cancers suggests a possible tumor suppressor function for this microRNA. Here, we show that miR-145 expression is reduced in bladder cancer and urothelial carcinoma in situ, compared with normal urothelium, using transcription profiling and in situ hybridization. Ectopic expression of miR-145 induced extensive apoptosis in urothelial carcinoma cell lines (T24 and SW780) as characterized by caspase activation, nuclear condensation and fragmentation, cellular shrinkage, and detachment. However, cell death also proceeded upon caspase inhibition by the pharmacological inhibitor zVAD-fmk and ectopic expression of anti-apoptotic Bcl-2, indicating the activation of an alternative caspase-independent death pathway. Microarray analysis of transcript levels in T24 cells, before the onset of cell death, showed destabilization of mRNAs enriched for miR-145 7mer target sites. Among these, direct targeting of CBFB, PPP3CA, and CLINT1 was confirmed by a luciferase reporter assay. Notably, a 22-gene signature targeted on enforced miR-145 expression in T24 cells was significantly (P<0.00003) upregulated in 55 Ta bladder tumors with concomitant reduction of miR-145. Our data indicate that reduction in miR-145 expression may provide bladder cancer cells with a selective advantage by inhibition of cell death otherwise triggered in malignant cells.

Dysregulation of the transcription factors SOX4, CBFB and SMARCC1 correlates with outcome of colorectal cancer.

The aim of this study was to identify deregulated transcription factors (TFs) in colorectal cancer (CRC) and to evaluate their relation with the recurrence of stage II CRC and overall survival. Microarray-based transcript profiles of 20 normal mucosas and 424 CRC samples were used to identify 51 TFs displaying differential transcript levels between normal mucosa and CRC. For a subset of these we provide in vitro evidence that deregulation of the Wnt signalling pathway can lead to the alterations observed in tissues. Furthermore, in two independent cohorts of microsatellite-stable stage II cancers we found that high SOX4 transcript levels correlated with recurrence (HR 2.7; 95% CI, 1.2-6.0; P=0.01). Analyses of approximately 1000 stage I-III adenocarcinomas, by immunohistochemistry, revealed that patients with tumours displaying high levels of CBFB and SMARCC1 proteins had a significantly better overall survival rate (P=0.0001 and P=0.0275, respectively) than patients with low levels. Multivariate analyses revealed that a high CBFB protein level was an independent predictor of survival. In conclusion, several of the identified TFs seem to be involved in the progression of CRC.

SMARCC1 expression is upregulated in prostate cancer and positively correlated with tumour recurrence and dedifferentiation.

BACKGROUND: The identification of new prognostic markers in prostate cancer (PC) is essential to improve patient treatment and management. Data suggest that SMARCC1 protein, a part of the intranuclear SWI/SNF complex which enhances the transactivation of the androgen receptor, is upregulated in PC and therefore a possible candidate marker for PC progression. MATERIALS: Expression of SMARCC1 immunostaining was analysed on a tissue microarray containing specimens from 327 patients with prostate cancer and clinical follow-up information. Furthermore, 30 specimens from patients with benign prostate hyperplasia were included as controls as well as 30 specimens of benign prostate tissue from PC patients. Also, 18 specimens from lymph node metastases were analysed. RESULTS: All benign specimens showed no or minimal staining for SMARCC1. In contrast, 20% of the specimens from patients with non-metastatic and non-recurrent disease showed moderate to marked staining. In 31% of the patients with recurrent disease and in 31% of the patients with metastatic disease we found moderate to strong SMARCC1 immunostaining. In total, 23% of lymph node metastases expressed SMARCC1. SMARCC1 expression was also positively correlated to Gleason score (p<0.05), clinical T stage (p<0.01) and time to recurrence (p<0.001). In a logistic regression analysis, patients with a marked SMARCC1 immunostaining had a significantly elevated odds ratio (OR) of 16 for recurrent cancer and an OR of 4.5 for metastatic disease. Conclusions. Our present results demonstrate an increased expression of SMARCC1 protein in prostate cancer and reveal a positive correlation with tumour dedifferentiation, progression, metastasis and time to recurrence.