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Purpose:The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches.Experimental Design:We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer–specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer (n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq.Results:Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type–specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19. In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion.Conclusions:Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829–40. ©2016 AACR.

Supplementary Data from Pan-Cancer Analysis of the Mediator Complex Transcriptome Identifies CDK19 and CDK8 as Therapeutic Targets in Advanced Prostate Cancer

Purpose Clinical stage I (CSI) testicular germ cell tumors (TGCT) represents disease confined to the testis without metastasis and CSIS is defined as persistently elevated tumor markers (TM) after orchiectomy, indicating subclinical metastatic disease. This study aims at assessing clinical characteristics and oncological outcome in CSIS. Methods Data from five tertiary referring centers in Germany were screened. We defined correct classification of CSIS according to EAU guidelines. TM levels, treatment and relapse-free survival were assessed and differences between predefined groups (chemotherapy, correct/incorrect CSIS) were analyzed with Fisher’s exact and Chi-square test. Results Out of 2616 TGCT patients, 43 (1.6%) were CSIS. Thereof, 27 were correctly classified (cCSIS, 1.03%) and 16 incorrectly classified (iCSIS). TMs that defined cCSIS were in 12 (44.4%), 10 (37%), 3 (11.1%) and 2 (7.4%) patients AFP, ß-HCG, AFP plus ß-HCG and LDH, respectively. In the cCSIS group, six patients were seminoma and 21 non-seminoma. Treatment consisted of active surveillance, carboplatin-mono AUC7 and BEP (bleomycin, etoposide and cisplatin). No difference between cCSIS and iCSIS with respect to applied chemotherapy was found (p = 0.830). 5-year relapse-free survival was 88.9% and three patients (11%) in the cCSIS group relapsed. All underwent salvage treatment (3xBEP) with no documented death. Conclusion Around 1% of all TGCT were classified as cCSIS patients. Identification of cCSIS is of critical importance to avoid disease progression and relapses by adequate treatment. We report a high heterogeneity of treatment patterns, associated with excellent long-term survival irrespective of the initial treatment approach.

421 Background: Characteristics and outcome of non-seminomatous testicular germ cell tumours (NSGCT) with teratoma-containing primaries are still under debate. Methods: We performed a retrospective analysis including 557 patients with metastatic NSGCT as a registry study within the "German Testicular Cancer Study Group". Results: Of the eligible 557 patients with NSGCT, 237 (42%) of all orchiectomy specimens had teratoma-containing primaries, while 320 (58%) were teratoma-free. Teratoma-containing primaries had a significantly higher clinical stage (p=0.002) and worse prognosis (p=0.051) compared to teratoma-free specimens. Lymph node metastasis were significantly larger before (4.5 vs 2.5cm; p

Objective : Circulating microRNAs have clear potential for improving malignant germ-cell-tumor (MGCT) diagnosis. Here, we address the central issue of whether measurement of a single microRNA is sufficient for detecting testicular MGCTs, or whether there is added benefit in quantifying other members of the four-microRNA panel previously identified (miR-371a-3p/miR-372-3p/miR-373-3p and miR-367-3p). Patients/Methods : We performed a pooled analysis of available published data where all four panel miRNAs had been assessed using pre-amplification PCR technology (four studies; total 329 patients). Two studies using identical methodology (and identical normalization using endogenous miR-30b-5p) were used in the discovery phase (n=51 patients: 17 MGCT, 34 controls). The two other studies (n=278 patients: 140 MGCT, 138 controls), which assessed the same test panel but with different normalization approaches (endogenous miR-93-5p, exogenous cel-miR-39-3p), were used for the validation phase. We derived sensitivity, specificity, positive- and negative-predictive-values (PPV/NPV) for the detection thresholds that maximised the Youden Index (YI). Results : In the discovery-phase, the YI was 0.97 for miR-371a-3p (sensitivity=1, specificity=0.97), 0.71 (miR-367-3p), 0.68 (miR-372-3p), and 0.50 (miR-373-3p). These findings were confirmed in the validation-phase, with YI of 0.75 for miR-371a-3p (sensitivity=0.90, specificity 0.85), 0.55 (miR-367-3p), 0.47 (miR-372-3p), and 0.51 (miR-373-3p). Importantly, no combination of markers added additional diagnostic benefit to miR-371a-3p alone, in either the discovery or the validation phase. Conclusion : Quantifying circulating miR-371a-3p alone is sufficient for testicular MGCT diagnosis. PCR measurement of this single miRNA marker will be more cost-effective and easier to interpret, facilitating future incorporation into routine clinical practice. MicroAbstract Circulating microRNA testing is likely to transform future management for testicular cancer patients. Here, we undertook a pooled analysis (329 patients) to test whether measurement of a single microRNA is sufficient for detecting testicular cancer, or whether it is necessary to quantify other members of the four microRNA panel previously identified (miR-371a-3p/miR-372-3p/miR-373-3p/miR-367-3p). We show that quantifying circulating miR-371a-3p alone is sufficient for testicular cancer diagnosis.

BACKGROUNDNovel targeted treatments and immunotherapies have substantially changed therapeutic options for advanced and metastatic renal cell carcinomas (RCCs). However, accurate diagnostic tests for the identification of high-risk patients are urgently needed. Here, we analyzed SHOX2 mRNA expression in RCC tissues and SHOX2 gene body methylation quantitatively in circulating cell-free DNA (ccfDNA) and RCC tissues with regard to risk stratification.METHODSThe clinical performance of SHOX2 methylation was tested retrospectively and prospectively in a training and testing cohort of RCC tissue samples (n = 760 in total). SHOX2 mRNA expression analysis was included in the training cohort. In matched blood plasma samples from the testing cohort (n = 100), we prospectively examined the capability of pretherapeutic quantitative SHOX2 ccfDNA methylation to assess disease stage and identify patients at high risk of death.RESULTSSHOX2 gene body methylation was positively correlated with mRNA expression in RCC tissues (training cohort: Spearman ρ = 0.23, P < 0.001). SHOX2 methylation in tissue and plasma strongly correlated with an advanced disease stage (training cohort: ρ = 0.28, P < 0.001; testing cohort/tissue: ρ = 0.40, P < 0.001; testing cohort/plasma: ρ = 0.34, P = 0.001) and risk of death after initial partial or radical nephrectomy [training cohort: hazard ratio (HR) = 1.40 (95% CI, 1.24–1.57), P < 0.001; testing cohort/tissue: HR = 1.16 (95% CI, 1.07–1.27), P = 0.001; testing cohort/plasma: HR = 1.50 (95% CI, 1.29–1.74), P < 0.001].CONCLUSIONSPretherapeutic SHOX2 ccfDNA methylation testing allows for the identification of RCC patients at high risk of death after nephrectomy. These patients might benefit from an adjuvant treatment or early initiation of a palliative treatment.

Background/aim FGF-2, HGF, MIF and PTN have been suggested as biomarkers for testicular germ cell cancer patients in earlier studies. Our study was designed to validate these potential novel tumor markers. Materials and methods Serum FGF-2, HGF, MIF and PTN levels were analysed using an ELISA technique in a screening cohort of 20 testicular germ cell cancer patients and 10 healthy men. MIF levels were measured in a validation cohort of 84 patients with testicular cancer, 24 with non-malignant testicular tumors and 64 healthy men. Results Serum FGF-2, HGF and PTN levels did not differ in cancer patients and healthy males within the screening cohort, whereas MIF was significantly increased among cancer patients. Within the validation cohort, a modest but insignificant increase of serum MIF was observed in TGCT patients compared to healthy men. MIF levels were not correlated with adverse clinical-pathological parameters. Conclusion FGF-2, HGF, MIF and PTN are not suitable as non-invasive biomarkers for testicular germ cell cancer patients.

// Anne Offermann 1, * , Ignacija Vlasic 1, * , Isabella Syring 2, 6, 7 , Wenzel Vogel 1 , Christian Ruiz 3 , Tobias Zellweger 4 , Cyrill A. Rentsch 5 , Susanne Hagedorn 1 , Jochen Behrends 8 , Michael Nowak 6, 7 , Axel Merseburger 9 , Lukas Bubendorf 3 , Jutta Kirfel 7 , Stefan Duensing 10 , Zaki Shaikhibrahim 1, * , Sven Perner 1, * 1 Pathology of the University Medical Center Schleswig-Holstein, Campus Luebeck and Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany 2 Department of Urology, University Hospital Bonn, Bonn, Germany 3 Institute for Pathology, University Hospital Basel, Basel, Switzerland 4 Department of Urology, St. Claraspital, Basel, Switzerland 5 Department of Urology, University Hospital Basel, Basel, Switzerland 6 Section of Prostate Cancer Research, Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn, Bonn, Germany 7 Institute of Pathology, Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn, Bonn, Germany 8 Core Facility Fluorescence Cytometry, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany 9 Department of Urology, University Hospital Luebeck, Luebeck, Germany 10 Section of Molecular Urooncology, Department of Urology, University of Heidelberg School of Medicine, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Sven Perner, email: sven.perner@uksh.de Keywords: mediator complex, MED15, castration-resistant prostate cancer, androgen deprivation, PI3 kinase Received: April 15, 2016 Accepted: November 21, 2016 Published: December 10, 2016 ABSTRACT Androgen deprivation therapy (ADT) is the main therapeutic option for advanced prostate cancer (PCa). After initial regression, most tumors develop into castration-resistant PCa (CRPC). Previously, we found the Mediator complex subunit MED15 to be overexpressed in CRPC and to correlate with clinical outcome. Therefore, we investigated whether MED15 is implicated in the signaling changes taking place during progression to CRPC. Immunohistochemistry (IHC) for MED15 on matched samples from the same patients before and after ADT reveals significantly increased MED15 expression after ADT in 72%. A validation cohort comprising samples before and after therapy confirmed our observations. Protein analysis for pAKT and pSMAD3 shows that MED15 correlates with PI3K and TGFs activities, respectively, and that hyper-activation of both pathways simultaneously correlates with highest levels of MED15. We further show that MED15 protein expression increases in LNCaP cells under androgen deprivation, and via EGF mediated PI3K activation. PI3K/mTOR and TGFs-receptor inhibition results in decreased MED15 expression. MED15 knockdown reduces LNCaP cell viability and induces apoptosis during androgen deprivation, while cell cycle is not affected. Collectively, MED15 overexpression arises during ADT via hyper-activation of PI3K/mTOR signaling, thus MED15 may serve as a predictive marker for response to PI3K/mTOR inhibitors. Furthermore, MED15 is potentially a therapeutic target for the treatment of CRPC.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking. We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

5052 Background: Testicular germ cell tumors (TGCT) in clinical stage I (CSI) are tumors confined to the testis without evidence of metastasis. Around 50% of all TGCT patients present with elevated serum tumor markers (TM) such as alpha-feto protein (AFP), beta-humanochoriongonadotropin (ß-HCG) and lactate-dehydrogenase (LDH). After ablatio testis, TMs usually return to normal according to half-life kinetics, however, in clinical stage IS (CSIS) TMs remain elevated or increase after surgery. Follow-up data on CSIS is scarce and our study aims to assess clinical characteristics and oncologic outcomes in a large TGCT cohort. Methods: In this multicenter study we collected data from 5 tertiary referring hospitals in Germany, included patients with CSIS and evaluated TM levels, treatment and the primary outcome relapse-free survival. False CSIS was defined as documented CSIS but TMs that returned to normal after respective half-life kinetics. Differences between predefined groups (chemotherapy, TM, true/false CSIS) was analyzed with fisher’s exact and chi-square test. Results: Overall, 2616 patient data files were analyzed. Forty-three patients (1.6%) were documented as CSIS of which 27 (63%) were true and 16 (37%) false CSIS. Six (14%) had seminomas and 37 (86%) non-seminomas. In the true CSIS group AFP, ß-HCG, AFP plus ß-HCG and LDH were elevated in 13, 6, 3 and 2 cases. Four true CSIS patients received surveillance, 21 had 3x or 2x courses of BEP (bleomycin, etoposide and cisplatin) and 2 carboplatin. Within the false CSIS group, 2 patients were treated with surveillance, 10 received 3x BEP, one 3x PEI (cisplatin, etoposide and ifosfamid) and 3 had carboplatin. Chi-Square test revealed no difference between true or false CSIS classification in respect to application of chemotherapy (any chemotherapy, p = 0.83). Relapse-free survival after 5 and 10 years was 88.9% and 77.8%, respectively. Three patients in the true CSIS group relapsed (2 seminomas had carboplatin, 1 non-seminoma had surveillance). All relapses were treated with 3 courses of BEP with no documented death in the CSIS population. Conclusions: Overall, less than 2% of all TGCT were documented CSIS of which 37% were falsely classified. We report a high proportion of relapse-free survival in CSIS treated with surveillance or BEP with a high heterogeneity in treatment patterns. Correct classification of CSIS remains of critical importance to avoid toxicity for patients that could be safely treated with surveillance.

The Mediator complex, a multi-subunit protein complex, plays an integral role in regulating transcription. Genetic alterations of the mediator subunit 15 (MED15) in separate tumor entities have been described previously. However, till now, not much is known about the role of MED15 in urothelial bladder cancer (BCa). Using cBioPortal, database analysis was executed for the mRNA expression and survival analysis of MED15 in BCa. Immunohistochemistry (IHC) analysis against MED15 was performed on tissue microarrays with 18 benign, 126 BCa, and 38 metastases samples. The intensity evaluation was performed using a staining intensity score from 0 to 3 and associated with clinicopathological data. The BCa cell lines T24 and TCCSUP were used for the functional investigation. After the MED15 knockdown by small interfering (si)RNA, cell proliferation, migration and invasion were investigated. On the mRNA level, only a low number of alterations (2%) was found for MED15 in BCa. Due to the small count of events, there were no significant differences or tendencies in survival. For IHC, MED15 was found to have a higher expression in non-muscle invasive BCa compared with benign and muscle invasive BCa. For survival analysis, no significant differences between samples with or without overexpression of MED15 were found. In the functional analysis, proliferation, migration, and invasion were significantly reduced in BCa-cells following the transient siRNA-mediated MED15 knockdown. In summary, MED15 appears to play a role in the tumor parameters proliferation, migration, and invasion in BCa, but further investigations are necessary.

BACKGROUND/OBJECTIVE MED15 is a part of the multiprotein Mediator complex which is involved in the transcription of polymerase (Pol) II-dependent genes. Several studies in this field have reported altered expressions of distinct subunits in human malignancy. However, the role of MED15 in renal cell carcinoma (RCC) has not be investigated yet. METHODS First, we performed an RNA expression and survival analysis of MED15 in RCC by using the database cBioPortal. To confirm these data on the protein level, we executed immunohistochemical (IHC) staining against MED15 on a tissue microarray containing 184 samples of the most common subtypes of the tumour at the various stages. Further, we performed functional analysis including proliferation, migration, and invasion assays on the RCC cell lines A-498 and ACHN following the siRNA-mediated MED15 knockdown. RESULTS On the mRNA level, higher expression of MED15 was associated with worse patient survival rates. IHC staining validated this tendency, unfortunately the results were not significant. However, supporting this tendency, in vitro-assays showed a significant decrease in proliferation, migration, and invasion after knockdown of MED15. CONCLUSION The research concludes that MED15 does seem to play a tumour promoting role in the progression and metastatic spread of renal cell carcinoma.

Background/Aims/Objectives: To evaluate the influence of body mass index (BMI) on complications and oncological outcomes in patients undergoing radical cystectomy (RC). Methods: Clinical and histopathological parameters of patients have been prospectively collected within the “PROspective MulticEnTer RadIcal Cystectomy Series 2011”. BMI was categorized as normal weight (2), overweight (≥25–29.9 kg/m2) and obesity (≥30 kg/m2). The association between BMI and clinical and histopathological endpoints was examined. Ordinal logistic regression models were applied to assess the influence of BMI on complication rate and survival. Results: Data of 671 patients were eligible for final analysis. Of these patients, 26% (n = 175) showed obesity. No significant association of obesity on tumour stage, grade, lymph node metastasis, blood loss, type of urinary diversion and 90-day mortality rate was found. According to the ­American Society of Anesthesiologists score, local lymph node (NT) stage and operative case load patients with higher BMI had significantly higher probabilities of severe complications 30 days after RC (p = 0.037). The overall survival rate of obese patients was superior to normal weight patients (p = 0.019). Conclusions: There is no evidence of correlation between obesity and worse oncological outcomes after RC. While obesity should not be a parameter to exclude patients from cystectomy, surgical settings need to be aware of higher short-term complication risks and obese patients should be counselled ­accordingly.

Classic serum tumor markers (human chorionic gonadotropin, α1-fetoprotein and lactate dehydrogenase) have an important role in managing testicular germ cell tumor. Since only 60% of all patients with testicular germ cell tumor have elevations of these markers, there is a need for new biomarkers with greater sensitivity/specificity. miRNAs are deregulated in cancer and could serve as noninvasive serum biomarkers. We explored the role of serum miRNAs as a novel biomarker in patients with testicular germ cell tumor.Total RNA was isolated from serum. miRNA levels were quantified by quantitative real-time polymerase chain reaction. We assessed the miRNAs miR-302a-3p, 302b-3p, 302c-3p, 367-3p, 371a-3p, 372-3p and 373-3p in a subcohort of 30 patients with testicular germ cell tumor and 18 healthy subjects. Validation was performed in 76 patients treated with inguinal exploration due to suspicion of testicular germ cell tumor, of whom 59 had cancer and 17 had benign disease, and in 84 healthy male subjects.Serum miR-367-3p, 371a-3p, 372-3p and 373-3p levels were significantly increased in patients with testicular germ cell tumor compared to healthy individuals and patients with nonmalignant testicular disease. In particular miR-371a-3p allowed for sensitive (84.7%) and specific (99%) identification of patients with testicular germ cell tumor, thus, outperforming human chorionic gonadotropin or α1-fetoprotein testing. Furthermore, miR-367-3p was increased in nonseminoma compared to seminoma cases. Serum miRNA levels were increased in patients with advanced local stage and metastasis. In 9 patients with localized (clinical stage 1A) testicular germ cell tumor serum miR-371a-3p levels decreased postoperatively, indicating tumor specific release.miR-371a-3p allows for better identification of testicular germ cell tumor than α1-fetoprotein and human chorionic gonadotropin. It could be helpful for clinically managing testicular germ cell tumor, especially for monitoring surveillance therapy and residual disease after chemotherapy.

BACKGROUND/AIM The Mediator complex is a key regulator of gene transcription, and several studies have demonstrated altered expression of particular subunits in diverse human diseases, especially cancer. To date, nothing is known about the role of MED30 in bladder cancer. MATERIALS AND METHODS We, therefore, performed an RNA expression and survival analysis of the subunit MED30 in 537 samples of bladder cancer by using the database cBioPortal. To validate these data on the protein level, we practiced immunohistochemical staining against MED30 on a tissue microarray containing 210 samples of all tumour stages and performed survival analyses. For functional analysis, the siRNA-mediated knockdown of MED30 was performed in the cell lines T24 and TCCSUP followed by proliferation, migration, and invasion assays. RESULTS On the mRNA and protein levels, higher expression of MED30 is associated with better patient survival. In accordance with this, advanced T- and N-stages showed lower expression of MED30. In contrast, knockdown of MED30 led to reduction of the tumour parameters proliferation, migration, and invasion in the BCa cell lines. CONCLUSION MED30 appears to be integrated in the progression of the urothelial tumour in the bladder.

Background Case volume has been suggested to affect surgical outcomes in different arrays of procedures. We aimed to delineate the relationship between case volume and surgical outcomes and quality of care criteria of radical cystectomy (RC) in a prospectively collected multicenter cohort. Patients and Methods This was a retrospective analysis of a prospectively collected European cohort of patients with bladder cancer treated with RC in 2011. We relied on 479 and 459 eligible patients with available information on hospital case volume and surgeon case volume, respectively. Hospital case volume was divided into tertiles, and surgeon volume was dichotomized according to the median annual number of surgeries performed. Binomial generalized estimating equations controlling for potential known confounders and inter-hospital clustering assessed the independent association of case volume with short-term complications and mortality, as well as the fulfillment of quality of care criteria. Results The high-volume threshold for hospitals was 45 RCs and, for high-volume surgeons, was > 15 cases annually. In adjusted analyses, high hospital volume remained an independent predictor of fewer 30-day (odds ratio, 0.34; P = .002) and 60- to 90-day (odds ratio, 0.41; P = .03) major complications but not of fulfilling quality of care criteria or mortality. No difference between surgeon volume groups was noted for complications, quality of care criteria, or mortality after adjustments. Conclusion The coordination of care at high-volume hospitals might confer a similar important factor in postoperative outcomes as surgeon case volume in RC. This points to organizational elements in high-volume hospitals that enable them to react more appropriately to adverse events after surgery.

Purpose: The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches. Experimental Design: We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer–specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer (n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq. Results: Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type–specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19. In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion. Conclusions: Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829–40. ©2016 AACR.

Background Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. Methods In order to provide better understanding of the molecular biology of ccRCC and to identify potential diagnostic/prognostic biomarker and therapeutic targets, we utilized a microarray to profile mRNA expression of corresponding normal and malignant renal tissues. Real-time PCR, Western Blot and immunohistochemistry were applied to study the expression of candidate biomarkers. ccRCC cell lines were treated with sertraline to inhibit the dopamine transporter SLC6A3. Results Differential expression of fourteen mRNAs, yet not studied in ccRCC in depth, was confirmed using qPCR (upregulation: SLC6A3, NPTX2, TNFAIP6, NDUFA4L2, ENPP3, FABP6, SPINK13; downregulation: FXYD4, SLC12A1, KNG1, NPHS2, SLC13A3, GCGR, PLG). Up-/downregulation was also confirmed for FXYD4, KNG1, NPTX2 and SLC12A1 by Western Blot on the protein level. In contrast to the mRNA expression, protein expression of the dopamine transporter SLC6A3 was lower in ccRCC compared to normal renal tissue. Immunohistochemistry indicated that this decrease was due to higher concentrations of SLC6A3 in the proximal tubules. Immunohistochemical analyses further demonstrated that high SLC6A3 expression in ccRCC tissue was correlated with a shorter period of recurrence-free survival following surgery. Treatment of ccRCC cells with the SLC6A3 inhibitor sertraline induced dose-dependent cell-death. Conclusion Our study identified several novel biomarkers with diagnostic potential and further investigations on sertraline as therapeutic agent in ccRCC patients are warranted. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0495-5) contains supplementary material, which is available to authorized users.

Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. Long non-coding RNAs (lncRNA) regulate various cellular functions and have been implicated in ccRCC pathogenesis. In order to decipher the molecular biology of this tumor and to identify potential prognostic biomarkers and therapeutic targets, we re-evaluated published lncRNA expression profiling data. An expression profile of 49 lncRNAs allowed discrimination of localized and advanced ccRCC. The expression profile of six lncRNAs transcripts (lnc-ACO1625, lnc-CYP4A22-2/3, lnc-PEAK1.1-1, lnc-PCYOX1L, lnc-VCAN-1, lnc-ZNF180-2) with potential prognostic interest were validated in a cohort of 50 normal renal, 57 localized ccRCC and 45 advanced ccRCC tissues. lnc-ZNF180-2 levels were similar in localized ccRCC and normal renal tissue, but we observed a significant increase of lnc-ZNF180-2 expression in advanced ccRCC tissue. Furthermore, lnc-ZNF180-2 expression levels were an independent predictor of progression-free survival, cancer-specific survival and overall survival in ccRCC patients. We also observed that lnc-CYP4A22-2/3 expression levels allowed discrimination of ccRCC and normal renal tissue. In conclusion, lncRNAs are involved in renal carcinogenesis, and quantification of lnc-ZNF180-2 may be useful for the prediction of ccRCC patients outcome following nephrectomy.

Background Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. Methods Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. Results In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. Conclusions In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15 expression in the preinvasive precursor cells, which may provide diagnostic value to distinguish between benign and pre-malignant testicular specimen, and may indicate a role for MED15 in carcinogenesis in TGCT.

INTRODUCTION AND OBJECTIVES: Epigenetic changes, including promoter CpG island hypermethylation, occur frequently in bladder cancer (BC) and may be exploited as a means for BC detection and distinction between high-grade (HG) and low-grade (LG) disease. METHODS: To determine epigenetic differences between LG and HG BC we performed genome-wide methylation analysis with Agilent Human CpG Island Microarrays on a panel of fresh frozen BC tissue samples. Using Linear Models for Microarray Data (LIMMA) and local-pooled-error (LPE) approaches and unsupervised hierarchical clustering we identified 990 probes comprising a 32-gene panel that completely distinguished LG from HG BC based on methylation status. We selected five representative differentially methylated genes (DMGs) from our 32-gene panel for methylation validation by real-time PCRbased MethyLight in a series of 40 LG cases ageand sex-matched to 40 HG cases. RESULTS: Among the DMGs tested, including EOMES, GP5, PAX6, TCF4, and ZSCAN12, we identified that EOMES, GP5, and ZSCAN12 methylation significantly differs between normal, LG, and HG disease. GP5 and ZSCAN12, two novel methylated genes in BC, are significantly hypermethylated in HG versus LG BC (p1⁄40.006 and 0.028, respectively). Pathway enrichment analysis and functional annotation determined the most frequently methylated pathways in HG BC were enriched for anterior/posterior pattern specification, embryonic skeletal system development, and neuron fate commitment. The molecular functions of the most enriched genes were involved in DNA binding and transcription factor activity. CONCLUSIONS: These results indicate the ability to distinguish normal tissue from cancer, as well as LG from HG tumours, and reveal important pathways dysregulated in HG BC. Ultimately, the creation of a methylation panel able to distinguish between disease phenotypes will improve disease management and patient outcomes.