Niklas Klümper

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

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.

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC. To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen. The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts. MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen.

Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types

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.

Ercc1 Deficiency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Specific Manner.

KRAS-mutant lung adenocarcinoma is among the most common cancer entities and, in advanced stages, typically displays poor prognosis due to acquired resistance against chemotherapy, which is still largely based on cisplatin-containing combination regimens. Mechanisms of cisplatin resistance have been extensively investigated, and ERCC1 has emerged as a key player due to its central role in the repair of cisplatin-induced DNA lesions. However, clinical data have not unequivocally confirmed ERCC1 status as a predictor of the response to cisplatin treatment. Therefore, we employed an autochthonous mouse model of Kras-driven lung adenocarcinoma resembling human lung adenocarcinoma to investigate the role of Ercc1 in the response to cisplatin treatment. Our data show that Ercc1 deficiency in Tp53-deficient murine lung adenocarcinoma induces a more aggressive tumor phenotype that displays enhanced sensitivity to cisplatin treatment. Furthermore, tumors that relapsed after cisplatin treatment in our model develop a robust etoposide sensitivity that is independent of the Ercc1 status and depends solely on previous cisplatin exposure. Our results provide a solid rationale for further investigation of the possibility of preselection of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients might benefit from sequential cisplatin and etoposide chemotherapy. Implications: This study provides a solid rationale for the stratification of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients could benefit from sequential cisplatin and etoposide chemotherapy. Mol Cancer Res; 14(11); 1110–23. ©2016 AACR.

Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors

BackgroundTesticular 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.MethodsImmunohistochemical 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.ResultsIn 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.ConclusionsIn 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.

Mediator Complex Subunit MED1 Protein Expression Is Decreased during Bladder Cancer Progression

Introduction Bladder cancer (BCa) is among the most frequent cancer entities and relevantly contributes to cancer-associated deaths worldwide. The multi-protein Mediator complex is a central regulator of the transcriptional machinery of protein-coding genes and has been described to be altered in several malignancies. MED1, a subunit of the tail module, was described to negatively modulate expression of metastasis-related genes and to be downregulated in melanoma and lung cancer. In contrast, MED1 hyperactivity was described in breast and prostate cancer, likely due its function as a hub for nuclear hormone receptors. So far, only little is known about the function of the Mediator complex in BCa. The aim of this study was therefore to investigate the role of MED1 in BCa as a prognostic biomarker and a biomarker of disease progression. Methods The protein expression of MED1 was assessed by immunohistochemistry (IHC) on tissue microarrays from 224 patients: benign urothelium n = 31, non-muscle invasive BCa (pTis, pT1) n = 72, and muscle invasive BCa (pT2–T4) n = 121. Comprehensive clinicopathological information including follow-up were available. Quantification of MED1 protein expression was evaluated by the semiquantitative image analysis program Definiens. Results MED1 expression significantly decreased during BCa progression from benign urothelium to advanced BCa. Muscle invasion, the crucial step in BCa progression, was associated with low MED1 protein expression. Accordingly, decreased MED1 expression was found in primary BCa samples with positive lymphonodal status and distant metastases. Furthermore, cancer-specific survival was significantly worse in the group of low MED1 expression. Conclusion Our findings show that the downregulation of MED1 is associated with muscle invasion, metastatic spread, and shorter overall survival in BCa.

756 The Mediator complex subunit MED12 is implicated in the progression of urothelial cell carcinoma of the urinary bladder

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.