The Diminishment of Novel Endometrial Carcinoma-Derived Stem-like Cells by Targeting Mitochondrial Bioenergetics and MYC.

Cancer stem cells (CSCs) are a small subpopulation of tumor cells harboring properties that include self-renewal, multi-lineage differentiation, tumor reconstitution, drug resistance and invasiveness, making them key players in tumor relapse. In the present paper, we develop new CSC models and analyze the molecular pathways involved in survival to identify targets for the establishment of novel therapies. Endometrial carcinoma-derived stem-like cells (ECSCs) were isolated from carcinogenic gynecological tissue and analyzed regarding their expression of prominent CSC markers. Further, they were treated with the MYC-signaling inhibitor KJ-Pyr-9, chemotherapeutic agent carboplatin and type II diabetes medication metformin. ECSC populations express common CSC markers, such as Prominin-1 and CD44 antigen as well as epithelial-to-mesenchymal transition markers, Twist, Snail and Slug, and exhibit the ability to form free-floating spheres. The inhibition of MYC signaling and treatment with carboplatin as well as metformin significantly reduced the cell survival of ECSC-like cells. Further, treatment with metformin significantly decreased the mitochondrial membrane potential of ECSC-like cells, while the extracellular lactate concentration was increased. The established ECSC-like populations represent promising in vitro models to further study the contribution of ECSCs to endometrial carcinogenesis. Targeting MYC signaling as well as mitochondrial bioenergetics has shown promising results in the diminishment of ECSCs, although molecular signaling pathways need further investigations.

Identification of clinically relevant cytomegalovirus infections in patients with inflammatory bowel disease.

Several lines of evidence indicate that cytomegalovirus infection can be substantially associated with onset of inflammatory bowel disease, especially in patients refractory to immunosuppressive treatment. As cytomegalovirus is widely spread in the population, here we present a quantitative detection system suitable to differentiate clinically relevant cytomegalovirus infection from common latent cytomegalovirus. Using a quantitative real-time PCR approach, cytomegalovirus viral load was evaluated in 917 formalin-fixed and paraffin-embedded colon biopsy samples of 136 patients diagnosed with inflammatory bowel disease. Besides initial cytomegalovirus testing, the PCR system was also used to monitor therapy response after antiviral treatment. Cytomegalovirus DNA was detected in 37 patients (27%) with varying viral loads ranging from 5 to 8.7 × 105 copies/105 cells. Thereof, 13 patients (35%) received an antiviral treatment with 12 of them going into remission (92%). Later, five patients displayed a relapse and three patients who agreed to restart antiviral treatment again showed positive therapy response. A retrospective comparison of viral loads with antiviral therapy response revealed a threshold of 600 cytomegalovirus copies/105 cells as indicative for clinically relevant infection. Of note, sensitivity of cytomegalovirus detection by immunohistochemistry was found to be insufficient to reliably identify antiviral therapy responders. In conclusion, quantitative real-time PCR using formalin-fixed biopsy samples is suitable for detection of cytomegalovirus infection in tissue samples of patients with inflammatory bowel disease. Moreover, it allows the definition of a viral load threshold, predictive for clinical relevance concerning antiviral therapy response.

Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-ß and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-ß and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-ß and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-ß and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-ß/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-ß and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

Association of Tumor Suppressor Protein Pdcd4 With Ribosomes Is Mediated by Protein-Protein and Protein-RNA Interactions.

The Pdcd4 (programmed cell death gene 4) gene has been implicated as a novel tumor suppressor gene in the development of several types of human cancer. The Pdcd4 protein is believed to act as a translation suppressor of mRNAs containing structured 5' UTRs. Pdcd4 contains 2 copies of so-called MA3 domains that mediate tight interactions with the translation initiation factor eIF4A, resulting in the inhibition of the eIF4A helicase activity. The N-terminal part of Pdcd4, which has been less well characterized, binds RNA in vitro, but as yet, it has not been clear whether RNA binding by Pdcd4 plays a role in vivo. Here, the authors have identified 2 highly conserved clusters of basic amino acid residues that are essential for the RNA binding activity of Pdcd4. They also show that a substantial fraction of Pdcd4 is present, together with small ribosomal subunits, in translation preinitiation complexes. Using mutants that disrupt RNA binding or the Pdcd4-eIF4A interaction, they demonstrate that the ribosomal association of Pdcd4 is dependent on its RNA binding activity as well as on its ability to interact with eIF4A. Their work provides the first direct evidence for an essential role of the Pdcd4 RNA binding activity in vivo and suggests that RNA binding is required for recruiting Pdcd4 to the translation machinery.

Daxx is a transcriptional repressor of CCAAT/enhancer-binding protein beta.

CCAAT/enhancer-binding Protein beta (C/EBPbeta) is a member of the bZIP transcription factor family that is expressed in various tissues, including cells of the hematopoietic system. C/EBPbeta is involved in tissue-specific gene expression and thereby takes part in fundamental cellular processes such as proliferation and differentiation. Here, we show that the activity of C/EBPbeta is negatively regulated by the transcriptional co-repressor Daxx. C/EBPbeta was found to directly interact with Daxx after overexpression as well as on the endogenous level. Glutathione S-transferase pulldown assays showed that Daxx binds via amino acids 190-400 to the C-terminal part of C/EBPbeta. Co-expression of C/EBPbeta changed the sub-nuclear Daxx distribution pattern from predominantly POD-localized to nucleoplasmic. Daxx suppressed basal and p300-enhanced transcriptional activity of C/EBPbeta. Furthermore, Daxx decreased the C/EBPbeta-dependent phosphorylation of p300, which in turn was associated with a diminished level of p300-mediated C/EBPbeta acetylation. Co-expression of promyelocytic leukemia protein abrogated the repressive effect of Daxx on C/EBPbeta as well as the direct interaction of Daxx and C/EBPbeta, presumably by re-recruiting Daxx to PML-oncogenic domains. In acute promyelocytic leukemia (APL) cells, C/EBPbeta activity is known to be required for all-trans-retinoic acid-induced cell differentiation and disease remission. We show that all-trans-retinoic acid as well as arsenic trioxide treatment leads to a reduced C/EBPbeta fraction associated with Daxx suggesting a relief of Daxx-dependent C/EBPbeta repression as an important molecular event leading to APL cell differentiation. Overall, our data identify Daxx as a new negative regulator of C/EBPbeta and provide first clues for a link between abrogation of Daxx-C/EBPbeta complex formation and APL remission.

Disturbed expression of the apoptosis regulators XIAP, XAF1, and Smac/DIABLO in gastric adenocarcinomas.

Dysregulation of apoptosis plays an important role in carcinogenesis and tumor progression. Whereas x-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of apoptosis, its antagonists second mitochondria-derived activator of caspases/direct IAP binding protein with low PI (Smac/DIABLO), and XIAP-associated factor 1 (XAF1) promote apoptosis. To explore the relevance of XIAP, Smac/DIABLO, and XAF1 for carcinogenesis and tumor progression, we analyzed 46 primary gastric adenocarcinomas and non-neoplastic gastric mucosa samples by quantitative real-time polymerase chain reaction. XIAP, Smac/DIABLO, and XAF1 expression was found in all non-neoplastic gastric mucosa samples and all adenocarcinomas. XIAP expression levels did not change between non-neoplastic gastric mucosa and adenocarcinomas or between carcinomas of early and advanced stages. Although Smac/DIABLO expression was significantly (P=0.01) higher in carcinomas, the ratio of XIAP to Smac/DIABLO expression remained stable between non-neoplastic mucosa and carcinomas. XAF1 expression had the tendency to decrease from non-neoplastic mucosa to advanced adenocarcinomas. Importantly, the ratio of XIAP to XAF1 expression significantly (P=0.03) increased from non-neoplastic mucosa to adenocarcinomas and the increase was even higher in carcinomas of advanced stage (P=0.01). Moreover, expression of the XAF1 splice variants differing in the zinc-finger domain essential for XIAP-binding was analyzed and revealed a significant higher (P=0.03) variant-2/variant-1 ratio in advanced carcinomas. In conclusion, an increased expression ratio of XIAP to XAF1 in combination with a disturbed expression of the XAF1 splice variants could be shown in gastric adenocarcinomas. These marked imbalances probably result in an impaired ability for XAF1 to antagonize the effects of XIAP thereby contributing to apoptosis-resistance and generating an important growth advantage.

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.