miRNAs act as oncogenes or tumor suppressors in a wide variety of human cancers, including prostate cancer (PCa). We found a severe and consistent downregulation of miRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 region in metastatic cell lines as compared with normal prostatic epithelial cells (PrEC). In specimens of human prostate (28 normals, 99 primary tumors and 13 metastases), lower miRNA levels correlated significantly with a higher incidence of metastatic events and higher prostate specific antigen (PSA) levels, with similar trends observed for lymph node invasion and the Gleason score. We transiently transfected 10 members of the 14q32.31 cluster in normal prostatic epithelial cell lines and characterized their affect on malignant cell behaviors, including proliferation, apoptosis, migration and invasion. Finally, we identified FZD4, a gene important for epithelial-to-mesenchymal transition in (PCa), as a target of miR-377.

Post-translational modifications provide a fine-tuned control of protein function(s) in the cell. The well-known tumour suppressor p53 is subject to many post-translational modifications, which alter its activity, localization and stability, thus ultimately modulating its response to various forms of genotoxic stress. In this review, we focus on the role of recently discovered lysine-specific modifications of p53, methylation and acetylation in particular, and their effects on p53 activity in damaged cells. We also discuss a possibility of mutual influence of covalent modifications in the p53 and histone proteins located in the vicinity of p53 binding sites in chromatin and propose important ramifications stemming from this hypothesis.

During the recent years lysine methyltransferase Set7/9 ((Su(var)-3-9, Enhancer-of-Zeste, Trithorax) domain containing protein 7/9) has emerged as an important regulator of different transcription factors. In this study, we report a novel function for Set7/9 as a critical co-activator of E2 promoter-binding factor 1 (E2F1)-dependent transcription in response to DNA damage. By means of various biochemical, cell biology, and bioinformatics approaches, we uncovered that cell-cycle progression through the G1/S checkpoint of tumour cells upon DNA damage is defined by the threshold of expression of both E2F1 and Set7/9. The latter affects the activity of E2F1 by indirectly modulating histone modifications in the promoters of E2F1-dependent genes. Moreover, Set7/9 differentially affects E2F1 transcription targets: it promotes cell proliferation via expression of the CCNE1 gene and represses apoptosis by inhibiting the TP73 gene. Our biochemical screening of the panel of lung tumour cell lines suggests that these two factors are critically important for transcriptional upregulation of the CCNE1 gene product and hence successful progression through cell cycle. These findings identify Set7/9 as a potential biomarker in tumour cells with overexpressed E2F1 activity. Cell Death and Differentiation (2014) 21, 1889-1899; doi:10.1038/cdd.2014.108; published online 15 August 2014Lysine methylation of non-histone proteins has recently emerged as a novel regulatory mechanism to control protein functions. 1-4Set7/9 ((Su(var)-3-9, Enhancer-of-Zeste, Trithorax) domain containing protein 7/9) is a founding member of the family of non-chromatin lysine methyltransferases (KMTases). Set7/9 was initially identified as a monomethylase of histone H3 lysine 4 (H3K4) in vitro. 4,5However, we and others showed that the recombinant Set7/9 failed to target nucleosomes for methylation, [6][7][8] suggesting that Set7/9 functions as a factor-specific KMTase. There have been several non-histone proteins reported as the substrates for Set7/9, including TAF10 (TATA box binding protein (TBP)-associated factor, 30 kDa), 9 oestrogen receptor a (ERa), 10 RelA, 11 PCAF (P300/CBP-associated factor), 12 Stat3,13 Yap, 14 and Suv39h1. 15 However, in most cases the functional significance of this methylation is still not clear. The beststudied targets of Set7/9-mediated methylation are p53 16 and E2 promoter-binding factor 1 (E2F1), 17 transcription factors involved in regulation of DNA damage response (DDR).In response to genotoxic stress cancer cells undergo cell-cycle arrest either in G1/S or G2/M or in both checkpoints. The presence of intact p53 in cancer cells mediates transient G1/S checkpoint arrest, 18,19 which allows cells to repair the damaged DNA before replication or, if the amount of damage is insurmountable, drives cells into apoptosis. 20,21On the contrary, the activity of the E2F family transcription factors, especially E2F1, drives cells from the G1/S block to mitosis. 22,23 Transcriptional activity of E2F1, in turn, is repressed by the retin...

The tumour suppressor p53 is a crucial regulator of cell cycle arrest and apoptosis by acting as a transcription factor to regulate a variety of genes. At least in part, this control is exerted by p53 via regulating expression of numerous microRNAs. We identified two abundantly expressed microRNAs, miR-16 and miR-26a, whose expression is regulated by p53 during the checkpoint arrest induced by the genotoxic drug, doxorubicin. Importantly, among the targets of these miRs are two critical checkpoint kinases, Chk1 and Wee1. The p53-dependent augmentation of miR-16 and miR-26a expression levels led to the cell cycle arrest of tumour cells in G1/S and increased apoptosis. Strikingly, the bioinformatics analysis of survival times for patients with breast and prostate cancers has revealed that co-expression of mir-16 and miR-26a correlated with a better survival outcome. Collectively, our data provide a novel mechanism whereby p53 represses Chk1 and Wee1 expression, at least partially, via upregulation of miR-16 and miR-26a and thus sensitizes tumour cells to genotoxic therapies.

Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.

p63 is a p53 family transcription factor, which besides unique roles in epithelial development, shares tumor suppressive activity with its homolog p53. The p63 gene has different transcriptional start sites, which generate two N-terminal isoforms (transactivation domain (TA)p63 and amino terminal truncated protein(ΔN)p63); in addition alternative splicing at the 5′-end give rise to at least five C-terminal isoforms. This complexity of gene structure has probably fostered the debate and controversy on p63 function in cancer, with TP63-harboring two distinctive promoters, codifying for the TAp63 and ΔNp63 isoforms, and having discrete functions. However, ΔNp63 also drives expression of target genes that have a relevant role in cancer and metastasis. In this study, we identified a novel p63 transcriptional target, caspase-1. Caspase-1 is proinflammatory caspase, which functions in tumor suppression. We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter. Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets. In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers. Overall, our data report a novel p63 target gene involved in tumor suppression, and the clinical analysis underlines the biological relevance of this finding and suggests a further clinically predictive biomarker.

The Cullin4A (cul4A)-dependent ligase (CDL4A) E3 has been implicated in a variety of biological processes, including cell cycle progression and DNA damage response. Remarkably, CDL4A exerts its function through both proteolytic and non-proteolytic events. Here, we show that the p53 family member p73 is able to interact with the CDL4A complex through its direct binding to the receptor subunit DNA-binding protein 1 (DDB1). As a result, the CDL4A complex is able to monoubiquitylate p73. Modification of p73 by CDL4A-mediated ubiquitylation does not affect p73 protein stability, but negatively regulates p73-dependent transcriptional activity. Indeed, genetic or RNA interference-mediated depletion of DDB1 induces the expression of several p73 target genes in a p53-independent manner. In addition, by exploiting a bioinformatic approach, we found that elevated expression of Cul4A in human breast carcinomas is associated with repression of p73 target genes. In conclusion, our findings add a novel insight into the regulation of p73 by the CDL4A complex, through the inhibition of its transcriptional function.

Fused pyrimidine derivatives R 0515 Novel Alkylations of Cyclic Thioureas by α-Halocarboxylic Acids and Their Esters. Part 4. Alkylation of 1-Methyltetrahydropyrimidine-2(1H)-thione. -Alkylation of the cyclic thiourea (I) with α-chloroor bromoacetic acids or esters affords chloro or bromo salts of type (III). The chloro salts are readily hydrolyzed to the open-chain ammonium salt of type (IV) whereas the bromo salts are considerably more stable towards hydrolysis. -(KUSHAKOVA, P. M.; BASAN, Y. V.; YULISOVA, A. I.; LIFONTOVA, V. V.; RAMSH, S. M.; GARABADGIU, A. V.; BELOBRZECKAJA COSTA, L. N.; Chem. Heterocycl.