Overexpression of the scaffold WD40 protein WRAP53ß enhances the repair of and cell survival from DNA double-strand breaks.

Altered expression of the multifunctional protein WRAP53ß (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53ß function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53ß disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53ß exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53ß, attenuated this effect, suggesting that overexpression of WRAP53ß leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53ß and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53ß may contribute to genomic stability in and survival of cancer cells.

Downregulation of the cancer susceptibility protein WRAP53ß in epithelial ovarian cancer leads to defective DNA repair and poor clinical outcome.

Alterations in the scaffold protein WRAP53ß have previously been linked to carcinogenesis and, in particular, associated with an increased risk for epithelial ovarian cancer. Here, we investigated the pathogenic impact and prognostic significance of WRAP53ß in connection with epithelial ovarian cancer and examined the underlying mechanisms. We find that reduced expression of WRAP53ß in ovarian tumors correlated with attenuated DNA damage response and poor patient survival. Furthermore, in ovarian cancer cell lines, WRAP53ß was rapidly recruited to DNA double-strand breaks, where it orchestrated the recruitment of repair factors involved in homologous recombination and non-homologous end joining, including RNF168, 53BP1, BRCA1 and RAD51. Mechanistically, WRAP53ß accomplishes this by facilitating the necessary ubiquitinylation at DNA breaks. Finally, we demonstrate that loss of WRAP53ß significantly impairs the repair of DNA double-strand breaks, resulting in their accumulation. Our findings establish WRAP53ß as a regulator of homologous recombination and non-homologous end joining repair in ovarian cancer cells, suggesting that loss of this protein contributes to the development and/or progression of ovarian tumors. Moreover, our current observations identify the nuclear levels of WRAP53ß as a promising biomarker for the survival of patients with ovarian cancer.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis.

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.

Abrogation of Wip1 expression by RITA-activated p53 potentiates apoptosis induction via activation of ATM and inhibition of HdmX.

Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 - HdmX and Wip1, leading to efficient elimination of tumour cells.

A method for assembling a collaborative research team from multiple disciplines and academic centers to study the relationships between ECG estimation and MRI measurement of myocardial infarct size.

A method has been developed for establishing a "University Without Walls" for the purpose of studying the relationship between electrocardiographic estimation and magnetic resonance imaging measurements of myocardial infarct size. The research team includes faculty and students from 4 medical centers, with expertise extending from clinical to technical. Weekly interactive videoconferences provide the key research communication method. Study patients are recruited from 2 of the sites, and the correlations between their electrocardiographic and magnetic resonance imaging data are considered by the research team in conference. Outcomes of this program are both scientific publications in international peer-review journals and formal postdoctoral degree attainment by the research trainees.