Lisa Lirussi

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

SIGNIFICANCE An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. RECENT ADVANCES After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. CRITICAL ISSUES A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. FUTURE DIRECTIONS A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

Nucleolar accumulation of APE1 depends on charged Lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells

The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells.

SIRT1 gene expression upon genotoxic damage is regulated by APE1 through nCaRE-promoter elements

APE1 is recruited to the transcription initiation site of the SIRT1 promoter during early cell response to oxidative stress. This reveals the importance of BER enzyme involvement in controlling specific gene expression at the transcriptional level.

Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins

NPM1 is a novel modulator of the BER pathway. NPM1 depletion results in BER protein up-regulation; NPM1 has a stimulatory effect on BER capacity and promotes accumulation of BER proteins within nucleoli. Cisplatin leads to redistribution of NPM1 and BER proteins from nucleoli.

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an α-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization.

APE1 polymorphic variants cause persistent genomic stress and affect cancer cell proliferation.

Apurinic/apyrimidinic endonuclease 1 (APE1) is the main mammalian AP-endonuclease responsible for the repair of endogenous DNA damage through the base excision repair (BER) pathway. Molecular epidemiological studies have identified several genetic variants associated with human diseases, but a well-defined functional connection between mutations in APE1 and disease development is lacking. In order to understand the biological consequences of APE1 genetic mutations, we examined the molecular and cellular consequences of the selective expression of four non-synonymous APE1 variants (L104R, R237C, D148E and D283G) in human cells. We found that D283G, L104R and R237C variants have reduced endonuclease activity and impaired ability to associate with XRCC1 and DNA polymerase β, which are enzymes acting downstream of APE1 in the BER pathway. Complementation experiments performed in cells, where endogenous APE1 had been silenced by shRNA, showed that the expression of these variants resulted in increased phosphorylation of histone H2Ax and augmented levels of poly(ADP-ribosyl)ated (PAR) proteins. Persistent activation of DNA damage response markers was accompanied by growth defects likely due to combined apoptotic and autophagic processes. These phenotypes were observed in the absence of exogenous stressors, suggesting that chronic replication stress elicited by the BER defect may lead to a chronic activation of the DNA damage response. Hence, our data reinforce the concept that non-synonymous APE1 variants present in the human population may act as cancer susceptibility alleles.

Mammalian APE1 controls miRNA processing and its interactome is linked to cancer RNA metabolism

Mammalian apurinic/apyrimidinic endonuclease 1 is a DNA repair enzyme involved in genome stability and expression of genes involved in oxidative stress responses, tumor progression and chemoresistance. However, the molecular mechanisms underlying the role of apurinic/apyrimidinic endonuclease 1 in these processes are still unclear. Recent findings point to a novel role of apurinic/apyrimidinic endonuclease 1 in RNA metabolism. Through the characterization of the interactomes of apurinic/apyrimidinic endonuclease 1 with RNA and other proteins, we demonstrate here a role for apurinic/apyrimidinic endonuclease 1 in pri-miRNA processing and stability via association with the DROSHA-processing complex during genotoxic stress. We also show that endonuclease activity of apurinic/apyrimidinic endonuclease 1 is required for the processing of miR-221/222 in regulating expression of the tumor suppressor PTEN. Analysis of a cohort of different cancers supports the relevance of our findings for tumor biology. We also show that apurinic/apyrimidinic endonuclease 1 participates in RNA-interactomes and protein-interactomes involved in cancer development, thus indicating an unsuspected post-transcriptional effect on cancer genes.APE1 plays an important role in the cellular response to oxidative stress, and mutations are linked to tumor progression and chemoresistance. Here, the authors characterize the interactions of APE1 with RNA and demonstrate a role in microRNA processing.

Abstract 3337: Modulating the APE1/NPM1 interaction in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA repair pathways protect the genome from endogenous and environmental DNA damage. Such defensive mechanisms, however, enable tumor cells to survive DNA damage caused by chemo- and radio-therapy. Moreover, alterations of DNA repair pathways that may occur during tumorigenesis can make cancer cells reliant on a subset of repair enzymes for survival. Hence, knowledge of the mechanisms involved in the control of DNA repair proteins is of great interest for cancer diagnosis and treatment. The Apurinic/apyrimidinc endonuclease 1 (APE1) is an essential protein in eukaryotic cells. As the main AP-endonuclease in mammals, this protein is central to the base excision DNA repair pathway (BER). Beside its role in the maintenance of genomic stability, APE1 modulates gene expression by tuning the redox status of several transcription factors. Moreover, recent reports suggest that APE1 may take part in RNA processing and degradation, thus controlling the transcriptional output of the cell. Overexpression and aberrant localization of APE1 are recurrent phenotypes in tumors. Both situations are prognostic indicators of aggressiveness and onset of resistance. Interestingly, downregulation or inhibition of APE1 sensitizes cells to pharmacologically relevant gentoxins. We identified and characterized the molecular association between APE1 and Nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability, rRNA maturation and chromatin remodeling. The association with NPM1 modulates subcellular localization and endonuclease activity of APE1. Accordingly, NPM1−/− cells show lower BER capacity, and expression of an APE1 mutant unable to interact with NPM1 severely impairs cell proliferation. We recently reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, along with a poorer prognosis for patients having higher NPM1 levels. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Targeting the APE1/NPM1 interaction therefore represents a novel strategy for the development of anticancer drugs, as tumor cells which rely on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than normal cells. We exploited the AlphaScreen technology to screen for small molecules that act as inhibitors of the APE1/NPM1 interaction. Our approach detected a set of compounds able to interfere with the APE1/NPM1 association in vivo. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to genotoxins. To the best of our knowledge, this is the first attempt to target the APE1 interactome. Given the prognostic significance of the APE1 and NPM1 overexpression, these compounds might prove particularly effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas. Citation Format: Mattia Poletto, Dorjbal Dorjsuren, Lisa Lirussi, Carlo Vascotto, Pasqualina L. Scognamiglio, Daniela Marasco, Ajit Jadhav, David J. Maloney, Anton Simeonov, David M. Wilson, Gianluca Tell. Modulating the APE1/NPM1 interaction in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3337. doi:10.1158/1538-7445.AM2013-3337