Group Ferrari

DNA DAMAGE AND THE CELL CYCLE


Introduction

The increased survival of cancer patients treated with aggressive radio- or chemotherapy comes at a price, with secondary leukemia being one of the most common causes of death. In recent years, the rational design of molecules targeting oncogenic pathways that are hyper-functional in cancer cells or to which cancer cells become addicted has provided an alternative and promising approach to the use of “dirty” drugs. The established adaptability of cancer cells to external insults, however, constantly demands the implementation of novel strategies. Basic research aims to satisfy this demand through the continuous discovery of novel targets.

On one front, we are investigating the cellular response to genotoxic stress, to elucidate the impact of DNA damage checkpoint activation on the onset of mitosis.

On another front, we are engaged in the identification and characterization of small molecule inhibitors of mitotic ATPases and dual-specificity phosphatases.

Mitotic ATPases and genome stability

Inappropriate detection or untimely repair of DNA damage before the onset of mitosis may lead to chromosome breaks as well as rearrangements or fusions – comprehensively know as “structural abnormalities” - that lead to the development of cancer and have been focus of intense research in the last decades. On the other hand, loss or gain of entire chromosomes – known as “numerical abnormalities” - is a characteristic of tumors described more than a century ago and named “aneuploidy”. The most common causes of aneuploidy are: (i) defective attachment of sister chromatids by spindle microtubules (merotelic attachment) in pro-metaphase and metaphase, often linked to centrosome amplification, (ii) malfunction of the spindle assembly checkpoint; (iii) defects in chromosome cohesion.

RUVBL1 and RUVBL2 are essential genes belonging to the AAA+ superfamily (ATPases Associated with various cellular Activities). The two polypeptides form homo- and hetero-hexamers and as such are part of large multiprotein complexes involved in transcription, chromatin remodeling and DNA mismatch repair. Interestingly, expression of both RUVBL1 and RUVBL2 is upregulated in cancer.

In this study we addressed the mitotic role of RUVBL1. We observed that at anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, partially co-localizing with polo-like kinase 1 (PLK1). Interestingly, RUVBL1 and RUVBL2 separate during cytokinesis, with only RUVBL1 remaining co-localized with PLK1. PLK1 is able to phosphorylate RUVBL1 - but not RUVBL2 - at a single site (T239). This, along with the evidence of physical association between RUVBL1 and PLK1 in vivo,suggests that the latter may differentially regulate the function of the RuvB-like proteins during mitosis. Regarding the function of RUVBL1/2 in mitosis, we observed that depletion of RUVBL1 or RUVBL2 expression by RNAi causes extension of metaphase and retardation of the anaphase onset, with polar chromosomes failing to align at the metaphase plate and ultimately lagging at anaphase. Finally, we could show that the ATPase activity of RUVBL1 is indispensable for cell proliferation.

Current studies aim at elucidating the role of RUVBL1 in the molecular mechanism that facilitates polar chromosome congression at metaphase and the identification of RUVBL1 small molecule inhibitors.

Figure 1. RUVBL1 depletion affects the length of mitosis and results in lagging chromosomes.
RNAi-mediated knock-down studies followed by confocal live imaging of HeLa cells. Stills of control (Luc) or RUVBL1 siRNA-treated cells are shown. Lagging chromosomes are indicated with arrowheads. DNA is shown in cyan and α-tubulin in red.

Control of Exo1 by phosphorylation

Double-strand breaks (DSBs) are among the most cytotoxic DNA lesions generated by ionizing radiation (IR), certain chemotherapeutic drugs, collapse of stalled DNA replication forks or during physiological processes such as meiotic recombination. DSBs repair occurs through error-prone (Non-Homologous-End-Joining) or error-free (Homologous-Directed-Repair) pathways. Rapid and reversible control of pathways components is exerted through post-translational modifications (PTMs). To shed light on the role of PTMs that control DNA end-resection guiding error-free repair of DSBs, we focused on a key factor of HDR pathways, the 5’-to-3’ exonuclease-1 (EXO1). EXO1 is a Rad2 family DNA repair nuclease originally identified in S. pombe and shown to participate in several post-replication DNA repair pathways including mismatch repair, DSBs repair, meiotic and mitotic recombination. Additionally, yeast Exo1 was shown to localize at stalled replication forks where it counteracts fork reversal.

Studies that we conducted in the past showed that human EXO1 is regulated by sumoylation, which likely facilitates its recruitment at stalled DNA replication forks where it undergoes phosphorylation-dependent poly-ubiquitylation, which results in its proteasome-mediated degradation, thus avoiding pathological resection of DNA. At DSBs, human EXO1 functionally interacts with CtIP/RBBP8 that modulates both EXO1 recruitment at DNA ends and its resection activity. On the other hand, we showed that yeast Exo1 is regulated through interaction with 14-3-3 proteins and others provided evidence for inhibition of Exo1 activity by phosphorylation.

In this study we identified kinase and phosphatase controlling Exo1 phosphorylation upon stalled DNA replication as well as the entire complement of checkpoint-dependent phosphorylation sites in Exo1. Functional studies conducted in exo1Δ sgs1Δ strains and addressing the role of phosphorylation on Exo1 resection activity at DSBs are ongoing.

 

Figure 2. Phosphorylation-dependent control of yeast Exo1 at stalled replication forks.
Upon stalled DNA replication, Exo1 is phosphorylated in a checkpoint-dependent manner at five major sites. During checkpoint recovery, Exo1 dephosphorylation is likely controlled by the protein phosphatases Glc7 and Pph3.

CDC25 inhibitors

The network of signals originating at sites of damage and triggering cell cycle checkpoints involves the action of protein kinases and protein phosphatases. The protein phosphatase CDC25 is an oncogene overexpressed in a number of cancers that controls activation of Cyclin-Dependent Kinases (CDKs) at key cell cycle transitions. Aim of this study is the identification and characterization of small molecule CDC25 inhibitors. To this end, we first identified a common pharmacophore (i.e., a set of structural features necessary for molecular recognition of a ligand by a macromolecule) through in silico fragmentation of all known CDC25 inhibitors. Next, we conducted a similarity search of a molecule databases (>2000 structures) with the obtained fragments and finally we performed molecular docking of selected scaffolds into the catalytic pocket of CDC25, the crystal structure of which is available. Enzymatic assays followed by cellular studies led to the identification of 8 scaffolds that are undergoing modeling-guided synthesis to provide second-generation compounds that will be profiled in enzymatic assays and tested in 2D and 3D cell culture systems for their efficacy as CDC25 inhibitors.


Figure 3. Control of cell cycle transitions.
The point of action of different Cyclin-CDK complexes during transition through the cell cycle and the network of positive and negative inputs are shown.

Photo-activatable metal-based complexes and cancer therapy

Photodynamic therapy (PDT) is an attractive alternative to chemotherapy. Among the different photosensitizers (PSs) employed, Ru(II) polypyridyl complexes were found to be valid substitutes to porphyrin-based or phtalocyanine-based PSs. In this study we obtained evidence that one such complex, namely [Ru(bipy)2-dppz-7-methoxy][PF6]2 (Ru65), localized in the nucleus of various cancer and normal cells and displayed cytotoxicity only upon UV-A irradiation. Importantly, we demonstrated that Ru65 intercalated in DNA and, upon light irradiation, promoted guanine oxidation, resulting in nicks in the double helix. We confirmed this mechanism of action in living cells showing that UV-A irradiation of cells loaded with Ru65 resulted in a transient DNA damage response. Strikingly, photo-irradiation of Ru65 triggered cell death upon resolution of DNA damage and with distinct mechanisms in interphase or mitotic cells. Interphase cells underwent cell cycle arrest at the G2/M phase and massive cytoplasmic vacuolation, which was paralleled by an unfolded-protein stress-response, resulting in reduction of viability and cell death through a paraptosis-like mechanism. On the other hand, UV-A irradiation of Ru65 in G2/M synchronized cells did not allow completion of mitosis and rapid cell death through classic apoptotic pathways ensued. Importantly, targeting mitotic cells with Ru65 allowed to significantly increase its photo-toxicity. Overall, our findings allow to draw two important conclusions: (i) PSs targeting the nucleus and causing a transient DNA damage response upon photoactivation represent an excellent alternative to current chemotherapeutics; (ii) combination of cell cycle inhibitors and PSs appears to be an even better alternative to the use of PSs alone for an effective clearance of cancer cells.

Figure 4. Dual mode of cell death upon photo-irradiation of Ru65 in interphase and mitosis.
Scheme of the different cell death modes triggered by UV-A irradiation of Ru65 in interphase or at mitosis.

Publications

Anstaett, Philipp; Pierroz, Vanessa; Ferrari, Stefano; Gasser, Gilles (2015). Two-photon uncageable enzyme inhibitors bearing targeting vectors. Photochemical & Photobiological Sciences, 14(10):1821-1825.

Bologna, Serena; Altmannova, Veronika; Valtorta, Emanuele; Koenig, Christiane; Liberali, Prisca; Gentili, Christian; Anrather, Dorothea; Ammerer, Gustav; Pelkmans, Lucas; Krejci, Lumir; Ferrari, Stefano (2015). Sumoylation regulates EXO1 stability and processing of DNA damage. Cell Cycle, 14(15):2439-2450.

Gentili, Christian; Castor, Dennis; Kaden, Svenja; Lauterbach, David; Gysi, Mario; Steigemann, Patrick; Gerlich, Daniel W; Jiricny, Josef; Ferrari, Stefano (2015). Chromosome Missegregation Associated with RUVBL1 Deficiency. PLoS ONE, 10(7):e0133576.

Mari, Cristina; Pierroz, Vanessa; Ferrari, Stefano; Gasser, Gilles (2015). Combination of Ru(II) Complexes and Light: New Frontiers in Cancer Therapy. Chemical Science, 6:2660-2686.

Leonidova, Anna; Anstaett, Philipp; Pierroz, Vanessa; Spingler, Bernhard; Ferrari, Stefano; Gasser, Gilles (2015). Induction of Cytotoxicity through Photo-release of Aminoferrocene. Inorganic Chemistry, 54:9740-9748.

Mari, Cristina; Pierroz, Vanessa; Leonidova, Anna; Ferrari, Stefano; Gasser, Gilles (2015). Towards Selective Light Activated Ru(II)-based Prodrug Candidates. European Journal of Inorganic Chemistry, 23:3879-3891.

Mari, Cristina; Pierroz, Vanessa; Rubbiani, Riccardo; Patra, Malay; Hess, Jeannine; Spingler, Bernhard; Oehninger, Luciano; Schur, Julia; Ott, Ingo; Salassa, Luca; Ferrari, Stefano; Gasser, Gilles (2014). DNA Intercalating Ru(II) Polypyridyl Complexes as Effective Photosensitizers in Photodynamic Therapy. Chemistry - A European Journal, 20(44):14421-14436.

Leonidova, Anna; Pierroz, Vanessa; Adams, Luke A; Barlow, Nicholas; Ferrari, Stefano; Graham, Bim; Gasser, Gilles (2014). Enhanced Cytotoxicity through Conjugation of a "Clickable" Luminescent Re(I) Complex to a Cell-Penetrating Lipopeptide. ACS Medicinal Chemistry Letters, 5(7):809-814.

Joshi, Tanmaya; Pierroz, Vanessa; Ferrari, Stefano; Gasser, Gilles (2014). Bis(dipyridophenazine)(2-(2'-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II) hexafluorophosphate: a lesson in stubbornness. ChemMedChem, 9(7):1419-1427.

Gianferrara, Teresa; Spagnul, Cinzia; Alberto, Roger; Gasser, Gilles; Ferrari, Stefano; Pierroz, Vanessa; Bergamo, Alberta; Alessio, Enzo (2014). Towards matched pairs of porphyrin-Re(I) /(99m) Tc(I) conjugates that combine photodynamic activity with fluorescence and radio imaging. ChemMedChem, 9(6):1231-1237.

Hess, Jeannine; Konatschnig, Sandro; Morard, Sandra; Pierroz, Vanessa; Ferrari, Stefano; Spingler, Bernhard; Gasser, Gilles (2014). Novel, mercury-free synthetic pathway for trifluoromethylthio-substituted metallocenes. Inorganic Chemistry, 53(7):3662-3667.

Kitanovic, Igor; Can, Suzan; Alborzinia, Hamed; Kitanovic, Ana; Pierroz, Vanessa; Leonidova, Anna; Pinto, Antonio; Spingler, Bernhard; Ferrari, Stefano; Molteni, Roberto; Steffen, Andreas; Metzler-Nolte, Nils; Wölfl, Stefan; Gasser, Gilles (2014). A deadly organometallic luminescent probe: anticancer activity of a ReI bisquinoline complex. Chemistry - A European Journal, 20(9):2496-2507.

Hess, Jeannine; Konatschnig, Sandro; Morard, Sanda; Pierroz, Vanessa; Ferrari, Stefano; Spingler, Bernhard; Gasser, Gilles (2014). A Novel, Mercury-Free Synthetic Pathway for Trifluoromethylthio-Subsituted Metallocenes. Inorganic Chemistry, 53(7):3662-3667.

Joshi, Tanmaya; Pierroz, Vanessa; Mari, Cristina; Gemperle, Lea; Ferrari, Stefano; Gasser, Gilles (2014). A bis(dipyridophenazine)(2-(2-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II) complex with anticancer action upon photodeprotection. Angewandte Chemie (International ed. in English), 53(11):2960-2963.

Leonidova, Anna; Pierroz, Vanessa; Rubbiani, Riccardo; Lan, Yanjun; Schmitz, Anita G; Kaech, Andres; Sigel, Roland K O; Ferrari, Stefano; Gasser, Gilles (2014). Photo-Induced Uncaging of a Specific Re(I) Organometallic Complex in Living Cells. Chemical Science, 5:4044-4056.

Leonidova, Anna; Pierroz, Vanessa; Rubbiani, Riccardo; Heier, Jakob; Ferrari, Stefano; Gasser, Gilles (2014). Towards cancer cell-specific phototoxic organometallic rhenium(I) complexes. Dalton Transactions, 43(11):4287-4294.

Gianferrara, Teresa; Spagnul, Cinzia; Alberto, Roger; Gasser, Gilles; Ferrari, Stefano; Pierroz, Vanessa; Bergamo, Alberta; Alessio, Enzo (2014). Towards matched Pairs of Porphyrin-Re(I)/99mTc(I) Conjugates that combine Photodynamic Activity with Fluorescence- and Radio-Imaging. ChemMedChem, 9(6):1231-1237.

Patra, Malay; Joshi, Tanmaya; Pierroz, Vanessa; Ingram, Katrin; Kaiser, Marcel; Ferrari, Stefano; Spingler, Bernhard; Keiser, Jennifer; Gasser, Gilles (2013). DMSO-Mediated ligand dissociation: renaissance for biological activity of n-heterocyclic-[Ru(η(6) -arene)Cl2 ] Drug Candidates. Chemistry - A European Journal:14768-14772.

Patra, Malay; Ingram, Katrin; Leonidova, Anna; Pierroz, Vanessa; Ferrari, Stefano; Robertson, Murray N; Todd, Matthew H; Keiser, Jennifer; Gasser, Gilles (2013). In vitro metabolic profile and in vivo antischistosomal activity studies of (η(6)-Praziquantel)Cr(CO)3 derivatives. Journal of Medicinal Chemistry, 56(22):9192-9198.

Bologna, Serena; Ferrari, Stefano (2013). It takes two to tango: Ubiquitin and SUMO in the DNA damage response. Frontiers in Genetics, 4:106.

Stölting, Meline N L; Ferrari, Stefano; Handschin, Christoph; Becskei, Attila; Provenzano, Maurizio; Sulser, Tullio; Eberli, Daniel (2013). Myoblasts inhibit prostate cancer growth by paracrine secretion of TNF alpha. Journal of Urology, 189(5):1952-1959.

Spagnul, Cinzia; Alberto, Roger; Gasser, Gilles; Ferrari, Stefano; Pierroz, Vanessa; Bergamo, Alberta; Gianferrara, Teresa; Alessio, Enzo (2013). Novel water-soluble Tc-99m(I)/Re(I)-porphyrin conjugates as potential multimodal agents for molecular imaging. Journal of Inorganic Biochemistry, 122:57-65.

Patra, Malay; Ingram, Katrin; Pierroz, Vanessa; Ferrari, Stefano; Spingler, Bernhard; Gasser, Robin B; Keiser, Jennifer; Gasser, Gilles (2013). [(η(6)-Praziquantel)Cr(CO)3] derivatives with remarkable in vitro anti-schistosomal activity. Chemistry - A European Journal, 19(7):2232-2235.

Andersen, S D; Keijzers, G; Rampakakis, E; Engels, K; Luhn, P; El-Shemerly, M; Nielsen, F C; Du, Y; May, A; Bohr, V A; Ferrari, S; Zannis-Hadjopoulos, M; Fu, H; Rasmussen, L J (2012). 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif. DNA Repair, 11(3):267-277.

Patra, Malay; Ingram, Katrin; Pierroz, Vanessa; Ferrari, Stefano; Spingler, Bernhard; Keiser, Jennifer; Gasser, Gilles (2012). Ferrocenyl Derivatives of the Anthelmintic Praziquantel: Design, Synthesis, and Biological Evaluation. Journal of Medicinal Chemistry, 55(20):8790-8798.

Pierroz, Vanessa; Joshi, Tanmaya; Leonidova, Anna; Mari, Cristina; Schur, Julia; Ott, Ingo; Spiccia, Leone; Ferrari, Stefano; Gasser, Gilles (2012). Molecular and cellular characterization of the biological effects of ruthenium(II) complexes incorporating 2-Pyridyl-2-Pyrimidine-4-Carboxylic acid. Journal of the American Chemical Society, 134(50):20376-20387.

Engels, K; Giannattasio, M; Muzi-Falconi, M; Lopes, M; Ferrari, S (2011). 14-3-3 proteins regulate exonuclease 1-dependent processing of stalled replication forks. PLoS Genetics, 7(4):e1001367.

Ruiz-Sánchez, P; König, C J; Ferrari, S; Alberto, R (2011). Vitamin B₁₂ as a carrier for targeted platinum delivery: in vitro cytotoxicity and mechanistic studies. Journal of Biological Inorganic Chemistry, 16(1):33-44.

Eid, W; Steger, M; El-Shemerly, M; Ferretti, L P; Pena-Diaz, J; König, C J; Valtorta, E; Sartori, A A; Ferrari, S (2010). DNA end resection by CtIP and exonuclease 1 prevents genomic instability. EMBO Reports, 11(12):962-968.

Bhatia, P; Menigatti, M; Brocard, M; Morley, S J; Ferrari, S (2010). Mitotic DNA damage targets the Aurora A/TPX2 complex. Cell Cycle, 9(22):4592-4599.

Ferrari, S. Regulation of cell cycle transitions. 2010, University of Zurich, Faculty of Science.

El-Shemerly, M; Hess, D; Pyakurel, A K; Moselhy, S; Ferrari, S (2008). ATR-dependent pathways control hEXO1 stability in response to stalled forks. Nucleic Acids Research, 36(2):511-519.

Pani, E; Menigatti, M; Schubert, S; Hess, D; Gerrits, B; Klempnauer, K H; Ferrari, S (2008). Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity. Biochimica et Biophysica Acta, 1783(6):1121-1128.

Bathia, P; Ferrari, S (2008). Role of cell cycle controllers in the development of cancer. In: Mondello, C. Multiple Pathways in Cancer Development. Kerala, India: Transworld Research Network, 69-86.

Pani, E; Ferrari, S (2008). p38MAPK delta controls c-Myb degradation in response to stress. Blood Cells Molecules and Diseases, 40(3):388-394.

Pani, E; Stojic, L; El-Shemerly, M; Jiricny, J; Ferrari, S (2007). Mismatch repair status and the response of human cells to cisplatin. Cell Cycle, 6(14):1796-1802.

Krystyniak, A; Garcia-Echeverria, C; Prigent, C; Ferrari, S (2006). Inhibition of Aurora A in response to DNA damage. Oncogene, 25(3):338-348.

Ferrari, S (2006). Protein kinases controlling the onset of mitosis. Cellular and Molecular Life Sciences, 63(7-8):781-795.

Ferrari, S; Marin, O; Pagano, M A; Meggio, F; Hess, D; El-Shemerly, M; Krystyniak, A; Pinna, L A (2005). Aurora-A site specificity: a study with synthetic peptide substrates. Biochemical Journal, 390(1):293-302.

El-Shemerly, M; Janscak, P; Hess, D; Jiricny, J; Ferrari, S (2005). Degradation of human exonuclease 1b upon DNA synthesis inhibition. Cancer Research, 65(9):3604-3609.

Mueller, A; Schäfer, B W; Ferrari, S; Weibel, M; Makek, M; Höchli, M; Heizmann, C W (2005). The calcium-binding protein S100A2 interacts with p53 and modulates its transcriptional activity. Journal of Biological Chemistry, 280(32):29186-29193.

Stojic, L; Mojas, N; Cejka, P; Di Pietro, M; Ferrari, S; Marra, G; Jiricny, J (2004). Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase. Genes and Development, 18(11):1331-1344.

Metzger, E; Müller, J M; Ferrari, S; Buettner, R; Schüle, R (2003). A novel inducible transactivation domain in the androgen receptor: implications for PRK in prostate cancer. EMBO Journal, 22(2):270-280.

Hill, M M; Andjelkovic, M; Brazil, D P; Ferrari, S; Fabbro, D; Hemmings, B A (2001). Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase. Journal of Biological Chemistry, 276(28):25643-25646.

Charrasse, S; Carena, I; Brondani, V; Klempnauer, K H; Ferrari, S (2000). Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway. Oncogene, 19(26):2986-2995.