Biochemistry and Molecular Biology (BMB)
The research of the team consists of two major lines, aimed respectively at metabolic and toxicity studies, mainly addressed to the sterol biosynthetic pathway (coordinator G. Balliano) and tumor angiogenesis (coordinator E. Giraudo).
Metabolic (sterol biosyntetic pathway) and toxicity studies:
the research is focused on the enzymes of the post-squalene steps of sterol biosynthesis in different organism (plants, yeasts and mammals). Our academic environment (a Drug Science and Technology Department) led us to steadily pursue the connections between biochemical and pharmaceutical features of sterol biosynthesis, often developing effective and specific inhibitors to target the post-squalenic enzymes [1-3]. Recently, we have discovered, in the yeast Saccharomyces cerevisiae, the first chaperonine-like interaction between enzymes of sterol biosynthesis. This interaction occurs between oxidosqualene cyclase and sterol-3-ketoreductase (Erg27p), one of the proteins of the C-4 sterol demethylase complex (C-4DC): in ERG27-deficient yeast strains, oxidosqualene cyclase is completely inactive .
Presently, we are studying the metabolites that accumulate in post-squalene cholesterol disorders and the effects of the alteration of cholesterol biosynthesis on some signalling cascades (such as Hedgehog proteins cascade) in order to find new druggable targets for antitumoral theraphy. These interests led us to start collaboration with the following international groups:
- Dr. Matti Poutanen (Turku Center for Disease Modelling, Turku, Finland)
- Dr. Gail Herman (Herman Lab. Center for Human and Molecular Genetics, the Research Institute at Nationwide Children's Hospital, Columbus, USA)
- Dr. Donald Poirier (Department of Molecular Medicine, Research Center and Laval University, Quebec City, Canada)
The toxicity studies concern cellular, protein and DNA damage caused by new nanomaterial (nanosized mesoporous silica, carbon and titania-based nanoparticles) designed for nanomedicine or other industrial applications .
- tracer experiments with radiolabelled intermediates (often obtained by biological synthesis) to assay either a single step or a more complex biosynthetic sequence
- inhibition of enzymatic activity with specifically-designed molecules
- use of knock-out organisms
- site-directed mutagenesis
- expression of mutated proteins
- cell culture
- authorized radioisotopes laboratory equipped for synthesis and
experiments with labelled substrates in animals and cell culture
(liquid scintillator, radioscanner, safety hood)
- authorized MOGM laboratory
- laboratory equipped for protein and DNA technology
(electrophoresis, PCR) and cell culture
The aim of this second arm of the projects is to discover new targets and molecular mechanisms for anti-angiogenic therapies.
Angiogenesis is required for invasive tumor growth and metastasis . Despite the development of innovative anti-angiogenic strategies, resistance to these treatments have been recently observed in both preclinical and clinical trials and there is a need to identify new angiogenic modulators and mechanisms.
We have demonstrated that Semaphorin 3A, a member of axon guidance cues that regulate both nervous system and blood vessel functions , was an endogenous angiogenesis inhibitor lost during tumor progression in transgenic mouse models of tumorigenesis. Re-expression of Sema3As in tumors, using adeno-associated virus (AAV)-8 delivery system inhibited angiogenesis, cancer growth, normalized tumor vessels, restored tumor normoxia, with the final result of overcoming the resistance induced by angiogenesis inhibition [8,9].
The current and future project of the group are the following:
- Design, synthesis, and employment in mouse model of cancer Sema3A super-agonist recombinant mutants as new “tumor vessel normalizing agents” able to inhibit tumor angiogenesis and to halt metastasis formation
- Identification of the mechanisms by which Semaphorins, their receptors and other axon guidance cues regulate “stroma” normalization, tumor invasiveness and metastases formation
- Evaluation of the role of the oxidosqualene cyclase (OSC), key enzyme regulating the synthesis of 2,3-oxidosqualene in the post-squalenic part of sterol synthesis, and of other related products, as a new regulators of tumor angiogenesis and metastasis formations in different mouse models of cancer
- Study of the role of specific microRNA in regulating metastasis formations and invasion in different mouse models of cancer
Collaborations with international groups:
- Prof. Doug Hanahan (ISREC and EPFL, Lausanne, Switzerland)
- Dr. Oriol Casanovas (Catalan Institute of Oncology, ICO, Barcelona, Spain)
- Dr. Sara Zanivan (Beatson Institute, Glasgow University, UK)
Technologies used ( present at IRCC in Candiolo -TO):
a) animal models:
- transgenic mouse model of spontaneous pancreatic (RipTag2), cervical (K14-HPV16/E2) and skin (K14-HPV16) carcinogenesis
- orthotopic mouse models of pancreatic ductal adenocarcinoma (PDAC) and of breast tumor (4T1)
- xenograft mouse models of PDAC and colon carcinogenesis (HPAF-II and HCT-116 respectively)
- In vivo gene delivery using adeno-associated virus (AAV) and retrovirus
- confocal microscope analysis and video-lapse microscopy for in vivo imaging
- In vivo imaging using luciferase, fluorescence and IVIS technology
Facilities (present at IRCC in Candiolo -TO):
- The Genomic Facility equipped with Agilent Bioanalyzer (Agilent Technologies), HTP-realtime PCR station (Applera 7900 HT) and DNA microarray platform (Illumina Beadstation), DNA Microarrays Illumina platform
- The Cell Separation facility is equipped with MoFlo cell sorter and two FACS analyzers
- The Animal Facility may host up to 7500 mice, transgenic and nude mice, and includes a BSL3 Lab and a breed section
Sterol biosynthesis, Oxidosqualene synthase, Enzyme inhibition, Recombinant protein, Site-directed mutagenesis, Anticancer drugs, Cholesterol disorders, Anti-angiogenic therapy, Transgenic mouse models of cancer, Tumor microenvironment, Inflammatory cells, Semaphorins, MicroRNAs, Tumor invasiveness, Metastatic dissemination
1. Oliaro-Bosso S., Viola F., Taramino S., Tagliapietra S., Barge A., Cravotto G. and Balliano G. Inhibitory Effect of Umbelliferone Aminoalkyl Derivatives on Oxidosqualene Cyclases fromSaccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii, Homo sapiens and Arabidopsis thaliana: a structure-activity study. Chem. Med. Chem. 2, (2007),226-233.
2. Balliano G., Dehmlow H., Oliaro-Bosso S., Scaldaferri M.,Taramino S.,Viola F., Caron G., Aebi J., Ackermann J. Oxidosqualene cyclase from Saccharomyces cerevisiae, Trypanosoma cruzi,Pneumocystis carinii and Arabidopsis thaliana expressed in yeast: A model for the development of novel antiparasitic agents. Bioorg. Med. Chem. Lett.19(2009), 718–723.
3. S. Oliaro-Bosso, G. Caron, S. Taramino, G. Ermondi, F. Viola, G. Balliano. Characterization of the channel constriction allowing the access of the substrate to the active site of yeast oxidosqualene cyclase. PLoS ONE (2011), 6(7): e22134. doi:10.1371/journal.pone.0022134.
4. C. Mo, P. Milla, K. Athenstaedt, R. Ott, G. Balliano, G. Daum, and M. Bard. In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity.Biochim. Biophys. Acta 1633 (2003), 68-74.
5. M. Ghiazza, E. Carella, S. Oliaro-Bosso, I. Corazzari, F. Viola, I. Fenoglio. (2013) Predictive tests to evaluate oxidative potential of engineered nanomaterials. Nanosafe 2012: International Conferences on Safe Production and Use of Nanomaterials, Journal of Physics: Conference Series 429, 012024-012032.
6. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 144 (2011), 646-74.
7. Serini G, Bussolino F, Maione F, Giraudo E. Class 3 semaphorins: physiological vascular normalizing agents for anti-cancer therapy. J Intern Med. 273 (2013), 138-55.
8. Maione F, Molla F, Meda C, Latini R, Zentilin L, Giacca M, Seano G, Serini G, Bussolino F, Giraudo E. Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models. J Clin Invest. 119 (2009), 3356-72.
9. Maione F, Capano S, Regano D, Zentilin L, Giacca M, Casanovas O, Bussolino F, Serini G, Giraudo E. Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages. J Clin Invest. 122 (2012), 1832-48.