Disfunzioni mitocondriali nella crescita tumorale, farmaco-resistenza e neurotossicità dei chemioterapici.

Responsabili di progetto

Sergio Giannattasio, Vladimir Todorovic

Accordo

MONTENEGRO - MoS-not in force - Ministero della Scienza del Montenegro

Bando

CNR-MoS 2017-2018

Dipartimento

Scienze biomediche

Area tematica

Scienze biomediche

Stato del progetto

Nuovo

Proposta di ricerca

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome (mtDNA) can contribute to development of chemoresistance in malignant tumors. Germline mutations in BRCA2 are associated with increased susceptibility to breast and ovarian cancer in women and prostate cancer (PC) in men. Loss of BRCA2 culminates in defective DNA repair and genome instability, which when prolonged can lead to accumulation of a lethal amount of damaged DNA ultimately resulting in cancer. Recent studies have shown a potential role of BRCA2 in modulating apoptosis mechanisms, but how this occurs is still controversial and has yet to be clarified.
There are similarities between glucose-induced repression of oxidative metabolism of yeast and metabolic reprogramming of tumor cells. The glucose-induced repression of oxidative metabolism is regulated by oncogene homologues in yeast, such as RAS and Sch9p, the yeast homologue of Akt. Yeast also undergoes an apoptosis-like programmed cell death process sharing several features with mammalian apoptosis, including oxidative stress and mitochondrial dysfunction. Evasion of apoptosis and sustained proliferative signaling are hallmarks of cancer. The similarities between tumor and yeast cells, together with the possibility of heterologous expression of human genes in yeast, make yeast a valuable model organism in cancer research, allowing for new insights into the function of mammalian oncogenes/oncosuppressors [Guaragnella et al. FEMS Yeast Res, 2014; Guaragnella et al. Biochem. Pharmacol., 2014]. In our laboratory at the Institute of Biomembranes and Bioenergetics of CNR (IBBE-CNR) a successful example of complementation between human and yeast cell models has recently revealed a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily conserved molecular mechanism involving regulation of ROS production and/or detoxification by BRCA2 during PCD processes [Guaragnella et al. Apoptosis, 2014]. In addition, the finding that mtDNA depletion activates a mitochondrial retrograde signaling pathway, conserved in mammals and yeast, that promotes reduction of BRCA2 protein levels has direct clinical implications because it makes tumors featuring mtDNA large deletions or depletion, such as prostate carcinomas [Arbini et al. Oncogenesis, 2013], promising candidates for tailored and personalized therapeutic regimens encompassing PARP inhibitors. In this project we will give insights into the role of mitochondrial dysfunction in BRCA2-driven cancer using both human prostate cancer cell lines and yeast cells expressing or non-expressing the oncosuppressor BRCA2. In collaboration with the Faculty of Medicine of the University of Montenegro we shall extend our investigations on mitochondrial dysfunction in another BRCA2-dependent tumor, breast cancer. We will perform NGS to select breast tumor tissues having BRCA2 mutations and to investigate whether pathogenic BRCA2 mutations are implicated in the regulation mtDNA mutation status and/or depletion.
The molecular mechanism involved in mtDNA-mediated drug resistance is not well understood. Prostate cancers in patients with a mutation in BRCA2 have earlier disease onset and an aggressive course, often necessitating the use of systemic therapy (Vesprini et al. Can Urol Assoc J 2011). The chemotherapeutic drugs approved for the treatment of castration-resistant PC include docetaxel and cabazitaxel, antineoplastic agents belonging to the taxane class. The first taxane developed in 1970s, paclitaxel, is approved for treatment of refractory ovarian cancer. Paclitaxel interacts with tubulin causing a mitotic block in cells by stabilizing microtubules, thereby decreasing their dynamic nature (Jordan, Curr Med Chem Anti-Cancer Agents 2002). 6-thioguanine (6-TG) is a chemotherapeutic drug that induces DNA damage and is used for treatment of acute leukemias and chronic myeloid leukemia (Elgemeie, Curr. Pharm. Des. 2003). However, its potential role as chemotherapeutic drug in prostate cancer as well as in other solid tumors has not been studied. To this aim yeast is a suitable model organism for a pre-clinical study on the anticancer activity of such compounds. Indeed, although purified yeast tubulin does not interact with paclitaxel, a paclitaxel-sensitive S. cerevisiae yeast strain has been constructed and it has been shown that paclitaxel treatment in this strain causes mitotic block and apoptotic-like cell death (Foland et al. Yeast 2005). In the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, a second aim of this project will be the analysis of the effects of paclitaxel and 6-TG or its analogues, alone and in combination, as for their toxicity in yeast cells expressing or not BRCA2 protein to identify new molecule/s to be tested in humane prostate cell lines alone, and in combination with paclitaxel. In addition, the effect of BRCA2 expression in conferring sensitivity to 6-TG and its analogs will be assessed. In collaboration with the University of Montenegro, we will make use of yeast as a model to study the role of mitochondrial dysfunction in paclitaxel toxicity and its related neurotoxicity. A key point of this project will be scientific and technological know-how transfer between the IBBE-CNR and the University of Montenegro, setting up the basis for future collaborations for the preparation of research project proposals.

Obiettivi della ricerca

1. Analysis of mitochondrial dysfunction through mtDNA characterization, analysis of mitochondrial respiration and structure (mitochondrial fission/fusion), reactive oxygen species accumulation, cytochrome c release, retrograde-target gene expression analysis, in human cancer cells and in BRCA2-expressing yeast cells;
2. Characterization of mitochondrial dysfunction and mitochondrial retrograde pathway activation in paclitaxel-treated human cancer cell lines and BRCA2-expressing yeast cells;
3. Functional genomics and NGS technology transfer from Italy to Montenegro.

Ultimo aggiornamento: 14/12/2024