Lay Summary

Mitochondria are subcellular, double-membrane organelles. From a functional point of view they can be considered as hubs where cellular energy metabolism and cell death signalling converge and are finely regulated. Functional mitochondria are essential for cancer cells.

Many of the agents with anti-cancer activity that act on Mitochondria (Mi), hold a substantial promise to be developed into efficient anti-cancer drugs, based on their selectivity for cancer cells. Despite the central role of Mi in human health and disease, there has been no successful therapy for defective mitochondrial function, which is devastating and often fatal. At the end of 2015, there were more than 200 trials on ClinicalTrials.gov that used "mitochondria" as key word. In this context, this proposal aims to provide a novel approach targeting Mi in cancer, making available new strategies to target drug-resistant and aggressive cancers such as Neuroblastoma (NB). Furthermore, we aim to identify novel mitochondrial biomarkers to be used as companion diagnostics, to predict clinical outcomes, as well as the patient response to therapy. This should allow a more successful and personalized approach to cancer diagnosis and therapy.

NB is the most common extracranial solid tumour in childhood, with a poor prognosis in advanced stages. NB causes 15% of childhood cancer-related mortality and overall survival rate for metastatic tumours is considerably low, 40% after 5 years. Most incidences are diagnosed during the first year of life, which also gives a better prospect for the outcome, whereas older patients have a poorer prognosis. In some NB cases, spontaneous regression has also been detected; however, underlying mechanisms remain unclear. Moreover, NB is a complex disease that has high genetic, biological, clinical, and morphological heterogeneity, and is therefore difficult to target for successful therapy. NBs require intact mitochondrial apoptosis for chemotherapy-induced cell death to occur. Evasion of apoptosis contributes to its aggressive phenotype and patients frequently succumb to chemoresistant disease. 

Understanding the molecular events that regulate mitochondrial apoptosis induced by cytotoxic therapies and how neuroblastoma cells evade apoptotic events may provide a new paradigm for NB therapy. Thus, novel strategies targeting resistance of neuroblastoma cells will be based on insights into the molecular mechanisms of apoptosis as well as other forms of cell death.

Executive Summary

1. Scientific Merit

Acquired resistance to apoptosis is one of the hallmarks of cancer. Thus, a thorough understanding of apoptotic signalling pathways and insights into apoptosis resistance mechanisms are imperative to identify novel drug targets for the design of more effective and target-selective therapeutic strategies. The intrinsic, or mitochondrial, pathway of apoptosis is an evolutionarily conserved and highly regulated form of cell death (CD) critical for development and homeostasis of multicellular organisms. Mitochondria (Mi) are essential intracellular organelles fundamentally implicated in cancer biology, including initiation, growth, metastasis, relapse, and acquired drug resistance [1].

The proposed project aims to provide a novel approach targeting Mi in cancer, by elucidating at molecular level a novel CD signalling pathway and by evaluating its implication in cancer therapeutics. The novel mitochondrial pathway (MiPa) has been first described at the Telethon Institute of Genetics and Medicine (TIGEM; www.tigem.it) (Host Institution) by the group of Prof. Franco (PI), who showed that genetic alterations in Holocytochrome c-type synthase (HCCS), a component of the mitochondrial respiratory chain (MRC), cause apoptosis [2]. They also provided evidence that mutations in HCCS induce activation of a non-canonical Mi apoptosis, triggered by: a) activation of a Mi-resident CASP9, independently from Apoptosome formation; b) MRC impairment and c) ROS overproduction [2]. Preliminary experiments performed by the applicant’s team, also provided evidence on the role of the new MiPa in cancer (Carotenuto P, Franco B, in preparation; see preliminary data reported in following sections). The hypothesis underlying this project is that molecular elucidation of the novel MiPa could provide directions to understand chemo-resistance, identify novel therapies and key targets to surpass or supplement current NB treatments. The novel strategy proposed by this project will be investigated in neuroblastome (NB), exploiting the clinical relevance of the preliminary data obtained, with particular attention to resistance to common therapeutic approaches. NB is mainly characterized by poor prognosis, high-mortality rate, multi-drug resistance, limited therapeutic options with lack of safe and efficacious treatments [3-5]. Understanding the molecular events that regulate apoptosis induced by cytotoxic therapies and how NB cells evade apoptotic events may provide a new paradigm for NB therapy.

2. Feasibility

Preliminary data described in the research design section, strongly support the feasibility of the project; the role of the novel MiPa in cancer has started to be elucidated and targets identified using the proposed approach. In addition, 2 hit compounds have already been identified and validated in in vitro and in vivo cancer models. The specific aims we propose to achieve our objective build up on robust preliminary data generated in our laboratory that demonstrate the validity of our hypothesis. In addition, our preliminary work also demonstrates that the research group has all the necessary expertise and technology to timely achieve the proposed tasks. In addition, recently at TIGEM, an important role for TFEB and MiT-TFE genes in tumour growth has been discovered [6] showing a considerable expertise of the Institute in cell metabolism and autophagy and a cancer-related environment that will facilitate the proposed research.

The feasibility will be also assured by the available core facilities. The TIGEM institute comprises 18 independent research groups with 5000 square meters converted in laboratories and research services. TIGEM’s research portfolio spans three strategic programs: Cell Biology of Genetic Diseases, Genomics Medicine, and Molecular Therapy. Research activity at TIGEM is supported by core facilities (http://www.tigem.it/core-facilities) that provide at TIGEM state-of-the-art technology as well as “house-keeping” assistance. Each core is supervised by a TIGEM investigator and is operated by specialized technical staff. Cores offer high-quality and rapid scientific and technical services that help to improve and speed up the work of TIGEM investigators. Cores facilities that will be critical to achieve the research goals of the proposed project include the High content Screening facility (HCS), the Next generation sequencing (NGS) Facility, Bioinformatics and Advanced microscopy and Imaging facility.

- The HCS facility makes use of two automated confocal microscopes (OPERA and Operetta systems from PerkinElmer), robotic liquid handling stations (Hamilton STARLet and PerkinElmer Zephir) to manipulate compound libraries, 96- and 384-well plate washers and dispensers, and dedicated cell culture room.

- The NGS facility (http://www.tigem.it/core-facilities/next-generation-sequencing-facility) performs in-house whole-exome sequencing (WES), RNA sequencing and preferential exome sequencing.

- Advanced Microscopy and Imaging (http://www.tigem.it/core-facilities/advanced-microscopy-and-imaging). Provides technical assistance, expertise, and qualitative and quantitative evaluation of microscopy data. The Core is composed of Light Microscopy (LM) and Electron Microscopy (EM) divisions.

3. Expertise

The network of scientific collaborations, developed by the applicant’s group with international teams of excellence, guarantees the best available expertise to carry out the programmed activities within the scheduled timeline. Brunella Franco, the PI is a human geneticist with a strong track record (130 publications in international peer-reviewed journals) in functional characterization of genes responsible for genetic disorders. The PI has formed numerous PhD students and Postdocs that have then obtained faculty appointments in internationally recognized research institutes. Dr. Carotenuto is the Co-investigator; he joined TIGEM one year ago after 4 years’ experience at The Institute of Cancer Research, London, UK. In the group of Dr. Braconi and subsequently under the supervision of Paul Workman, he produced several publications in top-journals. As showed in the references list of this application [7-11], his research work led to the identification of novel biomarkers in PAN-BTC cancers. Dr. Carotenuto provided significant scientific and technical support for projects involving molecular (CRISPR, RNAi, library screening, PCR, WB, protein Assays, etc...) and cell biology techniques (from 2D cell, 3D cell, organoids culture, primary cell culture and co-culture to cell functional assays; from flow cytometry and FACS to microscopy and high content imaging techniques). Dr Carotenuto will be an important asset for this ambitious project considering his long experience in operating high-throughput screening genomic facilities combined with experience in cancer genomics and with the expertise and research facilities of the hosting institution. Recently Dr Carotenuto was awarded a Marie Skłodowska-Curie Research Fellow reintegration grant co-funded by Horizon 2020 and AIRC. The proposed project will be developed with solid and lasting partnerships with International Cancer Clinical Centres (Dr Mirco Ponzoni, Gaslini Institute, Genova, IT, see collaboration letter). Specifically, Dr. Mirco Ponzoni, the scientific director of Experimental Therapies Laboratory in Oncology of Gaslini Institute in Genova, contributed to several publications in high impact journals obtaining results as following: Study of the "evasion" mechanisms used by cancer cells to escape the control of the immune system and the development of new immunotherapy strategies capable of circumventing the stratagems put in place by the tumour; development of biologically and clinically relevant animal models, called patient derived xenografts (PDX), for the study of experimental cancer therapies; development of NB 3D models in order to reduce the use of animals in preclinical testing, in accordance with the 3R principle (reduce, refine, replace). The research activity of the Experimental Therapies Laboratory in Oncology is mainly focused on neuroblastoma (NB) and other neuroectodermal tumours. Neuroblastoma studies absorb more than two thirds of the laboratory's personal and economic resources. This concentration of forces on this tumour is justified by the fact that Gaslini is the national Reference Center for the treatment of the NB and at the Institute there is an optimal situation for research (integrated bank of tissue and biological material, database, etc. ...).

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest