Lay Summary

Glioblastoma (GBM) is the most common malignant brain tumor, being highly invasive and not responsive to standard treatment (surgery, radiotherapy, and chemotherapy). Moreover, a major determinant of the high lethality of GBM is the presence of self-renewing, tumorigenic glioblastoma stem-like cells (GSCs) that threateningly support the development and the progressive growth of tumors.

Recent studies in vivo have demonstrated the anti-neoplastic effects of CNF1, a bacterial protein that specifically modulates regulatory proteins, thus affecting fundamental cellular processes. CNF1 is detrimental for GBM cells since it is able to block cell proliferation, leading to cell death, while displaying a beneficial activity on peritumoral neurons, enhancing their plasticity and maintaining their functional properties. However, it is unknown whether CNF1 could act on GSCs, and the mechanism exploited by CNF1 to act on GBM cells is also not well understood. To address this question, we will analyze the effect of CNF1 on different GSCs and GBM cell lines in the activity and the mechanism used to induce cytotoxicity, with the final goal of verifying if CNF1 can represent a potential new strategy against incurable brain tumors.

The proposed study could lead to the identification of a new potential successful strategy for the treatment of GBM. To date, GBM remains incurable, due to its heterogeneity and complex pathogenesis, and affected individuals have a very poor life expectancy, with a median survival of 15 months. In the present research, we will establish whether the bacterial protein CNF1 could act on GSCs and GBM cell lines, and, consequently, the mechanism underlying will be defined. Our hypothesis is that CNF1 acts on GSCs and, in general, on GBM via the rewiring of mitochondrial metabolism and ROS production. CNF1 is being increasingly recognized as a promising potential drug for a certain number of pathologic conditions of the central nervous system. In fact, it has been already proven that CNF1 acts on mitochondria, the powerhouse of the cell, and that it restores the correct behavior in a plethora of diseases in which mitochondria are affected. We have already reported that CNF1 is able to improve the behavioral phenotype and rescue cognitive deficit, in both Rett syndrome and Alzheimer’s disease mouse models, counteracting mitochondrial dysfunction and favoring cell energy restore. In addition, in a mouse model of epileptic seizures, where mitochondrial dysfunctions are considered a potential cause, CNF1 enhances brain energy and counteracts spontaneous epileptiform phenomena. Moreover, recent studies have demonstrated anti-neoplastic effects of CNF1 in a murine glioma model, blocking cell proliferation and leading to cell death, and significantly prolonging animal survival, with no obvious toxicity. Furthermore, CNF1 potently reduces tumor volume in mice, displaying a beneficial activity on peritumoral neurons, enhancing their plasticity and maintaining their functional properties. The maintenance of behavioral functions has been demonstrated after treatment with CNF1 at a symptomatic stage. These different results in vitro and in vivo point at mitochondria, stimulated by CNF1, as the key players. Therefore, we hypothesize that CNF1 acts on GSCs and, in general, on GBM via the rewiring of mitochondrial metabolism and ROS production.

We have identified three aims to be developed in 12 months.

• Aim 1 will be devoted to the characterization of the activity of CNF1 on human GSCs and GBM cells from a morphological and biochemical point of view.
• Aim 2 will be focused on the study of the impact of CNF1 on the metabolic activity of mitochondria, in human GSCs and GBM cells.
• Aim 3 will deal with the definition of the CNF1-engaged molecular pathways that link mitochondria to human GBM death.

Currently, finding a treatment to totally eradicate GBM is an important achievement for health care.  We are confident that to study the mechanisms by which CNF1 acts on GBM will help this goal achievement. Our studies will improve the knowledge of CNF1 activity on GBM and consequently, the scientific community will be provided with new tools to pursue the development of a novel potential drug for a so devastating disease. Indeed, a new therapeutic option in an oncological ambit should bring benefits to cancer patients and also economic output in terms of healthcare services.

Executive Summary

Gliomas are a diverse group of malignant primary neoplasms of the central nervous system (CNS), derived from glial cells, with a worldwide incidence of approximately 7 out of 100,000 individuals per year. According to the recent World Health Organization (WHO) classification, glioblastoma (GBM) is identified as WHO grade IV glioma and is the most common and aggressive malignant brain tumor, characterized by tissue ischemic necrosis, strong invasiveness, and microvascular proliferation. GBM is not responsive to standard treatment (surgery and combined radio- and chemotherapy), thus affected individuals having a very poor life expectancy. A major determinant of the high lethality of GBM is the presence of self-renewing, tumorigenic glioblastoma stem-like cells (GSCs) characterized by a relative quiescence, which renders them resistant to conventional therapies that target the proliferative population, supporting the development and progressive growth of tumors.

The cytotoxic necrotizing factor 1 (CNF1), a bacterial protein produced by some pathological strains of Escherichia coli, specifically modulates Rho GTPases, thus affecting fundamental cellular processes. CNF1, in fact, promotes new activities mainly linked to the actin cytoskeleton remodeling and to the activation of transcription factors. It has already proven that the protein causes changes in the mitochondrial architecture that mainly consists in the formation of a complex network of elongated mitochondria, and in the enhancement of mitochondrial activity that leads to an increase in cellular ATP content. Interestingly, the CNF1 activity on mitochondria allows CNF1 to restore the correct behavior in a plethora of diseases in which mitochondria are affected. In fact, CNF1 is able to improve the behavioral phenotype and rescue cognitive deficit in mouse models of both Rett syndrome and Alzheimer’s disease, where CNF1 is able to counteract mitochondrial dysfunction and favor cell energy restore. In addition, in a mouse model of epileptic seizures, where mitochondrial dysfunctions are considered a potential cause, CNF1 enhances brain energy and counteracts spontaneous epileptiform phenomena. Recent studies have demonstrated the anti-neoplastic effects of CNF1 in a mouse model of glioma. CNF1 is detrimental for GMB cells since it is able to block cytodieresis in proliferating cells, leading them to senescence and death, while displaying a beneficial activity on peritumoral neurons, enhancing their plasticity and maintaining their functional properties. These abilities point at CNF1 as a possible new strategy for the treatment of brain tumors.

However, it is unknown whether CNF1 could act on GSCs and also the mechanism exploited by CNF1 to act on GBM is not well understood. To address this question, we will analyze the effect of CNF1 on different cell lines of GBM and GSCs with the aim of studying the CNF1 activity and in particular, the mechanism used to induce cytotoxicity.

We hypothesize that CNF1 acts on GSCs and, in general, on GBM via the rewiring of mitochondrial metabolism and ROS production. We have already reported, in fact, that CNF1 acts on mitochondria and restores the correct behavior in a plethora of diseases in which mitochondria are affected.

Our studies will improve the knowledge of CNF1 activity on GBM, thus rendering CNF1 a potential and innovative tool against incurable brain tumors. Thus, to verify the hypothesis underlying this project, we have identified three aims to be exploited in 12 months:

•  Aim 1. Characterization of the activity of CNF1 on human GSCs and GBM cells, in term of cytotoxicity, Rho GTPases activation, cytoskeletal and mitochondrial modification;
•  Aim 2. Study of the impact of CNF1 on the metabolic activity in human GSCs and GBM cells, in term of the fundamental function of mitochondria, such as ATP production, oxygen consumption, extracellular acidification, and ROS production, to characterize the metabolic rewiring of CNF1 on GSCs and GBM cells;
• Aim 3. Definition of the CNF1-engaged molecular pathways that link mitochondria to human GBM senescence, by dissecting the mTOR and DNA damage response pathways that link mitochondria and senescence.

All three aims will be in charge of ISS – PI and Co-investigator.

We expect that CNF1 will exert its activity also on GSCs, causing an alteration in cell metabolism and ROS production in both human GSCs and GBM cells, ultimately leading to the death of cells.

Currently, finding a treatment to totally eradicate GBM could be an important achievement for health care. The proposed study, by establishing whether CNF1 could act on GSCs, will provide greater insight into the mechanism used by CNF1 to affect GBM cells, and the research findings could be a step forward the clinical use of CNF1 as an innovative treatment against a so devastating disease, with a great impact in reducing mortality in GBM.

The investigators and all the researchers involved in the study have the skills and competencies needed to meet the research goals, with long-lasting experience in the field of bacterial toxins, glioma, mitochondrial analyses as well as cellular and molecular pathways dissection.  

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest