The bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1) provides long-term survival in a murine glioma model

Vannini et al. BMC Cancer 2014, 14:449 http://www.biomedcentral.com/1471-2407/14/449 RESEARCH ARTICLE Open Access The bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1) provides long-term survival in a murine glioma model Eleonora Vannini1,2†, Anna Panighini1†, Chiara Cerri1, Alessia Fabbri3, Simonetta Lisi2, Enrico Pracucci2, Nicola Benedetto4, Riccardo Vannozzi4, Carla Fiorentini3, Matteo Caleo1*† and Mario Costa1† Abstract Background: Glioblastomas are largely unresponsive to all available treatments and there is therefore an urgent need for novel therapeutics. Here we have probed the antineoplastic effects of a bacterial protein toxin, the cytotoxic necrotizing factor 1 (CNF1), in the syngenic GL261 glioma cell model. CNF1 produces a long-lasting activation of Rho GTPases, with consequent blockade of cytodieresis in proliferating cells and promotion of neuron health and plasticity. Methods: We have tested the antiproliferative effects of CNF1 on GL261 cells and human glioma cells obtained from surgical specimens. For the in vivo experiments, we injected GL261 cells into the adult mouse visual cortex, and five days later we administered either a single intracerebral dose of CNF1 or vehicle. To compare CNF1 with a canonical antitumoral drug, we infused temozolomide (TMZ) via minipumps for 1 week in an additional animal group. Results: In culture, CNF1 was very effective in blocking proliferation of GL261 cells, leading them to multinucleation, senescence and death within 15 days. CNF1 had a similar cytotoxic effect in primary human glioma cells. CNF1 also inhibited motility of GL261 cells in a scratch-wound migration assay. Low dose (2 nM) CNF1 and continuous TMZ infusion significantly prolonged animal survival (median survival 35 days vs. 28 days in vehicle controls). Remarkably, increasing CNF1 concentration to 80 nM resulted in a dramatic enhancement of survival with no obvious toxicity. Indeed, 57% of the CNF1-treated animals survived up to 60 days following GL261 glioma cell transplant. Conclusions: The activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors. Keywords: Glioma, Mouse, Cerebral cortex, CNF1, Temozolomide Background Gliomas are primary central nervous system tumors that arise from astrocytes, oligodendrocytes or their precursors. Following the World Health Organization (WHO) classification, gliomas can be classified in 4 groups according to their histological characteristics and the most malignant form is glioblastoma multiforme (GBM). GBM is uniformly fatal and largely unresponsive to all available treatments. Despite intensive therapy including surgery, radiotherapy and chemotherapy, the average of survival of patients with glioblastoma usually is 15 months from the time of first * Correspondence: † Equal contributors 1 CNR Neuroscience Institute, Via Moruzzi 1, 56124 Pisa, Italy Full list of author information is available at the end of the article diagnosis [1,2]. Conventional surgical excision, generally limited to the main tumor mass, does not remove the microscopic foci of neoplastic cells that invade the surrounding normal brain substance beyond the main tumor mass, and that are responsible for the inevitable tumor recurrence. Radiotherapy and chemotherapy, often associated to surgery, cannot ablate completely these tumors, since this would require unacceptably high radiation/chemotherapic doses that result in severe brain-neuron damage. There is therefore a clear need to accelerate progress in the development of new strategies for treatment of glioma. Several therapeutic approaches for glioma are currently being investigated in animal models and patients. They include delivery of cytotoxic genes and proteins to glioma © 2014 Vannini et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vannini et al. BMC Cancer 2014, 14:449 http://www.biomedcentral.com/1471-2407/14/449 cells, suppression of angiogenesis, and immune stimulation [3]. Concerning chemotherapy, alkylating agents such as temozolomide (TMZ) are widely used in the treatment of brain tumours [4]. As a cytotoxic alkylating agent, TMZ is converted at physiologic pH to the short-lived active compound, monomethyl triazeno imidazole carboxamide (MTIC). The cytotoxicity of MTIC is primarily due to methylation of DNA at the O6 and N7 positions of guanine, resulting in inhibition of DNA replication. Chemotherapics have substantial side effects and limited efficacy, and this further underlies the need of innovative approaches for glioma treatment. In this paper we describe a potential novel therapy for glioma, based on intracerebral administration of cytotoxic necrotizing factor 1 (CNF1), a bacterial protein toxin produced by specific strains of Escherichia coli. CNF1 is a single-chain protein, consisting of a Nterminal domain involved in cell binding, a middle region mediating membrane translocation, and a Cterminal catalytic domain. The C-terminal part of CNF1 is released into the cytosol where it catalyzes the deamidation of a single glutamine residue of the Rho GTPases (RhoA, Rac1 and Cdc42). Rho GTPases are molecular switches that cycle between a GDP-bound inactive and a GTP-bound active state to control a multitude of cellular events, like actin cytoskeleton organization as well as gene transcription, cell proliferation, and survival [5]. Rho GTPases deamidated by CNF1 are not able to hydrolyse GTP and remain in a persistent activated state [6,7] which is followed by partial deactivation of these regulatory proteins via degradation by the ubiquitin–proteasome pathway [8]. The persistent activation of Rho GTPases by CNF1 causes a remarkable reorganization of the actin cytoskeleton with dramatic functional consequences. In particular, cultured proliferating cells exposed to CNF1 acquire a multinucleated phenotype (cytotoxic effect), due to stabilization of the actin network and prevention of cytodieresis despite ongoing nuclear division [9]. On the other hand, our recent studies have demonstrated “plasticizing” effects of CNF1 in neurons. Specifically, intracerebral administration of CNF1 improves neuronal function, learning and memory [10,11], and these effects are associated with a enhancement of brain plasticity, exemplified by the increase in spine density in cortical neurons [10]. In view of these striking, differential effects of CNF1 on proliferating cells and neurons, we have probed for the first time the potential antitumoral effects of this to (...truncated)