Nuno Bernardes

Engineering of bacterial strains and their products for cancer therapy

The use of live bacteria in cancer therapies offers exciting possibilities. Nowadays, an increasing number of genetically engineered bacteria are emerging in the field, with applications both in therapy and diagnosis. In parallel, purified bacterial products are also gaining relevance as new classes of bioactive products to treat and prevent cancer growth and metastasis. In the first part of the article, we review the latest findings regarding the use of live bacteria and products as anti-cancer agents, paying special attention to immunotoxins, proteins, and peptides. In particular, we focus on the recent results of using azurin or its derived peptide as anticancer therapeutic agents. In the second part, we discuss the challenges of using metagenomic techniques as a distinctive approach for discovering new anti-cancer agents from bacterial origin.

Microbial-based therapy of cancer: Current progress and future prospects

The use of bacteria in the regression of certain forms of cancer has been recognised for more than a century. Much effort, therefore, has been spent over the years in developing wild-type or modified bacterial strains to treat cancer. However, their use at the dose required for therapeutic efficacy has always been associated with toxicity problems and other deleterious effects. Recently, the old idea of using bacteria in the treatment of cancer has attracted considerable interest and new genetically engineered attenuated strains as well as microbial compounds that might have specific anticancer activity without side effects are being evaluated for their ability to act as new anticancer agents. This involves the use of attenuated bacterial strains and expressing foreign genes that encode the ability to convert non-toxic prodrugs to cytotoxic drugs. Novel strategies also include the use of bacterial products such as proteins, enzymes, immunotoxins and secondary metabolites, which specifically target cancer cells and cause tumor regression through growth inhibition, cell cycle arrest or apoptosis induction. In this review we describe the current knowledge and discuss the future directions regarding the use of bacteria or their products, in cancer therapy.

Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.

Recent patents on live bacteria and their products as potential anticancer agents.

This review intends to provide a comprehensive coverage of the various patents, published or issued, since 2007 on live or attenuated bacteria as potential anticancer agents, as well as microbial products including toxins, enzymes, antibiotics, various proteins and peptides as well as other small molecular weight products. Below is a list of such published/issued patents and a summary of the main contents of many such patents.

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased β1 integrin-mediated adhesion

ABSTRACT In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β1 in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young s module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response

Bacterial proteins and peptides in cancer therapy: today and tomorrow.

Cancer is one of the most deadly diseases worldwide. In the last three decades many efforts have been made focused on understanding how cancer grows and responds to drugs. The dominant drug-development paradigm has been the “one drug, one target.” Based on that, the two main targeted therapies developed to combat cancer include the use of tyrosine kinase inhibitors and monoclonal antibodies. Development of drug resistance and side effects represent the major limiting factors for their use in cancer treatment. Nowadays, a new paradigm for cancer drug discovery is emerging wherein multi-targeted approaches gain ground in cancer therapy. Therefore, to overcome resistance to therapy, it is clear that a new generation of drugs is urgently needed. Here, regarding the concept of multi-targeted therapy, we discuss the challenges of using bacterial proteins and peptides as a new generation of effective anti-cancer drugs.

Azurin interaction with the lipid raft components ganglioside GM-1 and caveolin-1 increases membrane fluidity and sensitivity to anti-cancer drugs

ABSTRACT Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts’ markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.

Bacterial protein azurin as a new candidate drug to treat untreatable breast cancers

Azurin is a blue copper protein produced by Pseudomonas aeruginosa. Azurin enters preferentially into cancer cells and forms a complex with p53, stabilizing it and inducing apoptosis. Azurin allows in vivo regression of human melanoma and breast cancer without toxic effects to the animal. Its immunoglobulin-like fold and a large exposed hydrophobic patch confer on it the property of a natural scaffold. This has already been proven for the receptor tyrosine kinase EphB2, since azurin prevents its binding to the ligand ephrinB2. We intend to extend the azurin interaction to cell adhesion molecules (CAMs), particularly P-cadherin. P-cadherin is frequently overexpressed in invasive breast carcinomas and it is a poor prognosis marker. Protein-protein docking results have validated this approach confirming an interaction between both proteins. Thus, natural azurin or derived proteins or peptides could be scaffolds against these proteins in breast cancer cells models, being an interesting new therapeutic tool.