Microbial-based therapy of cancer
Team: Arsénio M. Fialho, Nuno Bernardes
Several microorganisms have been used aiming the development of novel therapies for treating cancer. Despite encouraging results, a major disadvantage of their use has been the undesired infections caused by the bacteria. In order to overcome this drawback, novel approaches for anticancer therapies include the use of purified products from microbial origin such as proteins, enzymes, immunotoxins, antibiotics and other secondary metabolites. These approaches have been proved to be effective in anticancer therapy causing tumor regression through growth inhibition, cell cycle arrest or apoptosis induction.
Azurin is a low molecular weight (14kDa), water-solube protein produced by the bacterium Pseudomonas aeruginosa. Besides its well documented function as a redox partner in electron transfer reactions, azurin has been found to have cytotoxicity activity towards human cancer lines in vitro and in vivo. It has been demonstrated that azurin can enter preferentially into cancer cells and forms a complex with the tumor suppressor p53, stabilizing it and inducing apoptosis. A chemically-synthesized 28-amino acid peptide derived from azurin (p28) has been approved by the FDA and entered Phase I clinical trials as an anticancer agent.
Azurin has structural similarity with variable domains of immunoglobulins, thereby clearly demonstrating its single antibody-like structure. Interestingly, a zurin has a particular ability to mediate high-affinity interactions with unrelated proteins relevant in cancer, conferring on it the property of a natural scaffold for therapeutic purposes. This effect has been already proven for the receptor tyrosine kinase EphB2-mediated cell signaling, since azurin, due to its interaction with this receptor, prevents its binding to the ligand ephrinB2.
This line of research is being pursued in collaboration with Dr Joana Paredes and Dr Raquel Seruca, IPATIMUP.
1. Protein-protein interaction studies between azurin and members of the Cadherin superfamily (E-cadherin and P-cadherin)
We have demonstrated by docking analysis that the family of cadherins are good candidate targets to azurin binding. Surface Plasmon Resonance (SPR) experiments (in collaboration with Dr Cecília Arraiano, ITQB) have validated this approach. Therefore, we hypothesized that azurin could be a scaffold against P-cadherin in breast cancer cells (or even against its soluble forms), antagonizing its invasive effects and contributing to the inhibition of tumor progression.
2. In vitro cellular effects induced by azurin in P-cadherin overexpressing breast cancer cells
In vitro , azurin treatments of breast cancer cell models expressing different levels of P-cadherin caused a specific decrease of this protein shown by western blot analysis, whereas levels of E-cadherin remain unaltered. Additionally, the soluble form of P-cadherin was reduced after azurin treatment. These results were further confirmed by immunofluoresce assays. Moreover, Matrigel invasion assays were performed to test whether azurin can affect the invasiveness of P-cadherin overexpressing breast cancer cells. The results obtained demonstrate that azurin can significantly reduce the invasive phenotype of the cells. Studies are in progress aiming to elucidate the molecular mechanism(s) by which azurin interferes with P-cadherin in breast cancer cells.
3. Design and use of azurin's phage display libraries: optimizing the affinity and specificity of azurin to P-cadherin
A further development in the concept of "scaffold" implies the generation of azurin variants displaying sub-nanomolar affinities to pre-defined targets relevant in cancers. An ongoing strategy involves the construction of azurin's phage display libraries that can contain billions of unique azurin-engineered variants sequences.. Expression of the libraries through display technologies will allow the screening and selection of those with higher affinity than wild-type azurin to our target molecule (P-cadherin). This study will allow the determination of the inhibitory effect of azurin as a new anti-cancer drug to treat untreatable breast carcinomas that overexpress P-cadherin.
- Bernardes N., Ribeiro A. S., Abreu S., Vieira A.F., Carreto L., Santos M., Seruca R., Paredes J., Fialho A.M. “High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin”, International Journal of Biochemistry & Cell Biology, 50:1-9, 2014.
- Bernardes N., Ribeiro A.S., Abreu S., Mota B., Matos R.G., Arraiano C.M., Seruca R., Paredes J., Fialho A.M. “The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models” PLoS One, 8 (7), e69023, 2013.
- Bernardes N., Chakrabarty A.M., Fialho A.M. “Engineering of bacterial strains and their products for cancer therapy” Applied Microbiology and Biotechnology 97(12):5189-5199, 2013.
- Fialho, A.M., Chakrabarty, A.M. (editors), "Emerging Cancer Therapy: Microbial Approaches and Biotechnological tools" John Wiley & Sons , p. 432, 2010.
- Bernardes, N., Seruca, R., Chakrabarty, A.M., Fialho, A.M., "Microbial-based therapy of cancer: current progress and future prospects"Bioengineered Bugs , 3(1): 178-190, 2010.
- Fialho, A.M., Stevens, F.J., das Gupta, T.K., Chakrabarty, A.M., "Beyond host-pathogen interactions: microbial defense strategy in the host environmental", Current Opinion in Biotechnology , 18, 279-286, 2007.
- Chakrabarty, A.M., das Gupta, T.K., Yamada, T., Fialho, A.M., "Cupredoxin derived transport agents and methods of use thereof" US Patent 7,691,383 B", April 2010.