Felicitas Rataj
A novel multi-target cancer therapy based on destabilization of short-lived mRNAs
Published on 12 December 2014
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Thesis presented December 12, 2014
Abstract :
One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms.
In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the
N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated
N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9).
In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells.
In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9- ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies.
Keywords:
Multi-target therapy, tumor angiogenesis, TIS11b (ZFP36L1/BRF1), ARE-mediated mRNA decay, phosphorylation, protein engineering
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