Agliarulo, Ilenia (2016) Role of TRAP1 in stress adaptive response of cancer cells. [Tesi di dottorato]

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Quantitative and/or qualitative protein synthesis deregulation is a necessary event to realize and support malignant transformation. There are several molecular mechanisms that define the total amount of protein expression and assure cell homeostasis, such as signaling pathways regulation and a network of ribosome-bound chaperones that is involved in a protein quality control exerted on nascent chains. Starting from this background our research focuses on TRAP1, a member of the heat shock protein 90 family, and on his role in the regulation of protein synthesis and quality control. It has been previously demonstrated that TRAP1 interacts with a proteasomal subunit on the outer side of endoplasmic reticulum, and it has been found associated to ribosomes and to initiation/elongation translational factors. Then, this interaction with both machineries allows TRAP1 to modulate the expression of two mitochondrial proteins through a co-translational ubiquitination/degradation. Indeed, these two TRAP1 substrates are more ubiquitinated and less expressed in cancer cells upon TRAP1 silencing. Aims of PhD thesis: 1. to characterize the role of TRAP1 in protein synthesis regulation; 2. to identify molecular pathways through which TRAP1 performs this translational modulation in cancer cells; 3. to study the effects of TRAP1 translational regulation on migratory behavior. Firstly, we confirmed the association of TRAP1 to the translational machinery, since we found that the chaperone associates to polysomes. Moreover, an increase of the total amount of active polysomes upon TRAP1 silencing and the in vitro translational assays evidenced that TRAP1 is involved in protein synthesis process. Furthermore, we demonstrated that cap-dependent protein synthesis is decreased in presence of TRAP1, whereas the IRES-mediated one is enhanced. This attenuation of cap-dependent translation is achieved by TRAP1 through and indirect modulation of the expression/activity of two PI3K pathway members, AKT and p70S6K. Indeed, these two kinases result less expressed and less phosphorylated in TRAP1 expressing cells compared to TRAP1-knock down cells. A very interesting finding is that TRAP1-knock down cells are addicted to translation and to the AKT/p70S6K axis also for other biological processes. Actually, we found that in basal condition TRAP1 silenced cells are faster than control cells in migration assays, whereas treatments with translational drugs and a p70S6K inhibitor are able to abrogate this faster migratory behavior, with scarce effects on control cells. Moreover, we excluded that the observed differences are due to a cytoskeleton reorganization and epithelial marker downregulation. The involvement of TRAP1 in migration regulation was supported by a gene expression analysis performed in colorectal cancer cells stably interfered for TRAP1, where pro-motility genes were found regulated. Finally, as supported by preliminary data/observations, TRAP1 could be involved also in mitochondrial protein synthesis regulation, where it attenuates translation elongation inhibiting the release of the mitochondrial elongation factor from the ribosome.

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