TRIB2 confers resistance to MAPK and TOR1 inhibitors

Silva, Neuton Pedro Gorjão da

http://hdl.handle.net/10400.1/9833

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Authors

Silva, Neuton Pedro Gorjão da

Advisor(s)

Link, Wolfgang

Hill, Richard

Abstract(s)

Malignant melanoma is the deadliest form of skin cancer that leads to a median survival time of only 6 to 9 months. Besides the patients in early stages that could be successfully treated by surgery alone, actually, there is no efficient diagnostic procedure. It is imperative to find alternatives and better methods of diagnosis for metastatic melanoma. Acquisition of resistance to chemotherapy agents, namely cytotoxic drugs such as dacarbazine, remains a major problem in melanoma therapy. Since TRIB2 (tribbles2) protein has recently been implicated as a biomarker for melanoma, and as a mediator in the process of resistance to conventional chemotherapeutics or PI3K (Phosphoinositide 3-kinase) inhibitors, we hypothesised that resistance to the inhibition of different components of this and other signalling pathways such as mTOR (mammalian target of rapamycin) and MEK (MAPK/ERK Kinase) is mediated by TRIB2. In this study, we examined the impact of ectopic expression of TRIB2 in the presence of mTOR or MEK inhibitors. We demonstrated that in the presence of rapamycin, a potent inhibitor of mTORC1 (mammalian target of rapamycin complex 1 or mechanistic target of rapamycin complex 1), TRIB2 confers resistance to this kind of treatment. However, when we exposed cells to a mTOR2 inhibitor, TORIN1, resistance is not observed. We demonstrated that TRIB2 acts as an adaptor, recruits the mTOR complex 1 that phosphorylates AKT (also known as Protein Kinase B, PKB) on residue Serine 473. Furthermore, we investigated the effect of TRIB2 expression, when cells where exposed to pharmaceutical inhibition of MEK. We found that TRIB2 expression protects cells against the effect of the MEK inhibitor BAY 766. We have seen that TRIB2 and MEK co-localize, interact and form a protein-protein complex. We also observed that the inhibition of MEK/ERK and PI3K pathways regulated by TRIB2, represses FOXO3 (Forkhead box O3a) a binding to p27 promoter, a well-known and studied transcription factor. On the other hand, high levels of TRIB2 leads to an increase of NF-κB (factor nuclear K B) affinity for promotor. NF-κB enhances the transcription of different genes crucial for melanoma cells proliferation and survival. This study suggests that TRIB2 is a meaningful component of a drug resistance mechanism in melanoma cells. This molecular process determines and modulates important cell decisions which include cell growth, proliferation and survival.