The role of protein ITIH5 in resistance to vemurafenib targeted therapy, proliferation and activity of major signaling pathways in human melanoma cell lines

Vemurafenib je selektivni inhibitor mutiranog proteina BRAF V600E koji se javlja u 60% melanoma. Unatoč početnim obećavajućim rezultatima, ubrzo nakon liječenja vemurafenibom dolazi do pojave stečene otpornosti stanica melanoma na ovaj lijek. Stečena otpornost često je posljedica reaktivacije signalnog puta MAPK (engl. mitogen activated protein kinase) ili aktivacije alternativnog signalnog puta PI3K/AKT (engl. phosphoinositide3-kinase/Akt). U mnogim je tumorima, uključujući melanom, uočena smanjena ekspresija gena ITIH5. Unatoč tome, njegova uloga u stečenoj otpornosti stanica melanoma na vemurafenib još nije istražena. Cilj ovog diplomskog rada bio je istražiti učinak utišavanja ovoga gena na otpornost na vemurafenib, na signalne puteve MAPK i PI3K/AKT te na proliferaciju staničnih linija melanoma čovjeka WM793B i A375M. Utišavanje gena ITIH5 provedeno je prolaznom transfekcijom stanica malim interferirajućim RNA koje ciljaju ITIH5 koristeći kalcijev fosfat. Provjera ekspresije i uspješnosti utišavanja provedena je kvantitativnom polimeraznom lančanom reakcijom (qPCR). Nakon utišavanja ITIH5 provjerena je vijabilnost i proliferacija stanica metodom MTT te razina biljega aktivnosti istraživanih signalnih puteva i proliferacije metodom Western blot. Navedene su analize pokazale da tretman vemurafenibom znatno snižava ekspresiju gena ITIH5 te da snižena ekspresija ITIH5 povećava proliferaciju u navedenim staničnim linijama melanoma čovjeka. Vemurafenib is a selective inhibitor of the mutated protein BRAF V600E, which occurs in 60% of melanomas. Despite initial promising results, shortly after treatment with vemurafenib, acquired resistance of melanoma cells develops. Acquired resistance is often the result of the reactivation of the MAPK (mitogen activated protein kinase) signaling pathway or the activation of the alternative PI3K/AKT (phosphoinositide-3-kinase/Akt) signaling pathway. Decreased expression of the ITIH5 gene has been observed in many tumors, including melanoma. However, its role in the acquired resistance of melanoma cells to vemurafenib has not yet been investigated. The aim of this thesis was to investigate the effect of ITIH5 silencing on the resistance to vemurafenib, on the activity of MAPK and PI3K/AKT signaling pathways, and on the proliferation of human melanoma cell lines WM793B and A375M. ITIH5 silencing was performed by transfecting cells with small interfering RNA targeting ITIH5 using calcium phosphate. Verification of expression and success of silencing was performed by quantitative polymerase chain reaction (qPCR). After the silencing of ITIH5, cell viability and proliferation were checked using the MTT test, while the levels of specific markers that represent the activity of signaling pathways or the proliferation was checked using the Western blot method. The results showed that the reduced expression of the ITIH5 increases the proliferation of the human melanoma cell lines and is a consequence of vemurafenib treatment.