Studies on cell growth promoting AKT signaling pathway – a promising anti-cancer drug target
Kuupäev
2016-07-25
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
AKT kinaas on seriin/treoniinkinaas, mis kuulub AGC valkude perekonda. Sihtmärkvalke fosforüleerides reguleerib AKT rakkudes mitmeid olulisi füsioloogilisi protsesse nagu rakkude elulemus, rakutsükli edenemine, metabolism, transkriptsioon, valkude süntees, rakkude liikuvus jne. Kontrollimatult aktiivset PI3K/AKT signaalirada on kirjeldatud inimese maksa-, aju-, rinna-, soole- ja eesnäärmekasvajates ning seda loetakse mõnede kasvajate puhul halva prognoosi markeriks. Käesoleva töö raames leidsime, et aktiveeritud AKT signaalirada osaleb ka healoomulise hüperproliferatiivse patoloogia – Dupuytreni kontraktuuri - arengus. AKT kinaasi olulisuse ja sagedase seotuse tõttu kasvajate tekkimisega, peetakse teda heaks märklauaks kasvajavastaste terapeutikumide väljatöötamisel. Praeguseks on avastatud ja kliinilistesse katsetustesse võetud juba mitmeid AKT signaaliraja inhibiitoreid, kuid paraku on tugevad mittespetsiifilised kõrvaltoimed ja toksilisus takistanud nende edasise kasutamise raviskeemides. Käesoleva doktoritöö põhieesmärk oli leida ja iseloomustada AKT signaaliraja aktiivsust pärssivaid väikesemolekulaarseid aineid, mida võiks kasutada potentsiaalsete kasvajavastaste ravimitena. Otsitavate inhibiitorite täpsem sihtmärk oli AKT1 ja PDPK1 valkude omavaheline interaktsioon, mis on vajalik AKT1 esmaseks aktiveerimiseks. Väikesemolekulaarsete ainete skriinimine valgu komplementaarsusel põhinevat lähenemisviisi kasutades ning sellele järgnenud erinevatest meetoditest koosnenud valideerimisprotsess tuvastas ühe kemikaali – NSC156529 - mis pidurdas erinevat päritolu kasvajarakkude paljunemist nii koekultuuri tingimustes kui in vivo hiire kasvajamudelis. Tuumorirakkude töötlemine NSC156529 kemikaaliga mõjutas lisaks AKT kinaasi enda aktiivsusele ka AKT-i märklaudvalkude aktiivsust, viidates sellele, et antud kemikaal mõjutab AKT signaaliraja poolt reguleeritud raku biokeemilisi protsesse. Kuna in vivo kasvajamudelitest eraldatud tuumorite analüüsimisel leiti NSC156529 kemikaaliga töödeldud kasvajates rakkude diferentseerumisele viitavate markerite suurenenud ekspressioon, järeldati, et lisaks suudab NSC156529 tuumorirakkude paljunemist pärssida neid diferentseeruma suunates. Käesoleva töö kokkuvõtteks võib öelda, et AKT kinaasiga seotud signaalirada on sobiv märklaud kasvajavastaste terapeutikumide väljatöötamiseks ning testitud ühend NSC156529 võiks olla potentsiaalne kandidaat AKT signaaliraja aktiivsuse pärssimiseks kasvajarakkudes.
AKT protein is a serine/threonine kinase, which belongs to AGC group of protein kinases. By substrate phosphorylation AKT regulates several physiological processes in the cells, such as cell survival, cell cycle promotion, metabolism, transcription, translation, cell migration etc. Aberrantly activated PI3K/AKT signaling has been frequently found in several human tumors, like liver, brain, breast, colorectal and prostate cancers, and it is a poor prognostic marker for a number of cancer types. In this work we found that the activated AKT signaling might play a role in the progression of benign hyperproliferative pathology – the Dupuytren’s contracture. Due to AKT involvement in critical steps of human tumor pathogenesis, targeting AKT pathway has become a promising strategy in anti-cancer therapy. Although a number of small molecule AKT kinase inhibitors have been developed and tested in clinical trials, severe side effects have prevented their use in current treatment schemes. The main purpose of the present thesis was to identify and characterize the inhibitors of AKT signaling pathway that could be used as potential anti-cancer drugs. The exact target of the inhibitors was the AKT1-PDPK1 interaction, which is the first step in AKT activation cascade. Protein complementation-based screening and the following experiments revealed one chemical – NSC156529 – which inhibited the growth of tumor cells from different origin in cell culture as well as in a mouse tumor xenograft model. In addition to AKT activity inhibition, cancer cell incubation with NSC156529 chemical also reduced the phosphorylation of AKT downstream target proteins, which confirmed that NSC156529 treatment inhibited the key biochemical activities of the AKT signaling pathway. The analysis of NSC156529-treated xenografts revealed increased expression level of differentiation markers, suggesting that NSC156529 could limit tumor growth at least in part by directing cancer cells to differentiate. Conclusively, inhibiting activated AKT-regulated signaling pathway in tumor cells is a promising anti-cancer drug target and the small molecular compound NSC156529 could be a potent suppressor of AKT1 pathway in tumor cells harboring active AKT signaling.
AKT protein is a serine/threonine kinase, which belongs to AGC group of protein kinases. By substrate phosphorylation AKT regulates several physiological processes in the cells, such as cell survival, cell cycle promotion, metabolism, transcription, translation, cell migration etc. Aberrantly activated PI3K/AKT signaling has been frequently found in several human tumors, like liver, brain, breast, colorectal and prostate cancers, and it is a poor prognostic marker for a number of cancer types. In this work we found that the activated AKT signaling might play a role in the progression of benign hyperproliferative pathology – the Dupuytren’s contracture. Due to AKT involvement in critical steps of human tumor pathogenesis, targeting AKT pathway has become a promising strategy in anti-cancer therapy. Although a number of small molecule AKT kinase inhibitors have been developed and tested in clinical trials, severe side effects have prevented their use in current treatment schemes. The main purpose of the present thesis was to identify and characterize the inhibitors of AKT signaling pathway that could be used as potential anti-cancer drugs. The exact target of the inhibitors was the AKT1-PDPK1 interaction, which is the first step in AKT activation cascade. Protein complementation-based screening and the following experiments revealed one chemical – NSC156529 – which inhibited the growth of tumor cells from different origin in cell culture as well as in a mouse tumor xenograft model. In addition to AKT activity inhibition, cancer cell incubation with NSC156529 chemical also reduced the phosphorylation of AKT downstream target proteins, which confirmed that NSC156529 treatment inhibited the key biochemical activities of the AKT signaling pathway. The analysis of NSC156529-treated xenografts revealed increased expression level of differentiation markers, suggesting that NSC156529 could limit tumor growth at least in part by directing cancer cells to differentiate. Conclusively, inhibiting activated AKT-regulated signaling pathway in tumor cells is a promising anti-cancer drug target and the small molecular compound NSC156529 could be a potent suppressor of AKT1 pathway in tumor cells harboring active AKT signaling.
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