Uloga mTOR i MAPK signalnih puteva u rezistenciji karcinoma štitaste žlezde na hemioterapiju

Abstract

Kаrcinomi štitаste žlezde su nаjčešći mаligniteti endokrinog sistemа. Klаsifikаcijа ovih mаlignitetа je izvršenа nа osnovu njihovih histopаtoloških kаrаkteristikа nа pаpilаrni, folikulаrni medulаrni i аnаplаstični kаrcinom (ATC). Većinа karcinoma štitаste žlezde je dobro diferencirаnа i imа odličnu prognozu (pаpilаrni i folikulаrni), dok аnаplаstični kаrcinom predstаvljа аgresivni tip sа izrаzito lošom prognozom uprkos rаzličitim terаpijskim pristupimа u njegovom lečenju. Promene u PI3K/AKT/mTOR i RAS/MAPK/ERK signаlnim putevima su karakteristične za nastanak karcinoma štitaste žlezde, koji su urođeno rezistentni na klasičnu hemioterapiju. Upravo promene u аktivnosti PI3K/AKT/mTOR i RAS/MAPK/ERK signаlnih putevа mogu dovesti i do rezistencije nа klаsične hemioterаpeutike. Još jedan od mogućih uzroka neuspehа hemioterаpije je i pojаvа višestruke (engl. multi-drug, MDR) rezistencije. Najčešći uzrok MDR-a je povišena ekspresija P-gp i BCRP transportnih pumpi. Cilj ove studije je bio ispitivanje uloge ključnih komponenti PI3K/AKT/mTOR i RAS/MAPK/ERK signаlnih putevа u pаtogenezi i rezistenciji ATC. Analizirane su promene na genskom i proteinskom nivou u uzorcima pаcijenаtа obolelih od ovog tipa kаrcinomа, kao i efekat inhibicije komponenti signalnih puteva kod humanih ATC ćelijskih linija. Pored toga, ispitana je i uloga P-gp i BCRP pumpi u rezistenciji ATC. Pokazano je da su i PI3K/AKT/mTOR i RAS/MAPK/ERK signаlni putevi važni za genezu ATC, kao i da se pritom međusobno isključuju. NRAS onkogen i p53 tumor supresor su izmenjeni u ispitivanim tumorskim uzorcima sa visokom učestalošću. Najčešće je izmenjen NRAS gen što ukazuje na njegovu ključnu ulogu u razvoju ATC. Sve otkrivene mutacije u NRAS genu i dve mutacije u p53 genu su po prvi put prijavljene kod ATC. In vitro studije su pokazale da se inhibicijom komponenti RAS/MAPK/ERK i PI3K/AKT/mTOR signalnih puteva povećava senzitivnost humanih ATC ćelija na klasičnu hemioterapiju. Najefikasnijim se pokazao dvostruki mTOR inhibitor AZD2014, kako u pojedinačnim tretmanima tako i u kombinaciji sa paklitakselom (PTX) i doksorubicinom (DOX). Imunohistohemijska analiza P-gp i BCRP pumpi pokazala je njihovo značajno prisustvo kod ATC pacijenata što ukazuje na učešće ovih proteina u rezistenciji ATC. Sortiranjem ATC ćelija sa smanjenom akumulacijom rodamina 123 (Rho123), poznatog P-gp supstrata, uspostavljena je nova ATC ćelijska linija. Na taj način, dobijen je model koji više odgovara fenotipu uočenom kod ATC pacijenata nego komercijalne ATC ćelijske linije korišćene u ovoj studiji.. Na ovom modelu je ispitana efikasnost kombinovanog tretmanadvostrukim mTOR inhibitorom AZD2014 i PTX-om. Pokazano je da AZD2014 ne samo da povećava osetljivost ATC ćelija na PTX, već u kombinaciji sa ovim citostatikom efikasno inhibira i migraciju i invaziju ATC ćelija. Imajući u vidu da su rezistentnost i invazivnost ATC glavni uzroci loše prognoze, terapija kombinacijom dvostrukog mTOR inhibitora i PTX-a bi mogla doprineti efikasnijem lečenju pacijenata obolelih od ovog karcinoma.

Thyroid carcinoma is the most common malignancy of the endocrine system. Thyroid malignancies are classified according to their histopathological characteristic as papillary, follicular, medullary and anaplastic thyroid carcinoma (ATC). Most thyroid malignancies (papillary thyroid carcinoma and follicular thyroid carcinoma) are well differentiated and have favorable prognosis. On the other hand, ATC is one of the most aggressive human cancers, with an intrinsic resistance and dismal prognosis despite various therapeutic modalities. Changes in components of RAS/MAPK/ERK and PI3K/AKT/mTORpathways are common in thyroid cancer genesis which are resistant to classic chemotherapy agents. Changes in the activity of RAS/MAPK/ERK and PI3K/AKT/mTORsignaling pathways can lead to drug resistance. Besides these changes, possible cause of chemotherapy resistance is also multi-drug resistance (MDR). The most common cause of MDR is high expression of P-gp and BCRP proteins. The aim of this study was to investigate the role of the key components of RAS/MAPK/ERK and PI3K/AKT/mTOR pathways in the pathogenesis and chemoresistance of ATC. We analyzed gene and protein changes in set of ATC patient samples. We also investigated the role of inhibition of RAS/MAPK/ERK and PI3K/AKT/mTOR pathways in ATC chemosensitization using human ATC cell lines. The role of P-gp and BCRP proteins in ATC chemoresistance was also investigated. Analysis of alterations in RAS/MAPK/ERK and PI3K/AKT/mTOR pathways in ATC patients indicated that both pathways cooperate in the development of ATC. Our results revealed a negative correlation between the activity of RAS/MAPK/ERK and PI3K/AKT/mTOR pathways in the samples of ATC patients. NRAS oncogene and p53 tumor suppressor are mutated with high frequency in our set of ATC samples. NRAS is dominantly mutated gene, indicating the importance of this gene in ATC development. All detected mutations in NRAS gene, and two mutations in p53 gene, have never been reported in ATC genesis before. In vitro results suggest that the inhibition of either RAS/MAPK/ERK or PI3K/AKT/mTOR components may confer sensitivity of ATC cells to classic chemotherapeutics. Treatment with dual mTOR inhibitor, AZD2014, alone or in combination with paclitaxel (PTX) or doxorubicin (DOX) was shown to be the most effective. Immunohistochemical analysis showed high P-gp and BCRP expression in our ATC samples, which indicates the role of these proteins in ATC chemoresistance. We sorted ATC cells with the low Rhodamin123 (Rho123) accumulation which is substrate of P-gp protein and established new ATC cell line. In this way we obtained in vitro model system more similar to the patients’ phenotype, then comercial ATC cell lines used in this study. We investigated the potential of dual mTOR inhibitor, AZD2014 combined with PTX to sensitize this new ATC cell line. It was showed that treatment with AZD2014 not only sensitizes ATC cells to PTX, but also combined with this cytostatic, efficiently inhibits ATC cell migration and invasion. Taking into account that chemoresistance and invasiveness of ATC are the main causes of poor outcome, the application of dual mTOR inhibitor combined with PTX, seems to be a logical therapeutic strategy for patients with ATC.