Title Nove kombinirane terapije za BRAFV600E mutirani rak debelog crijeva sa stečenom rezistencijom na vemurafenib Title (english) Novel Combination Treatments for BRAFV600E Mutant Colon Cancer with Acquired Resistance to Vemurafenib Author Petra Grbčić Mentor Mirela Sedić (mentor)Committee member Ivana Ratkaj (predsjednik povjerenstva)Committee member Vesna Eraković Haber (član povjerenstva)Committee member Ines Mrakovčić-Šutić (član povjerenstva)Granter University of Rijeka Defense date and country 2021, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Mutacija BRAFV600E, zastupljena u oko 10% svih slučajeva raka debelog crijeva, povezana je sa slabim odgovorom na sistemsku terapiju i lošim preživljenjem pacijenata. Vemurafenib (PLX4032), specifični inhibitor proteina BRAFV600E, pokazao je potencijal u liječenju metastatskog melanoma s BRAFV600E mutacijom, ali isti klinički učinak nije zabilježen prilikom terapije pacijenata s rakom debelog crijeva koji imaju mutaciju BRAFV600E. Razlog tome može biti inicijalno slab odgovor na terapiju te brz razvoj rezistencije na vemurafenib. Stoga je cilj ovog doktorskog rada bio otkriti potencijalne nove terapijske mete i molekularne biljege odgovora i razvoja rezistencije stanica raka debelog crijeva sa mutacijom BRAFV600E na vemurafenib. U tu svrhu proveli smo proteomsko profiliranje praćeno bioinformatičkom analizom razlike izražaja proteina između stanica raka debelog crijeva s mutacijom BRAF/divljim tipom KRAS, divljim tipom BRAF/mutacijom KRAS i stanica koje sadrže divlji tip BRAF/KRAS. Validacija potencijalnih biljega karakterističnih za BRAFV600E mutaciju je bila provedena imunohistokemijskom analizom, kao i in silico analizom pomoću TCGA baze podataka. Obje analize su nam potvrdile značajno povećanje izražaja i aktivnosti nukleofozmina (NPM1) kao molekularne značajke specifične za BRAFV600E mutirani rak debelog crijeva. Farmakološka inhibicija NPM1 je potakla povećanu osjetljivost rezistentnih stanica na terapiju vemurafenibom. Inhibicija NPM1 bila je popraćena smanjenim izražajem i aktivnošću c-Myc te njegovih nizvodnih meta PSPH i RanBP1, uključenih u regulaciju biosinteze serina i duplikacije centrosoma. Kako su razine sfingolipida u stanici usko povezane sa sintezom serina, dodatno smo istražili ulogu sfingolipidnog metabolizma u regulaciji rezistencije na vemurafenib u stanicama raka debelog crijeva s BRAFV600E mutacijom. Otkrili smo značajno povećane razine izražaja aktivnih formi sfingozin kinaza 1 i 2 (SphK1, SphK2) u rezistentnim stanicama praćeno povećanjem razine njihovog produkta, sfingozin-1-fosfata (S1P). Farmakološka inhibicija SphK1 ili SphK2 te dodatak egzogenog ceramida C6 doveli su do povećanja osjetljivosti rezistentnih stanica na vemurafenib.Medutim, kombinirana inhibicija SphK2 i BRAF pomoću ABC294640 i PLX4032 je imala snažan sinergistički anti-proliferativni učinak pri najvećem broju kombinacija sub-toksičnih koncentracija ova dva terapeutika, te su stoga daljnja istraživanja bila provedena upravo na ovoj kombinaciji lijekova. Mehanistička studija provedena na kombiniranoj terapiji PLX4032 i ABC294640 ukazala je na inhibiciju AKT kinaze posredovanu smanjenom razinom S1P te posljedično smanjenje izražaja NPM1 i translacijski kontroliranog tumorskog proteina (TCTP). Zaključno, rezultati naše studije ukazuju na ulogu NPM1 kao potencijalnog biljega rezistencije BRAFV600E mutiranog raka debelog crijeva na vemurafenib. Također predlažemo da kombinirana terapija ABC294640 i PLX4032 predstavlja novi terapijski pristup uspješnom dokidanju rezistencije na BRAF inhibiciju u vemurafenib-rezistentnim stanicama raka debelog crijeva s BRAFV600E mutacijom.
Ključne riječi: rak debelog crijeva, BRAFV600E, PLX4032, vemurafenib, kemorezistencije, nukleofozmin, c-Myc, sfingolipidi, sfingozin-1-fosfat, sfingozin kinaza 2, ABC294640, Opaganib, translacijom kontroliran tumorski protein
Abstract (english) BRAFV600E mutations, present in about 10% of all colorectal cancer patients, are associated with poor responses to systemic therapy and low patient survival rates. Vemurafenib (PLX4032) is a specific BRAFV600E mutant inhibitor and has been applied in treatments of patients with metastatic melanoma exhibiting this mutation. However, when treating patients with BRAFV600E mutant colorectal cancer, the same response rate is not reached. One possible explanation is the fast-developing resistance to vemurafenib. The aim of this study was detecting novel potential targets that show correlation with the response and the development of resistance to vemurafenib in colon cancer cells and are therefore potential sites for targeted inhibition as a means of increasing sensitivity of cells to vemurafenib. We conducted proteomic and bioinformatic analyses of differences in proteome signatures between BRAFV600E mutant, KRAS mutant and BRAF/KRAS wild type colon cancer cells. Validation of obtained data was preformed using an immunohistochemistry analysis and an in silico analysis of TCGA data; both confirmed the abundance of nucleophosmin (NPM1) in BRAFV600E mutant colon cancer cells. Pharmacological inhibition of NPM1 increased the sensitivity of resistant cells to vemurafenib, which was related to the decrease in the expression and the activity of c-Myc, which in turn decreased the abundance of PSPH and RanBP1, both important for centrosome duplication and serine biosynthesis. Furthermore, we explored the role of sphingolipid metabolism in the regulation of resistance to vemurafenib in BRAFV600E mutated colon cancer cells. Sphingolipids are bioactive lipids whose role in mediating resistance to various therapeutics in colon cancer has already been proven. We detected increased activities of both sphingosine kinases (SphK1 and SphK2) in resistant cells, increased abundance of their product, sphingosine-1-phosphate (S1P), as well as changes in the ceramide metabolism. Pharmacological inhibition of either SphK1 or SphK2, as well as the addition of exogenous ceramide C6, increased sensitivity of resistant cells to vemurafenib. We presume that the inhibition of SphK2 via ABC294640-initiated decrease in AKT activity is due to decreased levels of S1P. In addition, the decrease in AKT activity was followed by the decrease in the translation and the activity of both NPM1 and translationally controlled tumour protein (TCTP). Results from our study suggest that the NPM1/c-Myc axis can be considered a potential therapeutic target to successfully evade resistance to vemurafenib in colon cancer harbouring BRAFV600E. Finally, we provided an insight into how a combined therapy of ABC294640 and PLX4032 can be a novel approach to successfully overcoming the resistance in BRAFV600E mutated colon cancer cells to specific BRAF inhibitor.
Keywords: colon cancer, BRAFV600E, PLX4032, vemurafenib, chemoresistance, nucleophosmin, c-Myc, sphingolipids, sphingosine-1-phosphate, sphingosine kinase 2, ABC294640, Opaganib, translationally controlled tumour protein
Keywords
kombinirana terapija
BRAFV600E
mutirani rak debelog crijeva
rezistencija
vemurafenib
Keywords (english)
BRAFV600E
Mutant Colon Cancer
Resistance
Vemurafenib
Language croatian URN:NBN urn:nbn:hr:193:558970 Promotion 2022 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Closed access Terms of use Created on 2022-03-14 09:44:23
