doktorska disertacija
Kaja Blagotinšek Cokan (Avtor), Damjana Rozman (Mentor), Katarina Trebušak Podkrajšek (Član komisije za zagovor), Tea Lanišnik-Rižner (Član komisije za zagovor), Gregor Majdič (Član komisije za zagovor)

Povzetek

Hepatocelularni karcinom (HCC) v svetovnem merilu predstavlja drugi najpogostejši z rakom povezani vzrok smrti. Kljub obsežni etiologiji HCC je vse več dokazov, da so moteni presnovni procesi v jetrih prevladujoči vzrok za nastanek HCC, ki prispeva k nastanku z več dejavniki in s presnovo povezanih maščobnih bolezni jeter (MAFLD), do pred kratkim imenovanih nealkoholne maščobne bolezni jeter (NAFLD). Ohranjanje ravnovesja holesterola sodi med najpomembnejše dejavnike presnove, vendar vloga holesterola v hepatokarcinogenezi še vedno ostaja nepojasnjena. Da bi se približali razumevanju molekularnih mehanizmov, ki uravnavajo presnovno odvisni potek HCC, smo spremljali spremembe v jetrih mišjega modela (Cyp51 KO) v poznem starostnem obdobju. Spremembe so posledica tarčnega izbitja gena za lanosterol 14?-demetilazo (CYP51) iz poznega dela sinteze holesterola. Miši Cyp51 KO so med 12 in 24 mesecem starosti razvile tumorje na jetrih. Pri 24 mesecih starosti je bila pojavnost tumorjev 77,8 % pri samicah in 50 % pri samcih. Poškodbe jetrnega tkiva so bile izrazitejše pri samicah, kjer smo opazili zmerno do hudo reakcijo žolčnih vodov s povezovanjem portalnih polj, spremljajoče blago vnetje in izrazitejšo fibrozo pri 12 mesecih starosti, jetrni fenotip pa se je še poslabšal do starosti 24 mesecev. Vpliv kronične presnovne bolezni, povzročene z izbitjem Cyp51 v hepatocitih, se je odrazil v številnih presnovnih prilagoditvah jeter, s katerimi smo pokazali pomembne razlike med spoloma. Opažene presnovne in transkripcijske spremembe motene presnove smo ovrednotili na tumorjih HCC Cyp51 KO miši in jih primerjali z literaturnimi podatki o HCC pri človeku. To je odprlo nova obzorja pri razumevanju od spola odvisnega razvoja hepatokarcinogeneze pri miših. Pri obeh spolih Cyp51 KO miši sta bili signalna pot PI3K/AKT in pot interakcije receptorja za ECM aktivirani preko različnih tarčnih genov, znižani so bili tudi pomembni presnovni procesi lipidov. Povišane vrednosti plazemskega holesterola in holesterola HDL, zavrta prepisovalna dejavnika FXR? ter LXR?:RXR?, in najpomembneje, medsebojni vpliv med zavrtim LXR? in aktivirano signalno potjo TGF-ß skupaj pojasnjujejo večjo dovzetnost za hepatokarcinogenezo pri Cyp51 KO samicah. Prepisovalna dejavnika (SOX9)2 in PPAR smo prepoznali kot pomembni tarči pri hepatokarcinogenezi samic, medtem ko bi povišano izražanje Cd36, tarčnega gena prepisovalnega dejavnika RORC, lahko predstavljal regulator signalne poti interakcije receptorja za ECM v hepatokarcinogenezi samcev. Od holesterola odvisno presnovno reprogramiranje predstavlja nov vidik razumevanja poteka molekularnih poti hepatokarcinogeneze pri ženskah, kjer se prevalenca za HCC s starostjo poviša, vendar še vedno ostaja nižja kot pri moških. Pomen presnove holesterola smo pokazali tudi s primerjalno funkcionalno genomsko analizo. Poleg tega pa smo pri tumorskih vzorcih žensk s HCC pokazali tudi znižano izražanje gena CYP51, ki še ni bil prikazan kot pomemben regulator presnovno odvisne hepatokarcinogeneze pri ženskah. V drugem delu doktorske naloge smo opredelili nov mišji model Cyp51LC, ki, v primerjavi z Cyp51 KO modelom, omogoča še časovno uravnavanje izbitja gena Cyp51. S tem ponuja možnost, da opazujemo le primarne spremembe, ki jih izbitje genov povzroči v jetrih odraslih živali. Spremembe na ultrastrukturni ravni hepatocitov Cyp51LC so pokazale obsežne poškodbe celičnih organelov. Presenetljivo smo opazili v celicah številne kristale različnih oblik, ki so bili prisotni v večjih količinah v Kupferjevih celicah. Ker pri mišjem modelu Cyp51LC ni bilo prisotne fibroze, intenzivnost poškodb jetrnega tkiva pa je bila nižja, smo lahko izolirali primarne hepatocite. Pokazali smo, da so primarni hepatociti močno izpostavljeni endoplazemskemu stresu in imajo aktivirano razgradnjo lipidnih kapljic. V hepatocitih smo ugotovili povečane koncentracije lanosterola in dihidrolanosterola, kar je pomembno z vidika nadaljnjih analiz zgradbe opaženih kristalov in pojasnjevanja začetnih znanilcev hepatokarcinogeneze znotraj parenhimskih celic jeter. Primarni hepatociti Cyp51LC mišjega modela tako predstavljajo pomemben celični model za poglobljene mehanistične študije presnovno odvisnega HCC. Naša študija tudi poudarja nujnost obravnave po spolu zaradi s spolom povezanih molekularnih dejavnikov, ki prispevajo k nastanku HCC tudi pri ljudeh.

Ključne besede

hepatocelularni karcinom;sinteza holesterola;gen CYP51;Bolezni prebavil;Disertacije;Bolezni jeter;Hepatocelularni karcinom;Presnova;Onkologija;Diseracije;Rak;Hepatocite;Mutacije;Geni;Kancerogeneza;Molekularna biologija;Translacija;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL MF - Medicinska fakulteta
Založnik: [K. Blagotinšek Cokan]
UDK: 616.36-006:575/577.2(043.3)
COBISS: 58909443 Povezava se bo odprla v novem oknu
Št. ogledov: 590
Št. prenosov: 148
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Hepatocellular carcinoma development in mice as a consequence of blocked cholesterol synthesis due to the CYP51 liver conditional knockout
Sekundarni povzetek: Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. Despite the extensive multifactorial etiology of HCC, there is growing evidence that disrupted metabolic processes in the liver are the predominant cause of HCC, contributing also to the development of metabolic fatty liver disease (MAFLD), previously termed non-alcoholic fatty liver disease (NAFLD). Maintaining cholesterol balance is one of the most important factors of metabolism, but the role of cholesterol in hepatocarcinogenesis still remains unexplained. To deepen the understanding of the molecular mechanisms that regulate the metabolic-dependent development of HCC, it is still necessary to use animal models since it is impossible, due to ethical and other reasons, to answer all questions in humans. One important aspect is also following the disease progression during the aging that would require following human subjects for several decades. To address the changes in the long term liver pathology related to disballanced cholesterol synthesis we monitored a mouse model (Cyp51 KO) of aging mice (12, 18 and 24 months). The mice represent a targeted knock-out of the lanosterol 14α-demethylase (CYP51) gene from the late part of cholesterol synthesis. Aging Cyp51 KO mice developed liver tumours between 12 and 24 months of age with an incidence of 77.8 % in females and 50 % in males at 24 months. We described that liver tissue damage was more prominent in Cyp51 KO females, where a moderate to severe ductular reaction, accompanied by mild inflammation and severe fibrosis were observed at 12 months of age, when the liver phenotype worsened until the age of 24 months. The influence of chronic metabolic disease caused by deletion of Cyp51 in hepatocytes was reflected in a number of mechanistic adjustments of the liver, which show a significant difference between sexes. All metabolic and transcriptional changes caused by disrupted cholesterol metabolism, which were evaluated in diagnostically confirmed HCC of Cyp51 KO mice and aligned with the latest human literature data, opened horizons for proposing a new sex-dependent mechanistic model for the hepatocarcinogenesis development in Cyp51 KO mice. In both sexes of Cyp51 KO mice, the PI3K/AKT signalling pathway and the ECM-receptor interaction pathway were activated by sex-specific altered genes and significant reduction of lipid-related metabolic processes was present. Elevated plasma cholesterol and cholesterol HDL, inhibited transcription factors FXRα and LXRα:RXRα, and most importantly, the crosstalk between inhibited LXRα and activated TGF-β signalling pathway explain the increased susceptibility to hepatocarcinogenesis in Cyp51 KO females. Additionally, transcription factors (SOX9)2 and PPARα were identified as important targets for female hepatocarcinogenesis, while the elevated expression of Cd36, a target gene of transcription factor RORC, could represent an important sex-dependent regulator of the ECM-receptor interaction signalling pathway in male hepatocarcinogenesis. Uncovered metabolic reprogramming of cholesterol-dependent pathways represents an important concept of understanding hepatocarcinogenesis in women, where the prevalence of HCC is higher but still lower in comparison to men. We used the comparative functional genomic analysis, as initial results indicated that cholesterol synthesis was reduced also in the evaluated publically available human HCC data sets, included in the analysis. While the inhibition of CYP51 expression has not yet been proven to be an important regulator of metabolically dependent hepatocarcinogenesis in women, our data indicate that the gene is under-expressed in some tumour samples from women with HCC. To dechipher, which cells of the liver are responsible for the pathological developments upon cholesterol disbalance, we developed another mouse model Cyp51LC. In comparison to the Cyp51 KO model, the Cyp51LC model allows not only the targeted gene deletion in hepatocytes, but also deletion in a time-controlled manner. In our case the doxycycline responsive promotor has been introduced that allows excision of the Cyp51 transgene upon application of the doxycycline at a certain time point, for a certain time period. With such approach we avoid the phenotype changes caused by gene deletion during animal development. Consequently, we can observe only the response of the organ/organism to the change in the adult animal, while still monitoring early phases of the disease. After application of doxycycline to mice major changes in the ultrastructure of hepatocytes have been observed, indicating extensive damage of cell organelles and, surprisingly, numerous crystals of various shapes whose content is still under investigation. They were more abundant in Kupffer cells compared to hepatocytes and likely include large amount of cholesterol synthesis intermediates. The liver inflammation and ductular reaction were in Cyp51LC livers weaker compared to Cyp51 KO model and fibrosis in liver parenchyma was not observed. The absence of fibrosis in the Cyp51LC mouse model is a large benefit since it allows primary hepatocyte isolation due to les overall liver tissue damage. Primary hepatocytes with depleted cholesterol synthesis showed an increase in endoplasmic reticulum stress and strongly activated lipid droplet degradation. The elevated concentrations of sterol intermedates lanosterol and dehydrolanosterol were found in hepatocytes, this is why we propose that they form crystals. The primary hepatocyte data is important to compare with the obtained on whole liver, since we can now evaluate which changes belong to hepatocyte and which to other cell types of the liver. At the level of sterol intermediates, where other sterol intermediates were also detected, this will be helpful to unravel the composition of crystals found in different liver cell types with depleted cholesterol synthesis. The primary hepatocytes of the Cyp51LC mouse model thus represent an important cellular model for in-depth mechanistic studies of cholesterol-dependent HCC.
Sekundarne ključne besede: hepatocellular carcinoma;cholesterol synthesis;CYP51 gene;Onkologija;Bolezni jeter;Kancerogeneza;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Doktorska disertacija
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Medicinska fak.
Strani: XVII f., 155 str.
ID: 12699928