Background: Sporadic clear-cell renal cell carcinoma (ccRCC) is associated with mutations in the VHL gene, upregulated mammalian target of rapamycin (mTOR) activity and glycolytic metabolism. Here, we analyze the effect of VHL mutational status on the expression level of mTOR, eIF4E-BP1, AMPK, REDD1, and PDK3 proteins.
Methods: Total proteins were isolated from 21 tumorous samples with biallelic inactivation, 10 with monoallelic inactivation and 6 tumors with a wild-type VHL (wtVHL) gene obtained from patients who underwent total nephrectomy. The expressions of target proteins were assessed using Western blot.
Results: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. Tumors with monoallelic inactivation of VHL underexpressed REDD1 in comparison to wtVHL tumors (P = 0.042), tumors with biallelic VHL inactivation (P < 0.005) and control tissue (P = 0.004). Additionally, REDD1 expression was higher in tumors with VHL biallelic inactivation than in control tissue (P = 0.008). Only in wt tumor samples PDK3 was overexpressed in comparison to tumors with biallelic inactivation of VHL gene (P = 0.012) and controls (P = 0.016). In wtVHL ccRCC, multivariate linear regression analysis revealed that 97.4% of variability in PDK3 expression can be explained by variations in AMPK amount.
Conclusion: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. We have shown for the first time VHL dependent expression of PDK3 and we provide additional evidence that VHL mutational status affects REDD1 expression in sporadic ccRCC.
Uvod: Sporadićni svetločelijski karcinom bubrega asociran je sa mutacijama u genu VHL, povišenom aktivnošču mTOR signalnog puta i glikolitićkim metabolizmom. Cilj ove studije bio je da se ispita efekat mutacionog statusa gena VHL na nivo ekspresije proteina mTOR, eIF4E-BP1, AMPK, REDD1 i PDK3.
Metode: Ukupni proteini izolovani su iz uzorka tumorskog tkiva sa bialelnom inaktivacijom gena VHL (n=21), uzoraka sa monoalelnom inaktivacijom (n=10) i tumora sa neizmenjenim genom VHL (wtVHL) (n=6) dobijenih od bolesnika sa karcinomom bubrega nakon totalne nefrektomije. Nivo ekspresije ispitivanih proteina utvrđen je Western blot metodom.
Rezultati: Nisu detektovane razlike u nivou ekspresije proteina mTOR, eIF4EBP1 i AMPK u zavisnosti od mutacionog statusa gena VHL. Tumori sa monoalelnom inaktivacijom gena VHL imali su povišen nivo ekspresije proteina REDD1 u poređenju sa wtVHL tumorima (P = 0,042), tumorima sa bialelnom inaktivacijom gena VHL (P < 0,005) i kontrolnim tkivom (P = 0,004). Dodatno, ekspresioni nivo REDD1 proteina je bio viši u tumorima sa bialelnom inaktivacijom gena VHL u odnosu na kontrolno tkivo (P = 0,008). Nivo ekspresije proteina PDK3 u wtVHL tumorima je bio povišen u odnosu na tumore sa bialelnom inaktivacijom gena VHL (P = 0,012) i kontrolno tkivo bubrega (P = 0,016). U tumorima sa wtVHL genom, multivarijantnom linearnom regresionom analizom pokazano je da se 97,4% varijabilnosti nivoa ekspresije PDK3 može objasniti varijabilnošču ekspresionog nivoa proteina AMPK.
Zaključak: Ekspresioni nivo proteina mTOR, eIF4EBP1 i AMPK nezavisan je od mutacionog statusa gena VHL. Ovom studijom je prvi put pokazano da nivo ekspresije proteina PDK3 zavisi od mutacionog statusa gena VHL i pruženi su dodatni dokazi da mutacioni status gena VHL utiće na nivo ekspresije proteina REDD1 u svetločelijskom karcinomu bubrega.
Keywords: VHL gene; clear-cell renal cell carcinoma; mammalian target of rapamycin; pyruvate dehydrogenase kinase 3; regulated in development and DNA damage responses.