Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site VSports app下载. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2015 Feb 10;6(4):2509-23.
doi: 10.18632/oncotarget.3191.

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

Lucía Barbier-Torres  1 , Teresa C Delgado  1 , Juan L García-Rodríguez  1 , Imanol Zubiete-Franco  1 , David Fernández-Ramos  1 , Xabier Buqué  2 , Ainara Cano  3 , Virginia Gutiérrez-de Juan  1 , Itziar Fernández-Domínguez  1 , Fernando Lopitz-Otsoa  1 , Pablo Fernández-Tussy  1 , Loreto Boix  4   5 , Jordi Bruix  4   5 , Erica Villa  6 , Azucena Castro  3 , Shelly C Lu  7 , Patricia Aspichueta  2 , Dimitris Xirodimas  8 , Marta Varela-Rey  1 , José M Mato  1 , Naiara Beraza  1 , María L Martínez-Chantar  1
Affiliations

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

Lucía Barbier-Torres et al. Oncotarget. .

Abstract

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma VSports手机版. .

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Global neddylation in HCC
(A) Nedd8 and NAE1 IHC analysis in human samples from normal liver (NL) (n=10) and hepatocarcinoma (HCC) (n=22) patients. (B) Box plots of Nedd8 and NAE1 gene levels in NL (n=200) and HCC (n=225) human samples from ONCOMINE database. (C) Nedd8 and NAE1 IHC and (D) mRNA expression analysis in good (n=16) and bad (n=13) prognosis HCC human samples. (E) Kaplan-Meier plot of overall survival of HCC patients grouped according to low or high Nedd8 expression. (F) Logistic regression to quantify the predictability of a Nedd8 model in better and poorer prognosis of HCC. Values are represented as mean ± SEM. *p<0.05, **p<0.01 (HCC vs NL; Bad vs good prognosis).
Figure 2
Figure 2. In vivo neddylation inhibition blocks tumor progression
(A) Representative Western blot analysis of global neddylation (shown as neddylated cullins) in whole extracts from WT and Phb1-KO livers and isolated hepatocytes. (B) Reduction of tumor size in Phb1-KO mice after MLN4924 treatment determined by caliper measurement of the longest diameter in the transverse view of the liver. (C) Staining of liver sections from Phb1-KO MLN4924 treated and Phb1-KO untreated mice with Nedd8 and HuR antibodies. Graphical representations are shown on the right of each panel. (D) Western blot analysis of Nedd8 and HuR on liver lysates from Phb1-KO MLN4924 and Phb1-KO untreated animals. (Values are mean ± SEM. *p<0.05; Phb1-KO MLN4924 vs Phb1-KO).
Figure 3
Figure 3. Oxidative Phosphorylation and Glycolysis are dependent on neddylation activity
(A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) values in Phb1-KO hepatocytes 48 hours after MLN4924 treatment or Nedd8 silencing. The energetic response in cells was measured in the presence of oligomycin, FCCP and rotenone. (C) Caspase-3 activity in WT and Phb1-KO hepatocytes after 48 hours of MLN4924 treatment. (D) ATP levels and (E) ROS production in Phb1-KO hepatocytes after 48 hours of MLN4924 treatment. (F) Phosphatidylethanolamine methyltransferase (PEMT) flux in Phb1-KO hepatocytes 48 hours after MLN4924 treatment or Nedd8 silencing. Values are represented as mean ± SEM. n=4 samples/time point. *p<0.05, **p<0.01, ***p<0.001 (MLN4924 or siNedd8 vs control).
Figure 4
Figure 4. Association between LKB1, Akt and neddylation in human HCC
(A) LKB1 and Akt IHC analysis in human samples from normal liver (NL) and HCC human samples from patients with good and bad prognosis. Graphical representations are shown on the right of each panel. (B) LKB1 and Akt mRNA expression in human samples from patients with HCC with good or bad prognosis. (C) Pearson's correlation between neddylation and LKB1 or Akt levels respectively. (D) ROC curves of LKB1 and Akt in good and bad prognosis of human HCC samples. The area under the ROC curve (AUC) measures the statistical potential of Akt and LKB1 to differentiate the two prognosis groups. Values are represented as mean ± SEM. *p<0.05 (Bad vs good prognosis).
Figure 5
Figure 5. LKB1 and Akt levels are dependent of neddylation in HCC mice models
(A) Representative Western blot analysis of LKB1 and Akt in whole extracts from WT and Phb1-KO livers and isolated hepatocytes. (B) Staining of liver sections from Phb1-KO MLN4924 treated and Phb1-KO untreated mice with LKB1 and Akt antibodies. Graphical representations are shown on the right of each panel. (C) Western blot analysis of LKB1 and Akt on liver lysates from Phb1-KO MLN4924 and Phb1-KO untreated animals. (D) Graphical representation of changes in tumor size in HepG2-xenografted nude mice. (E) Staining and graphical representation of quantitative analysis of each staining in HepG2-xenografted nude mice tumors. Values are represented as mean ± SEM. *p<0.05, **p<0.01 (Phb1-KO MLN4924 vs Phb1-KO; siNedd8 vs siControl).
Figure 6
Figure 6. LKB1 and Akt as new targets of neddylation
(A) Representative Western blot analysis of total lysates from WT and Phb1-KO hepatocytes treated with MLN4924 for 48 hours using Nedd8, LKB1 and Akt antibodies. LKB1 and Akt levels were also evaluated by Western blot in Phb1-KO hepatocytes after (B) Nedd8 silencing and (C) NEDP1-V5 overexpression. (D) The stability of LKB1 and Akt proteins as the percentage of protein level remaining after treated with cycloheximide (CHX). Vertical bars are indicative of the calculated half-life for every protein. (E) FLAG and HA immunoreactive proteins in WT hepatocytes expressing FLAG-LKB1 or HA-Akt and His6-Nedd8. Hepatocytes were cotransfected with FLAG-LKB1/HA-Akt and His6-Nedd8 plasmids, and FLAG/HA-containing proteins were purified using Ni2+-NTA and detected by Western blotting.
Figure 7
Figure 7. LKB1 and Akt stabilization play an important role on neddylation-induced metabolic disruptions in liver cancer
(A) Western blot analysis against Nedd8, LKB1 and Akt in Phb1-KO hepatocytes overexpressing Akt and LKB1 after 48 hours of MLN4924 treatment. (B) Basal oxygen consumption rate (OCR) and (C) extracellular acidification rate (ECAR) values in Phb1-KO hepatocytes after LKB1 and Akt overexpression and silencing. (D) OCR and ECAR were measured in Phb1-KO hepatocytes overexpressing LKB1 and Akt after 48 hours of MLN4924 treatment. The energetic response in cells was measured in the presence of oligomycin, FCCP and rotenone. (E) Caspase-3 activity in Phb1-KO hepatocytes overexpressing LKB1 and Akt after 48 hours of MLN4924 treatment. Values are mean ± SEM. *p<0.5, ***p<0.001 (LKB1/Akt overexpression and silencing vs control).
See this image and copyright information in PMC

References

    1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379(9822):1245–1255. - PubMed
    1. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31(4):339–346. - PubMed
    1. Zhou W, Liotta LA, Petricoin EF. Cancer metabolism and mass spectrometry-based proteomics. Cancer Lett. 2015;356(2):176–183. - PubMed
    1. Xirodimas DP. Novel substrates and functions for the ubiquitin-like molecule NEDD8. Biochem Soc Trans. 2008;36(Pt 5):802–806. - PubMed
    1. Xirodimas DP, Saville MK, Bourdon JC, Hay RT, Lane DP. Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell. 2004;118(1):83–97. - PubMed

Publication types

MeSH terms