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

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.


Measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Primary Phb1-KO mouse hepatocytes and BCLC5 cells were seeded respectively in a collagen I coated XF24 cell culture microplate (Seahorse Bioscience), at 2.0 × 10 4 cells per well. After 3 hours, 100 μl of growth media with DMSO or MLN4924 and control or Nedd8 siRNA were added. 48 hours after, growth medium was removed and replaced with 500 μl of assay medium prewarmed to 37°C, composed of DMEM without bicarbonate containing 1 mM sodium pyruvate, 2 mM l-glutamine, and cultured at 37°C in room air. Measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed after equilibration in assay medium for 1h. After an OCR and ECAR baseline measurement, sequential injections through ports in the XF Assay cartridges of pharmacologic inhibitors: Oligomycin (1mM), an inhibitor of ATP synthase, which allows a measurement of ATP-coupled oxygen consumption through oxidative phosphorylation (OXPHOS); carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) (300 nM), an uncoupling agent that allows maximum electron transport, and therefore a measurement of maximum OXPHOS respiration capacity; and finally Rotenone (1 µM), a mitochondrial complex 1 inhibitor were performed and changes in OCR and ECAR were analyzed. The normalize data were expressed as pmol of O2 per minute or milli-pH units (mpH) per minute, per μg protein for primary hepatocytes and viability measured by MTT assay for BCLC5 cells.
Briefly, four UPLC ® /time-of-flight mass spectrometry (TOF)-MS based platforms analyzing methanol; methanol/water and chloroform/methanol liver extracts were combined. Identified ion features in the methanol extract platform included fatty acyls, bile acids, and lysoglycerophospholipids. The extracts prepared for methanol platform were also derivatized for amino acid analysis. The chloroform/methanol extract platform provided coverage over glycerolipids, cholesteryl esters, sphingolipids and glycerophospholipids. Finally, the methanol/water extract platform comprised the study of polar metabolites, such as vitamins, nucleosides, nucleotides, carboxylic acids, coenzyme A derivatives, carbohydrate precursors/derivatives and redox-electron-carriers. For this platform, a mixture of methanol/water (60:40, v/v) containing non-endogenous internal standards was added to liver tissue (50:1, v/w) and homogenized using a Precellys 24 grinder. After 1 hour of incubation at -20ºC samples were centrifuged at 16,000 x g for 15 minutes. The supernatant was collected and chloroform was added. Polar phase was then transferred to a clean tube for solvent evaporation. Dried extracts were resuspended in water and, after centrifugation; supernatants were transferred to vials for UPLC ® -MS analysis. Lipid nomenclature follows the LIPID MAPS convention, www.lipidmaps.org.

Metabolomics Data processing and Normalization. Data obtained with the UPLC ® -MS
were processed with the TargetLynx application manager for MassLynx (Waters Corp.) as detailed (1,2). Intra-and interbatch normalization followed the procedure described in (3).
All the calculations were performed with R v2.13.0 (R Development Core Team, 2010).
Statistical Analysis. Liver metabolite concentrations were compared using unpaired Student´s or Welch´s t test where unequal variances were found. A number of 5 animals per group were studied. Hepatocytes isolated from Prohibitin 1 (Phb1)-KO mice display tumoral characteristics.

Supplementary
Neddylation inhibition using the small molecule inhibitor MLN4924 or Nedd8 siRNA accounts for decreased Nedd8 levels in these hepatic tumoral cells. The reduction in Nedd8 levels lowers liver kinase B1 (LKB1) and Akt levels, usually upregulated in tumoral cells. As a consequence of lowering LKB1 and Akt levels, a reduction in the OXPHOS/glycolysis metabolic ratio is observed. Under these circumstances, the metabolic slowdown observed after neddylation inhibition is associated with the negative impact on OXPHOS. As a consequence of decreased OXPHOS, tumoral cells need to look for alternative energetic pathways such as glycolysis. Interestingly, the metabolic switch from OXPHOS to glycolytic flux, usually advantageous to tumoral cells, in here is associated with increased tumoral cell apoptosis. We speculate that this is due to the fact that under neddylation inhibition the cellular energetic pool, already compromised by reduced OXPHOS, is being channeled to the high-demanding energetic process of DNA rereplication, previously shown to be induced under neddylation inhibition. Thereby, neddylation inhibition increased redox dysfunction and