FK866-induced NAMPT inhibition significantly decreased NAD levels in human hepatocarcinoma cells which could be ameliorated by NMN administration

We stimulated hepatocarcinoma cells with FK866 [10nM] and found significantly reduced NAMPT activity (-74.9±8.1% in Huh7 cells, -38.1±3.7% in Hep3B cells) (Fig.1A) which caused a sharp decline of NAD levels (Huh7 cells 3.3± 0.3μmol/g protein [con] vs. 0.3± 0.2μmol/g protein [10nM FK866]; Hep3B cells 2.2± 0.7μmol/g protein [con] vs. 0.2± 0.08μmol/g protein [10nM FK866]) (Fig.1B). Co-treatment with NMN restored intracellular NAD levels in all tested cell lines (Fig.1B). To investigate the sensitivity of non-cancerous human hepatocytes towards FK866, we used the same treatment conditions as for hepatocarcinoma cells and found that non- cancerous hepatocytes showed no significant reduction in NAMPT activity and NAD levels at 10nM FK866 after 48h (Supplement Fig.1A,B).

Emerging evidence suggests that the cellular acetylation state is associated with the energy state of a cell [30]. We could show that FK866-induced NAD depletion led to a decreased activity of NAD-dependent lysine deacetylases as measured by an increased global acetylation of lysine residues (+1.9-fold, p<0.001) (Fig.1C). The administration of NMN abrogated the FK866- induced hyperacetylation of lysine residues (p<0.001) (Fig.1C).

NAMPT inhibition by FK866 reduced cell viability, induced energy stress and led to delayed cell death in human hepatocarcinoma cells. We could detect a decreased cell viability in hepatocarcinoma cells (-49.4±4.6% in Huh7 cells, – 20.6±2.8% in Hep3B cells)(Fig.2A) after 24h of FK866 treatment. We wanted to investigate whether FK866-induced NAD depletion would result in a reduction of ATP generation and therefore would induce cellular energy stress in hepatocarcinoma cells. Time course studies revealed that ATP levels were lowered in Huh7 cells (-49.6±9.5%, p<0.01) and Hep3B cells (- 61.1±6.8%, p<0.001) after 48h of treatment with 10nM FK866 (Fig.2B). The ATP levels further declined after 72h in Huh7 cells (-90.2±2.5%, p<0.001) and Hep3B cells (-91.1±1.5%, p<0.001) (Fig.2C). The co-administration of NMN could ameliorate ATP levels in Huh7 and Hep3B cells after 48 and 72h (Fig.2B,C). After 72h, subsequent to the drop of NAD levels, the effects of FK866 on cell death became evident when measuring An /PI -stained cells. Hep3B cells, a p53 deficient cell line, already displayed an increase in An /PI cells after 48h of FK866 treatment (+1.8-fold, p<0.01) (Supplement Fig.2A) indicating that FK866-induced cell death did not depend on p53 function. Huh7 cells treated with FK866 [10nM] for 72h showed a 1.5-fold increase in An /PI cells compared to control cells (p<0.05) (Fig.2D) whereas the number of An /PI Hep3B cells increased further (+3.0-fold, p<0.01). Co-stimulation with NMN ameliorated the induction of cell death in Huh7 cells (p=0.09) and completely rescued FK866- induced cell death in Hep3B cells (p<0.01) (Fig.2D). Dysregulation of the AMPK/mTOR signalling pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes Growing evidence suggests that mTOR and AMPK dysregulation play an important role in hepatocellular carcinogenesis [20;31]. Therefore, we compared the protein amount of mTOR and its downstream target p70S6 kinase and also AMPKα activation in non-cancerous primary human hepatocytes and hepatocarcinoma cells. An increased protein level of total mTOR and p70S6 kinase was found in hepatocarcinoma cells compared to non-cancerous hepatocytes (Fig.3A). In contrast, AMPK activation was enhanced in non-cancerous primary human hepatocytes (PHH) compared to Huh7 and Hep3B cells despite equal AMPKα total protein amount (Fig.3A). This suggests that mTOR signalling and AMPK activation are involved in metabolic adaptation of hepatocarcinoma cells and might be interesting targets for prevention of cancer cell growth.

FK866-induced energy stress activated AMPKα and led to inhibition of mTOR complex1 signalling in hepatocarcinoma cells
To test the efficacy of FK866-induced NAD depletion to activate AMPK and inhibit the mTOR signalling pathway, we measured the phosphorylation state of different members of the AMPK/mTOR complex1 cascade. FK866 treatment increased the phosphorylation of AMPKα at Thr172 (+3.3-fold, p<0.01) in hepatocarcinoma cells (Fig.3B). This was associated with a significant down regulation of phosphorylated mTOR (Ser2448) by -50.7±0.1% (p<0.05) and the phosphorylation of its down-stream target p70S6 kinase (by -94.7±2.4%, p<0.001) and 4E-BP1 (by -30.0±.0.1%, p<0.05) indicating reduced protein synthesis and cell growth (Fig.3B). Co- treatment with NMN [500μM] completely reversed the FK866-induced effects on AMPK activation and mTOR complex1 signalling inhibition suggesting that the NMN biosynthetic activity of NAMPT is relevant in mediating the effects of FK866. NMN alone had no impact on AMPK activation and mTORC1 signalling in hepatocarcinoma cells (Fig.3B). Non-cancerous human hepatocytes treated with equal amounts of FK866 for 48h did not show significant changes in AMPK activation and mTOR phosphorylation (Supplement Fig.1C) verifying their lower sensitivity to FK866.

During malignant transformation the cellular metabolism undergoes multiple molecular and metabolic adaptations to support cell growth and survival. NAD is a key determinant in cancer cell biology as it is essential for redox reactions and key component of signalling pathways that regulate transcription, DNA repair, apoptosis and metabolism [1]. In mammals, NAMPT is a main regulator of the intracellular NAD pool [2;3]. Here, we investigated whether or not the NAMPT inhibitor, FK866, would affect intracellular NAD and ATP concentrations in hepatocarcinoma cells and consequently would be able to regulate the activity of the metabolic sensors AMPK and mTOR. Our study showed that FK866 rapidly reduced NAD levels in hepatocarcinoma cells and led to delayed ATP depletion which could be ameliorated by administration of Sublingual NMN. Break down of ATP levels was associated with increased cell death. In contrast to another study [6], we demonstrated that FK866 reduced NAMPT activity, depleted NAD and ATP content and induced cell death in p53-deficient Hep3B cells suggesting that FK866-mediated cell death does not depend on functional p53. Our results are in line with a study performed in chronic lymphocytic leukemia cells [14]. In our study, especially Hep3B cells showed a high sensitivity to FK866 and an increased number of dead cells occurred already after 48h of FK866 treatment. Interestingly, non-cancerous human hepatocytes subjected to the same FK866 treatment as hepatocarcinoma cells did not display reduced NAMPT activity and NAD content even at a FK866 concentration 10-fold of the EC50 (EC50 8.2nM) indicating a lower sensitivity of non-cancerous cells to FK866. This has also been described for normal blood cells [6;7]. Therefore, FK866 represents an interesting compound in cancer cell therapy as it progressively exhausts NAD content in cells with a high NAD turnover that mainly rely on nicotinamide and the NAMPT-mediated NAD salvage pathway as source of NAD. Cancer cells have a significantly higher NAD turnover than normal cells to sustain their rapid proliferation, relative genomic instability, permanently ongoing DNA repair, increased aerobic glycolysis and increased activity of NAD-dependent deacetylases [1;12;13]. This is in line with results of our previous study showing that the expression of SIRT1, a NAD-dependent deacetylase, was significantly higher in hepatocarcinoma cells than in non-cancerous hepatocytes [9].

In this study we could demonstrate that NAMPT inhibition by FK866 led to a sharp decline of intracellular ATP levels and therefore induced energy stress. As a key physiological energy sensor, AMPK is a major regulator of cellular energy homeostasis that coordinates multiple metabolic pathways to balance energy supply [24]. Several studies have shown that AMPK activators exhibit inhibitory effects on cancer cell growth [32;33]. AMPK is known to phosphorylate and activate tuberous sclerosis complex (TSC)2, a negative regulator of mTOR [34]. Therefore, the AMPK/mTOR pathway serves as a signalling nexus for regulating cellular metabolism, energy homeostasis, and cell growth, and dysregulation of each pathway may contribute to the development of HCC [20;26]. Since the discovery that the mTOR pathway is hyperactivated in many cancers including HCC [25;26;31;35], there is a great interest in finding molecular pathways and novel compounds that target AMPK/mTOR signalling as novel treatment option for HCC. We could show that components of the mTORC1 cascade were significantly higher expressed in hepatocarcinoma cells than in non-cancerous hepatocytes. Additionally, our data revealed that the activation of AMPK was significantly decreased in hepatocarcinoma cells. Reduced AMPK activity has also been detected in primary human breast cancer [36] and lymphoma [21] cells. Thus, a dysregulated AMPK activity may represent an important regulatory step during tumor initiation and progression, allowing cancer cells to gain a metabolic growth advantage by enhancing aerobic glycolysis (Warburg effect) [21]. We made the intriguing discovery that FK866 acts as an AMPK activator in cancer cells potentially through its ability to induce cellular energy stress. Activation of AMPK was associated with a down regulation of the mTORC1 pathway. All FK866 induced effects could be completely reversed by NMN suggesting that these effects were mediated by NAD. mTORC1 inhibition led to decreased activation of its two downstream targets, 70S ribosomal protein S6 kinase (p70S6K) and the eukaryotic initiation factor 4E binding protein 1 (4E-BP1). p70S6K and 4E-BP1 are major regulators of protein translation and cellular growth [35]. This contradicts a study performed in neuronal cells where FK866 or a NAMPT knock down was shown to reduce AMPK activation [37]. However, this can be explained by the use of non-cancerous neuronal cells compared to cancer cells in our study.

In summary, our study showed the importance of the NAMPT-mediated NAD salvage pathway for energy homeostasis in hepatocarcinoma cells. Furthermore, FK866-induced NAMPT inhibition led to activation of AMPK and inhibition of mTOR signalling suggesting a putative use of FK866 alone or as a chemotherapeutic sensitizing drug to reduce cancer cell growth. In every case of potential therapeutic use, administration of NMN as antidote may be useful to modulate or counteract FK866 toxicity. Only early stages of HCC are curable with today’s treatment protocols, therefore new therapeutic strategies are urgently needed and NAMPT inhibition represents a potential novel treatment approach.

 

 

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