Sorafenib is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs). Resistance to sorafenib develops frequently and could be mediated by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin (SIRT)1. We aimed to test whether sorafenib efficacy is influenced by cellular NAD levels and NAD-dependent SIRT1 function. We analyzed sorafenib effects on apoptosis induction, NAD salvage, mitochondrial function, and related signaling pathways in HCC cell lines (HepG2, Hep3B, und HUH7) overexpressing SIRT1 or supplemented with the NAD metabolite nicotinamide mononucleotide (NMN) compared to controls. Treatment of HCC cell lines with sorafenib dose-dependently induced apoptosis and a significant decrease in cellular NAD concentrations. The SIRT1 protein was downregulated in HUH7 cells but not in Hep3B cells. After sorafenib treatment, mitochondrial respiration in permeabilized cells was lower, citrate synthase activity was attenuated, and cellular adenosine triphosphate (ATP) levels were decreased. Concomitant to increased phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), sorafenib treatment led to decreased activity of the mechanistic target of rapamycin (mTOR), indicative of energy deprivation. Transient overexpression of SIRT1, as well as NAD repletion by NMN, decreased sorafenib-induced apoptosis. We can, therefore, conclude that sorafenib influences the NAD/SIRT1/AMPK axis. Overexpression of SIRT1 could be an underlying mechanism of resistance to sorafenib treatment in HCC.

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14C–nicotinamide. Lipids were stained by Oil red O staining. Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.

Germline mutations in the tumor suppressor gene PTEN cause PTEN Hamartoma Tumor Syndrome (PHTS). Pediatric patients with PHTS frequently develop lipomas. Treatment attempts with the mTORC1 inhibitor rapamycin were unable to reverse lipoma growth. Recently, lipomas associated with PIK3CA-related overgrowth syndrome were successfully treated with the novel PI3K inhibitor alpelisib. Here, we tested whether alpelisib has growth-restrictive effects and induces cell death in lipoma cells. We used PTEN-haploinsufficient lipoma cells from three patients and treated them with alpelisib alone or in combination with rapamycin. We tested the effect of alpelisib on viability, proliferation, cell death, induction of senescence, adipocyte differentiation, and signaling at 1–100 µM alpelisib. Alpelisib alone or in combination with rapamycin reduced proliferation in a concentration- and time-dependent manner. No cell death but an induction of senescence was detected after alpelisib incubation for 72 h. Alpelisib treatment led to a reduced phosphorylation of AKT, mTOR, and ribosomal protein S6. Rapamycin treatment alone led to increased AKT phosphorylation. This effect could be reversed by combining rapamycin with alpelisib. Alpelisib reduced the size of lipoma spheroids by attenuating adipocyte differentiation. Since alpelisib was well tolerated in first clinical trials, this drug alone or in combination with rapamycin is a potential new treatment option for PHTS-related adipose tissue overgrowth.