20), increased mitochondrial area (mean area 0.86 m2 in the ERCC1-WT cells versus 1.28 m2 in the ERCC1-KO cells, = 0.0002), and abnormal cristae (including distortion, stacking, or whirling) (Physique 7, A and B). Open in a separate window Figure 7 ERCC1-deficient cells present an abnormal mitochondrial structure associated CD295 with decreased respiratory capacity and increased glycolysis.(A) Representative transmission electron microscopy (TEM) pictures of the A549 ERCC1-WT, ERCC1-Hez, and ERCC1-KO cell lines. targeting DNA repair/metabolic crosstalks for cancer therapy. or had been inactivated by zinc finger gene targeting (5, 13). This panel, derived from the A549 NSCLC cell line, includes 8 cell populations: 1 parental ERCC1-WT, 1 ERCC1-heterozygous (Hez), 2 ERCC1-deficient (KO1 and KO2), and 4 rescue ERCC1-KO in which one of the 4 ERCC1 isoforms has been stably reintroduced (isoforms 201, 202, 203, or 204, respectively). As previously reported, this model is usually representative of the clinical and biological characteristics associated HS80 HS80 with ERCC1 deficiency (4, 5, 13). The ERCC1-proficient parental cell line and 1 ERCC1-deficient clone were produced in culture media only differing by isotopic amino acids (aa) (Physique 1A): the light culture contained unlabeled aa (Lys 0, HS80 Arg 0) whereas the heavy culture contained isotope-labeled aa (Lys 8, Arg 10). After 8 doubling occasions, proteins were extracted, digested, off-gel fractionated, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A forward and a reverse experiment in which isotope labeling was inverted between cell lines were performed in parallel in order to minimize false-positive results. This approach identified 45 hits (out of 5,051 known proteins), which were significantly differentially expressed between the ERCC1-WT and the ERCC1-KO cells (Physique 1B). Among these hits, 2 proteins were involved in the NAD+ biosynthesis pathway: NAMPT and NNMT (nicotinamide N-methyltransferase) (2- and 3.1-fold decrease in ERCC1-KO cells respectively, < 10C6) (Figure 1B). Three subunits of complex IV (cytochrome c oxidase) of the mitochondrial respiratory chain (COX4I1, COX5B, and COX6C) were also significantly decreased in ERCC1-KO populations (1.3-, 2.1-, and 2.5-fold, respectively, < 0.0001). Open in a separate window Physique 1 SILAC-based proteomic analysis identifies NAMPT decrease as a potentially targetable node in ERCC1-deficient clones.(A) Experimental workflow for quantitative proteomic analysis using SILAC. Two impartial experiments in the ERCC1-WT and ERCC1-KO cell lines from the A549 model with inverted labeling of the cell lines (forward and reverse experiments) were HS80 set up and run in parallel in order to limit the number of false-positive hits by having an internal control (= 2 impartial samples for each model). Protein lysates were mixed in a 1:1 proportion between the heavy- and light-labeled cells. After protein digestion and off-gel fractionation, fractions were analyzed by LC-MS/MS, and data analysis was performed by MaxQuant. (B) Scatter plot of the ratio of heavy over light amino acids normalized to the reverse (R) or forward (F) experiment, following a log2 normalization. Hits reaching significance (Benjamini-Hochberg adjusted value < 0.05) in both experiments are depicted in red. FP, false-positive. (C) Canonical NAD+ biosynthetic pathways. In humans, most NAD+ is usually synthesized from nicotinamide (NAm) through the salvage (recycling) pathway. NAMPT (NAm phosphoribosyltransferase) catalyzes the rate-limiting step in this pathway. Sirtuins (SIRTs) and poly(ADP-ribose) polymerase (PARPs) enzymes catalyze reactions that consume NAD+. The reaction scheme is based on the Biocyc pathway map for homo sapiens NAD+ biosynthesis (http://biocyc.org/), and adapted HS80 from Kim et al (15). NAPRT, nicotinic acid phosphoribosyltransferase; QPRT, quinolinic acid phosphoribosyltransferase; NMNATs, NMN adenylyltransferases; NADS, NAD+ synthetase. NAMPT catalyzes the rate-limiting step of the NAD+ biosynthesis salvage pathway (Physique 1C), thereby playing a crucial role in the maintenance of intracellular NAD+, an essential cofactor involved in several cellular reactions, including cellular energetics and DNA repair (14, 15). Growing evidence indicates that NAMPT exerts oncogenic activity in the context of cancer. Several NAMPT inhibitors have already been evaluated in early phase trials (16), with others being currently developed (17). NAMPT therefore appeared to be a targetable node in ERCC1-defective tumors, and we decided to further focus on this target. Decreased NAMPT expression is found in several ERCC1-deficient NSCLC models and in immunohistochemical study of lung adenocarcinomas. Because high-throughput experiments are.