p21-activated kinase 1: PAK'ed with potential

The p21-activated kinases (PAKs) are central players in growth factor signaling networks and morphogenetic processes that control proliferation, cell polarity, invasion and actin cytoskeleton organization. This raises the possibility that interfering with PAK activity may produce significant anti-tumor activity. In this perspective, we summarize recent data concerning the contribution of the PAK family member, PAK1, in growth factor signaling and tumorigenesis. We further discuss mechanisms by which inhibition of PAK1 can arrest tumor growth and promote cell apoptosis, and the types of cancers in which PAK1 inhibition may hold promise

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable

<div><p>Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth <i>in vivo</i>. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.</p></div

Ability to combat ROS varies across cell lines.

<p>NAD, NADH, NADP and NADPH in each cell line were quantitatively determined at 24, 48 and 72 hours following exposure to 0.4 μM GNE-617 (Ave ± SD, n = 5). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed). B) Schematic showing the cycle of oxidation and reduction of GSG and GSSG. C) Each cell line was exposed to 0.2 μM GNE-617 for 48 hours and labeled with [13C-6]glucose and levels of newly synthesized GSH and GSSG were determined (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. D) ROS levels were determined in each cell line at 24, 48 and 72 hours following exposure to 0.2 μM GNE-617 (shown is the Ave, n = 2). E) Levels of AMP and ATP in each cell line were quantitatively determined at 24, 48 and 72 hours following exposure to 0.4 μM GNE-617 (Ave ± SD, n = 5), and the ratio of the average AMP/ATP levels are shown. F) Western blot analysis showing levels of AMPK and activated AMPK (T172) at 24, 48 and 72 hours following exposure to 0.4 μM GNE-617. Full-length western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.s002" target="_blank">S2D Fig</a>.</p

Sensitive cell lines show defects in purine and pyrimidine biosynthesis.

<p>A-B) Cells were treated with 0.4 μM GNE-617 and levels of UMP and IMP (A), GMP and UDP (B) were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.g002" target="_blank">Fig 2A</a> (Ave ± SD, n = 5). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. C) Level of mono-nucleotides (n = 8), diphosphorylated-nucleotides (n = 7) and triphosphorylated-nucleotides (n = 7) in cells were determined following treatment with 0.4 μM GNE-617 (shown is the average and SD for each group). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant.</p

A549 cells increase reliance on the PPP following NAMPT inhibition.

<p>Schematic of key metabolites in glycolysis. B) Cells were treated with 0.4 μM GNE-617 and levels of each metabolite was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.g002" target="_blank">Fig 2A</a> (Ave ± SD, n = 5). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. C) Schematic of key metabolites in the PPP and in nucleotide biosynthesis. D) Cells were treated as in part B and levels of each metabolite was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.g002" target="_blank">Fig 2A</a> (Ave ± SD, n = 5). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. E) Each cell line was exposed to 0.4 μM GNE-617 for 48 hours and labeled with [13C-6]glucose. Levels of newly synthesized pentose-5-phosphate were determined relative to total metabolite levels (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. F) Schematic of the PPP with the step catalyzed by G6PD. Cells were treated with siRNA directed against G6PD and protein levels were examined after 5 days. Full-length western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.s002" target="_blank">S2C Fig</a>. G) Cells were treated either with siControl or siRNA directed against G6PD for 24 hours, and then treated with different concentrations of GNE-617 for an additional 4 days at which point cell viability was assessed (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant.</p

GNE-617 reduces NAD levels in sensitive and resistant cell lines.

<p>A) Structure of GNE-617 with its biochemical IC50 for NAMPT. B) Correlation between NAMPT protein levels and sensitivity to GNE-617 (IC50) determined in a 4-day viability assay. IC50 values were previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.ref015" target="_blank">15</a>]; protein levels were quantified from western blots shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.s001" target="_blank">S1A Fig</a>. Full-length western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.s002" target="_blank">S2A Fig</a>. C) NAD levels in each of the four cell lines was determined by mass spectrometry at various time after treatment with 0.4 μM GNE-617 (n = 3, ± SD). Insert shows data with a different y-axis scale at 72 and 96 hours. * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed). D) Relative viability of each cell line, as assessed by a CyQuant readout, was determined at various time after treatment with 0.4 μM GNE-617 (n = 3, ± SD). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed). E) LC-KJ cells were treated with a dose response of GNE-617 and viability determined after 7-days (CyQuant)(n = 3, ± SD). F) NAMPT protein levels were determined in each cell line at the indicated times after exposure to 0.4 μM GNE-617. Full western blots for this image are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164166#pone.0164166.s002" target="_blank">S2B Fig</a>. Shown below is quantitation of NAMPT levels (relative to GAPDH protein levels) for each time point.</p

H1334 cells increase OXPHOS in response to NAD depletion.

<p>Schematic of carbon flow from glucose or glutamine into OXPHOS. B) Each cell line was exposed to 0.2 μM GNE-617 for 48 hours and labeled with [13C-6]glucose. Levels of newly synthesized citrate (CIT) and α-ketoglutarate (α-KG) were determined (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. C) Each cell line was exposed to 0.2 μM GNE-617 for 48 hours and labeled with [13C-5]glutamine. Levels of newly synthesized glutamate (GLU), α-ketoglutarate (α-KG), citrate (CIT) and malate (MAL) were determined (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant. D) Each cell line was exposed to 0.2 μM GNE-617 for 48 hours and labeled with [13C-5]glutamine. Levels of newly synthesized malate (MAL) and aspartate (ASP) generated by reductive metabolism (quantify the M+3 isotopomers) were determined (Ave ± SD, n = 3). * indicates a p-value of <0.05, and statistical analysis was performed using a Mann Whitney t-test (two-tailed); “ns” indicates not-statistically significant.</p

Reduction of NAD results in large metabolic changes.

<p>A) Levels of 97 different metabolites were assessed in each cell line as described in the methods section at 24, 48 or 72 hours after treatment with 0.4 μM GNE-617 (n = 5, average of each metabolite is shown). Shown is the log2-fold change for the level of each metabolite relative to its level in untreated cells. B) Changes in different categories of metabolites at 72 hours in each cell line, as determined in panel A.</p