NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by ß-lapachone.

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD(+) synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD(+) pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, ß-lapachone (ß-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. ß-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD(+) consumption. Synergy with FK866+ß-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD(+) synthesis while increasing NAD(+) consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)(+) depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, µ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD(+)-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD(+) synthesis and ß-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD(+) pools in cancer cells to sensitize them to ROS-mediated cell death by ß-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.

Metabolic reprogramming during TGFß1-induced epithelial-to-mesenchymal transition.

Metastatic progression, including extravasation and micrometastatic outgrowth, is the main cause of cancer patient death. Recent studies suggest that cancer cells reprogram their metabolism to support increased proliferation through increased glycolysis and biosynthetic activities, including lipogenesis pathways. However, metabolic changes during metastatic progression, including alterations in regulatory gene expression, remain undefined. We show that transforming growth factor beta 1 (TGFß1)-induced epithelial-to-mesenchymal transition (EMT) is accompanied by coordinately reduced enzyme expression required to convert glucose into fatty acids, and concomitant enhanced respiration. Overexpressed Snail1, a transcription factor mediating TGFß1-induced EMT, was sufficient to suppress carbohydrate-responsive-element-binding protein (ChREBP, a master lipogenic regulator), and fatty acid synthase (FASN), its effector lipogenic gene. Stable FASN knockdown was sufficient to induce EMT, stimulate migration and extravasation in vitro. FASN silencing enhanced lung metastasis and death in vivo. These data suggest that a metabolic transition that suppresses lipogenesis and favors energy production is an essential component of TGFß1-induced EMT and metastasis.

Loss of the tumor suppressor Hace1 leads to ROS-dependent glutamine addiction.

Cellular transformation is associated with altered glutamine (Gln) metabolism. Tumor cells utilize Gln in the tricarboxylic acid (TCA) cycle to maintain sufficient pools of biosynthetic precursors to support rapid growth and proliferation. However, Gln metabolism also generates NADPH, and Gln-derived glutamate is used for synthesis of glutathione (GSH). As both NADPH and GSH are antioxidants, Gln may also contribute to redox balance in transformed cells. The Hace1 E3 ligase is a tumor suppressor inactivated in diverse human cancers. Hace1 targets the Rac1 GTPase for degradation at Rac1-dependent NADPH oxidase complexes, blocking superoxide generation by the latter. Consequently, loss of Hace1 increases reactive oxygen species (ROS) levels in vitro and in vivo. Given the link between Hace1 loss and increased ROS, we investigated whether genetic inactivation of Hace1 alters Gln metabolism. We demonstrate that mouse embryonic fibroblasts (MEFs) derived from Hace1(-/-) mice are highly sensitive to Gln withdrawal, leading to enhanced cell death compared with wild-type (wt) MEFs, and Gln depletion or chemical inhibition of Gln uptake blocks soft agar colony formation by Hace1(-/-) MEFs. Hace1(-/-) MEFs exhibit increased Gln uptake and ammonia secretion, and metabolic labeling using (13)C-Gln revealed that Hace1 loss increases incorporation of Gln carbons into the TCA cycle intermediates. Gln starvation markedly increases ROS levels in Hace1(-/-) but not in wt MEFs, and treatment with the antioxidant N-acetyl cysteine or the TCA cycle intermediate oxaloacetate efficiently rescues Gln starvation-induced ROS elevation and cell death in Hace1(-/-) MEFs. Finally, Gln starvation increases superoxide levels in Hace1(-/-) MEFs, and NADPH oxidase inhibitors block the induction of superoxide and cell death by Gln starvation. Together, these results suggest that increased ROS production due to Hace1 loss leads to Gln addiction as a mechanism to cope with increased ROS-induced oxidative stress.

Uncoupling hypoxia signaling from oxygen sensing in the liver results in hypoketotic hypoglycemic death.

As the ultimate electron acceptor in oxidative phosphorylation, oxygen plays a critical role in metabolism. When oxygen levels drop, heterodimeric hypoxia-inducible factor (Hif) transcription factors become active and facilitate adaptation to hypoxia. Hif regulation by oxygen requires the protein von Hippel-Lindau (pVhl) and pVhl disruption results in constitutive Hif activation. The liver is a critical organ for metabolic homeostasis, and Vhl inactivation in hepatocytes results in a Hif-dependent shortening in life span. While albumin-Cre;Vhl(F/F) mice develop hepatic steatosis and impaired fatty acid oxidation, the variable penetrance and unpredictable life expectancy has made the cause of death elusive. Using a system in which Vhl is acutely disrupted and a combination of ex vivo liver perfusion studies and in vivo oxygen measurements, we demonstrate that Vhl is essential for mitochondrial respiration in vivo. Adenovirus-Cre mediated acute Vhl disruption in the liver caused death within days. Deprived of pVhl, livers accumulated tryglicerides and circulating ketone and glucose levels dropped. The phenotype was reminiscent of inborn defects in fatty acid oxidation and of fasted PPARα-deficient mice and while death was unaffected by pharmacologic PPARα activation, it was delayed by glucose administration. Ex vivo liver perfusion analyses and acylcarnitine profiles showed mitochondrial impairment and a profound inhibition of liver ketone and glucose production. By contrast, other mitochondrial functions, such as ureagenesis, were unaffected. Oxygen consumption studies revealed a marked suppression of mitochondrial respiration, which, as determined by magnetic resonance oximetry in live mice, was accompanied by a corresponding increase in liver pO(2). Importantly, simultaneous inactivation of Hif-1ß suppressed liver steatosis and rescued the mice from death. These data demonstrate that constitutive Hif activation in mice is sufficient to suppress mitochondrial respiration in vivo and that no other pathway exists in the liver that can allow oxygen utilization when Hif is active precluding thereby metabolic collapse.

Q's next: the diverse functions of glutamine in metabolism, cell biology and cancer.

Several decades of research have sought to characterize tumor cell metabolism in the hope that tumor-specific activities can be exploited to treat cancer. Having originated from Warburg's seminal observation of aerobic glycolysis in tumor cells, most of this attention has focused on glucose metabolism. However, since the 1950s cancer biologists have also recognized the importance of glutamine (Q) as a tumor nutrient. Glutamine contributes to essentially every core metabolic task of proliferating tumor cells: it participates in bioenergetics, supports cell defenses against oxidative stress and complements glucose metabolism in the production of macromolecules. The interest in glutamine metabolism has been heightened further by the recent findings that c-myc controls glutamine uptake and degradation, and that glutamine itself exerts influence over a number of signaling pathways that contribute to tumor growth. These observations are stimulating a renewed effort to understand the regulation of glutamine metabolism in tumors and to develop strategies to target glutamine metabolism in cancer. In this study we review the protean roles of glutamine in cancer, both in the direct support of tumor growth and in mediating some of the complex effects on whole-body metabolism that are characteristic of tumor progression.

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

Determination of L1 retrotransposition kinetics in cultured cells.

L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase ( neo ) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1(RP)and L1.3. L1(RP)retrotransposed in the EGFP assay at a rate of approximately 0.5% of transfected cells/day, approximately 3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications.

Analysis of the promoter from an expanding mouse retrotransposon subfamily.

The mouse genome contains several subfamilies of the retrotransposon L1. One subfamily, TF, contains 4000-5000 full-length members and is expanding due to retrotransposition of a large number of active elements. Here we studied the TF 5' untranslated region (UTR), which contains promoter activity required for subfamily expression. Using reporter assays, we show that promoter activity is derived from TF-specific monomer sequences and is proportional to the number of monomers in the 5' UTR. These data suggest that nearly all full-length TF elements in the mouse genome are currently competent for expression. We aligned the sequences of 53 monomers to generate a consensus TF monomer and determined that most TF elements are truncated near a potential binding site for a transcription initiation factor. We also determined that much of the sequence variation among TF monomers results from transition mutations at CpG dinucleotides, suggesting that genomic TF 5' UTRs are methylated at CpGs.

Exon shuffling by L1 retrotransposition.

Long interspersed nuclear elements (LINE-1s or L1s) are the most abundant retrotransposons in the human genome, and they serve as major sources of reverse transcriptase activity. Engineered L1s retrotranspose at high frequency in cultured human cells. Here it is shown that L1s insert into transcribed genes and retrotranspose sequences derived from their 3' flanks to new genomic locations. Thus, retrotransposition-competent L1s provide a vehicle to mobilize non-L1 sequences, such as exons or promoters, into existing genes and may represent a general mechanism for the evolution of new genes.

Rapid amplification of a retrotransposon subfamily is evolving the mouse genome.

Retrotransposition affects genome structure by increasing repetition and producing insertional mutations. Dispersion of the retrotransposon L1 throughout mammalian genomes suggests that L1 activity might be an important evolutionary force. Here we report that L1 retrotransposition contributes to rapid genome evolution in the mouse, because a number of L1 sequences from the T(F) subfamily are retrotransposition competent. We show that the T(F) subfamily is large, young and expanding, containing approximately 4,800 full-length members in strain 129. Eleven randomly isolated, full-length T(F) elements averaged 99.8% sequence identity to each other, and seven of these retrotransposed in cultured cells. Thus, we estimate that the mouse genome contains approximately 3,000 active T(F) elements, 75 times the estimated number of active human L1s. Moreover, as T(F) elements are polymorphic among closely related mice, they have retrotransposed recently, implying rapid amplification of the subfamily to yield genomes with different patterns of interspersed repetition. Our data show that mice and humans differ considerably in the number of active L1s, and probably differ in the contribution of retrotransposition to ongoing sequence evolution.

Full-length L1 elements have arisen recently in the same 1-kb region of the gorilla and human genomes.

New copies of the mammalian retrotransposon L1 arise in the germline at an undetermined rate. Each new L1 copy appears at a specific evolutionary time point that can be estimated by phylogenetic analysis. In humans, the active L1 sequence L1.2 resides at the genomic locus LRE1. Here we analyzed the region surrounding the LRE1 locus in humans and gorillas to determine the evolutionary history of the region and to estimate the age of L1.2. We found that the region was composed of an ancient L1, L1Hs-Lrg, which was significantly divergent from all other L1 sequences available in the databases. We also determined that L1.2 was absent from the gorilla genome and arose in humans after the divergence of gorilla and human lineages. In the gorilla LRE1 region, we discovered a different full-length L1 element, L1Gg-1, which was allelic and present at a high gene frequency in gorillas but absent from other primates. We determined the nucleotide sequence of L1Gg-1 and found that it was 98% identical to L1.2, suggesting a close relationship between active L1s in gorillas and humans.

An actively retrotransposing, novel subfamily of mouse L1 elements.

Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations. Recent insertions of two full-length L1s, L1spa and L1Orl, caused the disease phenotypes of the spastic and Orleans reeler mice respectively. Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily. We have named this new subfamily TF (for transposable) and show that many full-length members of this family are present in the mouse genome. The TF 5' untranslated region has promoter activity, and TF-type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition. Both L1spa and L1Orl have reverse transcriptase activity in a yeast-based assay and retrotranspose at high frequency in cultured cells. Together, our data indicate that the TF subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome.

Many human L1 elements are capable of retrotransposition.

Using a selective screening strategy to enrich for active L1 elements, we isolated 13 full-length elements from a human genomic library. We tested these and two previously-isolated L1s (L1.3 and L1.4) for reverse transcriptase (RT) activity and the ability to retrotranspose in HeLa cells. Of the 13 newly-isolated L1s, eight had RT activity and three were able to retrotranspose. L1.3 and L1.4 possessed RT activity and retrotransposed at remarkably high frequencies. These studies bring the number of characterized active human L1 elements to seven. Based on these and other data, we estimate that 30-60 active L1 elements reside in the average diploid genome.

High frequency retrotransposition in cultured mammalian cells.

We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were variably 5' truncated, ended in poly(A) tracts, and were flanked by target-site duplications or short deletions. Point mutations in conserved domains of the L1.2-encoded proteins reduced retrotransposition by 100- to 1000-fold. Remarkably, L1.2 also retrotransposed in a mouse cell line, suggesting a potential role for L1-based vectors in random insertional mutagenesis.

Human enteric defensins. Gene structure and developmental expression.

Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5'-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19 24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.