Lipoprotein Lipase as a Candidate Target for Cancer Prevention/Therapy

Epidemiological studies have shown that serum triglyceride (TG) levels are linked with risk of development of cancer, including colorectal and pancreatic cancers, and their precancerous lesions. Thus, it is assumed that serum TG plays an important role in carcinogenesis, and the key enzyme lipoprotein lipase (LPL), which catalyzes the hydrolysis of plasma TG, may therefore be involved. Dysregulation of LPL has been reported to contribute to many human diseases, such as atherosclerosis, chylomicronaemia, obesity, and type 2 diabetes. Recently, it has been reported that LPL gene deficiency, such as due to chromosome 8p22 loss, LPL gene polymorphism, and epigenetic changes in its promoter region gene, increases cancer risk, especially in the prostate. In animal experiments, high serum TG levels seem to promote sporadic/carcinogen-induced genesis of colorectal and pancreatic cancers. Interestingly, tumor suppressive effects of LPL inducers, such as PPAR ligands, NO-1886, and indomethacin, have been demonstrated in animal models. Moreover, recent evidence that LPL plays important roles in inflammation and obesity implies that it is an appropriate general target for chemopreventive and chemotherapeutic agents

PPARγ Ligand as a Promising Candidate for Colorectal Cancer Chemoprevention: A Pilot Study

Activating synthetic ligands for peroxisome proliferator-activated receptor gamma (PPARγ), such as pioglitazone, are commonly used to treat persons with diabetes mellitus with improvement of insulin resistance. Several reports have clearly demonstrated that PPARγ ligands could inhibit colorectal cancer cell growth and induce apoptosis. Meanwhile, aberrant crypt foci (ACF) have come to be established as a biomarker of the risk of CRC in azoxymethane-treated mice and rats. In humans, ACF can be detected using magnifying colonoscopy. Previously, CRC and adenoma were used as a target for chemopreventive agents, but it needs a long time to evaluate, however, ACF can be a surrogate marker of CRC even for a brief period. In this clinical study, we investigated the chemopreventive effect of pioglitazone on the development of human ACF as a surrogate marker of CRC. Twenty-nine patients were divided into two groups, 20 were in the endoscopically normal control group and 9 were in the pioglitazone (15 mg/day) group, and ACF and adenoma were examined before and after 1-month treatment. The number of ACF was significantly decreased (5.8 ± 1.1 to 3.3 ± 2.3) after 1 month of pioglitazone treatment, however, there was no significant change in the number of crypts/ACF or in the number and size of adenomas. Pioglitazone may have a clinical application as a cancer-preventive drug. This investigation is just a pilot study, therefore, further clinical studies are needed to show that the PPARγ ligand may be a promising candidate as a chemopreventive agent for colorectal carcinogenesis

Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells

BACKGROUND: Many lines of evidence suggest that poly(ADP-ribose) polymerase-1 (Parp-1) is involved in transcriptional regulation of various genes as a coactivator or a corepressor by modulating chromatin structure. However, the impact of Parp-1-deficiency on the regulation of genome-wide gene expression has not been fully studied yet. RESULTS: We employed a microarray analysis covering 12,488 genes and ESTs using mouse Parp-1-deficient (Parp-1(-/-)) embryonic stem (ES) cell lines and the livers of Parp-1(-/- )mice and their wild-type (Parp-1(+/+)) counterparts. Here, we demonstrate that of the 9,907 genes analyzed, in Parp-1(-/- )ES cells, 9.6% showed altered gene expression. Of these, 6.3% and 3.3% of the genes were down- or up-regulated by 2-fold or greater, respectively, compared with Parp-1(+/+ )ES cells (p < 0.05). In the livers of Parp-1(-/- )mice, of the 12,353 genes that were analyzed, 2.0% or 1.3% were down- and up-regulated, respectively (p < 0.05). Notably, the number of down-regulated genes was higher in both ES cells and livers, than that of the up-regulated genes. The genes that showed altered expression in ES cells or in the livers are ascribed to various cellular processes, including metabolism, signal transduction, cell cycle control and transcription. We also observed expression of the genes involved in the pathway of extraembryonic tissue development is augmented in Parp-1(-/- )ES cells, including H19. After withdrawal of leukemia inhibitory factor, expression of H19 as well as other trophoblast marker genes were further up-regulated in Parp-1(-/- )ES cells compared to Parp-1(+/+ )ES cells. CONCLUSION: These results suggest that Parp-1 is required to maintain transcriptional regulation of a wide variety of genes on a genome-wide scale. The gene expression profiles in Parp-1-deficient cells may be useful to delineate the functional role of Parp-1 in epigenetic regulation of the genomes involved in various biological phenomena

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

金沢大学医薬保健研究域薬学系The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites. © 2006 The Japanese Biochemical Society

Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2.

細胞老化から癌化への変換のカギとなる解糖系制御機構解明に成功 -代謝を標的とした新しい抗がん剤開発に期待-. 京都大学プレスリリース. 2014-02-24.Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage-induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM

Stress-activated mitogen-activated protein kinases c-Jun NH 2-terminal kinase and p38 target Cdc25B for degradation

金沢大学医薬保健研究域薬学系Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFβ-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint. ©2009 American Association for Cancer Research

Binding of 14-3-3β but not 14-3-3σ controls the cytoplasmic localization of CDC25B: Binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3β, 14-3-3ε and 14-3-3σ, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3β bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3σ bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3β drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3σ and CDC25B did not affect the subeellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3β, even when other putative 14-3-3 binding sites were mutated. 14-3-3ε resembled 14-3-3β with regard to its binding to CDC25B and the control of CDC25B subcellujar localization. The results of the present study indicite that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3σ has effects on CDC25B other than the control of its subcellular localization

SCFβTrCP mediates stress-activated MAPK-induced Cdc25B degradation

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCFβTrCP-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH2-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCFβTrCP E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D94AGLCMDSPSP104. Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCFβTrCP, and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E82SS to D94AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E82SS to SPSP104 region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCFβTrCP requires the phosphorylation of S101PS103P in the βTrCP-binding-motif-like and adjacent PEST-like sequences. © 2011. Published by The Company of Biologists Ltd