Design and synthesis of versatile GSTP1-specific fluorogenic substrates for the highly sensitive detection of GSTP1 activity in living cells.

Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.

SMG6 regulates DNA damage and cell survival in Hippo pathway kinase LATS2-inactivated malignant mesothelioma.

Many genes responsible for Malignant mesothelioma (MM) have been identified as tumor suppressor genes and it is difficult to target these genes directly at a molecular level. We searched for the gene which showed synthetic lethal phenotype with LATS2, one of the MM causative genes and one of the kinases in the Hippo pathway. Here we showed that knockdown of SMG6 results in synthetic lethality in LATS2-inactivated cells. We found that this synthetic lethality required the nuclear translocation of YAP1 and TAZ. Both are downstream factors of the Hippo pathway. We also demonstrated that this synthetic lethality did not require SMG6 in nonsense-mediated mRNA decay (NMD) but in regulating telomerase reverse transcriptase (TERT) activity. In addition, the RNA-dependent DNA polymerase (RdDP) activity of TERT was required for this synthetic lethal phenotype. We confirmed the inhibitory effects of LATS2 and SMG6 on cell proliferation in vivo. The result suggests an interaction between the Hippo and TERT signaling pathways. We also propose that SMG6 and TERT are novel molecular target candidates for LATS2-inactivated cancers such as MM.

Pi-Class Glutathione S-transferase (GSTP1)-Selective Fluorescent Probes for Multicolour Imaging with Various Cancer-Associated Enzymes.

Pi-class glutathione S-transferase (GSTP1) is highly expressed in a wide variety of human cancer tissues compared to the corresponding normal counterpart. Therefore, GSTP1 is a potential target enzyme for overcoming resistance to chemotherapeutic agents or visualizing specific lesions such as cancer. Here, we present orange and red fluorescence-emitting probes selective for GSTP1. Carbofluorescein and TokyoMagenta fluorophores were modified with a previously described GSTP1-selective chromogenic compound to generate orange and red fluorescence probes, respectively. Of these probes, Ps-CF, the orange fluorescence-emitting probe, was confirmed to be highly specific for detecting GSTP1 exogenously or endogenously expressed in various cancer cells. Additionally, it was demonstrated that Ps-CF is applicable for the simultaneous detection of GSTP1 and another cancer-associated enzyme by using a green fluorescence emitting γ-glutamyl transpeptidase (GGT) probe. In conclusion, the fluorescent probes developed in this study enable the simultaneous detection of multiple tumour markers such as GSTP1 with other cancer-associated enzymes by concurrently using spectrally distinguished fluorescent probes, potentially broadening the scope of cancer detection.

A Covalent Inhibitor for Glutathione S-Transferase Pi (GSTP1-1 ) in Human Cells.

Glutathione S-transferase π (GSTP1-1 ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP1-1 both in vitro and in cellulo with a long duration of action.

A covalent G-site inhibitor for glutathione S-transferase Pi (GSTP1-1).

We herein report the first covalent G-site-binding inhibitor for GST, GS-ESF (1), which irreversibly inhibited the GSTP1-1 function. LC-MS/MS and X-ray structure analyses of the covalently linked GST-inhibitor complex suggested that 1 reacted with Tyr108 of GSTP1-1. The mechanism of covalent bond formation was discussed based on MD simulation results.

Statin suppresses Hippo pathway-inactivated malignant mesothelioma cells and blocks the YAP/CD44 growth stimulatory axis.

Malignant mesothelioma (MM) frequently exhibits Hippo signaling pathway inactivation (HPI) mainly due to NF2 and/or LATS2 mutations, which leads to the activation of YAP transcriptional co-activator. Here, we show antitumor effects of statin on MM cells with HPI, through the interplay of the mevalonate and Hippo signaling pathways. Statin attenuated proliferation and migration of MM cells harboring NF2 mutation by accelerating YAP phosphorylation/inactivation. CD44 expression was decreased by statin, in parallel with YAP phosphorylation/inactivation. Importantly, we discovered that YAP/TEAD activated CD44 transcription by binding to the CD44 promoter at TEAD-binding sites. On the other hand, CD44 regulated Merlin phosphorylation according to cell density and sequentially promoted YAP transcriptional co-activator, suggesting that CD44 plays two pivotal functional roles as an upstream suppressor of the Hippo pathway and one of downstream targets regulated by YAP/TEAD. Moreover, the YAP/CD44 axis conferred cancer stem cell (CSC)-like properties in MM cells leading to chemoresistance, which was blocked by statin. Together, our findings suggest that YAP mediates CD44 up-regulation at the transcriptional level, conferring CSC-like properties in MM cells, and statin represents a potential therapeutic option against MM by inactivating YAP.

SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells.

Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma.

Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.

Malignant mesothelioma (MM) shows inactivation of the BRCA1-associated protein 1 (BAP1) gene. In this study, we found BAP1 mutations in 5 (26%) of the 19 cell lines that we established from Japanese MM patients, and examined functional differences between the WT and mutant BAP1. First, we studied the subcellular localization of BAP1, demonstrating that the WT primarily resides in the nucleus and that the mutant BAP1 is found in the cytoplasm of the cells. Transduction of the WT BAP1 vector into MM cells with homozygous deletion at the BAP1 3' side resulted in both inhibition of cell proliferation and anchorage-independent cell growth, whereas BAP1 mutants of a missense or C-terminal truncated form showed impaired growth inhibitory effects. Next, we studied how BAP1 is involved in MM cell survival after irradiation (IR), which causes DNA damage. After IR, we found that both WT and mutant BAP1 were similarly phosphorylated and phospho-BAP1 localized mainly in the nucleus. Interestingly, BRCA1 proteins were decreased in the MM cells with BAP1 deletion, and transduction of the mutants as well as WT BAP1 increased BRCA1 proteins, suggesting that BAP1 may promote DNA repair partly through stabilizing BRCA1. Furthermore, using the MM cells with BAP1 deletion, we found that WT BAP1, and even a missense mutant, conferred a higher survival rate after IR compared to the control vector. Our results suggested that, whereas WT BAP1 suppresses MM cell proliferation and restores cell survival after IR damage, some mutant BAP1 may also moderately retain these functions.

Nucleolar protein PES1 is a marker of neuroblastoma outcome and is associated with neuroblastoma differentiation.

Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation.

Regulation of wee1(+) expression during meiosis in fission yeast.

In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

Inactivation of SMC2 shows a synergistic lethal response in MYCN-amplified neuroblastoma cells.

The condensin complex is required for chromosome condensation during mitosis; however, the role of this complex during interphase is unclear. Neuroblastoma is the most common extracranial solid tumor of childhood, and it is often lethal. In human neuroblastoma, MYCN gene amplification is correlated with poor prognosis. This study demonstrates that the gene encoding the condensin complex subunit SMC2 is transcriptionally regulated by MYCN. SMC2 also transcriptionally regulates DNA damage response genes in cooperation with MYCN. Downregulation of SMC2 induced DNA damage and showed a synergistic lethal response in MYCN-amplified/overexpression cells, leading to apoptosis in human neuroblastoma cells. Finally, this study found that patients bearing MYCN-amplified tumors showed improved survival when SMC2 expression was low. These results identify novel functions of SMC2 in DNA damage response, and we propose that SMC2 (or the condensin complex) is a novel molecular target for the treatment of MYCN-amplified neuroblastoma.

Discovering combinatorial interactions in survival data.

MOTIVATION: Although several methods exist to relate high-dimensional gene expression data to various clinical phenotypes, finding combinations of features in such input remains a challenge, particularly when fitting complex statistical models such as those used for survival studies. RESULTS: Our proposed method builds on existing 'regularization path-following' techniques to produce regression models that can extract arbitrarily complex patterns of input features (such as gene combinations) from large-scale data that relate to a known clinical outcome. Through the use of the data's structure and itemset mining techniques, we are able to avoid combinatorial complexity issues typically encountered with such methods, and our algorithm performs in similar orders of duration as single-variable versions. Applied to data from various clinical studies of cancer patient survival time, our method was able to produce a number of promising gene-interaction candidates whose tumour-related roles appear confirmed by literature.

The neuronal differentiation factor NeuroD1 downregulates the neuronal repellent factor Slit2 expression and promotes cell motility and tumor formation of neuroblastoma.

The basic helix-loop-helix transcription factor NeuroD1 has been implicated in the neurogenesis and early differentiation of pancreatic endocrine cells. However, its function in relation to cancer has been poorly examined. In this study, we found that NeuroD1 is involved in the tumorigenesis of neuroblastoma. NeuroD1 was strongly expressed in a hyperplastic region comprising neuroblasts in the celiac sympathetic ganglion of 2-week-old MYCN transgenic (Tg) mice and was consistently expressed in the subsequently generated neuroblastoma tissue. NeuroD1 knockdown by short hairpin RNA (shRNA) resulted in motility inhibition of the human neuroblastoma cell lines, and this effect was reversed by shRNA-resistant NeuroD1. The motility inhibition by NeuroD1 knockdown was associated with induction of Slit2 expression, and knockdown of Slit2 could restore cell motility. Consistent with this finding, shRNA-resistant NeuroD1 suppressed Slit2 expression. NeuroD1 directly bound to the first and second E-box of the Slit2 promoter region. Moreover, we found that the growth of tumor spheres, established from neuroblastoma cell lines in MYCN Tg mice, was suppressed by NeuroD1 suppression. The functions identified for NeuroD1 in cell motility and tumor sphere growth may suggest a link between NeuroD1 and the tumorigenesis of neuroblastoma. Indeed, tumor formation of tumor sphere-derived cells was significantly suppressed by NeuroD1 knockdown. These data are relevant to the clinical features of human neuroblastoma: high NeuroD1 expression was closely associated with poor prognosis. Our findings establish the critical role of the neuronal differentiation factor NeuroD1 in neuroblastoma as well as its functional relationship with the neuronal repellent factor Slit2.

Regulation of yeast forkhead transcription factors and FoxM1 by cyclin-dependent and polo-like kinases.

Members of the forkhead-box (Fox) family of transcription factors are present in many eukaryotes. More than 100 such proteins that share homology in the winged-helix DNA-binding domain have been identified in higher eukaryotes. This family of transcription factors is implicated in the regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA repair, stress resistance and metabolism. A subfamily of Fox proteins are required to activate expression of the genes encoding B-type cyclins, Cdc25 and Polo-like kinase (Plk) during the mitotic cell cycle and meiosis in organisms from yeast to mammals. These proteins are activators of cyclin-dependent kinase 1 (Cdk1). Cdk1 and Plk phosphorylate Fox and its associated proteins at different sites, resulting in activation or repression of Fox transcriptional activity, depending on the target genes. In addition to their documented transcriptional functions, Fox proteins are involved in the regulation of pre-mRNA processing, at least in yeast. In this review, we will focus on the role of Fox proteins in the fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae, in addition to the role of FoxM1 in mammals in the cell cycle and in pre-mRNA processing, as revealed in recent studies.

Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast.

The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2+. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

Cdc2p controls the forkhead transcription factor Fkh2p by phosphorylation during sexual differentiation in fission yeast.

In most eukaryotes, cyclin-dependent kinases (Cdks) play a central role in control of cell-cycle progression. Cdks are inactivated from the end of mitosis to the start of the next cell cycle as well as during sexual differentiation. The forkhead-type transcription factor Fkh2p is required for the periodic expression of many genes and for efficient mating in the fission yeast Schizosaccharomyces pombe. However, the mechanism responsible for coordination of cell-cycle progression with sexual differentiation is still unknown. We now show that Fkh2p is phosphorylated by Cdc2p (Cdk1) and that phosphorylation of Fkh2p on T314 or S462 by this Cdk blocks mating in S. pombe by preventing the induction of ste11+ transcription, which is required for the onset of sexual development. We propose that functional interaction between Cdks and forkhead transcription factors may link the mitotic cell cycle and sexual differentiation.

Mei4p coordinates the onset of meiosis I by regulating cdc25+ in fission yeast.

The kinase Cdc2p is a central regulator of entry into and progression through nuclear division during mitosis and meiosis in eukaryotes. Cdc2p is activated at the onset of mitosis by dephosphorylation on tyrosine-15, the phosphorylation status of which is determined mainly by the kinase Wee1p and the phosphatase Cdc25p. In fission yeast, the forkhead-type transcription factor Mei4p is required for expression of many genes during meiosis, with mei4 mutant cells arresting before meiosis I. The mechanism of cell cycle arrest in mei4 cells has remained unknown, however. We now show that cdc25(+) is an important target of Mei4p in control of entry into meiosis I. Forced dephosphorylation of Cdc2p on tyrosine-15 thus induced meiosis I in mei4 mutant cells without a delay, although no spores were formed. We propose that Mei4p acts as a rate-limiting regulator of meiosis I by activating cdc25(+) transcription in coordination with other meiotic events.

A checkpoint control linking meiotic S phase and recombination initiation in fission yeast.

During meiosis, high levels of recombination initiated by DNA double-strand breaks (DSBs) occur only after DNA replication. However, how DSB formation is coupled to DNA replication is unknown. We examined several DNA replication proteins for a role in this coupling in Schizosaccharomyces pombe, and we show that ribonucleotide reductase, the rate-limiting enzyme of deoxyribonucleotide synthesis and the target of the DNA synthesis inhibitor hydroxyurea (HU) is indirectly required for DSB formation linked to DNA replication. However, in cells in which the function of the DNA-replication-checkpoint proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, or Cds1p was compromised, DSB formation occurred at similar frequencies in the absence or presence of HU. The DSBs in the HU-treated mutant cells occurred at normal sites and were associated with recombination. In addition, Cdc2p is apparently not involved in this process. We propose that the sequence of meiotic S phase and initiation of recombination is coordinated by DNA-replication-checkpoint proteins.

DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast.

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.