Therapeutic Strategy for Targeting Aggressive Malignant Gliomas by Disrupting Their Energy Balance.

Although abnormal metabolic regulation is a critical determinant of cancer cell behavior, it is still unclear how an altered balance between ATP production and consumption contributes to malignancy. Here we show that disruption of this energy balance efficiently suppresses aggressive malignant gliomas driven by mammalian target of rapamycin complex 1 (mTORC1) hyperactivation. In a mouse glioma model, mTORC1 hyperactivation induced by conditional Tsc1 deletion increased numbers of glioma-initiating cells (GICs) in vitro and in vivo Metabolic analysis revealed that mTORC1 hyperactivation enhanced mitochondrial biogenesis, as evidenced by elevations in oxygen consumption rate and ATP production. Inhibition of mitochondrial ATP synthetase was more effective in repressing sphere formation by Tsc1-deficient glioma cells than that by Tsc1-competent glioma cells, indicating a crucial function for mitochondrial bioenergetic capacity in GIC expansion. To translate this observation into the development of novel therapeutics targeting malignant gliomas, we screened drug libraries for small molecule compounds showing greater efficacy in inhibiting the proliferation/survival of Tsc1-deficient cells compared with controls. We identified several compounds able to preferentially inhibit mitochondrial activity, dramatically reducing ATP levels and blocking glioma sphere formation. In human patient-derived glioma cells, nigericin, which reportedly suppresses cancer stem cell properties, induced AMPK phosphorylation that was associated with mTORC1 inactivation and induction of autophagy and led to a marked decrease in sphere formation with loss of GIC marker expression. Furthermore, malignant characteristics of human glioma cells were markedly suppressed by nigericin treatment in vivo Thus, targeting mTORC1-driven processes, particularly those involved in maintaining a cancer cell's energy balance, may be an effective therapeutic strategy for glioma patients.

Strong therapeutic potential of γ-secretase inhibitor MRK003 for CD44-high and CD133-low glioblastoma initiating cells.

The Notch signal regulates both cell viability and apoptosis, and maintains stemness of various cancers including glioblastoma (GBM). Although Notch signal inhibition may be an effective strategy in treating GBM initiating cells (GICs), its applicability to the different subtypes of GBM remains unclear. Here, we analyzed the effectiveness of MRK003, a preclinical γ-secretase inhibitor, on GICs. Nine patient-derived GICs were treated by MRK003, and its efficacy on cell viability, apoptosis, sphere forming ability and Akt expression level which might be related to Notch downstream and be greatly important signals in GBM was evaluated. MRK003 suppressed viability and sphere-formation ability, and induced apoptosis in all GICs in varying doses of MRK003. Based on their sensitivities to MRK003, the nine GICs were divided into "relatively sensitive" and "relatively resistant" GICs. Sensitivity to MRK003 was associated with its inhibitory effect on Akt pathway. Transgenic expression of the myristoylated Akt vector in relatively sensitive GICs partially rescued the effect of MRK003, suggesting that the effect of MRK003 was, at least in part, mediated through inhibition of the Akt pathway. These GICs were differentiated by the expression of CD44 and CD133 with flow cytometric analysis. The relatively sensitive GICs are CD44-high and CD133-low. The IC50 of MRK003 in a set of GICs exhibited a negative correlation with CD44 and positive correlation with CD133. Collectively, MRK003 is partially mediated by the Akt pathway and has strong therapeutic potential for CD44-high and CD133-low GICs.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo.

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

Association of a murine leukaemia stem cell gene signature based on nucleostemin promoter activity with prognosis of acute myeloid leukaemia in patients.

Acute myeloid leukaemia (AML) is a heterogeneous neoplastic disorder in which a subset of cells function as leukaemia-initiating cells (LICs). In this study, we prospectively evaluated the leukaemia-initiating capacity of AML cells fractionated according to the expression of a nucleolar GTP binding protein, nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model in which green fluorescent protein (GFP) is expressed under the control of a region of the NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukaemia colonies in vitro, and exhibited the highest leukaemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukaemia-initiating capacity that we termed the 'leukaemia stem cell gene signature'. This gene signature stratified human AML patients into distinct clusters that reflected prognosis, demonstrating that the mouse leukaemia stem cell gene signature is significantly associated with the malignant properties of human AML. Further analyses of gene regulation in leukaemia stem cells could provide novel insights into diagnostic and therapeutic approaches to AML.

Abundant nucleostemin expression supports the undifferentiated properties of germ cell tumors.

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation.

Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis.

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress.

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.

Foxo3a Inhibitors of Microbial Origin, JBIR-141 and JBIR-142.

JBIR-141 (1) and JBIR-142 (2) were discovered as potent Foxo3a inhibitors that consist of three quite unique substructures, a 1-((dimethylamino)ethyl)-5-methyl-4,5-dihydrooxazole-4-carboxylic acid that is originated from Ala-Thr amino acid residues, a 3-acetoxy-4-amino-7-(hydroxy(nitroso)amino)-2,2-dimethylheptanoic acid, and an α-acyl tetramic acid fused with a 2-methylpropan-1-ol moiety. Their structures involving absolute configurations were determined by spectroscopic data, chemical degradation, anisotropy methods, and LC-MS analyses of diastereomeric derivatives. Compounds 1 and 2 exhibited specific inhibition against Foxo3a transcriptional activity with IC50 values of 23.1 and 166.2 nM, respectively.

Loss of Tsc1 accelerates malignant gliomagenesis when combined with oncogenic signals.

Glioblastomas frequently harbour genetic lesions that stimulate the activity of mammalian target of rapamycin complex 1 (mTORC1). Loss of heterozygosity of tuberous sclerosis complex 1 (TSC1) or TSC2, which together form a critical negative regulator of mTORC1, is also seen in glioblastoma; however, it is not known how loss of the TSC complex affects the development of malignant gliomas. Here we investigated the role of Tsc1 in gliomagenesis in mice. Tsc1 deficiency up-regulated mTORC1 activity and suppressed the proliferation of neural stem/progenitor cells (NSPCs) in a serial neurosphere-forming assay, suggesting that Tsc1-deficient NSPCs have defective self-renewal activity. The neurosphere-forming capacity of Tsc1-deficient NSPCs was restored by p16(Ink4a)p19(Arf) deficiency. Combined Tsc1 and p16(Ink4a)p19(Arf) deficiency in NSPCs did not cause gliomagenesis in vivo. However, in a glioma model driven by an active mutant of epidermal growth factor receptor (EGFR), EGFRvIII, loss of Tsc1 resulted in an earlier onset of glioma development. The mTORC1 hyperactivation by Tsc1 deletion accelerated malignant phenotypes, including increased tumour mass and enhanced microvascular formation, leading to intracranial haemorrhage. These data demonstrate that, although mTORC1 hyperactivation itself may not be sufficient for gliomagenesis, it is a potent modifier of glioma development when combined with oncogenic signals.

Effects of aspirin and/or salicylate on hydrolysis and glucuronidation of indomethacin in rat erythrocytes and hepatocytes.

This study was conducted to explore the mechanism of the pharmacokinetic interaction between aspirin (ASP) and indomethacin (IND) using rat erythrocytes (RBCs) and hepatocytes. ASP was hydrolyzed to salicylic acid (SA) in both the RBCs and hepatocytes. Within RBCs, aspirin and/or salicylate (ASP/SA) increased the concentration of IND, accompanied by a constant hydrolysis of IND. In hepatocytes, a low dose of IND was subjected to glucuronidation rather than hydrolysis, and ASP/SA inhibited both the acylglucuronidation of IND and hydrolysis of IND glucuronide. A high dose of IND underwent hydrolysis with about double the glucuronidation, and ASP/SA decreased the ratio of hydrolysis to glucuronidation, accompanied by a loss of ASP, IND and their metabolites from the medium. Collectively, the results provide metabolic insight into the mechanism of drug-drug interaction between ASP/SA and IND in the hepatocytes and RBCs.

Correlation between the physicochemical property of some nonsteroidal anti-inflammatory drugs and changes in adenosine triphosphate, glutathione and hemoglobin in rat erythrocytes.

This study was conducted to explore the relationship between physicochemical property and toxic effectiveness using rat red blood cells (RBCs). The toxic effectiveness of acid nonsteroidal anti-inflammatory drugs (NSAIDs) was systemically examined by the depletion of intracorpuscular adenosine triphosphate (ATP), glutathione (GSH), and hemoglobin (Hb) at various doses, increased every 5 fmol/RBC. When the RBCs were incubated with NSAIDs, the drugs attained maximum levels within RBC, and the levels were then reduced. The ATP depletion seemed to be observed on the excretion of the drugs prior to the depletions of GSH and Hb. The physicochemical properties of NSAIDs were obtained from QMPRPlus, SMILES code, and CS ChemRaw Ultra. Correlation between their physicochemical properties and their doses for the depletions of ATP, GSH and Hb was performed in comparison with those of the membrane bound enzyme (MBE) inhibiting- and methemoglobin (MHb)-generating drugs. The ATP depletion by NSAIDs was correlated with the GSH depletion and intracorpuscular levels of the drugs, but not with the Hb depletion. The GSH depletion was correlated with the Hb depletion and participated in the lipophilicity of the drugs.