Inhibitory Effect of Pyra-Metho-Carnil on Cancer Spheroid Growth Through Decrease in Glycolysis-associated Molecules.

BACKGROUND/AIM: Pyra-Metho-Carnil (PMC) has been identified as a novel candidate compound for treating numerous malignancies; however, its mechanism of action remains unknown. In this study, we conducted RNA-sequencing (RNA-seq) analyses to elucidate the mechanism of PMC against human colorectal cancer cells harboring mutant KRAS (mtKRAS). MATERIALS AND METHODS: RNA-seq analyses of the HKe3-wild-type KRAS and HKe3-mtKRAS spheroids treated with DMSO or PMC for 6 days were performed. RESULTS: RNA-seq data suggested that PMC treatment suppresses the aerobic glycolysis pathway in HKe3-mtKRAS spheroids through the down-regulation of the HIF1 pathway. Indeed, treatment with PMC markedly suppresses the absorption of glucose by spheroids and the secretion of lactate from them. CONCLUSION: PMC suppresses growth of cancer spheroid through down-regulation of cancer-specific glucose metabolism.

Measurement of GLUT4 Traffic to and from the Cell Surface in Muscle Cells.

Elevated blood glucose following a meal is cleared by insulin-stimulated glucose entry into muscle and fat cells. The hormone increases the amount of the glucose transporter GLUT4 at the plasma membrane in these tissues at the expense of preformed intracellular pools. In addition, muscle contraction also increases glucose uptake via a gain in GLUT4 at the plasma membrane. Regulation of GLUT4 levels at the cell surface could arise from alterations in the rate of its exocytosis, endocytosis, or both. Hence, methods that can independently measure these traffic parameters for GLUT4 are essential to understanding the mechanism of regulation of membrane traffic of the transporter. Here, we describe cell population-based assays to measure the steady-state levels of GLUT4 at the cell surface, as well as to separately measure the rates of GLUT4 endocytosis and endocytosis. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Measuring steady-state cell surface GLUT4myc Basic Protocol 2: Measuring steady-state cell surface GLUT4-HA Basic Protocol 3: Measuring GLUT4myc endocytosis Basic Protocol 4: Measuring GLUT4myc exocytosis.

Death domain-associated protein DAXX regulates noncoding RNA transcription at the centromere through the transcription regulator ZFAT.

The centromere is an essential chromosomal structure for faithful chromosome segregation during cell division. No protein-coding genes exist at the centromeres, but centromeric DNA is actively transcribed into noncoding RNA (ncRNA). This centromeric transcription and its ncRNA products play important roles in centromere functions. We previously reported that the transcriptional regulator ZFAT (zinc-finger protein with AT hook) plays a pivotal role in ncRNA transcription at the centromere; however, it was unclear how ZFAT involvement was regulated. Here, we show that the death domain-associated protein (DAXX) promotes centromeric localization of ZFAT to regulate ncRNA transcription at the centromere. Coimmunoprecipitation analysis of endogenous proteins clearly reveals that DAXX interacts with ZFAT. In addition, we show that ectopic coexpression of ZFAT with DAXX increases the centromeric levels of both ZFAT and ncRNA, compared with ectopic expression of ZFAT alone. On the other hand, we found that siRNA-mediated depletion of DAXX decreases the centromeric levels of both ZFAT and ncRNA in cells ectopically expressing ZFAT. These results suggest that DAXX promotes the centromeric localization of ZFAT and ZFAT-regulated centromeric ncRNA transcription. Furthermore, we demonstrate that depletion of endogenous DAXX protein is sufficient to cause a decrease in the ncRNA levels at the centromeres of chromosomes 17 and X in which ZFAT regulates the transcription, indicating a physiological significance of DAXX in ZFAT-regulated centromeric ncRNA transcription. Taken together, these results demonstrate that DAXX regulates centromeric ncRNA transcription through ZFAT.

Cancer Spheroid Proliferation Is Suppressed by a Novel Low-toxicity Compound, Pyra-Metho-Carnil, in a Context-independent Manner.

BACKGROUND/AIM: In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated. MATERIALS AND METHODS: Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GI50) values were determined. RESULTS: From these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GI50 value of PMC in nude mice assay was 7.7 mg/kg and nude mice that were administered 40 mg/kg PMC for 7 days did not show any abnormal blood cell count values. CONCLUSION: PMC is a low-toxicity compound that inhibits the growth of different tumor cell types.

CENP-B promotes the centromeric localization of ZFAT to control transcription of noncoding RNA.

The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.

Growth Suppression of Cancer Spheroids With Mutated KRAS by Low-toxicity Compounds from Natural Products.

BACKGROUND/AIM: Among compounds from natural products selectively suppressing the growth of cancer spheroids, which have mutant (mt) KRAS, NP910 was selected and its derivatives explored. MATERIALS AND METHODS: The area of HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in three-dimensional floating (3DF) cultures treated with 18 NP910 derivatives. The 50% cell growth inhibition (GI50) was determined by long-term 3DF (LT3DF) culture and nude mice assay. RESULTS: We selected NP882 (named STAR3) as the most effective inhibitor of growth of HKe3-mtKRAS spheroids with the least toxicity among NP910 derivatives. GI50s of STAR3 in LT3DF and nude mice assay were 6 µM and 30.75 mg/kg, respectively. However, growth suppression by STAR3 was observed in 50% of cell lines independent of KRAS mutation, suggesting that the target of STAR3 was not directly associated with KRAS mutation and KRAS-related signals. CONCLUSION: STAR3 is a low-toxicity compound that inhibits growth of certain tumour cells.

The transcriptional regulator Zfat is essential for maintenance and differentiation of the adipocytes.

Adipocytes play crucial roles in the control of whole-body energy homeostasis. Differentiation and functions of the adipocytes are regulated by various transcription factors. Zfat (zinc-finger protein with AT-hook) is a transcriptional regulator that controls messenger RNA expression of specific genes through binding to their transcription start sites. Here we report important roles of Zfat in the adipocytes. We establish inducible Zfat-knockout (Zfat iKO) mice where treatment with tamoxifen causes a marked reduction in Zfat expression in various tissues. Tamoxifen treatment of Zfat iKO mice reduces the white adipose tissues (WATs) mass, accompanied by the decreased triglyceride levels. Zfat is expressed in both the adipose-derived stem cells (ADSCs) and mature adipocytes in the WATs. In ex vivo assays of the mature adipocytes differentiated from the Zfat iKO ADSCs, loss of Zfat in the mature adipocytes reduces the triglyceride levels, suggesting cell autonomous roles of Zfat in the maintenance of the mature adipocytes. Furthermore, we identify the Atg13, Brf1, Psmc3, and Timm22 genes as Zfat-target genes in the mature adipocytes. In contrast, loss of Zfat in the ADSCs impairs adipocyte differentiation with the decreased expression of C/EBPα and adiponectin. Thus, we propose that Zfat plays crucial roles in maintenance and differentiation of the adipocytes.

ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B-H4K8ac-BRD4 axis.

Centromeres are genomic regions essential for faithful chromosome segregation. Transcription of noncoding RNA (ncRNA) at centromeres is important for their formation and functions. Here, we report the molecular mechanism by which the transcriptional regulator ZFAT controls the centromeric ncRNA transcription in human and mouse cells. Chromatin immunoprecipitation with high-throughput sequencing analysis shows that ZFAT binds to centromere regions at every chromosome. We find a specific 8-bp DNA sequence for the ZFAT-binding motif that is highly conserved and widely distributed at whole centromere regions of every chromosome. Overexpression of ZFAT increases the centromeric ncRNA levels at specific chromosomes, whereas its silencing reduces them, indicating crucial roles of ZFAT in centromeric transcription. Overexpression of ZFAT increases the centromeric levels of both the histone acetyltransferase KAT2B and the acetylation at the lysine 8 in histone H4 (H4K8ac). siRNA-mediated knockdown of KAT2B inhibits the overexpressed ZFAT-induced increase in centromeric H4K8ac levels, suggesting that ZFAT recruits KAT2B to centromeres to induce H4K8ac. Furthermore, overexpressed ZFAT recruits the bromodomain-containing protein BRD4 to centromeres through KAT2B-mediated H4K8ac, leading to RNA polymerase II-dependent ncRNA transcription. Thus, ZFAT binds to centromeres to control ncRNA transcription through the KAT2B-H4K8ac-BRD4 axis.

Mutant KRAS Promotes NKG2D+ T Cell Infiltration and CD155 Dependent Immune Evasion.

BACKGROUND/AIM: Roles for mutant (mt) KRAS in the innate immune microenvironment in colorectal cancer (CRC) were explored. MATERIALS AND METHODS: Human CRC HCT116-derived, mtKRAS-disrupted (HKe3) cells that express exogenous mtKRAS and allogenic cytokine-activated killer (CAK) cells were co-cultured in 3D floating (3DF) culture. The anti-CD155 antibody was used for function blocking and immuno histochemistry. RESULTS: Infiltration of CAK cells, including NKG2D+ T cells, into the deep layer of HKe3-mtKRAS spheroids, was observed. Surface expression of CD155 was found to be up-regulated by mtKRAS in 3DF culture and CRC tissues. Further, the number of CD3+ tumor-infiltrating cells in the invasion front that show substantial CD155 expression was significantly larger than the number showing weak expression in CRC tissues with mtKRAS. CD155 blockade decreased the growth of spheroids directly and indirectly through the release of CAK cells. CONCLUSION: CD155 blockade may be useful for therapies targeting tumors containing mtKRAS.

Zfat Is Indispensable for the Development of Erythroid Cells in the Fetal Liver.

BACKGROUND/AIM: In mice, fetal liver is the first tissue of definitive erythropoiesis for definitive erythroid expansion and maturation. ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in primitive hematopoiesis and T cell development. The aim of this study was to examine whether or not Zfat is involved in definitive erythropoiesis in the fetal liver during mammalian development. MATERIALS AND METHODS: The role of Zfat during mouse fetal erythropoiesis in the fetal liver was examined using tamoxifen-inducible CreERT2 Zfat-deficient mice. RESULTS: Zfat-deficient mice exhibit moderate anemia with small and pale fetal liver through a decreased number of erythroblasts by E12.5. Apoptosis sensitivity in fetal liver erythroid progenitors was enhanced by Zfat-deficiency ex vivo. Moreover, Zfat knockdown partially inhibited CD71-/lowTer119- to CD71highTer119- transition of fetal liver erythroid progenitors with impairment in the elevation of CD71 expression. CONCLUSION: Zfat plays a critical role for erythropoiesis in the fetal liver.

Zfat expression in ZsGreen reporter gene knock­in mice: Implications for a novel function of Zfat in definitive erythropoiesis.

Zinc finger and AT­hook domain containing (Zfat) is a transcriptional regulator harboring an AT­hook domain and 18 repeats of a C2H2 zinc­finger motif, which binds directly to the proximal region of transcription start sites in Zfat­target genes. It was previously reported that deletion of the Zfat gene in mice yields embryonic lethality by embryonic day 8.5 and impairs primitive hematopoiesis in yolk sac blood islands. In addition, Zfat has been reported to be involved in thymic T­cell development and peripheral T­cell homeostasis. In the present study, in order to obtain a precise understanding of the expression and function of Zfat, a knock­in mouse strain (ZfatZsG/+ mice), which expressed ZsGreen in the Zfat locus, was established. ZsGreen signals in tissues and cells of ZfatZsG/+ mice were examined by flow cytometric and histological analyses. Consistent with our previous studies, ZsGreen signals in ZfatZsG/+ mice were detected in the embryo and yolk sac blood islands, as well as in thymocytes, B and T cells. In the ZfatZsG/+ thymus, ZsGreen+ cells were identified not only in T­cell populations but also in thymic epithelial cells, suggesting the role of Zfat in antigen­presenting cells during thymic T­cell development. ZsGreen signals were observed in definitive erythroid progenitor cells in the fetal liver and adult bone marrow of ZfatZsG/+ mice. The proportion of ZsGreen+ cells in these tissues was highest at the early stage of erythroid differentiation, suggesting that Zfat serves particular roles in definitive erythropoiesis. Histological studies demonstrated that ZsGreen signals were detected in the pyramidal cells in the hippocampal CA1 region and the Purkinje cells in the cerebellum, suggesting novel functions of Zfat in nervous tissues. Taken together, these results indicated that the ZfatZsG/+ reporter mouse may be considered a useful tool for elucidating the expression and function of Zfat.

Growth Suppression of Human Colorectal Cancer Cells with Mutated KRAS by 3-Deaza-cytarabine in 3D Floating Culture.

BACKGROUND/AIM: During screening for compounds that selectively suppress growth of human colorectal cancer (CRC) spheroids with mutant (mt) KRAS, the uridine analogue, 5-bromouridine (BrUrd) was identified and its derivatives were explored. MATERIALS AND METHODS: DNA incorporation in two-dimensional (2D) and three-dimensional floating (3DF) cultures was examined with the uridine analogue, 5-ethynyl-2'-deoxyuridine (EdU). The area of HKe3 CRC spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in 3DF culture with 11 BrUrd derivatives. RESULTS: EdU was strongly incorporated into newly-synthesized DNA from HKe3-mtKRAS cells compared to HKe3-wtKRAS in 2D and 3DF culture. 3-Deaza-cytarabine, which has properties of BrUrd and cytidine, was the most effective inhibitor of HKe3-mtKRAS spheroids with the least toxicity to HKe3-wtKRAS. Growth suppression of 3-deaza-cytarabine was stronger than cytarabine in 2D culture, and toxicity was lower than gemcitabine in long-term 3DF culture. CONCLUSION: 3-Deaza-cytarabine exhibits properties useful for the treatment of CRC patients with mtKRAS.

ROS-induced cleavage of NHLRC2 by caspase-8 leads to apoptotic cell death in the HCT116 human colon cancer cell line.

Excess production of reactive oxygen species (ROS) is known to cause apoptotic cell death. However, the molecular mechanisms whereby ROS induce apoptosis remain elusive. Here we show that the NHL-repeat-containing protein 2 (NHLRC2) thioredoxin-like domain protein is cleaved by caspase-8 in ROS-induced apoptosis in the HCT116 human colon cancer cell line. Treatment of HCT116 cells with the oxidant tert-butyl hydroperoxide (tBHP) induced apoptosis and reduced NHLRC2 protein levels, whereas pretreatment with the antioxidant N-acetyl-L-cysteine prevented apoptosis and the decrease in NHLRC2 protein levels seen in tBHP-treated cells. Furthermore, the ROS-induced decrease in NHLRC2 protein levels was relieved by the caspase inhibitor z-VAD-fmk. We found that the thioredoxin-like domain of NHLRC2 interacted with a proenzyme form of caspase-8, and that caspase-8 cleaved NHLRC2 protein at Asp580 in vitro. Furthermore, siRNA-mediated knockdown of caspase-8 blocked the ROS-induced decrease in NHLRC2 protein levels. Both shRNA and CRISPR-Cas9-mediated loss of NHLRC2 resulted in an increased susceptibility of HCT116 cells to ROS-induced apoptosis. These results suggest that excess ROS production causes a caspase-8-mediated decrease in NHLRC2 protein levels, leading to apoptotic cell death in colon cancer cells, and indicate an important role of NHLRC2 in the regulation of ROS-induced apoptosis.

Apremilast Induces Apoptosis of Human Colorectal Cancer Cells with Mutant KRAS.

BACKGROUND/AIM: We previously reported the crucial roles of oncogenic Kirsten rat sarcoma viral oncogene homologue (KRAS) in inhibiting apoptosis and disrupting cell polarity via the regulation of phosphodiesterase type 4B2 (PDE4B2) expression in human colorectal cancer (CRC) HCT116 cells in a three-dimensional culture (3DC). Here, we evaluated the effects of apremilast, a selective PDE4 inhibitor, on luminal apoptosis in 3DC and nude mice assay using HKe3 human CRC cells stably expressing wild-type (wt)PDE4B2 (HKe3-wtPDE4B2), mutant (mt)PDE4B2 (kinase dead) (HKe3-wtKRAS), wtKRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS). MATERIALS AND METHODS: Apoptosis was detected by immunofluorescence using confocal laser scanning microscopy or western blot in HKe3-wtPDE4B2, HKe3-mtPDE4B2, HKe3-wtKRAS and mtKRAS cells treated with or without apremilast in 3DC. Tumourigenicity was assessed in nude mice assay using these cells. RESULTS: Apremilast did not inhibit the proliferation of HKe3-wtPDE4B2 cells or HKe3-mtKRAS in two-dimensional cultures, whereas the number of apoptotic HKe3-wtPDE4B2 cells and HKe3-mtKRAS cells increased after apremilast treatment in 3DC, leading to formation of a luminal cavity. Tumour growth in nude mice was dramatically reduced by intraperitoneal injection of apremilast. Notably, a decreased level of caspase-1 expression was observed in HKe3-wtPDE4B2 and HKe3-mtKRAS cells. CONCLUSION: Apremilast induces tumour regression in nude mice, possibly by inducing caspase-1 expression.

An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids.

BACKGROUND/AIM: Alpha-kinase 2 (ALPK2), suggested to be a novel tumour-suppressor gene down-regulated by oncogenic KRAS, plays a pivotal role in luminal apoptosis in normal colonic crypts. The aim of this study was to determine the association between ALPK2 germline variants and colorectal cancer. MATERIALS AND METHODS: Missense single nucleotide variants in the exons of the ALPK2 gene in 2,343 consecutive autopsy cases (1,446 cases with cancer and 897 cases without cancer) were screened using HumanExome BeadChip arrays. To address the functional effect of a missense ALPK2 variant, a 3D floating cell culture was performed using HCT116-derived human colorectal cancer cells stably expressing wild-type (wt) ALPK2 (HCT116-wtALPK2) or amino acid-substituted (sub) ALPK2 (HCT116-subALPK2). RESULTS: We identified that one of the ALPK2 germline variants, rs55674018 (p.Q1853E), was significantly associated with the presence of cancer (adjusted odds ratio(OR)=4.39; 95% confidence interval(CI)=1.31-14.78, p=0.001). The p.Q1853E variant was present in the East Asian population and located in the immunoglobulin-like domain. Notably, the basolateral polarity of actin in the surface of HCT116-wtALPK2 spheroids was more attenuated compared to that of HCT116-subALPK2 spheroids. Furthermore, luminal apoptosis and cell aggregation were promoted by wtALPK2, but not by subALPK2 in 3D culture. CONCLUSION: The p.Q1853E variant of ALPK2, which had been accumulating in the Japanese population, induced a metastatic phenotype by disrupting ALPK2 function.

Sequential cleavage of insulin receptor by calpain 2 and γ-secretase impairs insulin signalling.

AIMS/HYPOTHESIS: Soluble insulin receptor (sIR), the ectodomain of the insulin receptor (IR), has been detected in human plasma and its concentration paralleled that of blood glucose. We have previously developed an in vitro model using HepG2 liver-derived cells, which mimics changes in sIR levels in plasma from diabetic patients and shows that calcium-dependent proteases cleave IR extracellularly (a process known as shedding). The present study aimed to reveal the mechanisms of IR cleavage. METHODS: Using the in vitro model, we investigated the molecular mechanisms of IR cleavage, which is accelerated by high-glucose treatment. We also analysed the relationship between IR cleavage and cellular insulin resistance, and the correlation between plasma sIR levels and insulin sensitivity, which was assessed by the euglycaemic-hyperinsulinaemic clamp technique. RESULTS: Here, we determined that calpain 2, which is secreted into the extracellular space associated with exosomes, directly cleaved the ectodomain of the IRß subunit (IRß), which in turn promoted intramembrane cleavage of IRß by γ-secretase. IR cleavage impaired insulin signalling and the inhibition of IR cleavage (by knockdown of calpain 2 and γ-secretase), restored IR substrate-1 and Akt, independent of IR. Furthermore, the glucose-lowering drug, metformin, prevented IR cleavage accompanied by inhibition of calpain 2 release in exosomes, and re-established insulin signalling. In patients with type 2 diabetes, plasma sIR levels inversely correlated with insulin sensitivity. CONCLUSIONS/INTERPRETATION: Sequential cleavage of IR by calpain 2 and γ-secretase may contribute to insulin signalling in cells and its inhibition may be partly responsible for the glucose-lowering effects of metformin. Thus, IR cleavage may offer a new mechanism for the aetiology of insulin resistance.

Molecular mechanisms of transcriptional regulation by the nuclear zinc-finger protein Zfat in T cells.

Zfat is a nuclear protein with AT-hook and zinc-finger domains. We previously reported that Zfat plays crucial roles in T-cell survival and development in mice. However, the molecular mechanisms whereby Zfat regulates gene expression in T cells remain unexplored. In this study, we analyzed the genome-wide occupancy of Zfat by chromatin immunoprecipitation with sequencing (ChIP-seq), which showed that Zfat bound predominantly to a region around a transcription start site (TSS), and that an 8-bp nucleotide sequence GAA(T/A)(C/G)TGC was identified as a consensus sequence for Zfat-binding sites. Furthermore, about half of the Zfat-binding sites were characterized by histone H3 acetylations at lysine 9 and lysine 27 (H3K9ac/K27ac). Notably, Zfat gene deletion decreased the H3K9ac/K27ac levels at the Zfat-binding sites, suggesting that Zfat may be related to the regulation of H3K9ac/K27ac. Integrated analysis of ChIP-seq and transcriptional profiling in thymocytes identified Zfat-target genes with transcription to be regulated directly by Zfat. We then focused on the chromatin regulator Brpf1, a Zfat-target gene, revealing that Zfat bound directly to a 9-bp nucleotide sequence, CGAANGTGC, which is conserved among mammalian Brpf1 promoters. Furthermore, retrovirus-mediated re-expression of Zfat in Zfat-deficient peripheral T cells restored Brpf1 expression to normal levels, and shRNA-mediated Brpf1 knockdown in peripheral T cells increased the proportion of apoptotic cells, suggesting that Zfat-regulated Brpf1 expression was important for T-cell survival. Our findings demonstrated that Zfat regulates the transcription of target genes by binding directly to the TSS proximal region, and that Zfat-target genes play important roles in T-cell homeostasis.

The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway.

Marked Reduction in FoxO1 Protein by its Enhanced Proteasomal Degradation in Zfat-deficient Peripheral T-Cells.

BACKGROUND: Zfat is a nuclear protein that harbours putative DNA-binding domains. T-cell specific deletion of Zfat in Zfat(f/f)-CD4Cre mice yields a significant decrease in the number of peripheral T-cells with a lower surface expression of interleukin-7 receptor-α (IL-7Rα). However, the molecular mechanism by which Zfat controls IL-7Rα expression remains unknown. MATERIALS AND METHODS: Expression levels of the molecules involved in IL-7Rα expression were determined by immunoblotting. RESULTS: Zfat-deficient peripheral T-cells showed a marked reduction in the FoxO1 protein that regulates IL-7Rα expression; however, the FoxO1 mRNA expression level was not affected by Zfat-deficiency. Furthermore, treatment of Zfat-deficient T-cells with a proteasome inhibitor, epoxomicin, restored FoxO1 expression levels, indicating that the loss of Zfat enhanced the proteasomal degradation of the FoxO1 protein. CONCLUSION: These results suggest that Zfat is required for peripheral T-cell homeostasis through IL-7Rα expression by controlling the FoxO1 protein.

Establishment of a Three-dimensional Floating Cell Culture System for Screening Drugs Targeting KRAS-mediated Signaling Molecules.

BACKGROUND/AIM: Oncogenic mutations in the KRAS gene are critically involved in many human tumors but drugs targeting oncogenic KRAS have not yet been clinically developed. Herein, we established a three-dimensional floating (3DF) culture system for screening drugs that target KRAS-mediated signaling molecules. MATERIALS AND METHODS: HKe3 cells, derived from colorectal cancer HCT116 cells and disrupted at mutated (mt) KRAS gene, were infected with a retrovirus expressing wild-type (wt) KRAS or mtKRAS to establish HKe3-derived cells expressing wtKRAS or mtKRAS. Established cells were cultured in 96-well plates with an ultra-low attachment surface and round bottom for 3DF culture. RESULTS: HKe3-wtKRAS and HKe3-mtKRAS cells in 3DF culture rapidly assembled into respective single spherical structures (spheroids). Furthermore, mtKRAS but not wtKRAS expression inhibited luminal apoptosis in spheroids indicating that the 3DF culture was compatible with the 3D matrigel culture. CONCLUSION: This 3DF culture system could be useful for screening drugs that target KRAS-mediated signaling molecules.