RAD52-mediated repair of DNA double-stranded breaks at inactive centromeres leads to subsequent apoptotic cell death.

Centromeres, where the kinetochore complex binds, are susceptible to damages including DNA double-stranded breaks (DSBs). Here, we report the functional significance and the temporally and spatially distinct regulation of centromeric DSB repair via the three pathways of non-homologous end joining (NHEJ), homologous recombination (HR) and single-strand annealing (SSA). The SSA factor RAD52 is most frequently recruited to centromeric DSB sites compared with the HR factor RAD51 and the NHEJ factor DNA ligase IV (LIG4), indicating that SSA plays predominant roles in centromeric DSB repair. Upon centromeric DSB induction, LIG4 is recruited to both active centromeres, where kinetochore complex binds, and inactive centromeres. In contrast, RAD51 and RAD52 are recruited only to inactive centromeres. These results indicate that DSBs at active centromeres are repaired through NHEJ, whereas the three pathways of NHEJ, HR and SSA are involved in DSB repair at inactive centromeres. Furthermore, siRNA-mediated depletion of either LIG4 or RAD51 promotes cell death after centromeric DSB induction, whereas RAD52 depletion inhibits it, suggesting that HR and NHEJ are required for appropriate centromeric DSB repair, whereas SSA-mediated centromeric DSB repair leads to subsequent cell death. Thus, SSA-mediated DSB repair at inactive centromeres may cause centromere dysfunction through error-prone repair.

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.

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.

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.

Epipharyngeal Abrasive Therapy (EAT) Reduces the mRNA Expression of Major Proinflammatory Cytokine IL-6 in Chronic Epipharyngitis.

The epipharynx, located behind the nasal cavity, is responsible for upper respiratory tract immunity; however, it is also the site of frequent acute and chronic inflammation. Previous reports have suggested that chronic epipharyngitis is involved not only in local symptoms such as cough and postnasal drip, but also in systemic inflammatory diseases such as IgA nephropathy and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID. Epipharyngeal Abrasive Therapy (EAT), which is an effective treatment for chronic epipharyngitis in Japan, is reported to be effective for these intractable diseases. The sedation of chronic epipharyngitis by EAT induces suppression of the inflammatory cytokines and improves systemic symptoms, which is considered to be one of the mechanisms, but there is no report that has proved this hypothesis. The purpose of this study was to clarify the anti-inflammatory effect of EAT histologically. The study subjects were 8 patients who were not treated with EAT and 11 patients who were treated with EAT for chronic epipharyngitis for 1 month or more. For immunohistochemical assessment, the expression pattern of IL-6 mRNA, which plays a central role in the human cytokine network, was analyzed using in situ hybridization. The expression of IL-6 in the EAT-treated group was significantly lower than those in the EAT nontreated group (p = 0.0015). In addition, EAT suppressed the expression of tumor necrosis factor alpha (TNFα), a crucial proinflammatory cytokine. As a result, continuous EAT suppressed submucosal cell aggregation and reduced inflammatory cytokines. Thus, EAT may contribute to the improvement of systemic inflammatory diseases through the suppression of IL-6 expression.

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.

Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRASG13D mutation.

BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRASG13D allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

A novel compound, ferulic acid-bound resveratrol, induces the tumor suppressor gene p15 and inhibits the three-dimensional proliferation of colorectal cancer cells.

Resveratrol, a phytoalexin present in grapes and other edible foods, has been reported to have beneficial effects against various diseases including cancer. We previously reported that resveratrol and its derivative, caffeic acid-adducted resveratrol, selectively inhibit the three-dimensional (3D) proliferation of a human colorectal cancer cell line, HCT116 with activating KRAS mutation. Herein, we demonstrated that a novel compound, ferulic acid-bound resveratrol, also represses the 3D proliferation of HCT116 cells. We observed that resveratrol conjugated to two ferulic acids represses the 3D proliferation of HCT116 cells more strongly than resveratrol and resveratrol conjugated to one ferulic acid. Resveratrol conjugated to two ferulic acids also inhibited the 3D proliferation of MCF7 human breast cancer cells. We further uncovered that the resveratrol derivative increases the mRNA level of the tumor suppressor p15, a CDK inhibitor that functions as a brake of cell proliferation in HCT116 cells. These results imply that the resveratrol derivative represses 3D proliferation via increasing p15 expression in HCT116 cells.

A novel resveratrol derivative selectively inhibits the proliferation of colorectal cancer cells with KRAS mutation.

Resveratrol is a polyphenolic compound in many edible foods including grapes, peanuts, and berries. Several studies have revealed the beneficial effects of resveratrol against various diseases such as heart disease, diabetes, obesity, neurological disorders, and cancer. A recent study showed that resveratrol inhibits the proliferation of HCT116 human colorectal cancer cells in three-dimensional culture (3DC) via induction of luminal apoptosis in HCT116 cell spheroids. In this study, we showed that a novel compound, caffeic acid-adducted resveratrol, has a stronger inhibitory effect on the growth of HCT116 cell spheroids in 3DC than resveratrol. It showed almost the same inhibitory efficacy as 5-fluorouracil, a conventional anticancer drug. We further showed that the resveratrol derivative did not affect the growth of HKe3 cell spheroids derived from HCT116 cells by disruption of the activating mutant KRAS gene. These results suggest that the resveratrol derivative inhibits the growth of HCT116 cell spheroids via inhibition of an oncogenic KRAS-mediated signaling pathway.

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.

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.

ANRIL regulates the proliferation of human colorectal cancer cells in both two- and three-dimensional culture.

ANRIL is a long noncoding RNA transcribed from the INK4 locus that encodes three tumor suppressor genes, p15, p16, and ARF. Previous studies demonstrated that ANRIL represses p15 and p16, which positively regulate the pRB pathway, leading to repression of cellular senescence of human normal fibroblasts. However, the role of ANRIL in cancer cell proliferation is less well understood. Here we report that ANRIL is involved in the proliferation of colorectal cancer HCT116 cells in two- and three-dimensional culture. Silencing ANRIL by both transfection with small interfering RNA and retrovirally produced small hairpin RNA reduced HCT116 cell proliferation in both two- and three-dimensional culture. HCT116 cells depleted for ANRIL were arrested in the S phase of cell cycle. Notably, silencing ANRIL did not result in the activation of expression of the INK4 locus. These results suggest that ANRIL positively regulates the proliferation of HCT116 cells in two- and three-dimensional culture in a p15/p16-pRB pathway-independent manner.

Zfat-deficient CD4⁺ CD8⁺ double-positive thymocytes are susceptible to apoptosis with deregulated activation of p38 and JNK.

Zfat, which is a nuclear protein harboring an AT-hook domain and 18-repeats of C2H2 zinc-finger motif, is highly expressed in immune-related tissues, including the thymus and spleen. T cell specific deletion of the Zfat gene by crossing Zfat(f/f) mice with LckCre mice yields a significant reduction in the number of CD4(+) CD8(+) double-positive (DP) thymocytes. However, physiological role for Zfat in T cell development in the thymus remains unknown. Here, we found that Zfat-deficient DP thymocytes in Zfat(f/f)-LckCre mice were susceptible to apoptosis both at an unstimulated state and in response to T cell receptor (TCR)-stimulation. The phosphorylation levels of p38 and JNK were elevated in Zfat-deficient thymocytes at an unstimulated state with an enhanced phosphorylation of ATF2 and with an over-expression of Gadd45α⋅ On the other hand, the activation of JNK in the Zfat-deficient thymocytes, but not p38, was strengthened and prolonged in response to TCR-stimulation. All these results demonstrate that Zfat critically participates in the development of DP thymocytes through regulating the activities of p38 and JNK.

Inhibitory Effect of Pyra-Metho-Carnil on the Differentiation and Maturation of Macrophages in Normal and Cancer Microenvironments.

BACKGROUND/AIM: In a previous study, we have demonstrated heightened Pyra-Metho-Carnil (PMC) efficacy in nude mice with intact innate immunity that lack T and B cells. This has prompted hypothesizing that PMC may target macrophages that promote cancer growth. In this study, we conducted co-culture experiments with macrophages derived from THP-1 human monocyte cell lines and spheroids representing normal and cancer microenvironments. We then performed RNA sequencing and flow cytometry analysis to elucidate the mechanisms by which PMC affects macrophage differentiation and maturation. MATERIALS AND METHODS: THP-1 cells were differentiated by phorbol 12-myristate 13-acetate (PMA) and matured by PMA and lipopolysaccharide (LPS) either with or without PMC. Co-cultures were performed using stimulated THP-1 cells and HKe3-wild-type KRAS or HKe3-mutant (mt) KRAS spheroids. We then performed RNA-seq analysis of THP-1 cells stimulated by PMA (either with or without PMC) and flow cytometry analysis of mice peripheral blood obtained after PMC administration. RESULTS: THP-1 cells matured by PMA and LPS specifically increased the area of HKe3-mtKRAS cancer spheroids and the addition of PMC to THP-1 cells was found to inhibit cancer spheroid growth. RNA-seq data suggested that PMC treatment of THP-1 cells stimulated with PMA suppressed cell motility regulatory functions via down-regulation of the NF[Formula: see text]B pathway. Furthermore, flow cytometry results showed that PMC treatment suppressed monocyte maturation in B6 mice. CONCLUSION: The high level of in vivo tumor suppression caused by PMC may be due to inhibition of the differentiation and maturation of tumor-associated macrophages via the NF[Formula: see text]B signaling pathway.

Immune-related zinc finger gene ZFAT is an essential transcriptional regulator for hematopoietic differentiation in blood islands.

TAL1 plays pivotal roles in vascular and hematopoietic developments through the complex with LMO2 and GATA1. Hemangioblasts, which have a differentiation potential for both endothelial and hematopoietic lineages, arise in the primitive streak and migrate into the yolk sac to form blood islands, where primitive hematopoiesis occurs. ZFAT (a zinc-finger gene in autoimmune thyroid disease susceptibility region/an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook) was originally identified as an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook, and is highly conserved among species. ZFAT is thought to be a critical transcription factor involved in immune-regulation and apoptosis; however, developmental roles for ZFAT remain unknown. Here we show that Zfat-deficient (Zfat(-/-)) mice are embryonic-lethal, with impaired differentiation of hematopoietic progenitor cells in blood islands, where ZFAT is exactly expressed. Expression levels of Tal1, Lmo2, and Gata1 in Zfat(-/-) yolk sacs are much reduced compared with those of wild-type mice, and ChIP-PCR analysis revealed that ZFAT binds promoter regions for these genes in vivo. Furthermore, profound reduction in TAL1, LMO2, and GATA1 protein expressions are observed in Zfat(-/-) blood islands. Taken together, these results suggest that ZFAT is indispensable for mouse embryonic development and functions as a critical transcription factor for primitive hematopoiesis through direct-regulation of Tal1, Lmo2, and Gata1. Elucidation of ZFAT functions in hematopoiesis might lead to a better understanding of transcriptional networks in differentiation and cellular programs of hematopoietic lineage and provide useful information for applied medicine in stem cell therapy.

MMP-2 Regulation of Emmprin on Tumour Cells and CD73 on Fibroblasts During Tumour-Stromal Interaction.

BACKGROUND/AIM: We previously found that binding between CD73 and extracellular matrix metalloproteinase (MMP) inducer (emmprin) and suppression of CD73 in both tumour cells and fibroblasts suppressed MMP-2 production when co-cultured. However, the importance of CD73 expression in either fibroblasts or cancer cells for cancer invasion remains unknown. In this study, we used siRNA to separately down-regulate CD73 in individual cells, and then performed a 3D co-culture to investigate tumour invasion. MATERIALS AND METHODS: siRNA was used for suppression of CD73 in either fibroblasts (ST353i, HDF) or tumour cells (FU-EPS-1, A431, CRL-2095). Immunoblotting was performed for detecting MMP-2 production after CD73 suppression. 3D-co-cultures were performed for examining tumour invasion. RESULTS: CD73 suppression revealed that CD73 expression on fibroblasts and emmprin on tumour cells were important in regulating MMP-2 production, suggesting that emmprin on tumour cells does not bind CD73 at the cis-manner, but rather at the trans-manner to CD73 present on fibroblasts. CD73 suppression also reduced MMP-2 production at the transcription level and reduced tumour invasion. CONCLUSION: CD73 on fibroblasts acts as a receptor for emmprin, which forms a complex that increases MMP-2 production, possibly resulting in increased invasiveness.

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.

Inhibition of phosphodiesterase-4 (PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model.

BACKGROUND: We previously established a three-dimensional (3-D) colonic crypt model using HKe3 cells which are human colorectal cancer (CRC) HCT116 cells with a disruption in oncogenic KRAS, and revealed the crucial roles of oncogenic KRAS both in inhibition of apoptosis and in disruption of cell polarity; however, the molecular mechanism of KRAS-induced these 3-D specific biological changes remains to be elucidated. RESULTS: Among the genes that were upregulated by oncogenic KRAS in this model, we focused on the phosphodiesterase 4B (PDE4B) of which expression levels were found to be higher in clinical tumor samples from CRC patients in comparison to those from healthy control in the public datasets of gene expression analysis. PDE4B2 was specifically overexpressed among other PDE4 isoforms, and re-expression of oncogenic KRAS in HKe3 cells resulted in PDE4B overexpression. Furthermore, the inhibition of PDE4 catalytic activity using rolipram reverted the disorganization of HCT116 cells into the normal physiologic state of the epithelial cell polarity by inducing the apical assembly of ZO-1 (a tight junction marker) and E-cadherin (an adherens junction marker) and by increasing the activity of caspase-3 (an apoptosis marker) in luminal cavities. Notably, rolipram reduced the AKT phosphorylation, which is known to be associated with the disruption of luminal cavity formation and CRC development. Similar results were also obtained using PDE4B2-shRNAs. In addition, increased expression of PDE4B mRNA was found to be correlated with relapsed CRC in a public datasets of gene expression analysis. CONCLUSIONS: These results collectively suggested that PDE4B is upregulated by oncogenic KRAS, and also that the inhibition of PDE4 catalytic activity can induce both epithelial cell polarity and luminal apoptosis in CRC, thus highlighting the utility of our 3-D culture (3 DC) model for the KRAS-induced development of CRC in 3-D microenvironment. Indeed, using this model, we found that PDE4B is a promising candidate for a therapeutic target as well as prognostic molecular marker in CRC. Further elucidation of the signaling network of PDE4B2 in 3 DC would provide a better understanding of CRC in vivo.

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response.

ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

Chitinase 3-like 1, Carcinoembryonic Antigen-related Cell Adhesion Molecule 6, and Ectopic Claudin-2 in the Carcinogenic Processes of Ulcerative Colitis.

BACKGROUND/AIM: The cumulative cancerous rate of colitis-associated cancer (CAC) has increased exponentially in patients with ulcerative colitis (UC). We have investigated the factors involved in the carcinogenic processes of CAC among UC patients. PATIENTS AND METHODS: A total of 42 UC patients who underwent surgical treatments between January 2001 and December 2010 at Kurume University Hospital (Fukuoka, Japan) were enrolled. We conducted this study using 3 cases of CAC out of 42 UC cases and 1 case of colorectal cancer. cDNA microarray analyses were performed using normal, inflamed, and cancerous tissues from surgical CAC specimens and protein expression was confirmed by immunohistochemical analyses. RESULTS: cDNA microarray revealed 32 genes that were dominantly expressed in tumorous regions of CAC. Gene ontology analysis revealed that these genes were involved in inflammatory responses and cytokine-cytokine receptor interactions. Chitinase 3-like1 (CHI3L1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), and Claudin-2 (CLND2) were selected from CAC-related genes as candidate molecules. Immunostaining revealed strong expression of each protein in cancerous regions. CONCLUSION: In this study, we identified CAC-related genes and found that CHI3L1, CEACAM6, and CLND2 were expressed in patient samples. All the above genes were associated with adherent invasive Escherichia coli (AIEC), which suggested that these molecules are likely involved in AIEC infection. Further analyses would be required to reveal unknown mechanisms of CAC-related genes in the tumor microenvironment.