HTLV-1 bZIP Factor-Induced Reprogramming of Lactate Metabolism and Epigenetic Status Promote Leukemic Cell Expansion.

Acceleration of glycolysis is a common trait of cancer. A key metabolite, lactate, is typically secreted from cancer cells because its accumulation is toxic. Here, we report that a viral oncogene, HTLV-1 bZIP factor (HBZ), bimodally upregulates TAp73 to promote lactate excretion from adult T-cell leukemia-lymphoma (ATL) cells. HBZ protein binds to EZH2 and reduces its occupancy of the TAp73 promoter. Meanwhile, HBZ RNA activates TAp73 transcription via the BATF3-IRF4 machinery. TAp73 upregulates the lactate transporters MCT1 and MCT4. Inactivation of TAp73 leads to intracellular accumulation of lactate, inducing cell death in ATL cells. Furthermore, TAp73 knockout diminishes the development of inflammation in HBZ-transgenic mice. An MCT1/4 inhibitor, syrosingopine, decreases the growth of ATL cells in vitro and in vivo. MCT1/4 expression is positively correlated with TAp73 in many cancers, and MCT1/4 upregulation is associated with dismal prognosis. Activation of the TAp73-MCT1/4 pathway could be a common mechanism contributing to cancer metabolism. SIGNIFICANCE: An antisense gene encoded in HTLV-1, HBZ, reprograms lactate metabolism and epigenetic modification by inducing TAp73 in virus-positive leukemic cells. A positive correlation between TAp73 and its target genes is also observed in many other cancer cells, suggesting that this is a common mechanism for cellular oncogenesis. This article is featured in Selected Articles from This Issue, p. 337.

Oral Health Promotion under the 8020 Campaign in Japan-A Systematic Review.

(1) Background: The aim of this study is to review the benefits of the 8020 Campaign since its inception. (2) Methods: We followed the PRISMA guideline and collected information regarding the 8020 Campaign through online database searches. (3) Results: Twenty-five studies met the inclusion criteria and were eligible for analysis. The main outcomes of the 25 included studies were reviewed. The quality evaluation demonstrated a range of studies showing a credible relationship between masticatory function, number of teeth, salivary secretion, frequent dental check-ups, and general health concerns. Due to the risk of bias, publication bias, and indirectness, 22 studies were considered that only had "fair" quality. (4) Conclusions: The 8020 Foundation funded several of the studies, and other research papers noted the 8020 Campaign in their papers, however there were no clear explanations for any direct relationship between their findings and the 8020 Campaign. As a result, evidence for the direct effectiveness and benefits assessment of the 8020 Campaign positive outcomes were compromised by confounding social and economic variables over the 30-year period. To fully elucidate how improvement in Japan's oral health was directly related to the 8020 Campaign, a more informed and systematic explanation of the campaign's structure and activities is required.

Whole-genome sequencing with long reads reveals complex structure and origin of structural variation in human genetic variations and somatic mutations in cancer.

BACKGROUND: Identification of germline variation and somatic mutations is a major issue in human genetics. However, due to the limitations of DNA sequencing technologies and computational algorithms, our understanding of genetic variation and somatic mutations is far from complete. METHODS: In the present study, we performed whole-genome sequencing using long-read sequencing technology (Oxford Nanopore) for 11 Japanese liver cancers and matched normal samples which were previously sequenced for the International Cancer Genome Consortium (ICGC). We constructed an analysis pipeline for the long-read data and identified germline and somatic structural variations (SVs). RESULTS: In polymorphic germline SVs, our analysis identified 8004 insertions, 6389 deletions, 27 inversions, and 32 intra-chromosomal translocations. By comparing to the chimpanzee genome, we correctly inferred events that caused insertions and deletions and found that most insertions were caused by transposons and Alu is the most predominant source, while other types of insertions, such as tandem duplications and processed pseudogenes, are rare. We inferred mechanisms of deletion generations and found that most non-allelic homolog recombination (NAHR) events were caused by recombination errors in SINEs. Analysis of somatic mutations in liver cancers showed that long reads could detect larger numbers of SVs than a previous short-read study and that mechanisms of cancer SV generation were different from that of germline deletions. CONCLUSIONS: Our analysis provides a comprehensive catalog of polymorphic and somatic SVs, as well as their possible causes. Our software are available at https://github.com/afujimoto/CAMPHOR and https://github.com/afujimoto/CAMPHORsomatic .

Systematic clustering algorithm for chromatin accessibility data and its application to hematopoietic cells.

The huge amount of data acquired by high-throughput sequencing requires data reduction for effective analysis. Here we give a clustering algorithm for genome-wide open chromatin data using a new data reduction method. This method regards the genome as a string of 1s and 0s based on a set of peaks and calculates the Hamming distances between the strings. This algorithm with the systematically optimized set of peaks enables us to quantitatively evaluate differences between samples of hematopoietic cells and classify cell types, potentially leading to a better understanding of leukemia pathogenesis.

Potential Effects of Indole-3-Lactic Acid, a Metabolite of Human Bifidobacteria, on NGF-induced Neurite Outgrowth in PC12 Cells.

Gut microbiota-derived tryptophan metabolites such as indole derivatives are an integral part of host metabolome that could mediate gut-brain communication and contribute to host homeostasis. We previously reported that infant-type Human-Residential Bifidobacteria (HRB) produced higher levels of indole-3-lactic acid (ILA), suggesting the former might play a specific role in microbiota-host crosstalk by producing ILA in human infants. Nonetheless, the biological meaning of bifidobacteria-derived ILA in infant health development remains obscure. Here, we sought to explore the potential role of ILA in neuronal differentiation. We examined the neurite outgrowth and acetylcholinesterase (AchE) activity of PC12 cells following exposure to ILA and NGF induction. We found that ILA substantially enhanced NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner, and had the most prominent effect at 100 nM. Significant increases in the expression of TrkA receptor, ERK1/2 and CREB were observed in ILA-treated PC12 cells, suggesting ILA potentiated NGF-induced neurite outgrowth through the Ras/ERK pathway. Additionally, ILA was found to act as the aryl hydrocarbon receptor (AhR) agonist and evoked NGF-induced neurite outgrowth in an AhR-mediated manner. These new findings provide clues into the potential involvement of ILA as the mediator in bifidobacterial host-microbiota crosstalk and neuronal developmental processes.

Heat-Killed Bifidobacterium breve B-3 Enhances Muscle Functions: Possible Involvement of Increases in Muscle Mass and Mitochondrial Biogenesis.

A previous clinical study on pre-obesity subjects revealed that Bifidobacterium breve B-3 shows anti-obesity effects and possibly increases muscle mass. Here, we investigated the effects of B-3 on muscle function, such as muscle strength and metabolism, and some signaling pathways in skeletal muscle. Male rodents were orally administered live B-3 (B-3L) or heat-killed B-3 (B-3HK) for 4 weeks. We found that administration of B-3 to rats tended to increase muscle mass and affect muscle metabolism, with stronger effects in the B-3HK group than in the B-3L group. B-3HK significantly increased muscle mass and activated Akt in the rat soleus. With regard to muscle metabolism, B-3HK significantly increased phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and cytochrome c oxidase (CCO) gene expression in the rat soleus, suggesting an effect on the AMPK-PGC1α-mitochondrial biogenesis pathway. Furthermore, B-3HK promoted oxidative muscle fiber composition in the gastrocnemius. We also observed a significantly higher level of murine grip strength in the B-3HK group than in the control group. These findings suggest the potential of heat-killed B-3 in promoting muscle hypertrophy and modifying metabolic functions, possibly through the Akt and AMPK pathways, respectively.

Identification of intermediate-sized deletions and inference of their impact on gene expression in a human population.

BACKGROUND: Next-generation sequencing has allowed for the identification of different genetic variations, which are known to contribute to diseases. Of these, insertions and deletions are the second most abundant type of variations in the genome, but their biological importance or disease association is not well-studied, especially for deletions of intermediate sizes. METHODS: We identified intermediate-sized deletions from whole-genome sequencing (WGS) data of Japanese samples (n = 174) with a novel deletion calling method which considered multiple samples. These deletions were used to construct a reference panel for use in imputation. Imputation was then conducted using the reference panel and data from 82 publically available Japanese samples with gene expression data. The accuracy of the deletion calling and imputation was examined with Nanopore long-read sequencing technology. We also conducted an expression quantitative trait loci (eQTL) association analysis using the deletions to infer their functional impacts on genes, before characterizing the deletions causal for gene expression level changes. RESULTS: We obtained a set of polymorphic 4378 high-confidence deletions and constructed a reference panel. The deletions were successfully imputed into the Japanese samples with high accuracy (97.3%). The eQTL analysis identified 181 deletions (4.1%) suggested as causal for gene expression level changes. The causal deletion candidates were significantly enriched in promoters, super-enhancers, and transcription elongation chromatin states. Generation of deletions in a cell line with the CRISPR-Cas9 system confirmed that they were indeed causative variants for gene expression change. Furthermore, one of the deletions was observed to affect the gene expression levels of a gene it was not located in. CONCLUSIONS: This paper reports an accurate deletion calling method for genotype imputation at the whole genome level and shows the importance of intermediate-sized deletions in the human population.

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone.

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a "cold" tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer.

HTLV-1 Alters T Cells for Viral Persistence and Transmission.

Human T-cell leukemia virus type 1 (HTLV-1) was the first retrovirus to be discovered as a causative agent of adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases. Two viral factors, Tax and HTLV-1 bZIP factor (HBZ), are thought to be involved in the leukemogenesis of ATL. Tax expression is frequently lost due to DNA methylation in the promoter region, genetic changes to the tax gene, and deletion of the 5' long terminal repeat (LTR) in approximately half of all ATL cases. On the other hand, HBZ is expressed in all ATL cases. HBZ is known to function in both protein form and mRNA form, and both forms play an important role in the oncogenic process of HTLV-1. HBZ protein has a variety of functions, including the suppression of apoptosis, the promotion of proliferation, and the impairment of anti-viral activity, through the interaction with several host cellular proteins including p300/CBP, Foxp3, and Foxo3a. These functions dramatically modify the transcriptional profiling of host T cells. HBZ mRNA also promotes T cell proliferation and viability. HBZ changes infected T cells to CCR4+TIGIT+CD4+ effector/memory T cells. This unique immunophenotype enables T cells to migrate into various organs and tissues and to survive in vivo. In this review, we summarize how HBZ hijacks the transcriptional networks and immune systems of host T cells to contribute to HTLV-1 pathogenesis on the basis of recent new findings about HBZ and tax.

Igß ubiquitination activates PI3K signals required for endosomal sorting.

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multi-system disease caused by expanded CTG repeats in dystrophia myotonica protein kinase (DMPK). The expanded CTG repeats are unstable and can increase the length of the gene with age, which worsens the symptoms. In order to establish a human stem cell system suitable for the investigation of repeat instability, DM1 patient-derived iPSCs were generated and differentiated into three cell types commonly affected in DM1, namely cardiomyocytes, neurons and myocytes. Then we precisely analysed the CTG repeat lengths in these cells. Our DM1-iPSCs showed a gradual lengthening of CTG repeats with unchanged repeat distribution in all cell lines depending on the passage numbers of undifferentiated cells. However, the average CTG repeat length did not change significantly after differentiation into different somatic cell types. We also evaluated the chromatin accessibility in DM1-iPSCs using ATAC-seq. The chromatin status in DM1 cardiomyocytes was closed at the DMPK locus as well as at SIX5 and its promoter region, whereas it was open in control, suggesting that the epigenetic modifications may be related to the CTG repeat expansion in DM1. These findings may help clarify the role of repeat instability in the CTG repeat expansion in DM1.

Epigenetic Variation between Human Induced Pluripotent Stem Cell Lines Is an Indicator of Differentiation Capacity.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocally repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

Bovine lactoferrin ingestion protects against inflammation via IL-11 induction in the small intestine of mice with hepatitis.

Accumulating evidence suggests that orally ingested lactoferrin protects against inflammation. To assess the efficacy of orally administered bovine lactoferrin (bLF) against hepatitis and to identify the underlying mechanism, in the present study, we used four mouse models of hepatitis induced by d-galactosamine (GalN), carbon tetrachloride (CCl4), GalN plus lipopolysaccharide (LPS) and zymosan plus LPS. Intraperitoneal (i.p.) injection of GalN (500 mg/kg body weight) in mice treated with bovine serum albumin (BSA) for 14 d significantly increased serum aspartate aminotransferase (AST) concentrations compared with the untreated mice. However, orally administered bLF reduced AST concentrations compared with BSA treatment. In mice that received a single injection (0·4 ml/kg) and twice-weekly injections (0·08 ml/kg) of CCl4 for 24 weeks and pretreated with bLF for 14 d and 24 weeks, respectively, significantly suppressed alanine aminotransferase and AST concentrations were observed compared with the BSA-treated control. Oral administration of bLF for 14 d before i.p. injection of LPS (5 mg/kg) plus GalN (1 g/kg) significantly improved the survival rate. In mice that received intravenous injection of zymosan (25 mg/kg) and LPS (15 µg/kg) at 7 d intervals, bLF reduced the elevation of AST concentrations and enhanced the production of IL-11 and bone morphogenetic protein 2 in the small intestine compared with the BSA-treated control. To evaluate the effects of IL-11, we used IL-11 receptor α-null mice treated with GalN, CCl4 and zymosan plus LPS. In this group, the activity of bLF was not significantly different from that of BSA. These data indicate that orally ingested bLF enhances the expression of IL-11 in the small intestine and up-regulates protective activity in mice with hepatitis.

Recent classification of renal epithelial tumors.

The recent classification of renal tumors is based on genetic evidence as well as on histologic features. Malignant tumor includes clear cell renal carcinoma (RCC), multilocular cystic RCC, papillary RCC, chromophobe RCC, carcinoma of the collecting duct of Bellini, renal carcinoma associated with Xp11.2 translocations/TFE3 gene fusions and mucinous tubular and spindle cell carcinoma. Benign tumor is subdivided into papillary adenoma, renal oncocytoma and metanephric adenoma. Recently, new disease entities such as acquired cystic disease-associated RCC, clear cell papillary RCC and renal carcinoma with t(6;11)(p21:q12) have been discovered. In this article, we briefly review and introduce the clinical, morphological and genetic features of these tumor entities.

Recent advances of immunohistochemistry for diagnosis of renal tumors.

The recent classification of renal tumors has been proposed according to genetic characteristics as well as morphological difference. In this review, we summarize the immunohistochemical characteristics of each entity of renal tumors. Regarding translocation renal cell carcinoma (RCC), TFE3, TFEB and ALK protein expression is crucial in establishing the diagnosis of Xp11.2 RCC, renal carcinoma with t(6;11)(p21;q12), and renal carcinoma with ALK rearrangement, respectively. In dialysis-related RCC, neoplastic cells of acquired cystic disease-associated RCC are positive for alpha-methylacyl-CoA racemase (AMACR), but negative for cytokeratin (CK) 7, whereas clear cell papillary RCC shows the inverse pattern. The diffuse positivity for carbonic anhydrase 9 (CA9) is diagnostic for clear cell RCC. Co-expression of CK7 and CA9 is characteristic of multilocular cystic RCC. CK7 and AMACR are excellent markers for papillary RCC and mucinous tubular and spindle cell carcinoma. CD82 and epithelial-related antigen (MOC31) may be helpful in the distinction between chromophobe RCC and renal oncocytoma. WT1 and CD57 highlights the diagnosis of metanephric adenoma. The combined panel of PAX2 and PAX8 may be useful in the diagnosis of metastatic RCC.

Hybrid peripheral nerve sheath tumors, including a malignant variant in type 1 neurofibromatosis.

The authors report a small case series of hybrid nerve sheath tumors occurring in the setting of type 1 neurofibromatosis. Four lesions were benign and consisted of plexiform neurofibromas with considerable areas of perineuriomatous differentiation in patients with type 1 neurofibromatosis. In these lesions, biphasic (Schwannian and perineuriomatous) differentiation was apparent on immunohistochemistry, with the perineuriomatous areas staining for epithelial membrane antigen, glut-1, and claudin-1 and being negative for S-100 protein. Three patients were members of a single family, with a history of various malignant neoplasms. Included in the series is 1 hybrid lesion in which neurofibromatous and perineuriomatous areas were clearly visible on hematoxylin- and eosin-stained slides. The lesion was unique in that it manifested malignant change in the S-100 protein-positive component, which was classified as malignant peripheral nerve sheath tumor. The malignant component showed areas with an epithelioid cell morphology.