Retrovirus-Derived RTL9 Plays an Important Role in Innate Antifungal Immunity in the Eutherian Brain.

Retrotransposon Gag-like (RTL) genes play a variety of essential and important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (also known as sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brain's innate immunity against viruses and bacteria through their removal of double-stranded RNA and lipopolysaccharide, respectively. In this work, we addressed the function of RTL9 (also known as SIRH10). Using knock-in mice that produce RTL9-mCherry fusion protein, we examined RTL9 expression in the brain and its reaction to fungal zymosan. Here, we demonstrate that RTL9 plays an important role, degrading zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Furthermore, in Rtl9 knockout mice expressing RTL9ΔC protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost. Thus, RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system: RTL9, RTL5 and RTL6, responding to fungi, viruses and bacteria, respectively.

Feeding a High-Fat Diet for a Limited Duration Increases Cancer Incidence in a Breast Cancer Model.

High-fat intake by young Asian women impacts the risk of breast cancer. Understanding the underlying molecular mechanisms may be essential for disease prevention in Asia as well as globally. We aimed to examine the effects of corn oil- and animal fat-based high-fat diets (32.9 and 31.4%, respectively, of fat energy ratio as compared to 12.3% in the standard diet) on mammary carcinogenesis and alterations in gene expression and epigenetic statuses in the mammary gland during the growth stages in a rat model. An increased incidence of carcinomas was observed after the cessation of high-fat feeding. In addition, rapid tumor growth and elevations in Celsr2 expression, which may be a result of DNA hypomethylation patterns in the 3' untranslated region of the gene were noted in the animal fat group. In the human breast carcinoma cell line MCF7, a marginal decrease in cell viability was observed following the knockdown of Celsr2, suggesting that the animal fat-associated risk of cancer is partly due to the deregulation of mammary cell proliferation via non-metabolic gene functions. The present results will contribute to the development of strategies for controlling the food-associated risk of breast cancer, particularly in younger age groups.

The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model.

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.

Different types of reactions to E7386 among colorectal cancer patient-derived organoids and corresponding CAFs.

Colorectal cancer (CRC) harbors genetic alterations in a component of the Wnt signaling pathway in ~90% of cases. In addition, the Wnt signaling pathway has been previously suggested to serve a notable role in the pathophysiology of CRC cells and cancer-associated fibroblasts (CAFs). In the present study, the possible effects of E7386, a selective inhibitor of the interaction between ß-catenin and the cAMP response element-binding protein-binding protein, were evaluated using organoids and the corresponding CAFs derived from patients with CRC. E7386 at 100 nM was revealed to decrease the viability of CRC organoids and CAFs. Analysis of the gene expression profiles revealed marked changes in the expression levels of different types of cancer-associated genes associated with E7386 concentrations in the organoids and/or CAFs, such as those regulating glucose and amino acid metabolism [phosphoenolpyruvate carboxykinase 2, asparagine synthetase (glutamine-hydrolyzing), phosphoserine aminotransferase 1 and phosphoglycerate dehydrogenase], stimulation of natural killer cell-mediated cytotoxicity (UL16-binding protein 1) and modification of the Wnt/ß-catenin signaling pathway (indicated by very low density lipoprotein receptor). Results of the hydrophilic metabolome analysis in the organoids were consistent with those of the transcriptomic analysis. In vivo experiments used corresponding xenograft models, although changes in volume of tumor tissues could not be observed at 50 mg/kg body weight twice a day for 14 days, results on the protein expression levels partially supported those in the in vitro experiments. In conclusion, different types of reactions, such as alterations in the glucose and amino acid metabolic pathways, stimulation of stress responses and NK-cell mediated cytotoxicity and components in the Wnt/ß-catenin signaling pathway, to E7386 in the CRC organoids and corresponding CAFs were observed under conditions with decreased cell viability. However, further mechanistic studies to clarify the relationships with Wnt/ß-catenin signaling pathway and these reactions using preclinical models and biomarker studies using clinical human samples are required for verification of the present preclinical biomarkers.

Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids.

Short-/middle-term and simple prediction studies for carcinogenesis are needed for the safety assessment of chemical substances. To establish a novel genotoxicity assay with an in vivo mimicking system, we prepared murine colonic/pulmonary organoids from gpt delta mice according to the general procedure using collagenase/dispase and cultured them in a 3D environment. When the organoids were exposed to foodborne carcinogens-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and acrylamide (AA)-in the presence of metabolic activation systems, mutation frequencies (MFs) occurring in the gpt gene dose-dependently increased. Moreover, the mutation spectrum analysis indicated predominant G:C to T:A transversion with PhIP, and A:T to C:G and A:T to T:A transversion with AA. These data correspond to those of a previous study describing in vivo mutagenicity in gpt delta mice. However, organoids derived from the liver, a non-target tissue of PhIP-carcinogenesis, also demonstrated genotoxicity with a potency comparable to colonic organoids. Organoids and PhIP were directly incubated in the presence of metabolic activation systems; therefore, there was a lack of organ specificity, as observed in vivo. Additionally, PhIP-DNA adduct levels were comparable in hepatic and colonic organoids after PhIP exposure. Taken together, the organoids prepared in the present study may be helpful to predict chemical carcinogenesis.

The Unique Genetic and Histological Characteristics of DMBA-Induced Mammary Tumors in an Organoid-Based Carcinogenesis Model.

Here, we report a model system using in vitro 7,12-dimethylbenz[a]anthracene (DMBA; 0.6 µM)-treated mammary tissue-derived organoids generated from heterozygous BALB/c-Trp53 knockout mice to induce tumors after injection into the nude mouse subcutis. In parallel, a single oral dose of DMBA (50 mg/kg bodyweight) to the same murine strain induced mammary adenocarcinomas, characterized by biphasic structures differentiated into luminal and myoepithelial lineages and frequent Hras mutations at codon 61. In the present study, the genetic and histological characteristics of DMBA-induced tumors in the organoid-based model were evaluated to validate its similarities to the in vivo study. The organoid-derived tumors were low-grade adenocarcinomas composed of luminal and basal/myoepithelial cells. When the organoid-derived carcinomas were passaged to other nude mice, they partly progressed to squamous cell carcinomas (SCCs). Whole exome sequencing revealed no mutations at Hras codon 61 in the organoid-derived tumors. However, various mutations were detected in other genes such as Tusc3 and Tgfbr2, which have been reported as cancer-associated or homeostatic squamous cell genes. The most common mutational pattern observed in these genes were the G:C to T:A transversions and G:C to A:T transitions, which are not typical of the mutations caused by DMBA treatment. In conclusion, DMBA exhibited carcinogenicity in the both the ex vivo and in vivo mammary carcinogenesis models, albeit with distinct histological and genetical alterations. Further studies are needed to clarify whether organoid-based carcinogenesis models generated following chemical treatment in vitro could be applied to the clarification of the novel mode of action of chemical carcinogenesis.

Re-expression of REG family and DUOXs genes in CRC organoids by co-culturing with CAFs.

Organoids derived from epithelial tumors have recently been utilized as a preclinical model in basic and translational studies. This model is considered to represent the original tumor in terms of 3D structure, genetic and cellular heterogeneity, but not tumor microenvironment. In this study, we established organoids and paired cancer-associated fibroblasts (CAFs) from surgical specimens of colorectal carcinomas (CRCs), and evaluated gene expression profiles in organoids with and without co-culture with CAFs to assess interactions between tumor cells and CAFs in tumor tissues. We found that the expression levels of several genes, which are highly expressed in original CRC tissues, were downregulated in organoids but re-expressed in organoids by co-culturing with CAFs. They comprised immune response- and external stimulus-related genes, e.g., REG family and dual oxidases (DUOXs), which are known to have malignant functions, leading tumor cells to proliferative and/or anti-apoptotic states and drug resistant phenotypes. In addition, the degree of differential induction of REG1 and DUOX2 in the co-culture system varied depending on CAFs from each CRC case. In conclusion, the co-culture system of CRC organoids with paired CAFs was able to partially reproduce the tumor microenvironment.

Novel hepatotoxicity biomarkers of extracellular vesicle (EV)-associated miRNAs induced by CCl4.

Recent findings have revealed that extracellular vesicles (EVs) are secreted from cells and circulate in the blood. EVs are classified as exosomes (40-100 nm), microvesicles (50-1,000 nm) or apoptotic bodies (500-2,000 nm). EVs contain mRNAs, microRNAs, and DNAs and have the ability to transfer them from cell to cell. Recently, especially in humans, the diagnostic accuracy of tumor cell type-specific EV-associated miRNAs as biomarkers has been found to be more than 90 %. In addition, microRNAs contained in EVs in blood are being identified as specific biomarkers of chemical-induced inflammation and organ damage. Therefore, microRNAs contained in the EVs released into the blood from tissues and organs in response to adverse events such as exposure to chemical substances and drugs are expected to be useful as novel biomarkers for toxicity assessment. In this study, C57BL/6 J male mice orally dosed with carbon tetrachloride (CCl4) were used as a hepatotoxicity animal model. Here, we report that not only the known hepatotoxicity biomarkers miR-122 and miR-192 but also 42 novel EV-associated biomarkers were upregulated in mice dosed with CCl4. Some of these novel biomarkers may be expected to be able to use for better understanding the mechanism of toxicity. These results suggest that our newly developed protocol using EV-associated miRNAs as a biomarker would accelerate the rapid evaluation of toxicity caused by chemical substances and/or drugs.

An organoid-based carcinogenesis model induced by in vitro chemical treatment.

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis.

Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition.

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event.

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.

Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality.

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

Variation in gene expression and aberrantly regulated chromosome regions in cloned mice.

DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9-40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4-4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.