Jun-ichi Miyazaki

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage ofxa0carcinogenesis.

Analysis of K-ras Codon 12 Mutation in Flat and Nodular Variants of Serrated Adenoma in the Colon

AbstractPURPOSE: The developmental process of serrated adenomas is obscure, and the importance of genetic alterations has not been elucidated clearly. The possibility that the developmental process and genetic alterations of serrated adenomas could differ from those of ordinary tubular adenomas was explored in this work. nMETHODS: Serrated adenomas were obtained by endoscopic resection (n = 57) and divided into two groups: flat (n = 10) and nodular (n = 47). Mutation of the K-ras gene was analyzed by enriched polymerase chain reaction–enzyme-linked mini-sequence assay, which can detect not only the presence of a mutation but also the mutation type of K-ras codon 12 with high sensitivity. Methylation-specific polymerase chain reaction was performed with specific primers for the DNA repair gene O6-methylguanine-DNA methyltransferase. nRESULTS: Serrated adenomas located in the rectum were more likely to have a K-ras mutation (9/12, 75 percent), whereas serrated adenomas of the flat type were less likely to have one (1/10, 10 percent). Furthermore, nodular serrated adenomas that occurred in the rectum possessed a high frequency of K-ras gene codon 12 point mutation (8/10, 80 percent) despite an overall frequency of 46.8 percent (22/47). A mutation of the K-ras codon 12 gene was detected in 23 (40.4 percent) of 57 serrated adenomas. Three types of point mutations of codon 12 were detected, with the mutation of GAT being observed most frequently. nCONCLUSIONS: This study shows that development of nodular serrated adenomas may depend on the mutation of the K-ras codon 12 gene, whereas development of flat serrated adenomas may not. Additionally, serrated adenomas that occur in the rectum are closely related to the mutation of the K-ras codon 12 gene. K-ras mutations in serrated adenomas may be unaffected by the epigenetic silencing of O6-methylguanine-DNA methyltransferase by promoter hypermethylation.

Evaluation of the type V pit pattern in the lesions of colonic Tis and T1 cancer

Background:u2002 The usefulness of magnifying videoendoscopic pit pattern diagnosis has been recognized in the differential diagnosis of colonic neoplasms. Also, the correspondence between lesions with a type V pit pattern and cancer has been emphasized. We evaluated the relationship between the type V pit pattern and carcinoma in situ or subdivided submucosal invading carcinomas.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis

The piRNA pathway is a piRNA‐guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI‐piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI‐piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI‐piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI‐piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI‐piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.

IRE1–XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells

In mammalian pancreatic &bgr; cells, the IRE1&agr;–XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed &bgr; cell–specific Ire1&agr; conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1&agr; was deleted using the Cre–loxP system. Ire1&agr; CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4–20 weeks. Ire1&agr; deletion in pancreatic &bgr; cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1&agr;–XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1&agr; functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic &bgr; cells.

Zfp296 negatively regulates H3K9 methylation in embryonic development as a component of heterochromatin

The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296−/− mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296−/− embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.

Olfactory receptors are expressed in pancreatic β-cells and promote glucose-stimulated insulin secretion

Olfactory receptors (ORs) mediate olfactory chemo-sensation in OR neurons. Herein, we have demonstrated that the OR chemo-sensing machinery functions in pancreatic β-cells and modulates insulin secretion. First, we found several OR isoforms, including OLFR15 and OLFR821, to be expressed in pancreatic islets and a β-cell line, MIN6. Immunostaining revealed OLFR15 and OLFR821 to be uniformly expressed in pancreatic β-cells. In addition, mRNAs of Olfr15 and Olfr821 were detected in single MIN6 cells. These results indicate that multiple ORs are simultaneously expressed in individual β-cells. Octanoic acid, which is a medium-chain fatty acid contained in food and reportedly interacts with OLFR15, potentiated glucose-stimulated insulin secretion (GSIS), thereby improving glucose tolerance in vivo. GSIS potentiation by octanoic acid was confirmed in isolated pancreatic islets and MIN6 cells and was blocked by OLFR15 knockdown. While Gαolf expression was not detectable in β-cells, experiments using inhibitors and siRNA revealed that the pathway dependent on phospholipase C-inositol triphosphate, rather than cAMP-protein kinase A, mediates GSIS potentiation via OLFR15. These findings suggest that the OR system in pancreatic β-cells has a chemo-sensor function allowing recognition of environmental substances obtained from food, and potentiates insulin secretion in a cell-autonomous manner, thereby modulating systemic glucose metabolism.

MUCINOUS ADENOCARCINOMA ASSOCIATED WITH RADIATION COLITIS

A 57‐year‐old woman complained of anal bleeding. She had undergone radical hysterectomy followed by radiation total 60u2003gray for the treatment of uterus cancer 8u2003years prior. Colonoscopic examination was performed, and laterally spreading tumors were detected in the transverse, descending, and sigmoid colon. Histopathological findings of operated specimen were compatible with radiation colitis, and mucinous adenocarcinoma existed in this region and invaded to the subserosa with lymphonodi metastasis. She died of peritonitis carcinomatosa 8u2003months later.