Altered microbiota caused by disordered gut motility leads to an overactivation of intestinal immune system in APC1638T mice.

Adenomatous polyposis coli (APC) is recognized as an antioncogene related to familial adenomatous polyposis and colorectal cancers. However, APC is a large protein with multiple binding partners, indicating APC has diverse roles besides as a tumor suppressor. We have ever studied the roles of APC by using APC1638T/1638T (APC1638T) mice. Through those studies, we have noticed stools of APC1638T mice were smaller than those of APC+/+ mice and hypothesized there be a disturbance in fecal formation processes in APC1638T mice. The gut motility was morphologically analyzed by immunohistochemical staining of the Auerbach's plexus. Gut microbiota was analyzed by terminal restriction fragment length polymorphism (T-RFLP). IgA concentration in stools was determined by enzyme-linked immunosorbent assay (ELISA). As results, macroscopic findings suggestive of large intestinal dysmotility and microscopic findings of disorganization and inflammation of the plexus were obtained in APC1638T mice. An alteration of microbiota composition, especially increased Bacteroidetes population was observed. Increases in IgA positive cells and dendritic cells in the ileum with high fecal IgA concentration were also confirmed, suggesting over-activation of gut immunity. Our findings will contribute to our understanding of APC's functions in the gastrointestinal motility, and lead to a development of novel therapies for gut dysmotility-related diseases.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

Circulating microRNA-92a-3p in colorectal cancer: a review.

Recent studies have found that microRNAs (miRNAs) are present in body fluids, including blood, cerebrospinal fluid, tears, saliva, breast milk, and urine in a stable form, and are called circulating miRNAs. Although their biological roles remain to be determined, circulating miRNAs are considered as mediators of intercellular communication like hormones and cytokines. Because circulating miRNAs can be collected in a non-invasive manner called as "liquid biopsy", they have also been studied as potential biomarkers for early detection, evaluation of therapeutic effects, and prediction of prognosis in various diseases, including cancers. In this review, we focus on the studies on circulating microRNA-92a-3p (miR-92a-3p) in colorectal cancer (CRC), considering their existence form, isolation methods, potential as biomarkers, and roles in CRC development and progression.

A further study on a disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

Adenomatous polyposis coli (APC), a well-known anti-oncogene, is considered to have multiple functions through its several binding domains. We have continuingly studied APC1638T/1638T mice (APC1638T mice) to elucidate the functions of APC other than tumor suppression. A distinctive feature of the APC1638T mice is they are tumor free and live as long as APC+/+ mice (WT mice). Previously, we found the length of crypt-villus axis in the jejunum was significantly elongated in APC1638T mice compared with that of WT mice. The populations of goblet cells, Paneth cells, and enteroendocrine cells were also disordered in APC1638T mice. Here, we further analyzed the intestinal dyshomeostasis in APC1638T mice, focusing on the proliferation and differentiation of intestinal stem cell (ISC) lineages, and apoptotic cell shedding at the villus tips. We found that the proliferation of ISC lineages was normally controlled; however, the shedding process of apoptosis cells was significantly delayed in the APC1638T mouse jejunum. Furthermore, the number of microfold cells (M cells) was significantly increased in the APC1638T mouse jejunum. Our data suggested both differentiation process of ISCs and turnover process of intestinal epithelia were disturbed in APC1638T mice, and that contributed to the villus elongation in the APC1638T mouse jejunum.

Morphological and functional abnormalities of hippocampus in APC1638T/1638T mice.

In the present study, we examined morphology and function of hippocampus in the APC1638T/1638T mouse. Expression levels of the APC mRNA and protein were both identical in the hippocampus of the APC+/+ and APC1638T/1638T mice. The dentate gyrus of the APC1638T/1638T hippocampus was thicker, and has more densely-populated granule cells in the APC1638T/1638T mouse hippocampus. Immunoelectron microscopy revealed co-localization of APC with alpha-amino-3- hydroxy-5-methyl- isoxazole-4-propionate receptor (AMPA-R) and with PSD-95 at post-synapse in the APC+/+ hippocampus, while APC1638T was co-localized with neither AMPA-R nor PSD-95 in the APC1638T/1638T hippocampus. By immunoprecipitation assay, full-length APC expressed in the APC +/+ mouse was co-immunoprecipitated with AMPA-R and PSD-95. In contrast, APC1638T expressed in the APC1638T/1638T mouse was not co-immunoprecipitated with AMPA-R and PSD-95. In the hippocampal CA1 region of the APC1638T/1638T mouse, c-Fos expression after electric foot shock was decreased compared with the APC+/+ mouse. The present study showed some abnormalities on morphology of the hippocampus caused by a truncated APC (APC1638T). Also, our findings suggest that failure in APC binding to AMPA-R and PSD-95 may bring about less activities of hippocampal neurons in the APC1638T/1638T mouse.

Two cases of bacterial meningitis due to meropenem-resistant Streptococcus pneumoniae: A threat of serotype 35B, ST 558 lineage.

Although the pneumococcal conjugate vaccine (PCV) has decreased the incidence of invasive pneumococcal disease (IPD) in children, cases of IPD caused by non-PCV serotypes have been increasing. Here, we report two cases of bacterial meningitis caused by meropenem-resistant Streptococcus pneumoniae; in both the cases, 13-valent PCV (PCV13) had been administered. The isolated S. pneumoniae strains were non-PCV13 serotype 35B and resistant to penicillin G, cefotaxime, and meropenem. In addition, multilocus sequence typing (MLST) revealed the sequence type (ST) to be 558. In case 1, a 6-month-old girl recovered without sequelae after antibiotic therapy comprising cefotaxime and vancomycin, whereas in case 2, a 9-month-old boy was treated with an empirical treatment comprising ceftriaxone and vancomycin administration. However, maintaining the blood concentration of vancomycin within the effective range was difficult, due to which the antibiotics were changed to panipenem/betamipron. During the treatment, he presented with seizures, which were effectively controlled with antiepileptic drugs. The rate of incidence of penicillin-susceptible IPD has been substantially increasing after the introduction of PCV. However, an upsurge in IPD cases due to multidrug-resistant (MDR) serotype 35B has been reported in countries where PCV13 was introduced before introducing in Japan. Moreover, an increase in the proportion of MDR serotype 35B and decrease in the susceptibility to broad-spectrum antimicrobials, including meropenem, have been reported. Hence, the number of meningitis cases caused by MDR serotype 35B/ST558 may increase in the future.

Extracellular Vesicles Containing MicroRNA-92a-3p Facilitate Partial Endothelial-Mesenchymal Transition and Angiogenesis in Endothelial Cells.

Extracellular vesicles (EVs) are nanometer-sized membranous vesicles used for primitive cell-to-cell communication. We previously reported that colon cancer-derived EVs contain abundant miR-92a-3p and have a pro-angiogenic function. We previously identified Dickkopf-3 (Dkk-3) as a direct target of miR-92a-3p; however, the pro-angiogenic function of miR-92a-3p cannot only be attributed to downregulation of Dkk-3. Therefore, the complete molecular mechanism by which miR-92a-3p exerts pro-angiogenic effects is still unclear. Here, we comprehensively analyzed the gene sets affected by ectopic expression of miR-92a-3p in endothelial cells to elucidate processes underlying EV-induced angiogenesis. We found that the ectopic expression of miR-92a-3p upregulated cell cycle- and mitosis-related gene expression and downregulated adhesion-related gene expression in endothelial cells. We also identified a novel target gene of miR-92a-3p, claudin-11. Claudin-11 belongs to the claudin gene family, which encodes essential components expressed at tight junctions (TJs). Disruption of TJs with a concomitant loss of claudin expression is a significant event in the process of epithelial-to-mesenchymal transition. Our findings have unveiled a new EV-mediated mechanism for tumor angiogenesis through the induction of partial endothelial-to-mesenchymal transition in endothelial cells.

Validation and application of a novel APC antibody in western blotting, immunoprecipitation, and immunohistochemistry.

Adenomatous polyposis coli (APC) is a large protein with multiple binding partners, suggesting diverse functions besides its well-known role in the destruction of ß-catenin. To elucidate these complex functions, it is crucial to evaluate the precise subcellular distribution of APC within a cell and tissue. However, most of the commercially available anti-APC antibodies can only be used for limited applications, resulting in the use of independently generated antibodies. This has led to various discrepancies between studies as a common antibody has not been established. In this study, we generated an antibody against the c-terminal domain of human APC, designated APC-C antibody, and evaluated its specificity and application in various immunological methods. Our data indicate that this novel APC-C antibody is a specific and versatile antibody that can be used in western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. Widespread use of this APC antibody will help enhance our understanding of APC's function in both normal and cancer cell biology.

A disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

The adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of APC, we explored APC 1638T/1638T (APC1638T) mice that express a truncated APC lacking the C-terminal domain. The APC1638T mice were tumor free and exhibited growth retardation. In the present study, we compared small intestinal crypt-villus cells homeostasis in APC +/+ (WT) mice and APC1638T mice. The body weight of APC1638T mice was significantly smaller than that of WT mice at all ages. The length of small intestine of APC1638T mice was significantly shorter than that of WT mice. The crypt-villus axis was significantly elongated, and the number of intestinal epithelial cells also increased in APC1638T mice compared with those in WT mice. However, the number of intestinal epithelial cells per 100 µm of villi was not different between WT and APC1638T mice. Migration and proliferation of intestinal epithelial cells in APC1638T mice were faster than that in WT mice. The population of Goblet cells, Paneth cells, and enteroendocrine cells was significantly altered in APC1638T mice. These results indicate that C-terminal domain of APC has a role in the regulation of intestinal epithelium homeostasis.

Positive feedback of DDX6/c-Myc/PTB1 regulated by miR-124 contributes to maintenance of the Warburg effect in colon cancer cells.

The human DEAD/H-box RNA helicase gene DDX6 is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein has been shown to cause malignant transformation. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, ribosome assembly, and more. However, details of the regulatory mechanism of DDX6 and functions of DDX6 in cancer cells are largely unknown. On the other hand, the Warburg effect is a well-known feature of cancer cells. Pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has 2 isoforms, PKM1 and PKM2. It has been frequently reported that PKM2 is a tumor-specific isoform and promotes the Warburg effect. However, the functions of the PKM1 gene have been hardly mentioned. Here, we showed that DDX6 was overexpressed in colorectal cancer specimens and regulated by microRNA (miR)-124 in colon cancer cells. Also, a DDX6/c-Myc/PTB1 positive feedback circuit regulated by miR-124 was shown to be established and to contribute to maintenance of the Warburg effect. Moreover, we showed that knockdown of DDX6 induced mainly apoptosis through an imbalance of PKM gene expression, especially causing down-regulation of PKM1 in colon cancer cells. These results suggest that miR-124 is a fine tuner of the Warburg effect and that DDX6 is one of the key molecules in Warburg effect-related miR-124 targeting various genes.

A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

Dickkopf-related protein 3 (Dkk-3) is a potential tumor suppressor reported in various cancer entities. However, we found that Dkk-3 was exceptionally upregulated in bladder cancer T24 cells. To validate the biological role of Dkk-3 other than a tumor suppressor, we examined the function of Dkk-3 in T24 cells. Gene silencing of Dkk-3 inhibited cell growth through inducing G0/G1 cell-cycle arrest. Furthermore, Dkk-3 knock-down caused macropinocytosis accompanied by autophagy, which were canceled in part by their inhibitors 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methyladenine (3-MA). The macropinocytosis was induced by the Dkk-3 knock-down when there were sufficient extracellular nutrients. On the other hand, when the nutritional condition was poor, the autophagy was mainly induced by the Dkk-3 knock-down. These data indicated that Dkk-3 has a role in modulating macropinocytotic and autophagic pathways, a distinct function other than a Wnt antagonist.

Anti-Oncogenic gem-Dihydroperoxides Induce Apoptosis in Cancer Cells by Trapping Reactive Oxygen Species.

Organic gem-dihydroperoxides (DHPs) and their derived peroxides have attracted a great deal of attention as potential anti-cancer agents. However, the precise mechanism of their inhibitory effect on tumors is unknown. To determine the mechanism of the inhibitory effects of DHPs, we examined the effects of DHPs on leukemia K562 cells. As a result, certain DHPs used in this study exhibited growth-inhibitory activity according to a clear structure-activity relationship. The most potent DHP, 12AC3O, induced apoptosis in K562 cells, but not in peripheral blood monocytes (PBMCs) or fibroblast cells. 12AC3O induced apoptosis through the intrinsic mitochondrial pathway and thereafter through the extrinsic pathway. The activity of the former pathway was partly attenuated by a JNK inhibitor. Interestingly, 12AC3O induced apoptosis by trapping a large amount of ROS, leading to an extremely lower intracellular ROS level compared with that in the cells in the steady-state condition. These results suggest that an appropriate level of intracellular ROS was necessary for the maintenance of cancer cell growth. DHPs may have a potential to be a novel anti-cancer agent with minimum adverse effects on normal cells.

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells.

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-ß were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.

Epigenetic regulation of microRNA-128a expression contributes to the apoptosis-resistance of human T-cell leukaemia jurkat cells by modulating expression of fas-associated protein with death domain (FADD).

Increased expression of miR-128a is often observed in acute lymphoblastic leukaemia (ALL) compared with its expression in acute myeloid leukaemia (AML). The objective of this study was to investigate the role of miR-128a, especially that in the Fas-signalling pathway, in T-cell leukaemia cells. The role of miR-128a in Fas-mediated apoptosis was examined by using Fas-activating antibody (CH-11)-susceptible Jurkat cells and -resistant Jurkat/R cells. Whereas ectopic expression of miR-128a conferred Fas-resistance on Jurkat cells by directly targeting Fas-associated protein with death domain (FADD), antagonizing miR-128a expression sensitized Jurkat/R cells to the Fas-mediated apoptosis through derepression of FADD expression. Myeloid leukaemia HL60 and K562 cells were also CH-11-resistant, sharing a similar resistant mechanism with Jurkat/R cells. Furthermore, CH-11 induced demethylation of the promoter region of miR-128a with resultant up-regulation of miR-128a expression in Jurkat/R cells, which was shown to be a mechanism for the resistance ofJurkat/R cells to Fas-mediated apoptosis. Our results indicate that the induction of miR-128a expression by DNA demethylation is a novel mechanism of resistance to Fas-mediated apoptosis.

MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases.

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

microRNA (miR)-205 is downregulated and acts as a tumor suppressor in human melanoma cells. Previously, for clinical application, we added aromatic benzene-pyridine (BP-type) analogs to the 3'-overhang region of the RNA-strand and changed the sequences of the passenger strand in the miR-143 duplex. Here, we demonstrated the antitumor effect in vitro and in vivo of miR-205 that was also chemically modified by BP and had altered passenger sequence. In in vitro experiments, transfection with the synthetic miR-205 (miR-205BP/S3) significantly inhibited the growth of human melanoma cells. Exogenous miR-205BP/S3 suppressed the protein expression levels of E2F1 and VEGF, which are validated targets of miR-205-5p, and BCL2, a transcribed molecule of E2F1, as did Pre-miR-205, used as a miR-205 mimic having the wild-type sequence. On the basis of the results of a luciferase activity assay, miR-205BP/S3 directly targeted E2F1, as did Pre-miR-205. However, miR-205BP/S3 was much more resistant to RNase than Pre-miR-205 in fetal bovine serum and to RNase in mice xenografted with human melanoma tissues. In addition, the intratumoral injection of miR-205BP/S3 exhibited a significant antitumor effect compared with the case of control miRNA or Pre-miR-205 in human melanoma cell-xenografted mice. These findings indicate that miR-205BP/S3 is a possible promising therapeutic modality for melanoma.

Extracellular disposal of tumor-suppressor miRs-145 and -34a via microvesicles and 5-FU resistance of human colon cancer cells.

The dysregulation of microRNA (miRNA) expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU)-resistant human colon cancer DLD-1 cells (DLD-1/5FU) from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs) before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells.

MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.

Colorectal cancer cell-derived extracellular vesicles induce phenotypic alteration of T cells into tumor-growth supporting cells with transforming growth factor-ß1-mediated suppression.

Emerging studies on tumor cell-derived extracellular vesicles (EVs) have shown the biological significance in tumor development and microenvironment through reprogramming immune cells around cancer cells. In this study, we used colorectal cancer cells as EVs donor, and T cells as recipients to examine whether EVs impair the T cell function. As a result, we found that colorectal cancer cell-derived EVs (CRC-EVs) were enriched with TGF-ß1. Interestingly, CRC-EVs induced phenotypic alteration of the T cells to Treg-like cells through activating TGF-ß/Smad signaling and inactivating SAPK signaling. Furthermore, the CRC-EVs-induced-Treg-like cells had a remarkable tumor-growth promoting activity in vitro and in vivo. These results suggest that colorectal cancer cells utilize EVs to tame immune cells for their prosperity.