Keiichi Tamai

Large noncoding RNA HOTAIR enhances aggressive biological behavior and is associated with short disease-free survival in human non-small cell lung cancer.

HOTAIR is one of long non-coding RNAs and its expression correlates with the prognosis and metastasis in various cancers. We showed that HOTAIR expression has an important role in the development of non-small cell lung cancer (NSCLC). In this study, we examined the expression of HOTAIR in 77 NSCLCs, their corresponding normal lung tissues and 6 brain metastases by quantitative real-time RT-PCR. High expression of HOTAIR (tumor/normal ratio ⩾2) was detected in 17 patients (22.1%) and was frequently found in patients with advanced stage, lymph node metastasis or lymph-vascular invasion and short disease free interval. Furthermore, brain metastases show significantly higher HOTAIR expression compared to primary cancer tissues. HOTAIR-expressing A549 cells showed induced cell migration and anchorage-independent cell growth in vitro. These results indicate the expression of HOTAIR enhanced the aggressive behavior of NSCLC cells.

Enhanced Expression of Long Non-Coding RNA HOTAIR Is Associated with the Development of Gastric Cancer

The long non-coding RNA HOTAIR has been reported to be a poor prognostic biomarker in a variety of malignant tumors. However, little is known about the association of HOTAIR with gastric cancer. We examined the expression of HOTAIR in 68 gastric cancer samples using quantitative real-time RT-PCR and analyzed its correlation with the clinical parameters. The functional role of HOTAIR was examined by generating human gastric cancer cell lines with increased or suppressed HOTAIR expression. The anchorage -independent growth was assessed by soft agar assay. The increased or suppressed HOTAIR expressing gastric cancer cells were injected into the tail vein or peritoneal cavity of immunodeficient mice to examine the effect of this molecule on metastasis and peritoneal dissemination. The expression of HOTAIR was significantly higher in cancer lesions than in adjacent non-cancerous tissues in human gastric cancers. In the diffuse type of gastric cancer, the High-HOTAIR group (HOTAIR/GAPDH > 1) showed significantly more venous invasion, frequent lymph node metastases and a lower overall survival rate compared to the Low-HOTAIR group (HOTAIR/GAPDH < 1). Colony formation on the soft agar was enhanced in a HOTAIR-dependent manner. HOTAIR-expressing MKN74 formed more liver metastasis compared to control when they were injected into the tail vein of mice. In addition, reduced expression of HOTAIR in KATO III suppressed peritoneal dissemination. These results suggest that HOTAIR plays a pivotal role in the development of gastric cancer.

Exosome secretion of dendritic cells is regulated by Hrs, an ESCRT-0 protein

Exosomes are nanovesicles derived from multivesicular bodies (MVBs) in antigen-presenting cells. The components of the ESCRT (endosomal sorting complex required for transport) pathway are critical for the formation of MVBs, however the relationship between the ESCRT pathway and the secretion of exosomes remains unclear. We here demonstrate that Hrs, an ESCRT-0 protein, is required for fascilitating the secretion of exosomes in dendritic cells (DCs). Ultrastructural analyses showed typical saucer-shaped exosomes in the culture supernatant from both the control and Hrs-depleted DCs. However, the amount of exosome secretion was significantly decreased in Hrs-depleted DCs following stimulations with ovalbumin (OVA) as well as calcium ionophore. Antigen-presentation activity was also suppressed in exsosomes purified from Hrs-depleted DCs, while no alteration in OVA degradation was seen in Hrs-depleted DCs. These data indicated that Hrs is involved in the regulation of antigen-presentation activity through the exosome secretion.

The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein

Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that α-synuclein (αSYN), a principal culprit for Lewy pathology in Parkinsons disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular αSYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of αSYN but facilitated αSYN secretion. The hypersecretion of αSYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of αSYN and thereby contribute to the intercellular propagation of Lewy pathology in PD.

Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway

The molecular mechanisms of assembly and budding of hepatitis C virus (HCV) remain poorly understood. The budding of several enveloped viruses requires an endosomal sorting complex required for transport (ESCRT), which is part of the cellular machinery used to form multivesicular bodies (MVBs). Here, we demonstrated that Hrs, an ESCRT-0 component, is critical for the budding of HCV through the exosomal secretion pathway. Hrs depletion caused reduced exosome production, which paralleled with the decrease of HCV replication in the host cell, and that in the culture supernatant. Sucrose-density gradient separation of the culture supernatant of HCV-infected cells revealed the co-existence of HCV core proteins and the exosome marker. Furthermore, both the core protein and an envelope protein of HCV were detected in the intraluminal vesicles of MVBs. These results suggested that HCV secretion from host cells requires Hrs-dependent exosomal pathway in which the viral assembly is also involved.

Possible involvement and the mechanisms of excess trans-fatty acid consumption in severe NAFLD in mice

BACKGROUND & AIMS Excessive trans-fatty acids (TFA) consumption has been thought to be a risk factor mainly for coronary artery diseases while less attention has been paid to liver disease. We aimed to clarify the impact of TFA-rich oil consumption on the hepatic pathophysiology compared to natural oil. METHODS Mice were fed either a low-fat (LF) or high-fat (HF) diet made of either natural oil as control (LF-C or HF-C) or partially hydrogenated oil, TFA-rich oil (LF-T or HF-T) for 24 weeks. We evaluated the liver and body weight, serological features, liver lipid content and composition, liver histology and hepatic lipid metabolism-related gene expression profile. In addition, primary cultures of mice Kupffer cells (KCs) were evaluated for cytokine secretion and phagocytotic ability after incubation in cis- or trans-fatty acid-containing medium. RESULTS The HF-T-fed mice showed significant increases of the liver and body weights, plasma alanine-aminotransferase, free fatty acid and hepatic triglyceride content compared to the HF-C group, whereas the LF-T group did not differ from the LF-C group. HF-T-fed mice developed severe steatosis, along with increased lipogenic gene expression and hepatic TFA accumulation. KCs showed increased tumor necrosis factor secretion and attenuated phagocytotic ability in the TFA-containing medium compared to its cis-isomer. CONCLUSIONS Excessive consumption of the TFA-rich oil up-regulated the lipogenic gene expression along with marked hepatic lipid accumulation. TFA might be pathogenic through causing severe steatosis and modulating the function of KCs. The quantity and composition of dietary lipids could be responsible for the pathogenesis of non-alcoholic steatohepatitis.

Circulating miR-483-3p and miR-21 is highly expressed in plasma of pancreatic cancer

Several recent studies have revealed that microRNAs (miRNAs) have a role in carcinogenesis and cancer development, and that it is stably detectable in plasma/serum. The aim of this study was to test whether miR-483-3p as well as miR-21 could be plasma biomarkers for PDAC. The plasma samples were obtained from three groups including 32 pancreatic ductal adenocarcinoma (PDAC) patients, 12 patients with intraductal papillary mucinous neoplasm (IPMN) patients and 30 healthy controls (HC). We evaluated the plasma miR-483-3p and miR-21 expression level by quantitative RT-PCR. We compared the differences in the plasma level of these miRNAs among the three groups, and investigated the relevance of their plasma expression level to the clinical factors in PDAC. The expressions of miR-483-3p and miR-21 were detected in all examined plasma samples. The plasma expression levels of these miRNAs were significantly higher in PDAC compared to HC (P<0.01). The plasma miR-483-3p expression was significantly higher in PDAC patients than IPMN patients (P<0.05). The plasma miR-21 level was associated with advanced stage (P<0.05), metastasis to lymph node and liver (P<0.01), and shorter survival (P<0.01) of the PDAC patients. Together, these findings suggest that measurement of the plasma miR-483-3p level is useful for discriminating PDAC from IPMN, and that the plasma miR-21 level predicts outcome of PDAC patients.

Branched chain amino acids enhance the maturation and function of myeloid dendritic cells ex vivo in patients with advanced cirrhosis

An imbalance of plasma amino acids is observed in patients with advanced cirrhosis. The aim of this study was to investigate the influence of the extracellular amino acid imbalance on the function of myeloid dendritic cells (DCs) in patients with advanced cirrhosis. We made a serum‐free culture medium consistent with the average concentration of plasma amino acids from healthy controls (HC, n = 25) or patients with advanced cirrhosis (LC, n = 43) to reflect more closely the actual environment of the living body. We compared the phenotypical and biological functions of blood dendritic cells antigen‐positive dendritic cells (BDCA+ DCs) and monocyte‐derived dendritic cells (MoDCs) from LC and HC with these media. After adding stimulants, the CD83 and CD86 expressions of DCs from LC were lower than those from HC. In both HC and LC, both CD83 and CD86 expressions of DCs stimulated under the cirrhotic medium were lower than under the control medium. This phenomenon was accompanied by a suppression of the mammalian target of rapamycin (mTOR)/S6K‐signaling pathways. The interleukin 12 (IL‐12) production in the cirrhotic medium was significantly lower than in the control medium and increased when valine or leucine was added to the medium. In patients with advanced cirrhosis, peripheral blood mononuclear cells stimulated in the autologous plasma after oral administration of branched‐chain amino acid (BCAA) granules had significantly increased interferon gamma production. Conclusion: In advanced cirrhosis, there is impairment of the function and maturation of DCs, which has been shown to be related to an imbalance in the extracellular amino acid profile. Elevating the extracellular concentration of BCAAs ex vivo in patients with advanced cirrhosis improved the function of DCs. (HEPATOLOGY 2009.)

Four‐year study of lamivudine and adefovir combination therapy in lamivudine‐resistant hepatitis B patients: influence of hepatitis B virus genotype and resistance mutation pattern

Summary.  To investigate the efficacy of long‐term lamivudine (3TC) and adefovir dipivoxil (ADV) combination therapy in 3TC‐resistant chronic hepatitis B virus (HBV) infected patients, we analysed 28 3TC‐resistant patients treated with the combination therapy during 47 months (range, 9–75). At 12, 24, 36, and 48 months, the rates of virological response with undetectable HBV DNA (≤2.6 log copies/mL) were 56, 80, 86, and 92%, respectively. Among 17 hepatitis B e antigen (HBeAg)‐positive patients, HBeAg disappeared in 24% at 12 months, 25% at 24 months, 62% at 36 months, and 88% at 48 months. When HBV genotypes were compared, patients with genotype B achieved virological response significantly more rapidly than those with genotype C (P = 0.0496). One patient developed virological breakthrough after 54 months, and sequence analysis of HBV obtained from the patient was performed. An rtA200V mutation was present in the majority of HBV clones, in addition to the 3TC‐resistant mutations of rtL180M+M204V. The rtN236T ADV‐resistant mutation was observed in only 25% clones. In vitro analysis showed that the rtA200V mutation recovered the impaired replication capacity of the clone with the rtL180M+M204V mutations and induced resistance to ADV. Moreover, rtT184S and rtS202C, which are known entecavir‐resistant mutations, emerged in some rtL180M+M204V clones without rtA200V or rtN236T. In conclusion, 3TC+ADV combination therapy was effective for most 3TC‐resistant patients, especially with genotype B HBV, but the risk of emergence of multiple drug‐resistant strains with long‐term therapy should be considered. The mutation rtA200V with rtL180M+M204V may be sufficient for failure of 3TC+ADV therapy.

Loss of hrs in the central nervous system causes accumulation of ubiquitinated proteins and neurodegeneration.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.