Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS.

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). We previously established Drosophila models of ALS targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. We aimed to determine whether Hippo pathway genes modify the ALS phenotype using Caz knockdown flies. We found a genetic link between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. Our results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS.

MicroRNA-196b is an independent prognostic biomarker in patients with pancreatic cancer.

Pancreatic cancer is a highly aggressive malignancy, with <50% patients surviving beyond 6 months after the diagnosis, and thus, there is an urgent need to explore new diagnostic and therapeutic approaches for this disease. Therefore, we conducted microRNA (miRNA) array analysis to detect miRNA molecules potentially associated with pancreatic cancer malignancy. To assess the identified miRNAs, we performed quantitative reverse transcription-PCR on 248 pancreatic ductal adenocarcinomas (UICC stage II). We also examined miRNA expression [microRNA-21 (miR-21) and microRNA-31 (miR-31)] and epigenetic alterations, including CpG island methylator phenotype (CIMP), potentially associated with the identified miRNAs. For functional analysis, we conducted proliferation and invasion assays using a pancreatic cancer cell line. miRNA array analysis revealed that microRNA-196b (miR-196b) was the most up-regulated miRNA in pancreatic cancer tissues compared with normal pancreatic duct cells. High miR-196b expression was associated with miR-21 (P = 0.0025) and miR-31 (P = 0.0001) expression. It was also related to poor prognosis in the multivariate analysis using overall survival (hazard ratio: 1.66; 95% confidence interval: 1.09-2.54; P = 0.019). Functional analysis demonstrated that miR-196b inhibitor decreased cell proliferation and that miR-196b mimic promoted cancer cell invasion. In conclusion, a significant association of high miR-196b expression with poor prognosis was observed in pancreatic cancer. Our data also revealed that miR-196b played an oncogenic role and that the transfection of the miR-196b inhibitor had an anti-tumour effect in the pancreatic cancer cell line. These results suggest that miR-196b is a promising diagnostic biomarker and therapeutic target in pancreatic cancer.

A case of adenosquamous carcinoma of the lower bile duct diagnosed preoperatively via transpapillary biopsy.

A 78-year-old man presented to our hospital with fever and brownish urine. Upon thorough examination, a diagnosis of obstructive jaundice and acute cholangitis associated with a lower bile duct tumor was made. Endoscopic retrograde cholangiopancreatography revealed entire circumferential stenosis of the lower bile duct. Examination of a transpapillary biopsy specimen of the lesion suggested adenosquamous carcinoma. The patient underwent subtotal stomach-preserving pancreaticoduodenectomy. Histopathological examination revealed adenocarcinoma of the lower bile duct and squamous cell carcinoma components;a case of adenosquamous carcinoma was accordingly diagnosed. The lower bile duct tumor directly extended into the pancreatic parenchyma for approximately 1mm. We performed radical surgery and administered adjuvant chemotherapy with gemcitabine because of advanced neural invasion after consulting with the patient. There was no sign of recurrence 46 months after surgery. As adenosquamous carcinoma of the extrahepatic bile duct is rare, it is difficult to preoperatively diagnose the condition. Only a few cases have been reported till date.

Association of the Low-density Lipoprotein Cholesterol/High-density Lipoprotein Cholesterol Ratio with Glecaprevir-pibrentasvir Treatment.

Objective The change in serum lipid levels by direct-acting antiviral (DAA) treatment for chronic hepatitis C varies depending on the type of DAA. How the lipid level changes induced by glecaprevir-pibrentasvir (G/P) treatment contribute to the clinical outcome remains unclear. We conducted a prospective observational study to evaluate the effectiveness of G/P treatment and the lipid level changes. Methods The primary endpoint was a sustained virologic response at 12 weeks (SVR12). The total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels and LDL-C/HDL-C (L/H) ratio were measured every two weeks. Patients This study included 101 patients. Seventeen cases of liver cirrhosis and nine cases of DAA retreatment were registered. The G/P treatment period was 8 weeks in 74 cases and 12 weeks in 27 cases. Results SVR12 was evaluated in 96 patients. The rate of achievement of SVR12 in the evaluable cases was 100%. We found significantly elevated TC and LDL-C levels over the observation period compared to baseline. The serum levels of HDL-C did not change during treatment but were significantly increased after treatment compared to baseline. The L/H ratio was significantly increased two weeks after the start of treatment but returned to the baseline after treatment. Conclusion The primary endpoint of the SVR12 achievement rate was 100%. G/P treatment changed the serum lipid levels. Specifically, the TC and LDL-C levels increased during and after treatment, and the HDL-C levels increased after treatment. G/P treatment may be associated with a reduced thrombotic risk. Therefore, validation in large trials is recommended.

Intramolecular acyl migration and enzymatic hydrolysis of a novel monoacylated ascorbic acid derivative, 6-O-dodecanoyl-2-O-alpha-d-glucopyranosyl-L-ascorbic acid.

A stable ascorbic acid derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), exhibits vitamin C activity in vitro and in vivo after enzymatic hydrolysis to ascorbic acid. AA-2G has been approved by the Japanese Government as a quasi-drug principal ingredient in skin care and as a food additive. In order to achieve efficient action as an ascorbic acid source, a pro-vitamin C agent, on a variety of cells or tissues, we have synthesized a series of monoacyl AA-2G derivatives. Our previous studies indicate that a series of the derivatives is a readily available source of AA activity in vitro and in vivo, and suggested that intramolecular acyl migration of the derivatives might have occurred in a neutral aqueous solution. In this study, intramolecular acyl migration and enzymatic hydrolysis of a monoacyl AA-2G derivative, 6-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (6-sDode-AA-2G), were investigated. 6-sDode-AA-2G underwent an intramolecular acyl migration to yield ca. 10% of an isomer in neutral aqueous solutions, and the acyl-migrated isomer was isolated and characterized as 5-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (5-sDode-AA-2G). In some tissue homogenates from guinea pigs as well as in neutral aqueous solutions, 6-sDode-AA-2G underwent partial acyl migration to give 5-sDode-AA-2G. 6-sDode-AA-2G and the resulting 5-sDode-AA-2G were predominantly hydrolyzed with esterase to AA-2G and then with alpha-glucosidase to ascorbic acid in the tissue homogenates. The results will provide a further basis for its use as an ingredient in skin care, as an effective pharmacological agent and as a promising food additive.

Cancer-related genes and ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by the progressive degeneration of both upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. Recent advances in human genetics have identified more than 30 ALS-causing genes or genetic loci that include the fused in sarcoma (FUS) gene. In addition, a set of studies suggested a mutual relationship between cancer and ALS. The hpo gene, Drosophila MST was newly identified as a novel genetic modifier of the cabeza (caz), Drosophila FUS. The Hippo pathway negatively regulates the control of organ growth and tumor suppression. Moreover, the p53 tumor suppressor was found to genetically interact with caz. Frontotemporal lobar degeneration (FTLD) is characterized by the degeneration of neurons in the frontal and temporal lobes, and consists of a spectrum with ALS. Fusion protein nucleophosmin-human myeloid leukemia factor 1 (NPM-hMLF1), which is associated with the pathologies of myelodysplastic syndrome and acute myeloid leukemia, was recently shown to suppress defects in the Drosophila FTLD model expressing the human FUS gene. Further studies in the field are expected to elucidate epidemiological, genetic, and histopathological links between cancer and ALS/FTLD, and will lead to the development of therapeutic strategies. We herein summarize previous and current findings that support mutual links between cancer and ALS/FTLD.

Overexpression of ter94, Drosophila VCP, improves motor neuron degeneration induced by knockdown of TBPH, Drosophila TDP-43.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterized by the motor neuron degeneration that eventually leads to complete paralysis and death within 2-5 years after disease onset. One of the major pathological hallmark of ALS is abnormal accumulation of inclusions containing TAR DNA-binding protein-43 (TDP-43). TDP-43 is normally found in the nucleus, but in ALS, it localizes in the cytoplasm as inclusions as well as in the nucleus. Loss of nuclear TDP-43 functions likely contributes to neurodegeneration. TBPH is the Drosophila ortholog of human TDP-43. In the present study, we confirmed that Drosophila models harboring TBPH knockdown develop locomotive deficits and degeneration of motoneurons (MNs) due to loss of its nuclear functions, recapitulating the human ALS phenotypes. We previously suggested that ter94, the Drosophila ortholog of human Valosin-containing protein (VCP), is a modulator of degeneration in MNs induced by knockdown of Caz, the Drosophila ortholog of human FUS. In this study, to determine the effects of VCP on TDP-43-assosiated ALS pathogenic processes, we examined genetic interactions between TBPH and ter94. Overexpression of ter94 suppressed the compound eye degeneration caused by TBPH knockdown and suppressed the morbid phenotypes caused by neuron-specific TBPH knockdown, such as locomotive dysfunction and degeneration of MN terminals. Further immunocytochemical analyses revealed that the suppression is caused by restoring the cytoplasmically mislocalized TBPH back to the nucleus. In consistent with these observations, a loss-of-function mutation of ter94 enhanced the compound eye degeneration caused by TBPH knockdown, and partially enhanced the locomotive dysfunction caused by TBPH knockdown. Our data demonstrated that expression levels of ter94 influenced the phenotypes caused by TBPH knockdown, and indicate that reagents that up-regulate the function of human VCP could modify MN degeneration in ALS caused by TDP-43 mislocalization.

NPM-hMLF1 fusion protein suppresses defects of a Drosophila FTLD model expressing the human FUS gene.

Fused in sarcoma (FUS) was identified as a component of typical inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In FTLD, both nuclear and cytoplasmic inclusions with wild-type FUS exist, while cytoplasmic inclusions with a mutant-form of FUS occur in many ALS cases. These observations imply that FUS plays a role across these two diseases. In this study, we examined the effect of several proteins including molecular chaperons on the aberrant eye morphology phenotype induced by overexpression of wild-type human FUS (hFUS) in Drosophila eye imaginal discs. By screening, we found that the co-expression of nucleophosmin-human myeloid leukemia factor 1 (NPM-hMLF1) fusion protein could suppress the aberrant eye morphology phenotype induced by hFUS. The driving of hFUS expression at 28 °C down-regulated levels of hFUS and endogenous cabeza, a Drosophila homolog of hFUS. The down-regulation was mediated by proteasome dependent degradation. Co-expression of NPM-hMLF1 suppressed this down-regulation. In addition, co-expression of NPM-hMLF1 partially rescued pharate adult lethal phenotype induced by hFUS in motor neurons. These findings with a Drosophila model that mimics FTLD provide clues for the development of novel FTLD therapies.

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives.

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

EZH2 expression is a prognostic biomarker in patients with colorectal cancer treated with anti-EGFR therapeutics.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that suppresses microRNA-31 (miR-31) in various human malignancies including colorectal cancer. We recently suggested that miR-31 regulates the signaling pathway downstream of epidermal growth factor receptor (EGFR) in colorectal cancer. Therefore, we conducted this study for assessing the relationship between EZH2 expression and clinical outcomes in patients with colorectal cancer treated with anti-EGFR therapeutics. We immunohistochemically evaluated EZH2 expression and assessed miR-31 and gene mutations [KRAS (codon 61/146), NRAS (codon 12/13/61), and BRAF (codon 600)] in 109 patients with colorectal cancer harboring KRAS (codon 12/13) wild-type. We also evaluated the progression-free survival (PFS) and overall survival (OS). In the result, low EZH2 expression was significantly associated with shorter PFS (log-rank test: P = 0.023) and OS (P = 0.036) in patients with colorectal cancer. In the low-miR-31-expression group and the KRAS (codon 61/146), NRAS, and BRAF wild-type groups, a significantly shorter PFS (P = 0.022, P = 0.039, P = 0.021, and P = 0.036, respectively) was observed in the EZH2 low-expression groups than in the high-expression groups. In the multivariate analysis, low EZH2 expression was associated with a shorter PFS (P = 0.046), independent of the mutational status and miR-31. In conclusion, EZH2 expression was associated with survival in patients with colorectal cancer who were treated with anti-EGFR therapeutics. Moreover, low EZH2 expression was independently associated with shorter PFS in patients with cancer, suggesting that EZH2 expression is a useful additional prognostic biomarker for anti-EGFR therapy.

Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid.

Topical treatment of skin with all-trans-retinoic acid (ATRA), the major biologically active form of vitamin A, results in hyperproliferation of basal keratinocytes, leading to an accelerated turnover of epidermis cells and thickening of the epidermis, probably via induction of production of paracrine growth factors for keratinocytes in epidermal suprabasal keratinocytes and/or dermal fibroblasts. Since hepatocyte growth factor (HGF) is a factor mitogenic to epidermal keratinocytes secreted from dermal fibroblasts, the effect of ATRA on basal and induced HGF production in human dermal fibroblasts in culture was examined. ATRA alone did not induce HGF production, but it significantly enhanced HGF production induced by the cAMP-elevating agent cholera toxin or the membrane-permeable cAMP analog 8-bromo-cAMP. Cholera toxin-induced activation of cAMP responsive element (CRE)-binding protein (CREB) was enhanced by pretreating cells with ATRA for 24 h. In contrast, HGF production induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) was potently inhibited by ATRA. These modulatory effects of ATRA were different from the effects of transforming growth factor-beta1 (TGF-beta) and dexamethasone, both of which inhibited HGF production induced by all of the four inducers. Up-regulation of HGF gene expression by cholera toxin and EGF was also enhanced and inhibited, respectively, by ATRA. Both 9-cis-retinoic acid (9-cis-RA) and 13-cis-retinoic acid (13-cis-RA), which are stereo-isomers of ATRA, showed a modulatory effect on HGF induction similar to that of ATRA. These results suggest that ATRA augments the induction of HGF production caused by increased intracellular cAMP.

Microvessel density: correlation with 18F-FDG uptake and prognostic impact in lung adenocarcinomas.

UNLABELLED: Although researched for many years, the prognostic value of tumor angiogenesis reflected by microvessel density (MVD) is still controversial, and there have been no previous reports regarding the correlation with 18F-FDG uptake in lung adenocarcinomas. Therefore, in the present study, we investigated the correlation between MVD determined with different endothelial cell antibodies and 18F-FDG uptake and compared the prognostic impact of those factors in lung adenocarcinomas. METHODS: Forty-four patients with 45 lung adenocarcinomas underwent 18F-FDG PET before surgery. Consecutive paraffin-embedded sections obtained from each resected tumor were immunostained for CD31 (a panendothelial cell marker), CD105 (a proliferation-related endothelial cell marker), and CD34/alpha-SMA (for double labeling of endothelial cells and mural cells). Four high-power fields in the area with the highest MVD were selected for analysis. Computer-assisted image analysis was used to assess MVD. RESULTS: MVD staining results for panendothelial cell markers can be classified into 3 microvessel patterns: diffuse, alveolar, and mixed. The highly ordered alveolar pattern is believed to represent preexisting alveolar vessels trapped in lung adenocarcinomas and may have no significant meaning for the aggressiveness of tumors. Preexisting alveolar cells also do not contribute to 18F-FDG uptake. CD105 staining of MVD (CD105-MVD) showed a significantly positive correlation with 18F-FDG uptake (P < 0.0001), whereas CD31 staining of MVD (CD31-MVD) showed a marginally negative correlation with it (P = 0.057). Although CD105-MVD correlated negatively with prognosis, patients with low CD105-MVD, compared with those with high or moderate CD105-MVD, had a much better prognosis in both disease-free and overall survival analyses (P = 0.017 and P = 0.013, respectively). Patients with low CD31-MVD had the worst prognosis (P = 0.032 for disease-free survival analysis and P = 0.179 for overall survival analysis). CONCLUSION: There is no positive correlation between 18F-FDG uptake and MVD determined with panendothelial cell markers (CD31 and CD34); in contrast, there is a marginally negative correlation between them. MVD determined with CD105, which is a proliferation-related endothelial cell marker, reflects active angiogenesis, correlates positively with 18F-FDG uptake, and is a better indicator of prognosis in lung adenocarcinomas.

An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites.

2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.

Knockdown of the Drosophila FIG4 induces deficient locomotive behavior, shortening of motor neuron, axonal targeting aberration, reduction of life span and defects in eye development.

Mutations in Factor-Induced-Gene 4 (FIG4) gene have been identified in Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varon syndrome and epilepsy with polymicrogyria. FIG4 protein regulates a cellular abundance of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a signaling lipid on the cytosolic surface of membranes of the late endosomal compartment. PI(3,5)P2 is required for retrograde membrane trafficking from lysosomal and late endosomal compartments to the Golgi. However, it is still unknown how the neurodegeneration that occurs in these diseases is related to the loss of FIG4 function. Drosophila has CG17840 (dFIG4) as a human FIG4 homolog. Here we specifically knocked down dFIG4 in various tissues, and investigated their phenotypes. Neuron-specific knockdown of dFIG4 resulted in axonal targeting aberrations of photoreceptor neurons, shortened presynaptic terminals of motor neurons in 3rd instar larvae and reduced climbing ability in adulthood and life span. Fat body-specific knockdown of dFIG4 resulted in enlarged lysosomes in cells that were detected by staining with LysoTracker. In addition, eye imaginal disk-specific knockdown of dFIG4 disrupted differentiation of pupal ommatidial cell types, such as cone cells and pigment cells, suggesting an additional role of dFIG4 during eye development.

Association of Fusobacterium nucleatum with immunity and molecular alterations in colorectal cancer.

The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6% (44/511), which was lower than that in United States cohort studies (13%). Similar to the United States studies, F. nucleatum positivity in Japanese colorectal cancers was significantly associated with microsatellite instability (MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets (i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain microRNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. MicroRNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in colorectal cancer cells. Thus, emerging evidence may provide insights for strategies to target microbiota, immune cells and tumor molecular alterations for colorectal cancer prevention and treatment. Further investigation is needed to clarify the association of Fusobacterium with T-cells and microRNA expressions in colorectal cancer.

The relationship between EZH2 expression and microRNA-31 in colorectal cancer and the role in evolution of the serrated pathway.

Polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that correlates with the regulation of invasion and metastasis and is overexpressed in human cancers such as colorectal cancer. MicroRNA-31 (miR-31) plays an oncogenic role and is associated with BRAF mutation and poor prognosis in colorectal cancer. EZH2 is functionally considered to suppress miR-31 expression in human cancers; however, no study has reported its relationship with colon cancer. We therefore evaluated EZH2 expression using immunohistochemistry and assessed miR-31 and epigenetic alterations using 301 colorectal carcinomas and 207 premalignant lesions. Functional analysis was performed to identify the association between EZH2 and miR-31 using cancer cell lines. In the current study, negative, weak, moderate, and strong EZH2 expressions were observed in 15%, 19%, 25%, and 41% of colorectal cancers, respectively. EZH2 was inversely associated with miR-31 (P < 0.0001), independent of clinicopathological and molecular features. In a multivariate stage-stratified analysis, high EZH2 expression was related to favorable prognosis (P = 0.0022). Regarding premalignant lesions, negative EZH2 expression was frequently detected in sessile serrated adenomas/polyps (SSA/Ps) (76%; P < 0.0001) compared with hyperplastic polyps, traditional serrated adenomas, and non-serrated adenomas (25-36%). Functional analysis demonstrated that the knockdown of EZH2 increased miR-31 expression. In conclusion, an inverse association was identified between EZH2 and miR-31 in colorectal cancers. Our data also showed that upregulation of EZH2 expression may be rare in SSA/Ps. These results suggest that EZH2 suppresses miR-31 in colorectal cancer and may correlate with differentiation and evolution of serrated pathway.

18F-FDG uptake by primary tumor as a predictor of intratumoral lymphatic vessel invasion and lymph node involvement in non-small cell lung cancer: analysis of a multicenter study.

UNLABELLED: Intratumoral lymphatic vessel invasion and lymph node involvement are important factors in the planning of therapeutic strategies, particularly limited surgical resection in patients with non-small cell lung cancer. (18)F-FDG uptake within the primary lesion correlates with aggressiveness on PET studies. The more metabolically active the tumor, the more aggressive are the findings. The aim of this multicenter study was to determine whether (18)F-FDG uptake of the primary tumor is a predictor of intratumoral lymphatic vessel invasion and lymph node metastasis in patients with non-small cell lung cancer. METHODS: One hundred thirty-two patients with lung cancer were studied. All patients underwent a thoracotomy within 4 wk of the (18)F-FDG PET study. A 3-point visual scoring system (low, moderate, or high grade in comparison with mediastinal activity) was used to interpret (18)F-FDG uptake within the primary lesions. The degree of (18)F-FDG uptake in the primary tumor was correlated with the incidence of intratumoral lymphatic vessel invasion and lymph node involvement. Multivariate analysis was performed with logistic multivariate analysis to assess the joint effects and interactions of the variables (age, sex, tumor size, histology, and (18)F-FDG uptake) on intratumoral lymphatic vessel invasion and lymph node involvement. RESULTS: Intratumoral lymphatic vessel invasion and lymph node involvement were found in 7.1% and 5.9%, respectively, of the patients classified in the low-grade group, and in 14.3% and 10.0%, respectively, of the patients classified in the moderate-grade group. In contrast, of the patients classified in the group with high (18)F-FDG uptake, intratumoral lymphatic vessel invasion and lymph node involvement were found in 39.7% and 38.9%, respectively. Multivariate analysis showed that only (18)F-FDG uptake was a significant factor for intratumoral lymphatic vessel invasion and that tumor size and (18)F-FDG uptake were significant factors for lymph node involvement. Of the patients in the high-grade group whose tumors were classified as > or =3 cm in size, lymph node involvement was found in 51.5%. In contrast, of the patients in the low- to moderate-grade group whose tumors were classified as <3 cm in size, lymph node involvement was found in only 9.1% (P < 0.0001). CONCLUSION: Patients with a low to moderate (18)F-FDG uptake in the primary lesion had a significantly lower risk of concurrent intratumoral lymphatic vessel invasion and nodal involvement than did patients with a high (18)F-FDG uptake. In patients with non-small cell lung cancer, (18)F-FDG uptake by the primary tumor is a strong predictor of intratumoral lymphatic vessel invasion and lymph node metastasis.

PKC- and PI3K-dependent but ERK-independent proliferation of murine splenic B cells stimulated by chondroitin sulfate B.

High molecular weight polyanions such as dextran sulfate are known to be weak polyclonal activators of murine B cells, but the molecular mechanism of their mitogenic activitiy is not fully elucidated. Although chondroitin sulfate A (CSA), B (CSB) and C (CSC) are highly charged polyanions, little is known about their effects on the proliferation of B cells. In this study, we demonstrated that CSB stimulated proliferation of murine B cells as markedly as did anti-IgM antibody, more markedly than did dextran sulfate and much more markedly than did CSA, CSC, heparin and hyaluronic acid. CSB caused translocation of protein kinase C (PKC) isoform beta from cytosol to membrane fractions and increased phosphorylation of Akt but not phosphorylation of extracellular signal-regulated kinase (ERK) of B cells. CSB-induced B cell proliferation was almost completely blocked by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or the PKC inhibitor GF109203X but was not significantly inhibited by the ERK kinase inhibitor PD98059. The mitogenic effect of anti-IgM was significantly inhibited by all the three inhibitors, while the mitogenic effect of LPS was inhibited only by LY294002. These findings indicate that CSB stimulated proliferation of murine B cells more markedly than did dextran sulfate and suggest that PKC and PI3K are crucial but that ERK is less important for the mitogenic activity of CSB, the signaling pathways of which may be at least partly distinct from those of anti-IgM and LPS.

Osteoblastic osteosarcoma of the mandible: findings on Tc-99m HMDP bone and Ga-67 citrate scintigraphy.

A 25-year-old woman presented with a disturbance in the opening of her mouth 5 months before admission. On admission, painful swelling of the right preauricular region was revealed. Computed tomography (CT) demonstrated a soft tissue density mass around the right condylar process of the mandible. Tc-99m hydroxymethylene diphosphonate (HMDP) bone scintigraphy and Ga-67 citrate scintigraphy showed avid uptake in the mass. The tumor was histologically identified as an osteoblastic osteosarcoma of the right mandible. There are few reports of Ga-67 citrate scintigraphy findings of osteoblastic osteosarcoma of the mandible. The accumulation patterns on Tc-99m HMDP bone scintigraphy and Ga-67 citrate scintigraphy are possibly characteristic of osteoblastic osteosarcoma of the mandible.

Analysis of the molecular features of rectal carcinoid tumors to identify new biomarkers that predict biological malignancy.

Although gastrointestinal carcinoid tumors are relatively rare in the digestive tract, a quarter of them are present in the rectum. In the absence of specific tumor biomarkers, lymphatic or vascular invasion is generally used to predict the risk of lymph node metastasis. We, therefore, examined the genetic and epigenetic alterations potentially associated with lymphovascular invasion among 56 patients with rectal carcinoid tumors. We also conducted a microRNA (miRNA) array analysis. Our analysis failed to detect mutations in BRAF, KRAS, NRAS, or PIK3CA or any microsatellite instability (MSI); however, we did observe CpG island methylator phenotype (CIMP) positivity in 13% (7/56) of the carcinoid tumors. The CIMP-positive status was significantly correlated with lymphovascular invasion (P = 0.036). The array analysis revealed that microRNA-885 (miR-885)-5p was the most up-regulated miRNA in the carcinoid tumors with lymphovascular invasion compared with that in those without invasion. In addition, high miR-885-5p expression was independently associated with lymphovascular invasion (P = 0.0002). In conclusion, our findings suggest that miR-885-5p and CIMP status may be useful biomarkers for predicting biological malignancy in patients with rectal carcinoid tumors.