Search

WNT/β-catenin-activated hepatocellular carcinoma (W/B subclass HCC) is considered as molecularly homogeneous entity and has been linked to resistance to immunotherapy. However, recent studies have indicated possible heterogeneity in the immuno-vascular microenvironment in this subclass. We set out to test the hypothesis that specific immuno-vascular features might stratify W/B subclass HCCs into tumors having distinct aggressive natures.In this study, we analyzed 352 resected HCCs including 78 immunohistochemically defined W/B subclass HCCs. The density of tumor-infiltrating CD3The T-cell density of W/B subclass HCCs was regionally heterogenous within tumor tissues, and focally reduced T-cell density was observed in areas with VETC. VETC-positivity (defined as VETC area ratio greater than 1%) and T-cell infiltration were inversely associated in both W/B subclass and non-W/B subclass HCCs. Fibroblast growth factor 2 (FGF2) gene expression was higher in W/B subclass than in non-W/B subclass HCCs. VETC positivity and low T-cell density correlated with increased expression of FGF2 in W/B subclass HCCs. VETC-positive HCCs showed significantly shorter DFS in W/B subclass HCCs.In conclusion, the immune and vascular microenvironments are interrelated and are also correlated with clinicopathological heterogeneity in W/B subclass HCC. These results may inform clinical practice and translational research on the development of therapeutic stratification of HCCs. This article is protected by copyright. All rights reserved.

Herein we describe the enantioselective intermolecular conjugate addition of nitroalkanes to unactivated α,β-unsaturated esters, catalyzed by a bifunctional iminophosphorane (BIMP) superbase. The transformation provides the most direct access to pharmaceutically relevant enantioenriched γ-nitroesters, utilizing feedstock chemicals, via fundamental reactivity and unprecedented selectivity. The methodology exhibits a broad substrate scope, including β-(fluoro)alkyl, aryl and heteroaryl substituted electrophiles, and was successfully applied on a gram scale with reduced catalyst loading, and additionally catalyst recovery was carried out. The formal synthesis of a range of drugs, and an enantioselective synthesis of (S)-rolipram were achieved. Additionally, computational studies revealed key reaction intermediates and transition states, and provided rationale for high enantioselectivities, in good agreement with experimental results.

The clinicopathological characteristics of steatosis in hepatocellular carcinoma (HCC) remain unclear. Here, we elucidate the features of macrovesicular steatosis (MaS) and microvesicular steatosis (MiS) in HCC and their relationships with background liver steatosis. A total of 165 HCC lesions were classified as MaS-HCC, MiS-HCC, or conventional HCC (cHCC) according to the cutoff value of 30% MaS or MiS in tumor cells. We analyzed the clinicopathological differences among these groups. MaS-HCC had less portal vein invasion, a higher proportion of HCC with intratumoral fibrosis, and a lower cumulative risk of recurrence than MiS-HCC or cHCC. Moreover, both MaS-HCC and MiS-HCC had lower incidences of hepatitis virus infection and higher levels of HbA1c than cHCC. Background liver steatosis was also higher in MaS-HCC than in cHCC. Immunohistochemical expression of perilipin (Plin1) and adipophilin (ADRP), major proteins expressed on lipid droplet membranes, revealed that almost all lipid droplets in HCC were Plin1 negative, whereas those in background liver were positive. In contrast, ADRP was expressed on lipid droplets in both HCC and background liver. We concluded that MaS-HCC and MiS-HCC were associated with metabolic abnormalities but exhibited different biologic behaviors. Furthermore, lipid droplets in HCC were pathophysiologically different from those in background liver.

AIM Sorafenib inhibits multiple kinase signaling pathways, including the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and is a promising therapy for hepatocellular carcinoma (HCC). However, the role of ERK activation in HCC remains unclear. This study was designed to investigate the potential link between ERK activation and aggressive HCC phenotypes. METHODS We evaluated nuclear ERK expression by immunohistochemistry in 154 resected HCC nodules from 136 patients. We then investigated the associations of ERK expression with the clinicopathological characteristics of HCC, c-MET expression, and the molecular subclass biomarkers Ki-67, keratin 19 (KRT19, CK19, or K19), and sal-like protein 4. Multivariate Cox regression analysis was carried out to determine independent prognostic factors for overall survival and recurrence-free survival. The effects of ERK activation by hepatocyte growth factor (HGF) on eight HCC cell lines were further examined. RESULTS High-level nuclear expression of ERK was observed in 20 (13%) of 154 nodules and was significantly associated with higher serum alpha-fetoprotein levels (P = 0.034), poorer differentiation (P = 0.003), a higher Ki-67 index (P

Epithelial-mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal-epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding "focal differentiation" in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E-cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co-cultured with Schwann cells to investigate cell morphology, motility, or EMT-related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E-cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co-cultured with Schwann cells demonstrated a sheet-like appearance, increased E-cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET-like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

Histological evaluation of fibrosis after a liver biopsy is crucial for evaluating the pathology of patients with chronic liver disease. Previous studies have reported quantitative analyses of fibrosis using images of collagen-stained sections. However, analysis of these studies requires manual selection of the region of interest. In addition, the quantification of elastic fibers is not considered. The present study was conducted in order to measure both the collagen and elastic fiber area ratios using Elastica van Gieson-stained whole-slide images (WSIs) of liver biopsy specimens. High-resolution WSIs provide precise color classification, enabling accurate detection of even fine collagen and elastic fibers. To minimize the influence of pre-existing fibrous tissue, median area ratios of the collagen and elastic fibers were independently calculated from the image tiles of the WSIs. These median area ratios were highly concordant with area ratios after the pre-existing fibrous tissues were manually trimmed from the WSI. Further, these median area ratios were correlated with liver stiffness as measured by transient elastography (collagen: r = 0.73 [P

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT-PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P = 0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P = 0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a 7-transmembrane receptor reportedly expressed in stem cells of the intestinal crypts and hair follicles of mice. Overexpression of LGR5 is observed in some types of cancer; however, there has been no specific assessment in colorectal tumorigenesis. We performed quantitative RT-PCR for LGR5 expression in 37 representative cancer cell lines, and showed that LGR5 mRNA was frequently overexpressed in colon cancer cell lines. Moreover, LGR5 expression was higher in colon cancer cell lines derived from metastatic tumors compared with those from primary tumors. In clinical specimens, there was significant overexpression of LGR5 in 35 of 50 colorectal cancers (CRCs), and in seven of seven sporadic colonic adenomas, compared with matched normal mucosa. This suggests up-regulation of LGR5 from the early stage of colorectal tumorigenesis. LGR5 expression showed marked variation among CRC cases and correlated significantly with lymphatic invasion, vascular invasion, tumor depth, lymph node metastasis, and tumor stage (IIIC vs. IIIB). In addition to cancer cells, crypt base columnar cells of the small intestine and colon were shown by in situ hybridization to express LGR5. This is the first report suggesting the involvement of LGR5, not only in early events but also in late events in colorectal tumorigenesis.

Two constituent proteases of the hatching enzyme of the medaka (Oryzias latipes), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish (Brachydanio rerio) and masu salmon (Oncorynchus masou) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.

The allelic division of the Na+/K(+)-ATPase alpha-subunit gene was found in eggs of the sea urchin Hemicentrotus pulcherrimus by polymerase chain reaction (PCR). Two PCR products of different lengths were detected from a genome in one embryo derived from a fertilized egg, although only one product in one embryo derived from an artificially activated egg by parthenogenesis was detected, indicating one copy of the gene in a haploid genome. One of the two PCR products from each fertilized egg was identical in size to the product from an artificially activated egg in the same batch. The other PCR product was the same in length as one of the products from the sperm with which the eggs were fertilized. These results indicate that recombination of the heteromeric alleles of the Na+/K(+)-ATPase alpha-subunit gene occurs in the sea urchin egg due to meiosis and fertilization. The sequencing of these products demonstrated that their exon sequences were identical and that the intron, inserted in the PCR products, generated polymorphism in length due to the frequency of the repeating 53 bp sequence and insertion/deletion of other two segments.

Most blind QAM equalization algorithms base their parameter adaptation on the minimization of non-convex cost functions. These cost functions may exhibit local minima which can cause undesirable convergence to equalizer parameter settings, resulting in insufficient removal of the intersymbol interference. In contrast, this work considers the minimization of a special convex cost function of the equalizer output in combination with an arbitrary non-degenerate linear constraint. We establish the property that the cost minimization in the space of the equalizer parameters is achieved on a compact set that contains at least one ideal zero-ISI equalizer parameter setting. Generically this compact set consists of a single point reflecting an ideal global convergence result, implying zero intersymbol interference. Further, through general abstract methods we demonstrate that this desirable convergence property is largely independent of the input constellation geometry and independent of the linear constraint chosen. The methods are developed for and motivated by the important classes of QAM constellations used in practice.

The hatching enzyme of the teleost, Oryzias latipes, is composed of two proteases, high choriolytic enzyme (choriolysin H, HCE) and low choriolytic enzyme (choriolysin L, LCE), which are similar in some enzymological characteristics and protein structure (55% identity in amino acid sequence) and belong to the astacin family. Two isoforms of HCE are detected. In the present study, the genes for HCE and LCE were isolated from the genomic library constructed from DNA of the inbred drR strain fish. In contrast to the close similarity of the enzymes, there was a marked difference in their gene organization. The LCE gene was a single copy gene and composed of eight exons interrupted by seven introns. The HCE genes were multicopy genes and lacked introns. In the haploid genome of the drR strain fish, there are eight HCE genes, seven of which were cloned. Each HCE gene was identified as that for either of the two isoforms of HCE. 5' flanking regions of the LCE gene and the HCE genes had consensus TATA box sequences, but not CAT box nor GC box sequences. The big difference in the exon-intron organization between the HCE genes and the LCE gene is discussed from an evolutionary viewpoint.

A marked increase in the Na+, K+-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na+, K+-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na+, K+-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na+, K+-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li+ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na+, K+-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.

Clinical observations of cases of ovarian metastasis suggest that there may be a unique mechanism underlying ovarian-specific metastasis. This study was undertaken to establish an in vivo model of metastasis to the ovary, and to investigate the mechanism of ovarian-specific metastasis. We examined the capacity for ovarian metastasis in eight different human carcinoma cell lines by implantation in female NOD/SCID mice transvenously and intraperitoneally. By transvenous inoculation, only RERF-LC-AI, a poorly differentiated carcinoma cell line, frequently demonstrated ovarian metastasis. By intraperitoneal inoculation, four of the eight cell lines (HGC27, MKN-45, KATO-III, and RERF-LC-AI) metastasized to the ovary. We compared E-cadherin expression among ovarian metastatic cell lines and others. All of these four ovarian metastatic cell lines and HSKTC, a Krukenberg tumor cell line, showed E-cadherin down-regulation and others did not. E-cadherin was then forcibly expressed in RERF-LC-AI, and inhibited ovarian metastasis completely. The capacity for metastasizing to the other organs was not affected by E-cadherin expression. We also performed histological investigation of clinical ovarian-metastatic tumor cases. About half of all ovarian-metastatic tumor cases showed loss or reduction of E-cadherin expression. These data suggest that E-cadherin down-regulation may be involved in ovarian-specific metastasis.