Published papers

Purpose: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC.

OBJECTIVE: The purpose of this study was to clarify the natural outcomes of hypoattenuating nodular lesions in patients with virus-related chronic liver disease depicted on dynamic CT. MATERIALS AND METHODS: Sixty lesions (mean size, 1.3 cm) exhibiting hypoattenuation or isoattenuation in the arterial and delayed phases of dynamic CT were retrospectively evaluated with additional CT (mean, six examinations) for a mean period of 838 days. The primary end point was emergence of hyperattenuating areas within hypoattenuating lesions, a phenomenon called attenuation conversion. Cumulative attenuation conversion rates suggesting rates of malignant transformation were calculated with the Kaplan-Meier method, and factors affecting attenuation conversion rate were analyzed with the Cox proportional hazard model. RESULTS: Thirty-six (60%) of 60 hypoattenuating lesions developed to hyperattenuating lesions, 21 were unchanged, and three disappeared spontaneously. The 36 lesions that became hyperattenuating were divided into two subgroups according to lesion enhancement pattern: hyper-in-hypoattenuating (n = 25) and entirely hyperattenuating (n = 11). The cumulative attenuation conversion rates for the 60 hypoattenuating lesions were 15.8%, 44.3%, and 58.7% at 1, 2, and 3 years. The hyper-in-hypoattenuating lesions showed more rapid progression to entirely enhanced lesions. Positive results for hepatitis C viral antibody (p = 0.028) and initial lesion size (p = 0.007) showed a positive correlation with attenuation conversion rate. CONCLUSION: Hypoattenuating hepatic nodular lesions in chronic liver disease depicted on dynamic CT have high malignant potential and should be followed with special attention to conversion from hypoattenuation to hyperattenuation to determine the optimal timing of treatment.

Background & Aims: In the current era of emerging molecular targeted drugs, it is necessary to identify before treatment the specific subclass to which a tumour belongs. Gadoxetic acid is a liver-specific contrast agent that is preferentially taken up by hepatocytes. Therefore, gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) should provide precise molecular information about hepatocellular carcinomas (HCCs). The aim of this study was to investigate the transporters of gadoxetic acid in HCC comprehensively and to analyse the molecular regulatory mechanism of such transporters.

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<0.01], elastic: r=0.53 [P<0.01]). Our approach to quantifying liver fibrosis will serve as an effective tool to evaluate liver diseases in routine practice.

The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), also known as GPR49, is a seven-transmembrane receptor that is expressed in stem cells of the intestinal crypts and hair follicles of mice. LGR5 is overexpressed in some types of human cancer, and is one of the target genes of the Wnt signaling pathway. To explore the function of LGR5 in cancer cells, stable hepatocellular carcinoma (HCC) cell lines expressing FLAG-tagged LGR5 were established. Overexpression of LGR5 resulted in changes in cell shape from an extended flat (mesenchymal) phenotype to a round aggregated (stem cell-like) phenotype. Cells transfected with LGR5 showed higher colony forming activity, and were more resistant to a cytotoxic drug than cells transfected with empty vector. Overexpression of LGR5 inhibited cell motility. LGR5-transfected cells formed nodule type tumors in the livers of immunodeficient mice, whereas empty vector-transfected cells formed more invasive tumors. Down-regulation of LGR5 changed the morphology of HCC cells from the aggregated phenotype to an extended spindle phenotype, and cell motility was increased. This is the first study reporting the functional role of LGR5 in the biology of HCC cells, and the results suggest that aberrant expression of LGR5 regulates epithelial cell phenotype and survival. (c) 2012 Elsevier Inc. All rights reserved.

Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min. The serum FastLec-Hepa counts increased with advancing fibrosis and illustrated significant differences in medians between all fibrosis stages. FastLec-Hepa is sufficiently sensitive and quantitative to evaluate the effects of PEG-interferon-alpha/ribavirin therapy in a short post-therapeutic interval. The obtained fibrosis progression is equivalent to -0.30 stages/year in patients with sustained virological response, and 0.01 stages/year in relapse/nonresponders. Furthermore, long-term follow-up of the severely affected patients found hepatocellular carcinoma developed in patients after therapy whose FastLec-Hepa counts remained above a designated cutoff value. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

Cyclase-associated protein 2 (CAP2) is a conserved protein that is found up-regulated in hepatocellular carcinoma (HCC). By using zebrafish, combined with HCC cell lines, we further investigated the role of CAP2. The zebrafish CAP2 sequence was 60% identical to human CAP2 with 77% homology in the C-terminal actin-binding domain, and 58% in the N-terminal cyclase-binding domain. CAP2 expression was observed during zebrafish development and was preferentially expressed in the skeletal muscle and heart. Knockdown using two different morpholinos against CAP2 resulted in a short-body morphant zebrafish phenotype with pericardial edema. CAP2 was observed co-localized with actin in zebrafish skeletal muscle, and in the leading edge of lamellipodium in HCC cell lines. CAP2 silencing resulted in a defect in lamellipodium formation and decreased cell motility in HCC cell lines. Strongly positive expression of CAP2 was observed in 10 of 16 (63%) poorly, 30 of 68 (44%) moderately, and 2 of 21 (10%) well differentiated HCC. CAP2 expression was significantly associated with tumor size, poor differentiation, portal vein invasion, and intrahepatic metastasis. Our results indicate that an important conserved function of CAP2 in higher vertebrates may be associated with the process of skeletal muscle development. CAP2 also played an important role in enhancing cell motility, which may promote a more invasive behavior in the progression of HCC. These findings highlight the link between development and cancer. (C) 2012 Elsevier Inc. All rights reserved.

Proneurotensin/neuromedin N (proNT/NMN), the precursor of neurotensin (NT) and neuromedin N (NMN), is produced by cancer tissues derived from the pancreas and colon. NT stimulates tumor growth and proliferation through its receptors; however, little is known about the precursor molecule in cancer tissues. We previously demonstrated that proNT/NMN is secreted from small cell lung carcinoma (SCLC) cell lines in serum-free conditioned medium, but not from non-small cell lung carcinoma (NSCLC) cell lines. It was suggested that this precursor may serve as a tumor marker for SCLC. In this study, we established in vivo xenograft models to evaluate the possibility of proNT/NMN as a specific tumor marker. SBC3 cells, derived from human SCLC, were inoculated into mice, and the proNT/NMN levels in plasma and tumor tissues were detected using specific antibodies. In contrast to control mouse plasma, the proNT/NMN levels in tumor-bearing mice increased as the tumors grew, and the elevated plasma proNT/NMN levels were decreased by tumor resection. Moreover, proNT/NMN was expressed in SBC3 tumors, suggesting that proNT/NMN was secreted into blood from the tumor, and this secretion may be specific to SCLC.

OBJECTIVE: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. METHODS: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. RESULTS: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). CONCLUSIONS: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

Objective: Keratin (K) 19 positivity has been reported to be a useful predictive marker for recurrence in patients with hepatocellular carcinoma (HCC) who have undergone hepatic resection. We investigated the clinical usefulness of K19 positivity in patients who had received curative radiofrequency ablation (RFA). Methods: We retrospectively evaluated the clinicopathological features, including imaging and K19 expression, in 246 patients with HCC who were within the Milan criteria and had received curative RFA. Using a two-step insertion method, tumor biopsies were obtained just prior to RFA and were evaluated histologically. Results: Tumor seeding due to liver biopsy and RFA was not observed. Ten patients (4.1%) had K19-positive HCC. Imaging findings were similar between K19-positive and -negative HCC (p = 0.187). Nine out of 10 patients (90%) who had K19-positive HCC had recurrence of HCC after RFA, and intrahepatic recurrences were observed within 12 months in 6 out of 10 (60.0%). K19 positivity was a significant risk factor for recurrence (p < 0.0001) and early recurrence (<1 year after RFA; p = 0.012). K19 expression (p = 0.016) was an independent risk factor for tumor status exceeding the Milan criteria after RFA. Conclusion: Expression of K19 is related to high recurrence of HCC after curative RFA. Copyright (C) 2011 S. Karger AG, Basel

The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.