Yoshihide Ueda

New p73 variants with altered C-terminal structures have varied transcriptional activities

p73 has been identified as a protein which shares significant homology with the tumor suppressor p53. We found two new types of splicing variant mRNAs for p73 expressed in MCF-7 cells which we named p73γ and ε. Sequence analysis revealed that these mRNAs encode variant p73 proteins bearing distinct carboxy-terminal structures, which are also different from the previously reported variants p73α and β. The mRNAs encoding p73γ and ε as well as α and β were confirmed to be expressed in normal human tissues in varied patterns. All of these splicing variants activated promoter with the p53-binding consensus sequence, but to different degrees. Furthermore, suppressive effects of p73α, γ and ε, but not β, on endogenous p53 activity were observed when transiently expressed in HepG2 and MCF-7 cells. These results suggested that the carboxy-terminal regions of p73 which were altered by alternative splicing affect these transactivation abilities and modulate the functions of p73 molecules.

Suppression of VEGFR-3 signaling inhibits lymph node metastasis in gastric cancer.

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF‐C or VEGF‐D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR‐3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR‐3 and other specific lymphatic markers, LYVE‐1 and prox‐1. Then we systemically administered anti‐VEGFR‐3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE‐1‐positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR‐3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.

Expression of activation-induced cytidine deaminase in human hepatocytes during hepatocarcinogenesis

Activation‐induced cytidine deaminase (AID) plays a role as a genome mutator in activated B cells, and inappropriate expression of AID has been implicated in the immunopathological phenotype of human B‐cell malignancies. Notably, we found that the transgenic mice overexpressing AID developed lung adenocarcinoma and hepatocellular carcinoma (HCC), suggesting that ectopic expression of AID can lead to tumorigenesis in epithelial tissues as well. To examine the involvement of AID in the development of human HCC, we analyzed the AID expression and its correlation with mutation frequencies of the p53 gene in liver tissues from 51 patients who underwent resection of primary HCCs. The specific expression, inducibility by cytokine stimulation and mutagenic activity of AID were investigated in cultured human hepatocytes. Only trace amounts of AID transcripts were detected in the normal liver; however, endogenous AID was significantly upregulated in both HCC and surrounding noncancerous liver tissues with underlying chronic hepatitis or liver cirrhosis (p < 0.05). Most liver tissues with underlying chronic inflammation with endogenous AID upregulation already contained multiple genetic changes in the p53 gene. In both hepatoma cell lines and cultured human primary hepatocytes, the expression of AID was substantially induced by TGF‐β stimulation. Aberrant activation of AID in hepatocytes resulted in accumulation of multiple genetic alterations in the p53 gene. Our findings suggest that the aberrant expression of AID is observed in human hepatocytes with several pathological settings, including chronic liver disease and HCC, which might enhance the genetic susceptibility to mutagenesis leading to hepatocarcinogenesis.

Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing.

Background and Aims The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. Methods Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy. Results Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents. Conclusion Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.

Organ-specific profiles of genetic changes in cancers caused by activation-induced cytidine deaminase expression.

Various molecular changes characterizing organ‐specific carcinogenesis have been identified in human tumors; however, the molecular mechanisms of the genomic changes specific for each cancer are not well defined. A transgenic (Tg) mouse model with constitutive expression of the nucleotide‐editing enzyme, activation‐induced cytidine deaminase (AID), develops tumors in various organs as a result of the mutagenic activities of AID. This phenotypic character of AID Tg mice allowed us to analyze the organ‐specific genetic changes in tumor‐related genes commonly triggered by AID‐mediated mutagenesis. Among the 80 AID Tg mice analyzed, 11 mice developed hepatocellular carcinomas, and 7 developed lung cancers. In addition, 1 developed the gastric cancer and 3 developed gastric adenomas. Organ‐specific preferences for nucleotide changes were observed in some of the tumor‐related genes in each epithelial tissue of the AID Tg mice. Of note, the c‐myc and K‐ras genes were the preferential targets of the mutagenic activity of AID in lung and stomach cancers, respectively, whereas mutations in the p53 and β‐catenin genes were commonly observed in all 3 organs. Quantitative RT‐PCR analyses revealed that alpha‐fetoprotein, insulin‐like growth factor2 and cyclin D1 genes were specifically upregulated in HCC, whereas upregulation of the matrix metalloproteinase7 gene was more marked in lung cancer. Our findings suggest that AID, a DNA mutator that plays a critical role linking inflammation to human cancers, might be involved in the generation of organ‐specific genetic diversity in oncogenic pathways during cancer development.

Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

Background and Aims Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. Methods To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. Results Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. Conclusion Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

Differentiation of Lymphatic Endothelial Cells From Embryonic Stem Cells on OP9 Stromal Cells

Objectives—The discovery of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3) has started to provide an understanding of the molecular mechanisms of lymphangiogenesis. The homeobox gene prox1 has been proven to specify lymphatic endothelial cells (ECs) from blood ECs. We investigated the process of lymphatic EC (LEC) differentiation using embryonic stem (ES) cells. Methods and Results—VEGFR-2+ cells derived from ES cells differentiated into LECs at day 3 on OP9 stromal cells defined by the expression of prox1, VEGFR-3, and another lymphatic marker podoplanin. VEGFR-2+ cells gave rise to LYVE-1+ embryonic ECs, which were negative for prox1 on day 1 but turned to prox1+ LECs by day 3. VEGFR-3-Fc or Tie2-Fc, sequestering VEGF-C or angiopoietin1 (Ang1), suppressed colony formation of LECs on OP9 cells. However, addition of VEGF-C and Ang1 in combination with VEGF to the culture of VEGFR-2+ cells on collagen-coated dishes failed to induce LECs. LEC-inducing activity of OP9 cells was fully reproduced on paraformaldehyde-fixed OP9 cells with the conditioned medium. Conclusion—We succeeded in differentiating LECs from ES cells and revealed the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation.

Primary hepatic carcinoid tumor with metachronous lymph node metastasis after long-term follow up.

A case of hepatic carcinoid tumor occurring in a 71‐year‐old man is reported. The tumor was diagnosed initially as a hepatocellular carcinoma, and was then shown after resection histologically to be a carcinoid tumor. The tumor cells formed small nests and trabeculae separated by fibrous septa and were positive for Grimelius staining. Immunohistochemically, most of the tumor cells stained positive with chromogranin A and neuron‐specific enolase. After a follow up for 5 years and 7 months, the patient developed tumors in lymph nodes between the remnant liver and the lesser curvature of the stomach with no tumors in other organs. Histologically, the tumor cells in the lymph nodes demonstrated a pattern of the immunostainings consistent with carcinoid tumor. After lymphadenectomy, the patient is free from recurrence in the regional lymph nodes for more than 1 year. This case is con‐sidered to be a primary hepatic carcinoid tumor with metachronous lymph node metastasis.

Risk Factors for Recurrence of Primary Sclerosing Cholangitis after Living Donor Liver Transplantation in Japanese Registry

The outcomes of primary sclerosing cholangitis (PSC) after living donor liver transplantation (LDLT) in a large series have not been reported. We aimed to determine long‐term patient and graft survival, risk factors for PSC recurrence, and the significance of recurrence after LDLT in a Japanese registry. Questionnaires concerning patient characteristics, treatments, and clinical courses were used. Data of 114 patients undergoing primary LDLT for PSC from July 1996 to December 2008 in 29 institutions were evaluated. For strict diagnoses of recurrence, patients with hepatic artery thrombosis (n = 8), ABO‐blood‐type‐incompatible transplantation (n = 8), and established ductopenic rejection (n = 2) were excluded and 96 patients were analyzed for risk factors. Recurrence was diagnosed in 26 patients (27%) at 8 to 79 months after transplantation. Patient, graft, and recurrence‐free survivals were 78, 74 and 57% at 5 years after LDLT, respectively. The graft loss rate was 69 versus 23% in patients with versus without recurrence, respectively. Multivariate analysis revealed that high MELD scores, first‐degree‐relative donors, postoperative CMV infection, and early biliary anastomotic complications were significant risk factors for recurrence. PSC recurrence was a significant risk factor of graft loss but not patient death. PSC recurrence was frequent and had significant impacts on outcomes after LDLT.

Wnt/β‐catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts

Wnt/β‐catenin signaling plays important roles in tumorigenesis in certain tumors as well as during development. However, the mechanism of tumorigenesis mediated by this signaling remains to be elucidated. We investigated the response of rodent fibroblasts to activation of Wnt/β‐catenin signaling by treatment with conditioned medium containing soluble Wnt‐3a protein (W3a‐CM) and by expression of a constitutive active β‐catenin gene harbored by an adenovirus vector. W3a‐CM induced transcriptional activation of a β‐catenin/T‐cell factor (Tcf)‐responsive promoter in rodent fibroblasts such as NIH3T3, Rat‐1, Swiss3T3 and Balb3T3 cells. In these cells, an increase in saturation density and an inhibition of apoptosis and/or promotion of growth in low‐serum medium were induced by treatment with W3a‐CM. In Rat‐1 cells, morphologic changes were also induced. All these alterations were reversible. Moreover, the inhibition of apoptosis of NIH3T3 cells in low‐serum medium and the morphologic changes in Rat‐1 cells, but not the increase in saturation density, were also induced by ectopic expression of a constitutive active β‐catenin gene. These results suggested that activation of Wnt/β‐catenin signaling induces inhibition of apoptosis and morphologic changes in these cells.