Kazuo Hatsuse

The correlation between cytoplasmic overexpression of epidermal growth factor receptor and tumor aggressiveness: poor prognosis in patients with pancreatic ductal adenocarcinoma.

Objectives: Recent studies have shown that some growth factor receptors with tyrosine kinase activity, eg, the epidermal growth factor receptor (EGFr) and the c-erbB-2 (HER-2) oncoprotein, are associated with aggressive biologic behavior of various cancer cell types. We examined the clinicopathological significance of the expression and localization of EGFr and HER-2 in both invasive and intraductal components of ductal adenocarcinomas of the pancreas. Methods: Tissue samples from 76 archival cases of pancreatic ductal adenocarcinoma were immunohistochemically analyzed for both membrane and cytoplasmic overexpression of EGFr and HER-2 oncoprotein. The rate of incidence between the invasive and intraductal components was analyzed and then their correlation with tumor differentiation and patient prognosis was analyzed. Results: Cytoplasmic EGFr overexpression was more frequent in invasive components (47 of 76, 62%) than in intraductal components (19 of 76, 25%), while membrane EGFr overexpression was more frequent in intraductal components (41 of 76, 54%) than in invasive components (11 of 76, 14%). The membrane HER-2 overexpression was also more frequent in intraductal components (15 of 76, 20%) than in invasive components (2 of 76, 3%), but the incidence of cytoplasmic HER-2 overexpression did not differ between intraductal components (12 of 76, 16%) and invasive components (8 of 76, 11%). The cytoplasmic EGFr overexpression in invasive components was more frequent in grade 3 group (32 of 33, 97%) than in grade 2 (15 of 32, 47%) and grade 1 groups (0 of 10, 0%) (P < 0.001). Patients with adenocarcinoma with cytoplasmic EGFr overexpression showed shorter overall survival than those with adenocarcinoma without cytoplasmic EGFr overexpression (P = 0.02). Conclusion: It is suggested that the cytoplasmic overexpression of EGFr plays a significant role in the progression of pancreatic ductal adenocarcinoma, especially in the invasion and acquisition of aggressive clinical behavior. Both membrane and cytoplasmic expression of HER-2 showed no significant correlation between tumor differentiation and poor survival.

Decrease of CD56+T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma

CD56+T cells and CD56+natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti‐CD3 Ab, IL‐2, or a combination of IL‐2 and IL‐12 and their IFN‐γ production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (−) patients contained 20% CD56+T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN‐γ production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH‐7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (−) patients. CD56+T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH‐7 cells. These results suggest that a decreased number of CD56+T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.

Potential crosstalk between insulin-like growth factor receptor type 1 and epidermal growth factor receptor in progression and metastasis of pancreatic cancer

The insulin-like growth factor receptor type 1 (IGF1R) and epidermal growth factor receptor (EGFR) are reportedly overexpressed in pancreatic cancer. However, the correlation between activated EGFR and IGF1R and their clinicopathological implications still remain unclear. The cellular localization and overexpression of IGF1R and EGFR were investigated immunohistochemically in primary invasive ductal pancreatic carcinomas obtained from 74 patients who underwent radical surgical resection. We also compared the status of IGF1R and EGFR overexpression between primary tumors and hepatic metastatic tumors obtained from 44 autopsied patients. Among the 74 surgically resected primary tumors, cytoplasm- and membrane-dominant EGFR overexpression was detected in 22 (30%) and 7 (9%), respectively, whereas cytoplasm- and membrane-dominant IGF1R overexpression was detected in 8 (11%) and 28 (38%), respectively. Membrane-dominant EGFR and cytoplasm-dominant IGF1R were more frequent in lower-grade tumors and correlated with favorable prognosis, whereas cytoplasm-dominant EGFR and membrane-dominant IGF1R were more frequent in higher-grade tumors and correlated with poor prognosis. In 36 autopsy specimens of pancreatic tumor with concurrent overexpression of IGF1R and EGFR, there was an inverse correlation between the IGF1R and EGFR localization patterns (P=0.001). In the hepatic metastatic tumors obtained by autopsy, the incidences of both IGF1R and EGFR overexpression were much higher than in the surgically resected primary tumors. More than half of the autopsy cases consistently showed membrane-dominant EGFR expression in both the primary tumor and hepatic metastases, whereas IGF1R expression showed considerable variation. Crosstalk between differently localized IGF1R and EGFR might play a role in determining the biological aggressiveness of pancreatic cancer, although their cellular localization may often alter during the process of metastasis.

p53/p66Shc-mediated signaling contributes to the progression of non-alcoholic steatohepatitis in humans and mice

BACKGROUND & AIMS The tumor suppressor p53 is a primary sensor of stressful stimuli, controlling a number of biologic processes. The aim of our study was to examine the roles of p53 in non-alcoholic steatohepatitis (NASH). METHODS Male wild type and p53-deficient mice were fed a methionine- and choline-deficient diet for 8 weeks to induce nutritional steatohepatitis. mRNA expression profiles in normal liver samples and liver samples from patients with non-alcoholic liver disease (NAFLD) were also evaluated. RESULTS Hepatic p53 and p66Shc signaling was enhanced in the mouse NASH model. p53 deficiency suppressed the enhanced p66Shc signaling, decreased hepatic lipid peroxidation and the number of apoptotic hepatocytes, and ameliorated progression of nutritional steatohepatitis. In primary cultured hepatocytes, transforming growth factor (TGF)-β treatment increased p53 and p66Shc signaling, leading to exaggerated reactive oxygen species (ROS) accumulation and apoptosis. Deficient p53 signaling inhibited TGF-β-induced p66Shc signaling, ROS accumulation, and hepatocyte apoptosis. Furthermore, expression levels of p53, p21, and p66Shc were significantly elevated in human NAFLD liver samples, compared with results obtained with normal liver samples. Among NAFLD patients, those with NASH had significantly higher hepatic expression levels of p53, p21, and p66Shc compared with the group with simple steatosis. A significant correlation between expression levels of p53 and p66Shc was observed. CONCLUSIONS p53 in hepatocytes regulates steatohepatitis progression by controlling p66Shc signaling, ROS levels, and apoptosis, all of which may be regulated by TGF-β. Moreover, p53/p66Shc signaling in the liver appears to be a promising target for the treatment of NASH.

Transformation-associated gene regulation by ATF6α during hepatocarcinogenesis

We have previously reported that the endoplasmic reticulum (ER) stress‐regulated transmembrane transcription factor 6 α (ATF6α) is implicated in the pathogenesis of hepatocellular carcinomas (HCCs). In order to further identify genes that are regulated by ATF6α, the global gene expression profiles of the ATF6α‐transfected and untransfected HCC cell line, HLF, were analyzed. These results were then compared with the differential gene expression patterns of poorly differentiated HCC and control non‐tumorous liver tissue. Our findings demonstrate that at least 18 genes are specifically upregulated by ATF6α, while another UPR mediator, XBP1 or ER‐stress inducer, thapsigargin could partially stimulate or even repress some of them in HCC cells. Moreover, six of these identified genes contain potential ER stress‐responsive elements and/or unfolded protein response elements in their 5′ regulatory regions.

Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells

BACKGROUND & AIMS Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. METHODS ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. RESULTS ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. CONCLUSIONS ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.

High-level Skp2 expression in pancreatic ductal adenocarcinoma: correlation with the extent of lymph node metastasis, higher histological grade, and poorer patient outcome.

Objectives: Recent studies have shown that overexpression of S-phase kinase-associated protein 2 (Skp2) occurs in many cancers at an advanced stage. We examined the clinicopathologic significance and prognostic implication of Skp2 expression in pancreatic invasive ductal carcinoma. Methods: Tissue samples from 46 pancreatic carcinomas were examined immunohistochemically for Skp2. The proportion of constituent tumor cells with Skp2 expression was analyzed and classified as high-level nuclear expression when more than 20% of the cancer cells were positive, or low-level nuclear expression otherwise. Results: High-level Skp2 overexpression was detected in 13 (28.3%) of the 46 tumors. The incidence of high-level Skp2 was correlated with higher histological grade (P = 0.0056), the extent of lymph node metastasis (P = 0.0086), the level of lymphatic permeation (P = 0.0024), and poorer patient outcome (P = 0.0189). Multivariate analysis showed that high-level Skp2 expression was an independent predictor of overall patient survival (P = 0.0140). Conclusions: It is suggested that examination of Skp2 expression might be clinically useful for prognostication in patients with pancreatic carcinoma and that Skp2 protein might be a novel therapeutic molecular target.

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.

The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells

Aims: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC).

Rim enhancement in colorectal metastases at CT during infusion hepatic arteriography: Does it represent liver parenchyma or live tumor cell zone?

Purpose: to evaluate the morphologic substrate of the rim enhancement of colorec-tal metastases seen at CT during infusion hepatic arteriography (CTIHA). Material and Methods: Eleven sector defects in the enhancing rim of 9 metastases at CTIHA were analyzed. the corresponding pathologic specimens were investigated for sector defects of liver parenchyma. We investigated whether there was a correlation between the central angle of the sector defects of rim enhancement at CTIHA and that of sector defects in the zone of liver parenchyma in histologic slices. the inner and outer diameters of the enhancing rim were also compared with the diameter of the metastases as seen at CT during arterial portography (CTAP). Results: There was a significant correlation between the central angle of sector defects of rim enhancement at CTIHA and the angle of sector defects in the zone of liver parenchyma in histologic slices (p=0.008, Spearmans test). the diameter of the metastases measured at CTAP was larger than the inner diameter and smaller than the outer diameter of the enhancing rim in CTIHA, i.e. the margins of the nodules as seen in CTAP are located in liver parenchyma and not in tumor tissue. Conclusion: the morphologic substrate of the rim enhancement of colorectal metastases seen at CTIHA is liver parenchyma.