Junpei Yamaguchi

The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma

Polycomb-group proteins Bmi1 and EZH2 are involved in the malignant transformation and biological aggressiveness of several human carcinomas. We herein examined the significance of the Bmi1 and EZH2 expression in hepatocellular carcinoma (HCC) and its preneoplastic lesions, dysplastic nodules. The expression of Bmi1 and EZH2 were examined immunohistochemically in HCC (n=27) and dysplastic nodules (n=14), and combined hepatocellular and cholangiocarcinoma (HC-CC) (n=14). The effect of Bmi1 and EZH2 knockdown was examined in cultured HCC cells (HuH7 and HepG2) using siRNA. It was determined that Bmi1 was constantly expressed in cholangiocytes, but not in hepatocytes, and EZH2 was detected in neither cholangiocytes nor hepatocytes. Bmi1 and EZH2 were overexpressed in HCC and more extensively in HC–CC (P<0.01). Interestingly, Bmi1 and EZH2 were not overexpressed in the dysplastic nodules. The expression of Bmi1 and EZH2 was heterogeneous and associated with vascular infiltration, the histological grades, and the cell proliferation activity in HCC and HC–CC. In cultured carcinoma cells overexpressing Bmi1 and EZH2, knockdown of Bmi1 and EZH2 resulted in decreased cell proliferation activities. Therefore, the overexpression of polycomb-group proteins Bmi1 and EZH2 is associated with the malignant progression of HCC, thereby reflecting the aggressive biological behavior in HCC and HC–CC.

Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence

Telomere shortening is a trigger of cellular senescence. Biliary epithelial cells in damaged small bile ducts in primary biliary cirrhosis (PBC) show senescent features such as the expression of senescence‐associated β‐galactosidase and the increased expression of p16INK4a and p21WAF1/Cip1. We investigated whether the telomere shortening is involved in the pathogenesis of biliary cellular senescence in PBC. We analyzed the telomere length of biliary epithelial cells using quantitative fluorescence in situ hybridization in livers taken from the patients with PBC (n = 13) and control livers (n = 13). We also assessed immunohistochemically the prevalence of DNA damage and the expression of p16INK4a and p21WAF1/Cip1. The study showed a significant decrease in telomere length in biliary epithelial cells in the damaged small bile ducts and bile ductules in PBC compared with normal‐looking bile ducts and bile ductules in PBC, chronic viral hepatitis, and normal livers (P < 0.01). γH2AX‐DNA‐damage‐foci were detected in biliary epithelial cells in damaged small bile ducts and bile ductules in PBC but were absent in biliary epithelial cells in chronic viral hepatitis and normal livers. The expression of p16INK4a and p21WAF1/Cip1 was increased corresponding to telomere shortening and γH2AX‐DNA‐damage‐foci in the damaged small bile ducts in PBC. Conclusion: Telomere shortening and an accumulation of DNA damage coincide with increased expression of p16INK4a and p21WAF1/Cip1 in the damaged bile ducts, characterize biliary cellular senescence, and may play a role in the following progressive bile duct loss in PBC. (HEPATOLOGY 2008.)

Over‐expression of polycomb group protein EZH2 relates to decreased expression of p16INK4a in cholangiocarcinogenesis in hepatolithiasis

Polycomb group protein EZH2 and Bmi1 are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi‐step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor gene p16INK4a. We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN‐1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN‐3 (carcinoma in situ), 12 BilIN‐2 (high‐grade dysplasia), 32 BilIN‐1 (low‐grade dysplasia) and 37 non‐neoplastic biliary epithelia from these livers. Expression of p16INK4a, EZH2 and Bmi1 were surveyed in these foci. P16INK4a promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT‐1 and TFK‐1) and small interfering RNA (siRNA), the effects of the knockdown of EZH2 on p16INK4a methylation of CC cells were examined. Expression of p16INK4a, which was frequent in BilIN1, was decreased in BilIN‐2/3 and invasive carcinoma, while EZH2 expression showed step‐wise increase from BilIN‐1, ‐2 and ‐3 to invasive carcinoma (p < 0.01). P16INK4a promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16INK4a methylation and decreased binding of EZH2 to the p16INK4a gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p16INK4a gene. Our data suggest that over‐expression of EZH2 may induce hypermethylation of p16INK4a promoter followed by decreased expression of p16INK4a in the multi‐step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis. Copyright

Expression of matrix metalloproteinase 7 is an unfavorable postoperative prognostic factor in cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts

Cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts (collectively referred to as the large bile ducts) is an intractable disease, and a papillary phenotype and well-differentiated histologic grade have been proposed as indicators of a favorable prognosis after surgical resection. In this study, we examined the significance of matrix metalloproteinases (MMPs) in cholangiocarcinoma with respect to clinicopathologic features. We subjected 66 surgically resected specimens of cholangiocarcinoma of the large bile ducts to clinicopathologic examination, including postoperative survival, papillary phenotype, and immunohistochemical expression of MMP-2,-7, -9, and membrane type 1 MMP (MT1-MP). Nonneoplastic biliary epithelium did not express these 4 MMPs, whereas cholangiocarcinoma frequently expressed MMP-2 (33.9%), -7 (75.8%), -9 (47.5%), and MT1-MMP (54.5%). In particular, conventional (nonpapillary) cholangiocarcinoma expressed MMP-7 and MT1-MMP more frequently than papillary cholangiocarcinoma. The expression of MMP-7 and MT1-MMP significantly correlated with the nonpapillary phenotype, poorly differentiated histologic grade, perineural invasion, and advanced TNM stage. In contrast, the expression of MMP-2 and -9 was not associated with any of the clinicopathologic features. Univariate analysis of disease-specific survival revealed that a papillary phenotype and expression of MMP-7 were prognostic factors of cholangiocarcinoma, in addition to TNM stage, poorly differentiated histologic grade, perineural invasion, and microscopic margin status. Multivariate analysis showed only TNM stage to be an independent prognostic factor. Expression of MMP-7 in cholangiocarcinoma is an unfavorable postoperative prognostic factor of cholangiocarcinoma arising from the large bile ducts. Underexpression of MMPs in papillary cholangiocarcinoma might be associated with a favorable prognosis.

Cyclooxygenase-2 Is Involved in the Up-Regulation of Matrix Metalloproteinase-9 in Cholangiocarcinoma Induced by Tumor Necrosis Factor-α

Matrix metalloproteinase-9 (MMP-9) is an important enzyme in tumor invasion and metastasis in malignant tumors, including cholangiocarcinoma (CC). Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, was recently reported to induce the up-regulation of MMP-9 in cultured CC cells. We examined whether cyclooxygenase-2 (COX-2) and prostaglandin-E2 (PGE2), another endogenous tumor promoter, are involved in the up-regulation of MMP-9 in CC using CC tissue specimens and a CC cell line, HuCCT-1. MMP-9 and COX-2 were immunohistochemically expressed in 58% and 89% of 110 CC cases, respectively; the expression of MMP-9 and COX-2 was correlated (r = 0.32, P = 0.00072). Using zymography, latent MMP-9 was detectable in all cases and active MMP-9 was detected in 24% of cases of the CC specimens. The TNF-alpha/TNF-receptor 1 (TNF-R1) interaction induced MMP-9 production and activation, as well as COX-2 overexpression and PGE2 production, and increased the migration of CC cells. MMP-9 up-regulation was inhibited by COX inhibitors, antagonists of EP2/4 (receptors of PGE2), and COX-1 and COX-2 siRNAs. Inhibitors of both MMP-9 and MMP-9 siRNA treatment abrogated the increase in the migration of CC cells induced by TNF-alpha. In conclusion, we propose a novel signaling pathway of MMP-9 up-regulation in CC cells such that TNF-alpha induces the activation of COX-2 and PGE2 via TNF-R1 followed by the up-regulation of MMP-9 via the PGE2 (EP2/4) receptor.

Spontaneous Regression of Inflammatory Pseudotumor of the Liver: Report of Three Cases

Inflammatory pseudotumor (IPT) of the liver is a rare benign hepatic lesion that sometimes mimics malignant tumors. An accurate diagnosis of IPT is important to avoid unnecessary surgery. We herein report three cases of IPT of the liver that spontaneously regressed and were successfully diagnosed without a surgical resection. Malignant tumors were initially suspected based on the initial imaging findings, including ultrasonography, computed tomography (CT), and magnetic resonance imaging. In particular, a delayed enhancement in the periphery of the masses was observed on dynamic CT scans, similar to the imaging results for metastatic tumors or intrahepatic cholangiocarcinomas. The serum levels of C-reactive protein were elevated in all three patients (6.6, 3.4, and 1.5 mg/dl), while the serum levels of tumor markers were almost within the normal ranges (carcinoembryonic antigen, <5 ng/ml; carbohydrate antigen 19-9, 201, 3, and 14 U/ml). Serial repeated imaging studies over the course of a month showed the spontaneous regression of the hepatic tumors, thus enabling us to make a diagnosis of IPT without a surgical resection. A percutaneous biopsy confirmed the histology in one case. The regression of tumors on repeated images should play a key role in making an accurate diagnosis of IPT.

Histone deacetylase inhibitor (SAHA) and repression of EZH2 synergistically inhibit proliferation of gallbladder carcinoma

Polycomb group protein EZH2, frequently overexpressed in malignant tumors, is the catalytic subunit of polycomb repressive complex 2 (PRC2). PRC2 interacts with HDACs in transcriptional silencing and relates to tumor suppressor loss. We examined the expression of HDAC isoforms (HDAC 1 and 2) and EZH2, and evaluated the possible use of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and EZH2 repressor for gallbladder carcinoma. We used 48 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs), gallbladder carcinoma (TGBC2TKB), and cholangiocarcinoma (HuCCT‐1 and TFK‐1) cell lines for examination. Immunohistochemically, EZH2 was overexpressed in gallbladder carcinoma, especially poorly differentiated carcinoma, but not in normal epithelium. In contrast, HDAC1/2 were expressed in both carcinoma and normal epithelium in vivo. This pattern was verified in cultured cells; EZH2 was highly expressed only in TGBC2TKB, whereas HDAC1/2 were expressed in HGECs and TGBC2TKB. Interestingly, SAHA treatment caused significant cell number decline in three carcinoma cells, and this effect was synergized with EZH2 siRNA treatment; however, HGECs were resistant to SAHA. In TGBC2TKB cells, the expression of EZH2 and HDAC1/2 were decreased by SAHA treatment, and p16INK4a, E‐cadherin, and p21were simultaneously activated; however, no such findings were obtained in HGECs, suggesting that the effect of SAHA depends on the EZH2‐mediated tumor suppressor loss. In conclusion, this study suggests a possible mechanism by which carcinoma cells but not normal cells are sensitive to SAHA and indicates the efficacy of this new anticancer agent in combination with EZH2 repression in gallbladder carcinoma. (Cancer Sci 2009)

Bile ductular cells undergoing cellular senescence increase in chronic liver diseases along with fibrous progression.

We investigated the pathologic significance of ductular reactions in chronic liver diseases with respect to cellular senescence. The expression of senescence-associated markers (p16(INK4a) and p21(WAF1/Cip1)), cell proliferation, cell cycle markers (cyclin D and cyclin A), and neural cell adhesion molecule (NCAM) was examined immunohistochemically in primary biliary cirrhosis (PBC, n = 37), chronic viral hepatitis (n = 39), nonalcoholic steatohepatitis (n = 25), and control normal livers (n = 12). The expression of p16(INK4a) and p21(WAF1/Cip1) was frequently found in ductular cells in the advanced stage of chronic liver diseases, especially in PBC (P < .05). Double immunostaining disclosed that most senescent cells expressed cyclin D (G(1)-phase marker). NCAM was frequently coexpressed in ductular cells showing senescence-associated markers. Some ductular cells in ductular reactions in chronic liver diseases were at G(1) arrest and undergoing cellular senescence. Such senescent cells may be involved in the progression of fibrosis of these diseases, particularly in PBC.

Papillary hyperplasia of the gallbladder in pancreaticobiliary maljunction represents a senescence-related lesion induced by lysolecithin.

Cellular senescence, an irreversible growth arrest, is considered to play as safeguard against malignant progression, though such a mechanism is speculative in human carcinogenesis. In gallbladder carcinoma, cholecystolithiasis and pancreaticobiliary maljunction (PBM) are major risk factors. Here, by using 113 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs) and gallbladder carcinoma cell line (TGBC2TKB), we examined carcinogenesis with respect to cellular senescence. Among 15 cases of PBM in which carcinoma was found in 4 cases, nonneoplastic gallbladder mucosa showed diffuse papillary hyperplasia (PHP). PHP was not found in gallbladders with cholecystolithiasis. Interestingly, PHP exhibited senescent features such as expression of p16INK4A and low cell proliferative activity. In contrast, EZH2, a polycomb group protein, was overexpressed in intraepithelial neoplasm and carcinoma in gallbladders with cholecystolithiasis. In PBM, EZH2 was expressed only in carcinoma foci but not in PHP. Cultured HGECs treated with lysolecithin, the level of which is elevated in gallbladder bile of PBM, showed increased expression of p16INK4A and senescence-associated β-galactosidase. Conversely, enforced overexpression of EZH2 in senescent HGECs reduced p16INK4A expression. A knockdown of EZH2 in cultured TGBC2TKB cells increased p16INK4a expression. In conclusion, PHP in PBM may act as a barrier to malignant transformation for decades. EZH2 may be responsible for the escape from cellular senescence followed by malignant transformation in the gallbladder of PBM.

Verification of the oncologic inferiority of percutaneous biliary drainage to endoscopic drainage: A propensity score matching analysis of resectable perihilar cholangiocarcinoma.

BACKGROUND Percutaneous transhepatic biliary drainage is an established biliary drainage method but is associated with a potential risk of seeding metastasis. The aim of this retrospective study was to evaluate whether percutaneous transhepatic biliary drainage really increases seeding metastasis and worsens the postoperative survival in patients with resectable perihilar cholangiocarcinoma. METHODS Patients who underwent resection for perihilar cholangiocarcinoma after percutaneous transhepatic biliary drainage or endoscopic biliary drainage were retrospectively reviewed. Seeding metastasis was defined as peritoneal/pleural dissemination and percutaneous transhepatic biliary drainage sinus tract recurrence. Univariate and multivariate analyses followed by propensity score matching were performed to adjust the data for the baseline characteristics of the percutaneous transhepatic biliary drainage and endoscopic biliary drainage patients. RESULTS Of 320 resected patients, 168 underwent percutaneous transhepatic biliary drainage and the remaining 152 received endoscopic biliary drainage before operation. The survival of the percutaneous transhepatic biliary drainage patients was significantly lower than that of the endoscopic biliary drainage patients (37.0% vs 44.3% at 5 years, P = .019). Multivariate analyses showed that percutaneous transhepatic biliary drainage was an independent predictor of poor survival (P = .011) and a risk factor for seeding metastasis (P = .005). After propensity score matching (71 patients in each group), the survival of the percutaneous transhepatic biliary drainage patients was significantly worse than that of the endoscopic biliary drainage patients (P = .018). The estimated cumulative recurrence rate of seeding metastasis was significantly higher in the percutaneous transhepatic biliary drainage patients than in the endoscopic biliary drainage patients (P = .005), while the recurrence rates at other sites were similar between the 2 groups (P = .413). CONCLUSION Percutaneous transhepatic biliary drainage increases the incidence of seeding metastasis and shortens the postoperative survival in patients with perihilar cholangiocarcinoma. Endoscopic biliary drainage is recommended as the optimal method for preoperative biliary drainage.