Yu-Ling Chen

Lin-28B expression promotes transformation and invasion in human hepatocellular carcinoma

MicroRNAs (miRNAs) play critical roles in embryonic development and are frequently deregulated in human cancers. The let-7 family members are tumor-suppressing miRNAs and are frequently downregulated in cancer cells. Lin-28 and Lin-28B are RNA-binding proteins highly expressed in embryonic tissues. Lin-28 proteins block let-7 precursors from being processed to mature miRNAs by inducing terminal uridylation and degradation of let-7 precursors. Here, we report that Lin-28B, but not Lin-28, is highly expressed in hepatocellular carcinoma (HCC). Lin-28B expression was more frequently noted in high-grade HCCs with high alpha-fetoprotein levels. Knockdown of Lin-28B by RNA interference in the HCC cell line HCC36 suppressed proliferation in vitro and reduced in vivo tumor growth in NOD/SCID mice. In contrast, overexpression of Lin-28B in the HCC cell line HA22T enhanced tumorigenicity. Overexpression of Lin-28B also induced epithelial-mesenchymal transition in HA22T cells and hence, invasion capacity. Large-scale real-time PCR array analysis revealed that, among 380 miRNAs, only let-7/mir-98 family members were regulated by Lin-28B. Lin-28B overexpression enhanced the expression of the known let-7 targets c-myc and HMGA2. It was also found that Lin-28B enhanced the expression of type 1 insulin-like growth factor receptor in a let-7-dependent manner. These results indicate that Lin-28B regulates tumor formation and invasion in HCC through coordinated repression of the let-7/mir-98 family and induction of multiple oncogenic pathways.

SIRT1 Promotes Tumorigenesis and Resistance to Chemotherapy in Hepatocellular Carcinoma and its Expression Predicts Poor Prognosis

BackgroundSIRT1 is a NAD+-dependent deacetylase that plays crucial roles in many biological processes, including stress response, apoptosis, cellular metabolism, adaptation to calorie restriction, aging, and tumorigenesis. The purpose of this study is to elucidate the clinicopathological and functional significance of SIRT1 expression in hepatocellular carcinoma (HCC).MethodsSIRT1 expression in HCC was determined by immunohistochemical staining. The results were correlated with clinicopathological parameters. SIRT1 was overexpressed in HCC cell line SK-Hep1 to study its role in tumorigenesis and resistance to chemotherapy.ResultsSIRT1 was overexpressed in 95 of 172 HCCs (55%). SIRT1 overexpression was associated with higher α-fetoprotein level, higher tumor grade, and absence of β-catenin mutation. SIRT1 expression predicted poor long-term survival for patients with resected HCC. The elevated SIRT1 protein level in HCC was not attributable to the elevation of mRNA level. The half-life of SIRT1 protein was longer in cell lines with higher expression of SIRT1. We further demonstrated that SIRT1 was degraded by the 26S proteasome in an ubiquitin-dependent manner. Overexpression of SIRT1 promoted tumorigenesis and resistance to chemotherapeutical agent and sorafenib.ConclusionsSIRT1 is an oncogenic protein for HCC and is a predictor of worse outcome after surgical resection of HCC.

Gas6/Axl pathway promotes tumor invasion through the transcriptional activation of Slug in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Other than the sorafenib treatment, no effective systemic therapy has been available thus far. Most targets in molecularly targeted therapy for cancer are receptor tyrosine kinases (RTKs). Therefore, identifying activated RTKs in HCC is critical for developing new molecularly targeted therapies. Using a phospho-RTK array, we found that Axl is one of the most frequently activated RTKs in liver cancer cell lines. The knockdown of Axl by RNA interference significantly reduced cell migration and invasion in the HCC cell lines HA22T and Mahlavu. Stimulation of HCC cell lines by Axl ligand growth arrest-specific 6 (Gas6) enhanced cell migration and invasion. The Gas6/Axl pathway enhanced the expression of the epithelial-mesenchymal transition-inducing transcription factor Slug, which is essential for the invasion-promoting activity of Axl. Treating HCC cells with the Axl inhibitor bosutinib suppressed Slug expression and decreased the invasiveness of HCC cell lines. These findings indicate that Gas6/Axl regulates tumor invasion through the transcriptional activation of Slug.

Overexpression of CTHRC1 in Hepatocellular Carcinoma Promotes Tumor Invasion and Predicts Poor Prognosis

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted glycoprotein that activates the planar cell polarity pathway of Wnt signaling. Using microarray analysis, we found that the CTHRC1 gene is overexpressed in hepatocellular carcinoma (HCC). The level of CTHRC1 mRNA was measured in 201 surgically resected HCCs using real time reverse transcription-polymerase chain reaction. Overexpression of CTHRC1 in HCC was associated with large tumor size and advanced tumor stage. Furthermore, expression of CTHRC1 as was identified as an independent prognostic factors in the multivariate analysis. Suppression of CTHRC1 expression inhibited tumor migration and invasion whereas overexpression of CTHRC1 promoted tumor invasion. Activation of RhoA, but not Rac1 or Cdc42, was found to play a crucial role in CTHRC1-induced cell migration. CTHRC1 promoted adhesion of cancer cells to extracellular matrix through induction of integrin β1 expression and activation of focal adhesion kinase. These results suggest CTHRC1 promotes tumor invasion and metastasis by enhancing the adhesion and migratory abilities of tumor cells. It is also a promising biomarker for predicting the prognosis of patients with HCC.

Viral hepatitis is associated with intrahepatic cholangiocarcinoma with cholangiolar differentiation and N-cadherin expression

Viral hepatitis-associated intrahepatic cholangiocarcinoma is thought to have common disease processes with hepatocellular carcinoma, but until now the histomorphological and genetic features of viral hepatitis-associated intrahepatic cholangiocarcinoma is still unknown. From 2000 to 2010, 170 patients with intrahepatic cholangiocarcinoma who received detailed pathological assessment and regular follow-up at the National Taiwan University Hospital were selected for this study. Of 170 patients, 69 (41%) were positive for hepatitis B and/or C virus. These patients were younger, were more frequently male, and had elevated serum α-fetoprotein levels as compared with seronegative intrahepatic cholangiocarcinoma patients. Grossly these tumors were mostly of the mass-forming type, and histologically, cholangiolar differentiation was more frequently seen. We identified N-cadherin as an immunohistochemical marker strongly associated with hepatitis virus infection. The prevalence of viral hepatitis in patients with N-cadherin-positive intrahepatic cholangiocarcinoma was 75%, and that in N-cadherin-negative patients was only 37%. N-cadherin-positive patients were younger, had elevated α-fetoprotein, and had no hepatolithiasis. All N-cadherin-positive intrahepatic cholangiocarcinomas were of the mass-forming type. N-cadherin positivity was strongly associated with cholangiolar morphology and lack of carcinoembryonic antigen and MUC2 expression, whereas K-RAS mutations were less frequent. Our results indicate that a subgroup of intrahepatic cholangiocarcinoma characterized by cholangiolar differentiation and N-cadherin expression is strongly associated with viral hepatitis.

Hepatocyte growth factor activates Wnt pathway by transcriptional activation of LEF1 to facilitate tumor invasion.

Hepatocyte growth factor (HGF) is a secretory protein that plays important roles in cancer growth and metastasis. Lymphoid-enhancing factor 1 (LEF1) is a transcription factor mediating Wnt/β-catenin signaling. Using microarray analysis, we found HGF induced expression of LEF1 in liver and breast cancer cell lines. HGF induced expression of LEF1 through phosphatidylinositol 3-kinase/Akt and nuclear factor-kappa B (NF-κB) signaling. Multiple NF-κB-binding sites were mapped within 3 kb upstream of LEF1 transcription initiation site. NF-κB binding to a site 2 kb upstream of LEF1 transcription initiation site was confirmed by chromatin immunoprecipitation assay. Knockdown of LEF1 inhibited the expression of Slug and Zinc finger E-box-binding homeobox 2 (ZEB2) and markedly attenuated HGF-induced tumor migration and invasion. Using immunohistochemical staining, we found LEF1 was frequently expressed in multiple types of carcinoma but not in the non-tumorous epithelial cells. Our finding suggest that transcriptional activation of LEF1 is a mechanism of cross talk between HGF/c-Met and Wnt/β-catenin pathways and is essential for HGF-induced tumor invasion.

TERT promoter mutation in resectable hepatocellular carcinomas: A strong association with hepatitis C infection and absence of hepatitis B infection

INTRODUCTIONnMutation in the core promoter of the telomerase reverse transcriptase (TERT) gene was determined to be a frequent event in malignant melanoma and other cancers. However, the role of TERT promoter mutation in hepatocellular carcinomas (HCCs) remains largely unknown.nnnMETHODSnGenomic DNA samples from the tumor tissue of 195 HCCs were analyzed for TERT promoter mutation at 2 hotspots (-124 and -146 bp from the ATG start site, g.1,295,228 and g.1,295,250, respectively) through direct sequencing.nnnRESULTSnThe TERT promoter mutation was identified in 57 of the 195 HCCs (29.2%) and was associated with old age (P = 0.0122), presence of anti-hepatitis C (HCV; P = 0.0048), and absence of hepatitis B surface antigen (HBsAg; P = 0.0007). However, the TERT promoter mutation did not correlate with serum α-fetoprotein levels, liver cirrhosis, tumor size, tumor grade, tumor stage, early tumor recurrence, β-catenin mutation or p53 mutation. A multivariate analysis confirmed that the absence of hepatitis B infection is an independent factor associated with TERT promoter mutation. Furthermore, among HCC patients infected with hepatitis C, those with concomitant hepatitis B infection exhibited infrequent TERT promoter mutation (P = 0.0435). Remarkably, patients presenting with TERT promoter mutation-positive and -negative HCCs exhibited similar disease-free and overall survival rates.nnnCONCLUSIONSnOur study indicated that the TERT promoter mutation frequently occurred in HCV-associated HCCs. The absence of Hepatitis B infection was significantly associated with the TERT promoter mutation. These findings suggest that various etiological factors may be involved in differing mechanisms to preserve telomeres during the carcinogenesis of HCCs.

GNAS Is frequently mutated in a specific subgroup of intraductal papillary neoplasms of the bile duct.

Intraductal papillary neoplasms of the bile duct (IPN-Bs) share clinicopathologic features with intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Approximately two thirds of IPMNs have activating point mutations of GNAS at codon 201. The role of GNAS mutation is unclear in IPN-B. In this study, we evaluated 41 patients diagnosed with IPN-B for clinicopathologic characteristics and follow-up information. Mutation analyses of GNAS and KRAS were performed. Twenty-three cases (56.1%) of IPN-B were categorized as the intestinal subtype, and 18 (43.9%) were considered the gastric/pancreatobiliary subtype. IPN-Bs showing intestinal differentiation demonstrated high immunohistochemical expressions of CK20, CDX2, and MUC2, as well as a significant association with macroscopic and microscopic mucin hypersecretions and villous architecture. GNAS and KRAS mutations were detected in 29% and 32% of IPN-Bs, respectively. All IPN-Bs with GNAS mutation showed intestinal differentiation. GNAS-mutated IPN-B was highly significantly associated with certain pathologic characteristics, including macroscopic and microscopic mucin hypersecretion and villous architecture. IPN-B with GNAS mutation tended to more frequently harbor KRAS mutation than those without GNAS mutation. IPN-Bs with intestinal differentiation, villous architecture, and mucin hypersecretion constitute a distinct subgroup of IPN-B, which frequently has GNAS mutation. This subtype shares common genetic alterations with IPMN of the pancreas.

S100P immunostaining identifies a subset of peripheral-type intrahepatic cholangiocarcinomas with morphological and molecular features similar to those of perihilar and extrahepatic cholangiocarcinomas.

Tsai J‐H, Huang W‐C, Kuo K‐T, Yuan R‐H, Chen Y‐L & Jeng Y‐M u2028(2012) Histopathologyu2028S100P immunostaining identifies a subset of peripheral‐type intrahepatic cholangiocarcinomas with morphological and molecular features similar to those of perihilar and extrahepatic cholangiocarcinomas

Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast.

Mutations in HER2 gene have been identified in a small subset of breast cancer cases. Identification of HER2 mutation has therapeutic implications for breast cancer, but whether a subgroup of breast cancer with a higher frequency of HER2 mutation exists, remains unknown. We analyzed HER2 mutation and pathologic factors on 73 formalin-fixed, paraffin-embedded samples, including 21 pleomorphic invasive lobular carcinoma (p-ILC) cases, 3 pleomorphic lobular carcinoma in situ (p-LCIS) cases, and 49 classic invasive lobular carcinoma (c-ILC) cases. Mutations were identified through direct sequencing. HER2 overexpression and amplification were determined through immunohistochemistry and fluorescent in situ hybridization. Six mutations were identified, including five in the 24 p-ILC or p-LCIS (p-ILC/p-LCIS) cases (20.8xa0%) and one in the 49 c-ILC cases (2.0xa0%), and the difference in frequency was significant (pxa0=xa00.013). Eight of the 24 (33.3xa0%) p-ILC/p-LCIS cases exhibited HER2 amplification or overexpression (amplification/overexpression), which was significantly higher than in the c-ILC cases (1/49, 2xa0%). Mutation and amplification/overexpression were mutually exclusive. HER2 mutations were identified more frequently in the p-ILC/p-LCIS cases with extensive apocrine change (pxa0=xa00.018). Combined HER2 alterations through mutation or amplification/overexpression were more frequently identified in p-ILC/p-LCIS cases without estrogen receptor expression. The high frequency (54.1xa0%, 13/24) of combined HER2 alterations in the p-ILC/p-LCIS cases suggests a crucial role of HER2 in the pathogenesis of p-ILC/p-LCIS. Because of the reported responsiveness of HER2 mutation to anti-HER2 therapy, p-ILC patients without HER2 amplification/overexpression should receive HER2 mutation analysis to identify this therapeutically relevant target.