Jeong Eun Yoo

Human Hepatocellular Carcinomas With ''Stemness''-Related Marker Expression: Keratin 19 Expression and a Poor Prognosis

There is a recently proposed subtype of hepatocellular carcinoma (HCC) that is histologically similar to usual HCC, but characterized by the expression of “stemness”‐related markers. A large‐scale study on two different cohorts of HCCs was performed to investigate the clinicopathologic features and epithelial‐mesenchymal transition (EMT)‐related protein expression status of this subtype of HCCs. The expression status of stemness‐related (e.g., keratin 19 [K19], cluster of differentiation [CD]133, epithelial cell adhesion molecule [EpCAM], and c‐kit) and EMT‐related markers (e.g., snail, S100A4, urokinase plasminogen activator receptor [uPAR], ezrin, vimentin, E‐cadherin, and matrix metalloproteinase [MMP]2) were examined using tissue microarrays from cohort 1 HCCs (n = 137). K19 protein expression in cohort 2 HCCs (n = 237) was correlated with the clinicopathologic parameters and messenger RNA (mRNA) levels of K19, uPAR, VIL2, Snail, Slug, and Twist. K19, EpCAM, c‐kit, and CD133 positivity were observed in 18.2%, 35.0%, 34.3%, and 24.8%, respectively. K19 was most frequently expressed in combination with at least one other stemness‐related marker (92.0%). K19‐positive HCCs demonstrated more frequent major vessel invasion and increased tumor size, compared to K19‐negative HCCs (P < 0.05). K19 was most significantly associated with EMT‐related protein expression (e.g., vimentin, S100A4, uPAR, and ezrin) (P < 0.05) and a poor prognosis (overall survival: P = 0.018; disease‐free survival: P = 0.007) in cohort 1. In cohort 2, HCCs with high K19 mRNA levels demonstrated higher mRNA levels of Snail, uPAR, and MMP2 (P < 0.05). K19‐positive HCCs demonstrated more frequent microvascular invasion, fibrous stroma, and less tumor‐capsule formation, compared to K19‐negative HCCs (P < 0.05). K19 expression was a significant independent predictive factor of poor disease‐free survival (P = 0.032). Conclusion: K19 was well correlated with clinicopathologic features of tumor aggressiveness, compared to other stemness‐related proteins. K19‐positive HCCs showed significantly increased EMT‐related protein and mRNA expression, suggesting that they may acquire more invasive characteristics, compared to K19‐negative HCCs through the up‐regulation of EMT‐associated genes. (HEPATOLOGY 2011;)

High telomerase activity and long telomeres in advanced hepatocellular carcinomas with poor prognosis

Telomerase reactivation and telomere maintenance are crucial in carcinogenesis and tumor progression. In this study, the relationships between telomere parameters, chromosomal instability and clinicopathological features were evaluated in hepatocellular carcinomas (HCCs). Telomere length (TL), telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA levels were measured in 49 hepatitis B virus (HBV)-related HCCs and corresponding non-tumorous tissues. The results were compared with clinicopathological data, including differentiation, multipolar mitosis (MM), anaphase bridge, immunohistochemical stain results for cytokeratin 19 (CK19) and patient outcome. TL of HCCs ranged from 4.7 to 13.1 kb, and 44.4% of HCCs showed telomere lengthening. hTERT mRNA levels and TA were closely related (P=0.008), and were significantly higher in HCCs than non-tumorous tissues. TL was significantly higher in HCCs with strong TA (P=0.048), high hTERT mRNA levels (P=0.001) and poor differentiation (P=0.041). Frequent MM was associated with poor differentiation (P=0.007) and advanced stage (P<0.001). TA was positively correlated with MM, anaphase bridges and advanced stage (P=0.019, P=0.017 and P=0.029). Thirteen (28.3%) HCCs were CK19+ and demonstrated longer telomeres than CK19− HCCs (P=0.046). Overall survival was poor in HCCs with MM >0.4 per field (P=0.016), high TA (P=0.009) and high TL ratio (HCC/non-HCC) >0.8 (P=0.044). Our results show that long telomeres, high TA and high mitotic instability are poor prognostic markers for HBV-related HCCs and their close association suggests that telomere maintenance may be important for the progression of HCCs with high chromosomal instability to more aggressive ones.

A fibrous stromal component in hepatocellular carcinoma reveals a cholangiocarcinoma-like gene expression trait and epithelial-mesenchymal transition.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the major primary liver cancers in adults. The phenotypic overlap between HCC and CC has been shown to comprise a continuous liver cancer spectrum. As a proof of this concept, a recent study demonstrated a genomic subtype of HCC that expressed CC‐like gene expression traits, such as CC‐like HCC, which revealed the common genomic trait of stem‐cell–like properties and aggressive clinical outcomes. Scirrhous HCC (S‐HCC), a rare variant of HCC, is characterized by abundant fibrous stroma and has been known to express several liver stem/progenitor cell markers. This suggests that S‐HCC may harbor common intermediate traits between HCC and CC, including stem‐cell traits, which are similar to those of CC‐like HCC. However, the molecular and pathological characteristics of S‐HCC have not been fully evaluated. By performing gene‐expression profiling and immunohistochemical evaluation, we compared the morphological and molecular features of S‐HCC with those of CC and HCC. S‐HCC expresses both CC‐like and stem‐cell–like genomic traits. In addition, we observed the expression of core epithelial‐mesenchymal transition (EMT)‐related genes, which may contribute to the aggressive behavior of S‐HCC. Overexpression of transforming growth factor beta (TGF‐β) signaling was also found, implying its regulatory role in the pathobiology of S‐HCC. Conclusion: We suggest that the fibrous stromal component in HCC may contribute to the acquisition of CC‐like gene‐expression traits in HCC. The expression of stem‐cell–like traits and TGF‐β/EMT molecules may play a pivotal role in the aggressive phenotyping of S‐HCC. (HEPATOLOGY 2012;55:1776–1786)

Large liver cell change in hepatitis B virus–related liver cirrhosis

Large liver cell change (LLCC) refers to microscopic lesions often found in various chronic liver diseases; however, its nature is still controversial. Thirty‐four formalin‐fixed and 19 fresh frozen hepatitis B virus (HBV)‐related cirrhosis samples were examined for the presence of LLCC, small liver cell change (SLCC), and hepatocellular carcinoma (HCC). The cell cycle checkpoint status (p21, p27, p16, Tp53), cell dynamics (proliferating cell nuclear antigen, Ki‐67, terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling, M30), DNA damage (γ‐H2AX [H2A histone family, member X]), telomere lengths, chromosomal instability (micronuclei index), and senescence‐associated β‐galactosidase (SA‐β‐Gal) activity were evaluated using an in situ approach and compared to those in normal liver (n = 5) and liver with chronic cholestasis (34 cases of hepatolithiasis and three cases of primary biliary cirrhosis). In HBV‐related cirrhosis, the p21, p27, and p16 cell cycle checkpoint markers were activated in normal‐looking cirrhotic hepatocytes (NLCH), but diminished gradually from LLCC, SLCC, to HCC, with an increase in Tp53 expression. There was a general decrease in telomere length from NLCH, LLCC, SLCC, to HCC. Micronuclei, γ‐H2AX foci, and net cellular gain were significantly increased from normal hepatocytes, NLCH, LLCC, SLCC, to HCC. The SA‐β‐Gal activity was weaker in LLCC compared to NLCH and absent in SLCC and HCC. In contrast, cholestatic LLCC showed retained expression of cell cycle checkpoint markers and decreased net cellular gain compared to adjacent normal‐looking hepatocytes. HBV‐related LLCC showed significantly higher Tp53 labeling index, γ‐H2AX labeling index, and micronuclei index; shorter telomere length; decreased SA‐β‐Gal activity; and increased net cellular gain compared to cholestatic LLCC. Conclusion: The nature of LLCC is rather heterogeneous depending on the biological setting. The characteristics of HBV‐related LLCC are more consistent with dysplastic rather than merely reactive hepatocytes, whereas cholestatic LLCC more likely represents reactive change with more stringent cell cycle checkpoint control. (HEPATOLOGY 2009.)

Notch1 differentially regulates oncogenesis by wildtype p53 overexpression and p53 mutation in grade III hepatocellular carcinoma

The tumor suppressor p53 is a key prognostic factor in hepatocellular carcinoma (HCC), yet only 35% of grade III tumors exhibit mutation of p53. Several other pathways have been implicated in HCC and, among these, the role of the Notch1/Snail pathway remains unclear. Therefore, we investigated the expression of p53, Notch1, and Snail proteins in HCC with regard to both clinical grade and p53 mutational status. Immunoblotting for p53 revealed that, whereas in many tumors increased p53 was a result of p53 mutation, wildtype p53 (p53WT) expression was also frequently elevated in HCCs. Coordinated evaluation of p53, Notch1, and Snail expression suggests that grade III HCC can be subdivided based on the expression of these three proteins. We found that Notch1 expression in HCC tissues and cell lines is differentially affected by p53WT and mutant p53 (p53Mut). Notch1 expression was correlated with p53 expression in cells expressing p53WT, but was not elevated in p53Mut‐expressing cells. Virally mediated expression or silencing of p53WT or p53Mut confirmed that p53WT overexpression causes Notch1 up‐regulation in HCC. Surprisingly, the consequence of Notch1 overexpression for the proliferative and invasive capacity of HCC cells depends on both the p53 mutational status and activation of the Snail pathway. Conclusion: In the presence of p53WT, Snail/Notch1 activation increased the invasiveness of HCC cells. In contrast, in the absence of p53WT, Notch1 decreased the invasiveness of HCC. Taken together, these findings shed new light on the complex role of the Notch1/Snail axis in HCC and provide a framework for further classifying HCC based on the expression and mutational status of p53 and the expression of Notch1 and Snail. (HEPATOLOGY 2011;53:1352‐1362)

Invasion and EMT-associated genes are up-regulated in B viral hepatocellular carcinoma with high expression of CD133-human and cell culture study☆

Hepatocellular carcinomas (HCCs) with expression of stem/progenitor cell markers including CD133 have been reported to have more aggressive biological behavior, and epithelial-mesenchymal transition (EMT), closely related invasion, has been suggested to generate cancer stem cells. To elucidate biological characteristics of HCCs expressing CD133, we evaluated migration assay and the mRNA expression levels of CD133, invasion-associated genes [urokinase plasminogen activator receptor (uPAR), villin 2 (VIL2), and MMP1 and MMP2], and EMT regulators (Snail, Slug, Twist, E-cadherin, and N-cadherin) by real-time PCR in HCC cell lines including HepG2, Hep3B, Huh7, PLC/RFP/6, SNU423, SNU449, and SNU475. Same genes and pathological features were also investigated in 49 samples of hepatitis B virus-related human HCCs. In all HCC cell lines studied, CD133-positive cells showed higher cell migration activity and up-regulated invasion- and EMT-associated genes with increased N-cadherin and decreased E-cadherin expressions compared to CD133-negative cells. The human HCCs were divided into the CD133-high group (top 40%) and the CD133-low group (bottom 40%) according to the level of CD133 mRNA. The CD133-high group showed relatively frequent vascular invasion and significantly higher expression of invasion-associated genes [uPAR (p=0.002), MMP1 (p=0.01), and MMP2 (p=0.003)] and EMT regulators [Snail (p=0.002) and Twist (p=0.0003)] compared to the CD133-low group. In conclusion, our results suggest that there is a subtype of HCC with high expression of CD133, which might have more invasive characteristics by up-regulation of invasion-associated genes and EMT-associated genes.

Chromosomal instability, telomere shortening, and inactivation of p21WAF1/CIP1 in dysplastic nodules of hepatitis B virus-associated multistep hepatocarcinogenesis.

Systemic analysis for chromosomal instability and inactivation of cell cycle checkpoints are scarce during hepatocarcinogenesis. We studied 24 patients with chronic B viral cirrhosis including 30 cirrhotic regenerative nodules, 35 low-grade dysplastic nodules, 15 high-grade dysplastic nodules, 7 dysplastic nodules with hepatocellular carcinoma foci, and 18 hepatocellular carcinomas. Eight normal livers were studied as the control group. Telomere length and micronuclei were detected by Southern blot and Feulgen-fast green dyeing technique, respectively, and p21WAF1/CIP1 expression was studied by immunohistochemistry. Micronuclei >1 per 3000 hepatocytes were found in 17% of low-grade dysplastic nodules, 87% of high-grade dysplastic nodules, and 100% of high-grade dysplastic nodules with hepatocellular carcinoma foci and hepatocellular carcinomas in contrast to those of all normal livers, and 90% of cirrhosis showed no micronuclei. The micronuclei index showed a gradual increase during hepatocarcinogenesis and there was a significant increase between cirrhosis and low-grade dysplastic nodules, low-grade dysplastic nodules and high-grade dysplastic nodules, and high-grade dysplastic nodules and hepatocellular carcinomas. Telomere length showed a gradual shortening during hepatocarcinogenesis and a significant reduction was found in high-grade dysplastic nodules (P=0.024) and hepatocellular carcinomas (P=0.031) compared with normal and cirrhotic livers. The micronuclei index was correlated with telomere shortening (P=0.016). The p21WAF1/CIP1 labeling index was significantly higher in cirrhosis than in normal livers (P=0.024) and markedly decreased in low-grade dysplastic nodules, high-grade dysplastic nodules, and hepatocellular carcinomas compared with cirrhosis (P<0.05). The p21WAF1/CIP1 labeling index was associated with telomere length (P<0.001) but not micronuclei index. This study shows that telomere shortening, chromosomal instability, and inactivation of p21WAF1/CIP1 checkpoint function occur in low-grade dysplastic nodules as well as in high-grade dysplastic nodules, and their cooperation is considered to be critical for malignant transformation during hepatitis B virus associated-multistep hepatocarcinogenesis.

Genomic Predictors for Recurrence Patterns of Hepatocellular Carcinoma: Model Derivation and Validation

In this study, Lee and colleagues develop a genomic predictor that can identify patients at high risk for late recurrence of hepatocellular carcinoma (HCC) and provided new biomarkers for risk stratification.

Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma

Metabolic activation is a common feature of many cancer cells and is frequently associated with the clinical outcomes of various cancers, including hepatocellular carcinoma. Thus, aberrantly activated metabolic pathways in cancer cells are attractive targets for cancer therapy. Yes‐associated protein 1 (YAP1) and transcriptional coactivator with PDZ‐binding motif (TAZ) are oncogenic downstream effectors of the Hippo tumor suppressor pathway, which is frequently inactivated in many cancers. Our study revealed that YAP1/TAZ regulates amino acid metabolism by up‐regulating expression of the amino acid transporters solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5). Subsequently, increased uptake of amino acids by the transporters (SLC38A1 and SLC7A5) activates mammalian target of rapamycin complex 1 (mTORC1), a master regulator of cell growth, and stimulates cell proliferation. We also show that high expression of SLC38A1 and SLC7A5 is significantly associated with shorter survival in hepatocellular carcinoma patients. Furthermore, inhibition of the transporters and mTORC1 significantly blocks YAP1/TAZ‐mediated tumorigenesis in the liver. These findings elucidate regulatory networks connecting the Hippo pathway to mTORC1 through amino acid metabolism and the mechanisms potential clinical implications for treating hepatocellular carcinoma. Conclusion: YAP1 and TAZ regulate cancer metabolism and mTORC1 through regulation of amino acid transportation, and two amino acid transporters, SLC38A1 and SLC7A5, might be important therapeutic targets. (Hepatology 2016;63:159–172)

Human PinX1 Mediates TRF1 Accumulation in Nucleolus and Enhances TRF1 Binding to Telomeres

Human PinX1 (hPinX1) is known to interact with telomere repeat binding factor 1 (TRF1) and telomerase. Here, we report that hPinX1 regulates the nucleolar accumulation and telomeric association of TRF1. In HeLa, HA-hPinX1 was co-localized with fibrillarin, a nucleolar protein, in 51% of the transfected cells and was present in the nucleoplasm of the remaining 48%. Mutant analysis showed that the C-terminal region was important for nucleolar localization, while the N-terminus exhibited an inhibitory effect on nucleolar localization. Unlike HA- and Myc-hPinX1, GFP-hPinX1 resided predominantly in the nucleolus. Nuclear hPinX1 bound to telomeres and other repeat sequences as well but, despite its interaction with TRF1, nucleolar hPinX1 did not bind to telomeres. Nucleolar hPinX1 forced endogenous TRF1 accumulation in the nucleolus. Furthermore, TRF1 binding to telomeres was upregulated in cells over-expressing hPinX1. In an ALT cell line, WI-38 VA-13, TRF1 did not co-localize with hPinX1 in the nucleoli. In summary, hPinX1 likely interacts with TRF1 in both the nucleolus and the nucleoplasm, and excess hPinX1 results in increased telomere binding of TRF1. The PinX1 function of mediating TRF1 nucleolar accumulation is absent from ALT cells, suggesting that it might be telomerase-dependent.