Hideyuki Kuroki

CD44s regulates the TGF-β-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

An Imbalance in TAZ and YAP Expression in Hepatocellular Carcinoma Confers Cancer Stem Cell–like Behaviors Contributing to Disease Progression

Transcriptional coactivator with PDZ-binding motif (TAZ) and yes-associated protein (YAP) are equivalently placed downstream effectors of the Hippo pathway with oncogenic roles in human cancers. However, the expression profiles of TAZ/YAP differ depending on the cancer cell type, suggesting that these proteins have different roles during cancer progression, yet no studies have examined the biologic significance of the balance between TAZ and YAP expression levels. Here we examined the functional roles of TAZ/YAP in hepatocellular carcinoma progression. We found that TAZ, but not YAP, was predominantly expressed in HCC. TAZ knockdown under normal conditions attenuated cell growth in HCC cells; however, TAZ knockdown combined with 5-fluorouracil treatment significantly increased chemoresistance compared with control cells. Notably, TAZ knockdown induced compensatory YAP expression and was accompanied by upregulation of CD90, a HCC-specific cancer stem cell marker. Continuous treatment with 5-fluorouracil also induced YAP expression and promoted tumor formation in vivo. Conversely, double knockdown of TAZ/YAP reduced chemoresistance and tumorigenicity. Moreover, YAP knockdown aggravated HCC cell growth to a greater degree than TAZ knockdown, and YAP overexpression was strongly associated with poor prognoses in patients with HCC. Collectively, these studies demonstrate that TAZ and YAP exhibit different functional roles in cancer progression, and a shift to predominant YAP expression upon TAZ depletion conferred cancer stem cell-like properties including chemoresistance and tumorigenicity in HCC. Therefore, targeting of both TAZ/YAP will be required for a complete antitumor response in HCC.

miR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells.

Background:The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC.Methods:MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines).Results:In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and β-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1.Conclusions:MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Background:Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).Methods:Circulating CD44+CD90+ cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.Results:CD44+CD90+ cells in the blood acquired epithelial–mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44high population showed higher anoikis resistance and sphere-forming ability than did the CD44low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.Conclusions:CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

Statins inhibit tumor progression via an enhancer of zeste homolog 2-mediated epigenetic alteration in colorectal cancer

While statin intake has been proven to reduce the risk of colorectal cancer (CRC), the mechanism of antitumor effects and clinical significance in survival benefits remain unclear. Statin‐induced antiproliferative effects and its underlying mechanism were examined using six CRC cell lines. Statins except pravastatin showed antiproliferative effects (simvastatin ≥ fluvastatin > atorvastatin) even though both of simvastatin and pravastatin could activate mevalonate pathways, suggesting the statin‐mediated antiproliferative effects depended on non‐mevalonate pathway. Indeed, statin induced p27KIP1 expression by downregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2), which acts as an epigenetic gene silencer. Additionally, the use of simvastatin plus classII histone deacetylase (HDAC) inhibitor (MC1568) induced further overexpression of p27KIP1 by inhibiting HDAC5 induction originated from downregulated EZH2 in CRC cells and synergistically led to considerable antiproliferative effects. In the clinical setting, Statin intake (except pravastatin) displayed the downregulated EZH2 expression and inversely upregulated p27KIP1 expression in the resected CRC by immunohistochemical staining and resulted in the significantly better prognoses both in overall survival (p = 0.02) and disease free survival (p < 0.01) compared to patients without statin intake. Statins may inhibit tumor progression via an EZH2‐mediated epigenetic alteration, which results in survival benefits after resected CRC. Furthermore, statin plus classII HDAC inhibitor could be a novel anticancer therapy by their synergistic effects in CRC.

Epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A inhibits the growth of cholangiocarcinoma cells

Enhancer of zeste homolog 2 (EZH2) is involved in malignant transformation and the biological aggressiveness of several human malignancies. Growing evidence indicates that EZH2 may be an appropriate therapeutic target for malignancies, including cholangiocarcinoma. Recently, an S-adenosyl-L-homocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep) was shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep and the combination of gemcitabine and DZNep in cholangiocarcinoma cells. The effects of DZNep and its combination with gemcitabine were assessed in the cholangiocarcinoma cell lines RBE and TFK-1. DZNep depleted the cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. DZNep treatment resulted in the inhibition of proliferation in the cholangiocarcinoma cell lines, and the combination of DZNep and gemcitabine showed synergistic inhibition of cell proliferation. DZNep induced apoptosis and G1 phase cell cycle arrest in cholangiocarcinoma cells, and the combination of DZNep and gemcitabine enhanced the induced apoptosis and G1 arrest when compared with gemcitabine alone. Inhibition of cell proliferation by DZNep was partially associated with upregulation of p16INK4a and p17KIP1. The present study shows that DZNep inhibits cell proliferation by inducing G1 arrest and apoptosis. These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach for the treatment of cholangiocarcinoma.

Functional assessment versus conventional volumetric assessment in the prediction of operative outcomes after major hepatectomy

PURPOSE In this study we examined whether conventional future remnant liver volume (FR volume) or function (FR function) better predicted the operative outcome after major hepatectomy. METHODS Of 510 patients who underwent hepatectomy for various indications, 133 patients with major hepatectomy were enrolled in this study. FR volume and the corresponding FR function routinely were measured with a 99mTc-GSA scintigraphy single-proton emission computed tomography fusion system. FR function was defined as the future remnant liver uptake ratio of 99mTc-GSA per whole liver by single-proton emission computed tomography. FR volume or function in cases with insufficient FR volume or function required for major hepatectomy were defined as marginal. RESULTS Morbidity, liver dysfunction-related morbidity, and mortality after major hepatectomy occurred in 40 (30%), 10 (7.5%), and 8 (5.7%) patients, respectively. Thirty-two of the 133 patients were diagnosed as marginal using FR volume, but only 11 patients were diagnosed as marginal using FR function. These results indicated that 21 patients (16%) were switched to the safe group using functional assessment. Operative outcomes in patients with safe FR function (n = 122) were equivalent to those of patients with safe FR volume (n = 101), but patients with marginal FR function (n = 11) had substantially worse outcomes than patients with marginal FR volumes (n = 32). Logistic regression analysis identified marginal FR function, but not volume, as a risk factor for worse operative outcome after major hepatectomy. Portal vein embolization induced the substantially greater FR function compared with FR volume. Although liver volume equally corresponds to liver function under normal conditions, liver cirrhosis was greatly associated with the major discrepancy (more than 10%) in patients without portal vein embolization. CONCLUSION Functional assessment for future remnant liver identified patients who were eligible for curative hepatectomy despite a marginal status based on conventional volumetric assessment. Thus, marginal FR function is a better predictor of operative outcome after major hepatectomy than FR volume, especially in patients with nonhealthy liver.

Triple positive tumor markers predict recurrence and survival in early stage hepatocellular carcinoma

Hepatectomy is feasible for patients with hepatocellular carcinoma (HCC) with good hepatic function who meet the Milan criteria. Several studies have indicated that tumor markers of HCC, α‐fetoprotein (AFP), Lens culinaris agglutinin‐reactive fraction of AFP percentage and protein induced by vitamin K absence/antagonist‐II were good predictors of malignant potential. It is important to identify highly malignant cases of HCC, and the aim of this study was to clarify the impact of triple positive tumor markers as the prognostic factors for early stage HCC within the Milan criteria.

EZH2 is associated with malignant behavior in pancreatic IPMN via p27Kip1 downregulation.

Background The epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression. Methods Fifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of EZH2 and p27Kip1 mRNA were analyzed by real time PCR. Results In immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma). EZH2 expression displayed a similar pattern (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma) with cell proliferative activity. EZH2 expression in malignant (CIS and invasive carcinoma) IPMNs was significantly higher than that in adenoma and borderline-atypia IPMNs. EZH2 expression level in IPMN lesions was positively correlated with the Ki-67 positive nuclear ratio (p<0.0001). EZH2-positive cells in malignant IPMN did not express p27Kip1. EZH2 mRNA expressions in malignant lesions were significantly higher than those in benign lesions (p<0.0001). In contrast, p27Kip1 mRNA in malignant lesions was significantly decreased compared to those in benign lesion (p<0.05), and there was an inverse correlation between EZH2 and p27Kip1 mRNA levels (p = 0.0109). Conclusion EZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27Kip1.

Effect of LSKL peptide on thrombospondin 1-mediated transforming growth factor β signal activation and liver regeneration after hepatectomy in an experimental model.

A strategy for accelerating liver regeneration after hepatectomy would offer great benefits in preventing postoperative liver failure and improving surgical outcomes. Transforming growth factor (TGF) β is a potent inhibitor of hepatocyte proliferation. Recently, thrombospondin (TSP) 1 has been identified as a negative regulator of liver regeneration by activation of local TGF‐β signals. This study aimed to clarify whether the LSKL (leucine–serine–lysine–leucine) peptide, which inhibits TSP‐1‐mediated TGF‐β activation, promotes liver regeneration after hepatectomy in mice.