Kouki Nio

Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma

Recent evidence suggests that hepatocellular carcinoma (HCC) is organized by a subset of cells with stem cell features (cancer stem cells; CSCs). CSCs are considered a pivotal target for the eradication of cancer, and liver CSCs have been identified by the use of various stem cell markers. However, little information is known about the expression patterns and characteristics of marker‐positive CSCs, hampering the development of personalized CSC‐targeted therapy. Here, we show that CSC markers EpCAM and CD90 are independently expressed in liver cancer. In primary HCC, EpCAM+ and CD90+ cells resided distinctively, and gene‐expression analysis of sorted cells suggested that EpCAM+ cells had features of epithelial cells, whereas CD90+ cells had those of vascular endothelial cells. Clinicopathological analysis indicated that the presence of EpCAM+ cells was associated with poorly differentiated morphology and high serum alpha‐fetoprotein (AFP), whereas the presence of CD90+ cells was associated with a high incidence of distant organ metastasis. Serial xenotransplantation of EpCAM+/CD90+ cells from primary HCCs in immune‐deficient mice revealed rapid growth of EpCAM+ cells in the subcutaneous lesion and a highly metastatic capacity of CD90+ cells in the lung. In cell lines, CD90+ cells showed abundant expression of c‐Kit and in vitro chemosensitivity to imatinib mesylate. Furthermore, CD90+ cells enhanced the motility of EpCAM+ cells when cocultured in vitro through the activation of transforming growth factor beta (TGF‐β) signaling, whereas imatinib mesylate suppressed TGFB1 expression in CD90+ cells as well as CD90+ cell‐induced motility of EpCAM+ cells. Conclusion: Our data suggest the discrete nature and potential interaction of EpCAM+ and CD90+ CSCs with specific gene‐expression patterns and chemosensitivity to molecular targeted therapy. The presence of distinct CSCs may determine the clinical outcome of HCC. (HEPATOLOGY 2013)

The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma.

BACKGROUND & AIMS Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. We investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. METHODS Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. RESULTS The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells. CONCLUSIONS SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features.

Oncostatin M Renders Epithelial Cell Adhesion Molecule–Positive Liver Cancer Stem Cells Sensitive to 5-Fluorouracil by Inducing Hepatocytic Differentiation

Recent evidence suggests that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells; CSC). Although normal stem cells and CSCs are considered to share similar self-renewal programs, it remains unclear whether differentiation programs are also maintained in CSCs and effectively used for tumor eradication. In this study, we investigated the effect of oncostatin M (OSM), an interleukin 6-related cytokine known to induce the differentiation of hepatoblasts into hepatocytes, on liver CSCs. OSM receptor expression was detected in the majority of epithelial cell adhesion molecule-positive (EpCAM(+)) HCC with stem/progenitor cell features. OSM treatment resulted in the induction of hepatocytic differentiation of EpCAM(+) HCC cells by inducing signal transducer and activator of transcription 3 activation, as determined by a decrease in stemness-related gene expression, a decrease in EpCAM, alpha-fetoprotein and cytokeratin 19 protein expressions, and an increase in albumin protein expression. OSM-treated EpCAM(+) HCC cells showed enhanced cell proliferation with expansion of the EpCAM-negative non-CSC population. Noticeably, combination of OSM treatment with the chemotherapeutic agent 5-fluorouracil (5-FU), which eradicates EpCAM-negative non-CSCs, dramatically increased the number of apoptotic cells in vitro and suppressed tumor growth in vivo compared with either saline control, OSM, or 5-FU treatment alone. Taken together, our data suggest that OSM could be effectively used for the differentiation and active cell division of dormant EpCAM(+) liver CSCs, and the combination of OSM and conventional chemotherapy with 5-FU efficiently eliminates HCC by targeting both CSCs and non-CSCs.

Gd-EOB-DTPA-enhanced magnetic resonance imaging and alpha-fetoprotein predict prognosis of early-stage hepatocellular carcinoma

The survival of patients with hepatocellular carcinoma (HCC) is often individually different even after surgery for early‐stage tumors. Gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd‐EOB‐DTPA)‐enhanced magnetic resonance imaging (MRI) has been introduced recently to evaluate hepatic lesions with regard to vascularity and the activity of the organic anion transporter OATP1B3. Here we report that Gd‐EOB‐DTPA‐enhanced MRI (EOB‐MRI) in combination with serum alpha‐fetoprotein (AFP) status reflects the stem/maturational status of HCC with distinct biology and prognostic information. Gd‐EOB‐DTPA uptake in the hepatobiliary phase was observed in ∼15% of HCCs. This uptake correlated with low serum AFP levels, maintenance of hepatocyte function with the up‐regulation of OATP1B3 and HNF4A expression, and good prognosis. By contrast, HCC showing reduced Gd‐EOB‐DTPA uptake with high serum AFP levels was associated with poor prognosis and the activation of the oncogene FOXM1. Knockdown of HNF4A in HCC cells showing Gd‐EOB‐DTPA uptake resulted in the increased expression of AFP and FOXM1 and the loss of OATP1B3 expression accompanied by morphological changes, enhanced tumorigenesis, and loss of Gd‐EOB‐DTPA uptake in vivo. HCC classification based on EOB‐MRI and serum AFP levels predicted overall survival in a single‐institution cohort (n = 70), and its prognostic utility was validated independently in a multi‐institution cohort of early‐stage HCCs (n = 109). Conclusion: This noninvasive classification system is molecularly based on the stem/maturation status of HCCs and can be incorporated into current staging practices to improve management algorithms, especially in the early stage of disease. (Hepatology 2014;60:1674–1685)

Defeating EpCAM+ liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma

BACKGROUND & AIMS Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. METHODS Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. RESULTS CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CONCLUSIONS CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.

Chronic hepatitis C infection–induced liver fibrogenesis is associated with M2 macrophage activation

The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV–induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV–induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.

The evolving concept of liver cancer stem cells

Liver cancer is an often fatal malignant tumor with a high recurrence rate and chemoresistance. The major malignant phenotypes of cancer, including recurrence, metastasis, and chemoresistance, are related to the presence of cancer stem cells (CSCs). In the past few decades, CSCs have been identified and characterized in many tumors including liver cancer. Accumulated evidence has revealed many aspects of the biological behavior of liver CSCs and the mechanism of their regulation. Based on these findings, a number of studies have investigated eradication of liver CSCs. This review focuses on the recent advances in our understanding of the biology of liver CSCs and the development of strategies for their treatment.

De Novo Emergence of Mesenchymal Stem-Like CD105+ Cancer Cells by Cytotoxic Agents in Human Hepatocellular Carcinoma

BACKGROUND: Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). Recently, we reported that the CSC markers epithelial cell adhesion molecule (EpCAM) and CD90 are expressed independently in primary HCCs and cell lines, and CD90+ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor-beta. METHODS: The EpCAM+ cell lines HuH1 and HuH7 were treated with 5-fluorouracil (5-FU) or epirubicin in vitro. Gene and protein expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting, respectively. The expression of CD105 in primary HCC was evaluated by immunohistochemistry. The relationship of CD105 expression status and HCC prognosis was evaluated using 85 surgically resected HCC tissues by Kaplan–Meier survival analysis. RESULTS: 5-FU or epirubicin treatment resulted in the generation of CD90+ and CD105+ cells in vitro in HuH1 and HuH7 cells, which originally contain no CD90+ or CD105+ cells. This phenomenon was validated by qRT-PCR analysis with activation of the epithelial-mesenchymal transition (EMT) program regulators Snail family zinc finger 1 (SNAI1) and SNAI2. Immunohistochemical analysis indicated that CD105+ cells were morphologically identical to vascular endothelial cells in untreated primary HCCs. However, surgically resected specimens after transcatheter arterial chemoembolization clearly indicated that CD105+ cancer cells survived at the peripheral edge of the tumor. Kaplan–Meier survival analysis indicated that HCCs expressing CD105 showed poor prognosis after surgery with statistical significance. CONCLUSIONS: Taken together, our data highlight the role of CD105+ HCC cells with activation of the EMT program generated de novo after cytotoxic therapy on the prognosis of HCC patients.

Sorafenib suppresses extrahepatic metastasis de novo in hepatocellular carcinoma through inhibition of mesenchymal cancer stem cells characterized by the expression of CD90

Cancer stem cells (CSCs) are a pivotal target for eradicating hepatocellular carcinoma (HCC). We previously reported that distinctive CSCs regulating tumorigenicity (EpCAM+ CSCs) and metastasis (CD90+ CSCs) have different epithelial/mesenchymal gene expression signatures. Here, we examined the influence of sorafenib, a multiple-receptor tyrosine kinase inhibitor used as a first-line treatment for advanced HCC, on EpCAM+ and CD90+ CSCs. CD90+ cells showed higher c-Kit gene/protein expression than EpCAM+ cells. Sorafenib treatment reduced the number of CD90+ cells with attenuated c-Kit phosphorylation, whereas it enriched the EpCAM+ cell population. We evaluated the role of CD90+ and EpCAM+ CSCs in vivo by subcutaneously injecting these CSCs together in immune-deficient mice. We observed that sorafenib subtly affected the suppression of primary tumor growth maintained by EpCAM+ CSCs, but completely inhibited the lung metastasis mediated by CD90+ CSCs. We further evaluated the effect of sorafenib on extracellular vesicle (EV) production and found that sorafenib suppressed the production of EVs containing TGF-β mRNA in CD90+ cells and inhibited the cell-cell communication and motility of EpCAM+ cells. Our data suggest the following novel effects of sorafenib: suppressing CD90+ CSCs and inhibiting the production of EVs regulating distant metastasis.

Chromodomain-helicase-DNA-binding protein 4: a novel therapeutic target in liver cancer stem cells

First of all, we appreciate the precious comments written by Professors Dr. Ochiya and Dr. Willoughby to our study (1,2).