Yi-Ming Zhao

Metadherin promotes hepatocellular carcinoma metastasis through induction of Epithelial-Mesenchymal transition

Purpose: To investigate the expression of metadherin (MTDH) for its prognostic value in hepatocellular carcinoma (HCC) and its role in promoting HCC metastasis. Experimental Design: This study employed a tissue microarray containing samples from 323 HCC patients to examine the expression of MTDH and its correlation with other clinicopathologic characteristics. The role of MTDH in the regulation of HCC metastasis was investigated both in vitro and in vivo using short hairpin RNA (shRNA)–mediated downregulation of MTDH in HCC cell lines with various metastatic potentials. Results: The expression of MTDH was markedly higher in HCC tumors than in normal liver tissue. Particularly high MTDH expression was observed in tumors with microvascular invasion, pathologic satellites, poor differentiation, or tumor-node-metastasis stages II to III. Furthermore, the clinical outcome was consistently poorer for the MTDHhigh group than for the MTDHlow group in the 1-, 3-, and 5-year overall survival (OS) rates and in the 1-, 3-, 5-year cumulative recurrence rates. In a nude mice model, the shRNA-mediated downregulation of MTDH resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary and abdominal metastasis. Furthermore, we found that the expression level of MTDH correlated with four epithelial–mesenchymal transition (EMT) markers. Knockdown of MTDH expression in HCC cell lines resulted in downregulation of N-cadherin and snail, upregulation of E-cadherin, and translocation of β-catenin. Conclusions: MTDH may promote HCC metastasis through the induction of EMT process and may be a candidate biomarker for prognosis as well as a target for therapy. Clin Cancer Res; 17(23); 7294–302. ©2011 AACR.

Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and indicates poor prognosis for hepatocellular carcinoma

Heterogeneous ribonucleoprotein (hnRNP) A1 is a member of the A/B subfamily of ubiquitously expressed hnRNPs, which have a wide variety of functions in gene expression and signal transduction. To investigate the biological function and clinical significance of hnRNP A1 in hepatocellular carcinoma (HCC), we measured hnRNP A1 expression in four HCC cell lines and two independent cohorts of HCC patients. We found that hnRNP A1 was overexpressed in the highly metastatic HCC cell lines and in tumor tissues of patients with recurrent HCC. Knockdown of hnRNP A1 in highly metastatic HCC cells caused a significant decrease in cell invasion, while upregulation of hnRNP A1 in poorly metastatic HCC cells led to a significant increase in their invasive capacity. We found that this effect may occur through the regulation of CD44v6 expression by hnRNP A1 in HCC cells. Both quantitative reverse transcription‐polymerase chain reaction (qRT‐RCR) and immunohistochemistry revealed that hnRNP A1 was upregulated in HCC tissues and coincided with overexpression of CD44v6. HCC patients with high hnRNP A1 tended to have higher levels of CD44v6, shorter overall survival (OS) and higher rates of tumor recurrence. Multivariate analyses revealed that hnRNP A1 alone or in combination with CD44v6 were independent prognostic indicators for OS and time to recurrence and have potential as therapeutic targets. In conclusion, overexpression of hnRNP A1 promotes HCC invasion by regulating the level of CD44v6 and indicates a poor prognosis for HCC patients after curative resection.

MicroRNA-26a suppresses epithelial-mesenchymal transition in human hepatocellular carcinoma by repressing enhancer of zeste homolog 2

BackgroundOur previous study reported that microRNA-26a (miR-26a) inhibited tumor progression by inhibiting tumor angiogenesis and intratumoral macrophage infiltration in hepatocellular carcinoma (HCC). The direct roles of miR-26a on tumor cell invasion remain poorly understood. In this study, we aim to explore the mechanism of miR-26a in modulating epithelial-mesenchymal transition (EMT) in HCC.MethodsIn vitro cell morphology and cell migration were compared between the hepatoma cell lines HCCLM3 and HepG2, which were established in the previous study. Overexpression and down-regulation of miR-26a were induced in these cell lines, and Western blot and immunofluorescence assays were used to detect the expression of EMT markers. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Immunohistochemical assays were conducted to study the relationships between miR-26a expression and enhancer of zeste homolog 2 (EZH2) and E-cadherin expression in human HCC samples.ResultsDown-regulation of miR-26a in HCCLM3 and HepG2 cells resulted in an EMT-like cell morphology and high motility in vitro and increased in tumor growth and pulmonary metastasis in vivo. Through down-regulation of EZH2 expression and up-regulation of E-cadherin expression, miR-26a inhibited the EMT process in vitro and in vivo. Luciferase reporter assay showed that miR-26a directly interacted with EZH2 messenger RNA (mRNA). Furthermore, the expression of miR-26a was positively correlated with E-cadherin expression and inversely correlated with EZH2 expression in human HCC tissue.ConclusionsmiR-26a inhibited the EMT process in HCC by down-regulating EZH2 expression.

MiR-146a enhances angiogenic activity of endothelial cells in hepatocellular carcinoma by promoting PDGFRA expression

Endothelial cells (ECs) are critical for angiogenesis, and microRNAs play important roles in this process. We investigated the regulatory role of microRNAs in ECs of hepatocellular carcinoma (HCC) by examining the microRNA expression profile of human umbilical vein endothelial cells (HUVECs) in the absence or presence of human HCC cells, and identified miR-146a as the most highly upregulated microRNA. Furthermore, we revealed that miR-146a promoted the expression of platelet-derived growth factor receptor α (PDGFRA) in HUVECs, and this process was mediated by BRCA1. Overexpression of PDGFRA in the ECs of HCC tissues was associated with microvascular invasion and predicted a poorer prognosis. These results suggest that miR-146a plays a key role in regulating the angiogenic activity of ECs in HCC through miR-146a-BRCA1-PDGFRA pathway. MiR-146a and PDGFRA may emerge as potential anti-angiogenic targets on ECs for HCC therapy.

Macrophage-secreted IL-8 induces epithelial-mesenchymal transition in hepatocellular carcinoma cells by activating the JAK2/STAT3/Snail pathway.

Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.

HNRNPAB Induces Epithelial–Mesenchymal Transition and Promotes Metastasis of Hepatocellular Carcinoma by Transcriptionally Activating SNAIL

Expression of heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) has been reported to be dysregulated in tumors, but its specific contributions to tumor formation and progression are not fully understood. Here, we demonstrate that HNRNPAB is overexpressed in highly metastatic cells and tumor tissues from patients with hepatocellular carcinoma (HCC) with recurrence. We found that HNRNPAB overexpression promoted epithelial-mesenchymal transition (EMT) in a manner associated with HCC metastasis in vitro and in vivo. RNA interference-mediated silencing of the EMT factor SNAIL attenuated HNRNPAB-enhanced cell invasion in vitro and lung metastasis in vivo. Mechanistically, HNRNPAB acted to transactivate SNAIL1 transcription, which in turn inhibited transcription of the pivotal SNAIL target gene E-cadherin. Overexpression of HNRNPAB in HCC samples correlated with higher SNAIL levels, shorter overall survival, and higher tumor recurrence. HNRNPAB overexpression, alone or in combination with SNAIL, was found to be a significant independent risk factor for recurrence and survival after curative resection. In conclusion, our findings define HNRNPAB as an activator of EMT and metastasis in HCC that predicts poor clinical outcomes.

First in-human intraoperative imaging of HCC using the fluorescence goggle system and transarterial delivery of near-infrared fluorescent imaging agent: a pilot study

Surgical resections remain the primary curative interventions for hepatocellular carcinoma (HCC). However, lack of real-time intraoperative image guidance confines surgeons to subjective visual assessment of the surgical bed, leading to poor visualization of small positive nodules and the extension of diffuse HCC. To address this problem, we developed a wearable fluorescence imaging and display system (fluorescence goggle) for intraoperative imaging of HCCs in human patients. In this pilot study, both intravenous (IV) and transarterial hepatic (TAH) delivery of indocyanine green (ICG) were explored to facilitate fluorescence goggle-mediated HCC imaging. The results show that all primary tumors in patients (n = 4) who received TAH delivery of ICG were identified successfully by the fluorescence goggle. In addition, 6 satellite tumors were also detected by the goggle, 5 of which were neither identifiable via preoperative magnetic resonance imaging (MRI) and computed tomography (CT) nor by visual inspection and palpation. In the group (n = 5) that received ICG intravenously, only 2 of 6 tumors visible by preoperative MRI or CT were identified with the fluorescence goggle, demonstrating the limitation of this delivery route for a non-tumor-selective imaging agent. Comparative analysis shows that the HCC-to-liver florescence contrast detected by the goggle was significantly greater in patients that received TAH than IV delivery of ICG (P = 0.013). This pilot study demonstrates the feasibility of using the fluorescence goggle to identify multifocal lesions and small tumor deposits using TAH ICG delivery in HCC patients.

Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK–Src signalling pathways

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over‐expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK–Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock‐down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK–Src signalling pathway. Furthermore, Capn4‐mediated invasion and metastasis of HCC cells required up‐regulation of matrix metalloproteinase‐2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho‐FAK, and over‐expression of both Capn4 and phospho‐FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK–Src signalling pathway and MMP2. Copyright

Validity of plasma macrophage migration inhibitory factor for diagnosis and prognosis of hepatocellular carcinoma.

We performed our study to determine whether plasma macrophage migration inhibitory factor (MIF) levels have diagnostic and prognostic value in hepatocellular carcinoma (HCC) patients. Enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the expression of MIF in plasma and tissues, respectively. Plasma MIF levels were compared to HCC occurrence, clinicopathological features and outcomes. Cutpoints of plasma MIF levels for diagnosis and prognosis were, respectively, determined by receiver operating characteristic analysis and X‐tile in corresponding training cohort, and then were confirmed in the validation cohort. The postoperative plasma MIF levels of HCC patients were detected in an independent cohort (80 HCC patients). As a result, MIF expression in situ was mainly observed in the cytoplasm of HCC cells. Intratumoral MIF expression was positively correlated with plasma MIF levels (r = 0.759, p < 0.001). Compared to serum α‐fetoprotein (AFP), plasma MIF had a higher diagnostic value for discrimination of HCC from controls at 35.3 ng/ml. With determined cutpoints, plasma MIF levels demonstrated a significant association with overall survival (OS) and disease‐free survival (DFS) of HCC patients even in patients with normal serum AFP levels and Tumor Node Metastasis (TNM) stage I. In addition, the plasma MIF levels were identified as an independent factor for OS [hazard ratio (HR) = 1.754; p = 0.012] and DFS (HR = 2.121; p < 0.001). Plasma MIF levels decreased markedly within 30 days after tumor resection (p < 0.001). Therefore, plasma MIF levels have potential as a diagnostic and prognostic factor for HCC.

Platelets promote the adhesion of human hepatoma cells with a highly metastatic potential to extracellular matrix protein: involvement of platelet P-selectin and GP IIb-IIIa.

Abstract.Purpose: To investigate the role and possible mechanisms of platelets in liver cancer metastasis. Methods: The optimum conditions of hepatoma cell adhesion to the extracellular matrix (ECM) were determined. The ability of cells to adhere to the ECM was compared between human hepatoma cell lines with a highly metastatic potential (MHCC97) and human hepatoma cell lines with a lower metastatic potential (SMMC7721). By using adhesion assays and inhibition studies in vitro, the effects of platelets and their specific adhesive molecules were compared via the ability of MHCC97 and SMMC7721 to adhere to ECM protein. Results: The SMMC7721 cell adhesion rate to vitronectin, fibronectin, and fibrinogen, respectively, was significantly lower than that of MHCC97 cells (44.9% vs 73.6%, 47.4% vs 76.4%, and 59.3% vs 80.6%, P <0.05). Both hepatoma cell adhesion to the ECM-bound platelets was unchanged whether the platelets were activated or not. SMMC7721 cell adhesion to the ECM was not affected by platelets, but MHCC97 cell adhesion to the ECM was significantly enhanced by platelets (P <0.01). In addition, this effect was significantly reduced when either P-selectin or GP IIb-IIIa was blocked by monoclonal antibodies (P <0.05, P <0.01). In the inhibition studies, the ability of SMMC7721 to adhere to the ECM-bound activated platelets was also lower than that of MHCC97 (P <0.05). However, when GP IIb was blocked by antibody, the adhesion ability of both cells was similar (P >0.05). Conclusions: Human hepatoma cells with a highly metastatic potential proved to have a highly adhesive ability. MHCC97 cell adhesion to the ECM was significantly enhanced by platelets. The interaction of MHCC97 cells with the ECM-bound activated platelets may be mediated by platelet P-selectin and GP IIb-IIIa.