Ju-Bo Zhang

Human Hepatocellular Carcinoma Tumor–derived Endothelial Cells Manifest Increased Angiogenesis Capability and Drug Resistance Compared with Normal Endothelial Cells

Purpose: Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype compared with endothelial cells (NEC) from adjacent normal tissue and may be able to acquire resistance to drugs. The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma (HCC). Experimental Design: TECs or NECs were isolated from HCC or adjacent normal liver tissue using anti-CD105 antibody coupled to magnetic beads. The phenotypic and functional properties of endothelial cells were characterized by testing the expression of CD105, CD31, CD144, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and von Willebrand factor, and the ability of DiI-Ac-LDL-uptake and tube formations. CD105+ TECs were compared with CD105+ NECs and human umbilical vein endothelial cells (HUVEC) by examining their ability to proliferate, motility, ability to adhere to tumor cells, response to tumor conditioned medium, and reactions to the chemotherapy drugs Adriamycin and 5-fluorouracil and the antiangiogenic drug sorafenib. Results: Compared with CD105+ NECs and HUVECs, CD105+ TECs showed increased apoptosis resistance and motility and proangiogenic properties. Meanwhile, CD105+ TECs had a greater ability to adhere to tumor cells and survive in the tumor environment. Moreover, CD105+ TECs acquired more resistance to Adriamycin, 5-fluorouracil, and sorafenib than CD105+ NECs and HUVECs. Conclusions: TECs possessed enhanced angiogenic activity and resistance to chemotherapeutic drugs and an angiogenesis inhibitor, and may provide a better tool for studying tumor angiogenesis and antiangiogenesis drugs in HCC.

Involvement of protein kinase C β–extracellular signal‐regulating kinase1/2/p38 mitogen‐activated protein kinase–heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion

To understand the molecular mechanism that underlies the role of various prominent signal pathways in hepatocellular carcinoma (HCC) metastasis, a human signal transduction oligonucleotide microarray analysis was carried out in cultured HCC cell models with increasing spontaneous metastatic potential (MHCC97L, MHCC97H, and HCCLM6). The results revealed that the mitogen‐activated protein kinase (MAPK) pathway is the prominently upregulated pathway in HCC metastasis. Further study showed that basal phosphorylated levels of extracellular signal‐regulating kinase (ERK)1/2 and p38 MAPK consecutively increased from MHCC97L to MHCC97H to HCCLM6 cells, but not c‐Jun N‐terminal kinase. The phosphorylation of ERK1/2 and p38 MAPK was regulated by upregulated protein kinase Cβ (PKCβ) in HCC cells through the integrated use of PKCβ RNA interference, the PKCβ specific inhibitor enzastaurin and a PKC activator phorbol‐12‐myristate‐13‐acetate. Heat shock protein 27 (HSP27) was also verified as a downstream common activated protein of PKCβ–ERK1/2 and PKCβ–p38 MAPK. In vitro migration and invasion assay further showed that the depletion of PKCβ or inhibition of PKCβ activation effectively decreased HCC cell motility and invasion. Moreover, the motility and invasion of phorbol‐12‐myristate‐13‐acetate‐stimulated PKCβ‐mediated HCC cells was significantly negated by an ERK inhibitor, 1.4‐diamino‐2.3‐dicyano‐1.4‐bis[2‐aminophenylthio] butadiene, or a p38 MAPK inhibitor, 4‐(4‐Fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole. It also showed that HSP27 is critical in PKCβ‐mediated HCC cell motility and invasion. Taken together, this study reveals the important role of this PKCβ‐ERK1/2/p38MAPK‐HSP27 pathway, which was verified for the first time, in modulating HCC cell motility and invasion. (Cancer Sci 2008; 99: 486–496)

Two pathologic types of hepatocellular carcinoma with lymph node metastasis with distinct prognosis on the basis of CK19 expression in tumor

Few studies have investigated the pathologic types and prognosis of hepatocellular carcinoma (HCC) with lymph node metastasis (LNM). The purpose was to explore pathologic types and pertinent therapy of HCC with LNM.

P48 is a predictive marker for outcome of postoperative interferon-α treatment in patients with hepatitis B virus infection-related hepatocellular carcinoma

Postoperative interferon‐α (IFN‐α) therapy improved survival in patients with hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). The identification of predictive markers of outcome will help to select patients who are most likely to benefit from treatment.

Evaluation of midkine as a diagnostic serum biomarker in hepatocellular carcinoma.

Purpose: To evaluate the value of serum midkine (MDK) as a diagnostic biomarker in hepatocellular carcinoma, particularly for those with negative alpha-fetoprotein (AFP) and at an early stage. Experimental Design: MDK expression in tumors was assessed by immunohistochemistry from 105 patients with hepatocellular carcinomas or liver cirrhosis. Serum MDK levels were detected by ELISA in 933 participants including hepatocellular carcinomas and hospital controls from different medical centers. Sensitivities and specificities of serum MDK in diagnosing hepatocellular carcinoma according to AFP level and Barcelona Clinic Liver Cancer (BCLC) stage were analyzed. Results: MDK levels were significantly elevated in hepatocellular carcinoma tissues as well as serum samples. The sensitivity of serum MDK for hepatocellular carcinoma diagnosis was much higher than that of AFP (86.9% vs. 51.9%) with similar specificities (83.9% vs. 86.3%). Notably, serum MDK had an outstanding performance in distinguishing AFP-negative hepatocellular carcinomas from different controls: In those AFP-negative hepatocellular carcinomas, the sensitivity could reach as high as 89.2%. Moreover, receiver operating characteristic (ROC) curve analysis also showed that serum MDK had a better performance compared with AFP in distinguishing early-stage hepatocellular carcinomas as well as small hepatocellular carcinomas. Even in very early-stage hepatocellular carcinomas, MDK showed an obviously higher sensitivity compared with AFP (80% vs. 40%). Furthermore, serum MDK level was significantly decreased in patients with hepatocellular carcinomas after curative resection and re-elevated when tumor relapse occurred. Conclusions: Serum MDK is significantly elevated in most hepatocellular carcinomas, including those with negative AFP and at an early stage, which may serve as a novel diagnostic marker in early diagnosis and postoperative monitoring of hepatocellular carcinomas. Clin Cancer Res; 19(14); 3944–54. ©2013 AACR.

PROX1 promotes hepatocellular carcinoma metastasis by way of up-regulating hypoxia-inducible factor 1α expression and protein stability.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of death from cancer worldwide. HCC has a very poor prognosis because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis. The molecular mechanism of HCC invasiveness and metastasis is poorly understood. The homeobox protein PROX1 is required for hepatocyte migration during mouse embryonic liver development. In this study, we show that high PROX1 protein expression in primary HCC tissues is associated with significantly worse survival and early tumor recurrence in postoperative HCC patients. Knockdown of PROX1 expression in HCC cells inhibited cell migration and invasiveness in vitro and HCC metastasis in nude mice while overexpression of PROX1 in HCC cells promoted these processes. PROX1s pro‐metastasis activity is most likely attributed to its up‐regulation of hypoxia‐inducible factor 1α (HIF‐1α) transcription and stabilization of HIF‐1α protein by recruiting histone deacetylase 1 (HDAC1) to prevent the acetylation of HIF‐1α, which subsequently induces an epithelial‐mesenchymal transition response in HCC cells. We further demonstrated the prognostic value of using the combination of PROX1 and HDAC1 levels to predict postoperative survival and early recurrence of HCC. Conclusion: PROX1 is a critical factor that promotes HCC metastasis. (Hepatology 2013;58:692‐705)

Prognostic significance of serum gamma-glutamyl transferase in patients with intermediate hepatocellular carcinoma treated with transcatheter arterial chemoembolization.

Objectives Not every unresectable hepatocellular carcinoma (HCC) could receive survival benefits from transcatheter arterial chemoembolization (TACE), even for intermediate HCC (Barcelona Clinic Liver Cancer stage B). The aim of this study was to investigate prognostic significance of serum &ggr;-glutamyl transferase (GGT) in patients with intermediate HCC treated with TACE. Methods A total of 277 patients with intermediate HCC were consecutively treated with TACE and overall survival (OS) was evaluated with the Kaplan–Meier method. Significant difference was estimated with the Log rank method according to GGT value before treatment. Univariate and multivariate analyses were used for the study of significance of prognostic factor. Results The median follow-up period was 18.7 months. The 1-year and 3-year OS rates were 71.6 and 38.5% in patients with normal GGT and 48.8 and 16.9% in patients with high GGT (P=0.002). High GGT, correlating with higher tumor size, &agr;-fetoprotein (AFP), and alanine aminotrasferase, was an independent prognostic factor for OS (P=0.009). Others included tumor size and ascites. Furthermore, in small HCC and normal AFP subgroup, serum GGT was also correlated with OS (P=0.013 and 0.041, respectively). The combination of GGT and AFP had a better power to predict the TACE effects. Conclusion GGT level was an important prognostic factor to predict prognosis of patients with intermediate HCC treated with TACE.

Biological characteristics of fluorescent protein-expressing human hepatocellular carcinoma xenograft model in nude mice.

Objectives To study biological characteristics of stable red fluorescent protein (RFP)-expressing or green fluorescent protein (GFP)-expressing HCCLM3 cell lines and those of their relevant xenograft models in nude mice. Methods HCCLM3, a human hepatocellular carcinoma cell line with high metastatic potential was infected with RFP or GFP full-length cDNA via lentivirus. Stable RFP-expressing or GFP-expressing HCCLM3 cells, namely HCCLM3-R and HCCLM3-G, were subcutaneously injected and two patient-like metastatic models of HCCLM3-R and HCCLM3-G in nude mice were established using surgical orthotopic implantation from subcutaneous tumor tissues. Cell proliferation, karyotype, biomarker expression, tumor growth, and metastasis of HCCLM3-R and HCCLM3-G were analyzed in vitro and in vivo. Results RFP and GFP genes were integrated in genomic DNA of HCCLM3. HCCLM3-R and HCCLM3-G expressed red and green fluorescence, stable and intense, 300 days after 60 consecutive passages, and also positively expressed CK8+, P16+, AFP+ and negatively expressed HBsAg−. Their biomarker expression and karyotype were found to be similar to those of the parental HCCLM3, and their tumorigenesis occurred in 10 nude mice without exception after a subcutaneous injection and did the same in 20 nude mice after an orthotopic implantation. The results showed that the rate of spontaneous metastasis to the liver and lung and peritoneal seeding was 100, 100, and 90%, respectively. Conclusion Stable fluorescent protein-expressing HCCLM3-R and HCCLM3-G xenografts in nude mice could be of two useful models for studying mechanisms of hepatocellular carcinoma growth and metastasis in real time.

High expression of macrophage colony-stimulating factor-1 receptor in peritumoral liver tissue is associated with poor outcome in hepatocellular carcinoma after curative resection.

BACKGROUND Macrophage colony-stimulating factor 1 receptor (CSF-1R) expression in hepatocellular carcinoma (HCC) and its prognostic values are unclear. This study evaluated the prognostic values of the intratumoral and peritumoral expression of CSF-1R in HCC patients after curative resection. METHODS Tissue microarrays containing material from cohort 1 (105 patients) and cohort 2 (32 patients) were constructed. Immunohistochemistry was performed and prognostic values of these and other clinicopathological data were evaluated. The CSF-1R mRNA level was assessed by quantitative real-time polymerase chain reaction in cohort 3 (52 patients). RESULTS Both the CSF-1R density and its mRNA level were significantly higher in peritumoral liver tissue than in the corresponding tumor tissue. CSF-1R was distributed in a gradient in the long-distance peritumoral tissue microarray, with its density decreasing as the distance from the tumor margin increased. High peritumoral CSF-1R was significantly associated with more intrahepatic metastases and poorer survival. Peritumoral CSF-1R was an independent prognostic factor for both overall survival and time to recurrence and affected the incidence of early recurrence. However, intratumoral CSF-1R did not correlate with any clinicopathological feature. Peritumoral CSF-1R was also associated with both overall survival and time to recurrence in a subgroup with small HCCs (< or =5 cm). CONCLUSIONS Peritumoral CSF-1R is associated with intrahepatic metastasis, tumor recurrence, and patient survival after hepatectomy, highlighting the critical role of the peritumoral liver milieu in HCC progression. CSF-1R may become a potential therapeutic target for postoperative adjuvant treatment.

GOLM1 Modulates EGFR/RTK Cell-Surface Recycling to Drive Hepatocellular Carcinoma Metastasis

The mechanism of cancer metastasis remains poorly understood. Using gene profiling of hepatocellular carcinoma (HCC) tissues, we have identified GOLM1 as a leading gene relating to HCC metastasis. GOLM1 expression is correlated with early recurrence, metastasis, and poor survival of HCC patients. Both gain- and loss-of-function studies determine that GOLM1 acts as a key oncogene by promoting HCC growth and metastasis. It selectively interacts with epidermal growth factor receptor (EGFR) and serves as a specific cargo adaptor to assist EGFR/RTK anchoring on the trans-Golgi network (TGN) and recycling back to the plasma membrane, leading to prolonged activation of the downstream kinases. These findings reveal the functional role of GOLM1, a Golgi-related protein, in EGFR/RTK recycling and metastatic progression of HCC.