Guo-Ming Shi

Association of Autophagy Defect with a Malignant Phenotype and Poor Prognosis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The role of autophagy and the prognostic value of autophagic genes are largely unknown in HCC. Here, we showed decreased expression of autophagic genes and their corresponding autophagic activity and increased expression of the antiapoptotic gene Bcl-xL in HCC cell lines compared with a normal hepatic cell line. We also found decreased expression of the autophagic gene Beclin 1 in 44 HCC tissue samples compared with adjacent nontumor tissues. In addition, we found that the most aggressive malignant HCC cell lines and HCC tissues with recurrent disease displayed much lower autophagic levels, especially when Bcl-xL was overexpressed. Interestingly, in a tissue microarray study consisting of 300 HCC patients who underwent curative resection, the expression of Beclin 1 was only significantly correlated with disease-free survival (DFS; P < 0.0001) and overall survival (OS; P < 0.0001) in the Bcl-xL(+) group. Multivariate and univariate analyses also revealed that Beclin 1 expression was an independent predictor for DFS and OS in Bcl-xL(+) patients. In addition, we found a significant correlation between Beclin 1 expression and tumor differentiation in Bcl-xL(+) but not in Bcl-xL(-) HCC patients. In conclusion, our data showed expression of autophagic genes and their corresponding autophagic activities were suppressed in HCC. The autophagy defects synergized with altered apoptotic activity might facilitate tumor malignant differentiation, which results in a more aggressive cancer cell phenotype and poor prognosis of HCC.

Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma†

It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c‐Met/hepatocyte growth factor signaling. However, the role of CD151 alone and coexpression of CD151/c‐Met in hepatocellular carcinoma (HCC) remains unclear. We found that expression of CD151 was positively related to metastatic potential of HCC cell lines, and modified cells with CD151high showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo. Furthermore, HCC patients with vascular invasion, large tumors, multiple tumors, high tumor‐node‐metastasis stage, and undifferentiated tumor were prone to have higher CD151 expression. The postoperative 3‐, 5‐, and 7‐year overall survival (OS) of patients in HCCs with CD151high were significantly lower than those in the CD151low group, and correspondingly cumulative recurrence rates in HCCs with CD151high were significantly higher than those in the CD151low group. Both CD151 and c‐Met were remarkably overexpressed in HCCs, compared with adjacent nontumorous and normal liver tissues. Pearson correlation analysis showed a slight correlation between CD151 and c‐Met in HCCs. Importantly, the 5‐ and 7‐year OS rates in CD151high/c‐Methigh patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151low/c‐Metlow patients (63.9% and 54.6%, respectively). Five‐ and 7‐year cumulative recurrence rates in CD151high/c‐Methigh patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151low/c‐Metlow patients (39.0% and 52.5%, respectively). Multivariate analysis revealed that CD151 and combination of CD151/c‐Met were independent prognostic indicators for OS and cumulative recurrence. Conclusion: CD151 is positively associated with invasiveness of HCC, and CD151 or combination of CD151/c‐Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target. (HEPATOLOGY 2008.)

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis.

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.

CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

Tetraspanin CD151 is involved in several pathological activities associated with tumor progression, including neoangiogenesis. However, the role and molecular mechanism of CD151 in the neoangiogenesis of hepatocellular carcinoma (HCC) remain enigmatic. We found that the level of expression of matrix metalloproteinase 9 (MMP9) was positively associated with CD151 expression in HCC cells. We developed a zone‐by‐zone blockade and demonstrated that overexpression of CD151 in HCC cells facilitated MMP9 expression through a phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK‐3β)/Snail signaling pathway. In contrast, down‐regulation of CD151 expression impaired the ability of HCC cells to form microvessels in vitro and reduced their in vivo metastatic potential. In a clinical setting, a significant correlation of the expression of CD151 with MMP9 expression and with microvessel density (MVD) was revealed by Pearson correlation analysis of HCC patients. The postoperative 3‐, 5‐, and 7‐year overall survival rates of HCC patients with CD151high/MMP9high/MVDhigh were significantly lower than those of the CD151low/MMP9low/MVDlow group or groups in which only one or two of CD151, MMP9, and MVD were highly expressed. Cumulative recurrence rates were also highest in HCC patients with CD151high/MMP9high/MVDhigh in comparison with the other groups. Multivariate Cox proportional hazards analysis showed that the concomitant overexpression of CD151, MMP9, and MVD was an independent marker for predicting poor prognosis of HCC. Conclusion: Overexpression of CD151 up‐regulated the expression of MMP9 through the PI3K/Akt/GSK‐3β/Snail pathway. CD151‐dependent neoangiogenesis appeared to promote the progression of HCC, and this suggests that CD151 may be useful as a high‐priority therapeutic target for antiangiogenesis in HCC. HEPATOLOGY 2010

CXCR6 Upregulation Contributes to a Proinflammatory Tumor Microenvironment That Drives Metastasis and Poor Patient Outcomes in Hepatocellular Carcinoma

CXC chemokines and their cognate receptors have been implicated widely in cancer pathogenesis. In this study, we report a critical causal relationship between CXCR6 expression and tumorigenesis in the setting of human hepatocellular carcinoma (HCC). Among the CXC chemokine receptors, only CXCR6 was detected in all the hepatoma cell lines studied. Moreover, in HCC tissue, CXCR6 expression was significantly higher than in noncancerous liver tissues. Reduction of CXCR6 or its ligand CXCL16 in cancer cells reduced cell invasion in vitro and tumor growth, angiogenesis, and metastases in vivo. Importantly, loss of CXCR6 led to reduced Gr-1+ neutrophil infiltration and decreased neoangiogenesis in hepatoma xenografts via inhibition of proinflammatory cytokine production. Clinically, high expression of CXCR6 was an independent predictor of increased recurrence and poor survival in HCCs. Human HCC samples expressing high levels of CXCR6 also contained an increased number of CD66b+ neutrophils and microvessels, and the combination of CXCR6 and neutrophils was a superior predictor of recurrence and survival than either marker used alone. Together, our findings suggest that elevated expression of CXCR6 promotes HCC invasiveness and a protumor inflammatory environment and is associated with poor patient outcome. These results support the concept that inhibition of the CXCR6-CXCL16 pathway may improve prognosis after HCC treatment.

αB-crystallin complexes with 14-3-3ζ to induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma.

The overall survival of patients with hepatocellular carcinoma (HCC) remains poor, and the molecular pathogenesis remains incompletely defined in HCC. Here we report that increased expression of αB‐Crystallin in human HCC predicts poor survival and disease recurrence after surgery. Multivariate analysis identifies αB‐Crystallin expression as an independent predictor for postoperative recurrence and overall survival. We show that elevated expression of αB‐Crystallin promotes HCC progression in vivo and in vitro. We demonstrate that αB‐Crystallin overexpression fosters HCC progression by inducing epithelial‐mesenchymal transition (EMT) in HCC cells through activation of the extracellular‐regulated protein kinase (ERK) cascade, which can counteract the effect of sorafenib. αB‐Crystallin complexes with and elevates 14‐3‐3ζ protein, leading to up‐regulation of ERK1/2 activity. Moreover, overexpression of αB‐Crystallin in HCC cells induces EMT progression through an ERK1/2/Fra‐1/slug signaling pathway. Clinically, our data reveal that overexpression of both αB‐Crystallin and 14‐3‐3ζ correlates with the HCC poorest survival outcomes, and sorafenib response is impaired in patients with αB‐Crystallin overexpression. Conclusion: These data suggest that the αB‐Crystallin‐14‐3‐3ζ complex acts synergistically to promote HCC progression by constitutively activating ERK signaling. This study reveals αB‐Crystallin as a potential therapeutic target for HCC and a biomarker for predicting sorafenib treatment response. (HEPATOLOGY 2013)

Proteasome inhibitor interacts synergistically with autophagy inhibitor to suppress proliferation and induce apoptosis in hepatocellular carcinoma.

The ubiquitin‐proteasome system and autophagy‐lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial.

Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling

Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial–mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment.

Margin-Infiltrating CD20+ B Cells Display an Atypical Memory Phenotype and Correlate with Favorable Prognosis in Hepatocellular Carcinoma

Purpose: The role of infiltrating B cells in hepatocellular carcinoma has been overlooked for many years. This study is aimed to delineate the distribution, prognostic value, and functional status of B cells in human hepatocellular carcinoma. Experimental design: Immunohistochemistry was used to investigate the distribution and clinical significance of infiltrating CD20+ B cells in a series of 120 patients with hepatocellular carcinoma. The results were further tested in an independent series of 200 patients with hepatocellular carcinoma. The functional status of CD20+ B cells was determined by flow cytometry, immunofluorescence, and in vitro coculture assay. Results: Infiltrating CD20+ B cells were predominantly concentrated in the tumor invasive margin, compared with the peri- and intratumor areas. High density of margin-infiltrating B lymphocytes (MIL-B) positively correlated with small tumor size, absence of vascular invasion, and increased density of CD8+ T cells (P < 0.05). Survival analyses revealed that increased number of MIL-Bs and their penetration through the tumor capsule were significantly associated with improved overall and recurrence-free survival, and were identified as independent prognosticators for patients with hepatocellular carcinoma (P < 0.05). Importantly, the results were further validated in another independent hepatocellular carcinoma cohort. Moreover, we found that MIL-Bs featured an atypical memory phenotype (IgD−IgG+CD27−CD38−), expressed surface markers characteristic of antigen-presenting cells, possessed tumor-killing potential by producing IFN-γ, interleukin 12p40 (IL-12p40), granzyme B, and TRAIL, and acted in cooperation with CD8+ T cells. Conclusions: The profile of CD20+ B cells in situ is a new predictor of prognosis for patients with hepatocellular carcinoma and provides a novel target for an optimal immunotherapy against this fatal malignancy. Clin Cancer Res; 19(21); 5994–6005. ©2013 AACR.

Overexpression of CD151 as an adverse marker for intrahepatic cholangiocarcinoma patients

Previous studies have indicated that CD151, a hydrophobic protein, forms a functional complex with the proto‐oncogene that encodes an N‐methyl‐N′‐nitro‐N‐nitroso‐guanidine (MET) protein (c‐Met), and CD151 overexpression reportedly is involved in metastasis/invasion of several tumors. The objective of the current study was to investigate the expression and role of CD151 and/or c‐Met in intrahepatic cholangiocarcinoma (ICC).