Ying-Hong Shi

Overexpression of PD-L1 Significantly Associates with Tumor Aggressiveness and Postoperative Recurrence in Human Hepatocellular Carcinoma

Purpose: The aberrant expression of programmed cell death 1 ligands 1 and 2 (PD-Ls) on tumor cells dampens antitumor immunity, resulting in tumor immune evasion. In this study, we investigated the expression of PD-Ls in human hepatocellular carcinoma (HCC) to define their prognostic significance after curative surgery. Experimental Design: Immunohistochemistry was used to investigate PD-Ls expression as well as granzyme B+ cytotoxic and FoxP3+ regulatory T cell infiltration on tissue microarrays containing 240 randomly selected HCC patients who underwent surgery. The results were further verified in an independent cohort of 125 HCC patients. PD-Ls expression on HCC cell lines was detected by Western blot assay. Results: Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-L2 also had a poorer survival, the difference in recurrence was not statistically significant. Multivariate analysis identified tumor expression of PD-L1 as an independent predictor for postoperative recurrence. No correlation was found between PD-Ls expression and granzyme B+ lymphocyte infiltration, whereas a significant positive correlation was detected between PD-Ls expression and FoxP3+ lymphocyte infiltration. In addition, tumor-infiltrating cytotoxic and regulatory T cells were also independent prognosticators for both survival and recurrence. The prognostic value of PD-L1 expression was validated in the independent data set. Conclusion: Our data suggest for the first time that PD-L1 status may be a new predictor of recurrence for HCC patients and provide the rationale for developing a novel therapy of targeting the PD-L1/PD-1 pathway against this fatal malignancy.

High expression levels of putative hepatic stem/progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of hepatocellular carcinoma

Background/aims To investigate the prognostic values of putative hepatic stem/progenitor cell (HSC/HPC) biomarkers in patients with hepatocellular carcinoma (HCC). Methods Fourteen biomarkers related to HSCs/HPCs or tumour angiogenesis were assessed by qRT-PCR and then validated by tissue microarrays (TMAs) in three independent cohorts of patients with HCC undergoing curative resection (n=67, 314 and 73). Results Most of the biomarkers were found to be overexpressed in patients with recurrent HCC by quantitative reverse transcription–PCR (qRT–PCR). The HSC/HPC biomarkers cytokeratin 19, ATP-binding cassette subfamily G member 2 (ABCG2), CD133, Nestin and CD44, and the markers of angiogenesis microvessel density (MVD, determined by CD34 immunostaining), vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) were confirmed as significant predictors for overall survival (OS) and/or relapse-free survival (RFS) in TMA analysis. As compared with the low HSC/HPC profile group, patients with a high HSC/HPC profile who had higher VEGF levels (p=0.012) and MVD (p=0.030) in tumours had significantly lower OS and RFS (p<0.0001). Based on Cox regression, a simplified model including CD133, CD44, Nestin and MVD was constructed and confirmed as an independent predictor for OS (p<0.0001) and RFS (p<0.0001), regardless of α-fetoprotein level, tumour stage and recurrence time (p<0.0001 for all). Conclusion High expression levels of HSC/HPC biomarkers are related to tumour angiogenesis and poor prognosis of HCC. The simplified model based on the HSC/HPC and tumour angiogenesis profile can be used to classify patients with HCC with a high risk of tumour recurrence after surgery.

Autophagy Activation in Hepatocellular Carcinoma Contributes to the Tolerance of Oxaliplatin via Reactive Oxygen Species Modulation

Purpose: Understanding the roles of mammalian autophagy in cancer highlights recent advances in the pharmacologic manipulation of autophagic pathways as a therapeutic strategy for cancer. However, autophagy status and corresponding functions in hepatocellular carcinoma (HCC) after therapeutic stress remain to be clarified. This study was to determine whether the autophagic machinery could be activated after chemotherapy and the contribution of autophagy to tolerance of oxaliplatin in HCC. Experimental Design: Autophagy activation and cell death induced by oxaliplatin were examined in two HCC cell lines as well as in vivo using an HCC model in nude mice. HCC tissue samples with or without locoregional chemotherapy before surgery were also examined by immunohistochemical and electron microscopic analysis. Results: Autophagy was functionally activated in HCC cell lines and xenografts after oxaliplatin treatment. Suppression of autophagy using either pharmacologic inhibitors or RNA interference of essential autophagy gene enhanced cell death induced by oxaliplatin in HCC cells. Generation of reactive oxygen species has an important role in the induction of cell death by oxaliplatin in combination with autophagy inhibitors. Critically, the combination of oxaliplatin with autophagy inhibitor chloroquine resulted in a more pronounced tumor suppression in HCC xenografts. Furthermore, autophagy-specific protein LC3 and autophagic autophagosome formation were induced to a significantly higher level in HCC specimens that had been subjected to locoregional chemotherapy. Conclusions: Autophagy activation under therapy stress contributes to HCC tumor cell survival. Targeting the autophagy pathway is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes in HCC patients. Clin Cancer Res; 17(19); 6229–38. ©2011 AACR.

CD24 Is a Novel Predictor for Poor Prognosis of Hepatocellular Carcinoma after Surgery

Purpose: To investigate the role of CD24 in tumor invasion and prognostic significance in hepatocellular carcinoma (HCC). Experimental Design: CD24 expression was measured in stepwise metastatic HCC cell lines, tumor, peritumoral tissues, and normal liver tissues by quantitative real-time PCR and Western blot. The role of CD24 in HCC was investigated by CD24 depletion using small interfering RNA. Tumor tissue microarrays of 314 HCC patients who underwent resection between 1997 and 2000 were used to detect expression of CD24, β-catenin, and proliferating cell nuclear antigen. Prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Results: CD24 was overexpressed in the highly metastatic HCC cell line and in tumor tissues of patients with recurrent HCC. Depletion of CD24 caused a notable decrease in cell proliferation, migration, and invasiveness in vitro. Univariate and multivariate analyses revealed that CD24 was a significant predictor for overall survival and relapse-free survival. CD24 expression was correlated with poor prognosis independent of α-fetoprotein, tumor-node-metastasis stage, and Edmondson stage. High CD24 expression was significantly associated with cytoplasmic and nuclear accumulation of β-catenin (P = 0.023), high tumor proliferative status (P = 0.018), and diffused intrahepatic recurrence and distant metastasis (P = 0.026). Adjuvant transcatheter arterial chemoembolization after surgery reduced the rate of early recurrence (≤1 year) in CD24+ HCC patients (P = 0.024) but had no significant effect in CD24− patients (P = 0.284). Conclusions: Overexpression of CD24 in HCC was associated with high invasiveness and metastatic potential, high tumor proliferation status, and activation of the Wnt/β-catenin pathway. CD24 may be a novel predictor for poor prognosis of HCC patients after surgery. (Clin Cancer Res 2009;15(17):5518–27)

FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3-CCL2 Signaling

Cancer-associated fibroblasts (CAF) are components of the tumor microenvironment whose contributions to malignant progression are not fully understood. Here, we show that the fibroblast activation protein (FAP) triggers induction of a CAF subset with an inflammatory phenotype directed by STAT3 activation and inflammation-associated expression signature marked by CCL2 upregulation. Enforcing FAP expression in normal fibroblasts was sufficient to endow them with an inflammatory phenotype similar to FAP(+)CAFs. We identified FAP as a persistent activator of fibroblastic STAT3 through a uPAR-dependent FAK-Src-JAK2 signaling pathway. In a murine liver tumor model, we found that FAP(+)CAFs were a major source of CCL2 and that fibroblastic STAT3-CCL2 signaling in this setting promoted tumor growth by enhancing recruitment of myeloid-derived suppressor cells (MDSC). The CCL2 receptor CCR2 was expressed on circulating MDSCs in tumor-bearing subjects and FAP(+)CAF-mediated tumor promotion and MDSC recruitment was abrogated in Ccr2-deficient mice. Clinically, we observed a positive correlation between stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma, a highly aggressive liver cancer with dense desmoplastic stroma, where elevated levels of stromal FAP predicted a poor survival outcome. Taken together, our results showed how FAP-STAT3-CCL2 signaling in CAFs was sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Cancer Res; 76(14); 4124-35. ©2016 AACR.

Sequestosome 1/p62 Protein Is Associated with Autophagic Removal of Excess Hepatic Endoplasmic Reticulum in Mice

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.

Alpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy

Backgroud RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment. Methods Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo. Results The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo. Conclusions An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.

Chemerin has a protective role in hepatocellular carcinoma by inhibiting the expression of IL-6 and GM-CSF and MDSC accumulation

Hepatocellular carcinoma (HCC) is linked to inflammation and immunosuppression. Chemerin is highly expressed in the liver and implicated in the regulation of inflammation. However, the role of chemerin in HCC remains unclear. In this study, we aimed to investigate whether chemerin is able to influence HCC progression by regulating tumor-associated inflammation. Here we demonstrated that chemerin significantly decreased in blood and tumor tissues of HCC patients, and tumor chemerin levels were inversely associated with the prognosis. In an orthotopic mouse model of HCC, Rarres2−/− mice exhibited aggressive tumor growth and lung metastasis, whereas chemerin overexpression greatly inhibited tumor growth. The tumor-inhibitory effect of chemerin was accompanied by a shift in tumor-infiltrating immune cells from myeloid-derived suppressive cells (MDSCs) to interferon-γ+T cells and decreased tumor angiogenesis. Furthermore, we demonstrated that the tumor-inhibitory effect of chemerin was partly dependent on T cells, as chemerin overexpression could inhibit tumor growth, albeit to a lesser extent, in Rag1−/− mice when compared with wild-type controls. Mechanistically, chemerin inhibited nuclear factor-κB activation and the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-6) by tumor cells and tumor-associated endothelial cell, respectively, via its receptors, and consequently, MDSC induction was impaired, leading to restoration of antitumor T-cell response and decreased tumor angiogenesis. Clinically, systemic and tumor levels of chemerin were found to inversely correlate with circulating concentrations of GM-CSF or IL-6 and tumor-infiltrating myeloid cells, respectively, in HCC patients. Moreover, neutralization of GM-CSF and IL-6 abrogated HCC progression and MDSC accumulation in Rarres2−/− mice. In conclusion, our study reveals the tumor-inhibitory effect of chemerin by suppressing inflammatory tumor microenvironment with therapeutic implications for inflammation-associated cancer-like HCC.

Genetic Variations in Plasma Circulating DNA of HBV-Related Hepatocellular Carcinoma Patients Predict Recurrence after Liver Transplantation

Background Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis. Methods Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphism were selected, and their prognostic value was assessed and validated in two independent cohorts of HCC patients (N = 102 and N = 77), using pretransplant plasma circulating DNA. Prognostic significance was assessed by Kaplan-Meier survival estimates and log-rank tests. Multivariate analyses were performed to evaluate prognosis-related factors. Results rs894151 and rs12438080 were significantly associated with recurrence (P = .003 and P = .004, respectively). Multivariate analyses demonstrated that the co-index of the 2 SNPs was an independent prognostic factor for recurrence (P = .040). Similar results were obtained in the third cohort (N = 77). Furthermore, for HCC patients (all the 3 cohorts) exceeding Milan criteria, the co-index was a prognostic factor for recurrence and survival (P<.001 and P = .002, respectively). Conclusions Our study demonstrated first that genetic variations of rs894151 and rs12438080 in pretransplant plasma circulating DNA are promising prognostic markers for tumor recurrence in HCC patients undergoing LT and identify a subgroup of patients who, despite having HCC exceeding Milan criteria, have a low risk of post-transplant recurrence.

Basal Autophagy and Feedback Activation of Akt Are Associated with Resistance to Metformin-Induced Inhibition of Hepatic Tumor Cell Growth

While accumulating evidence has shown that the use of the diabetic drug metformin may be beneficial against various tumors in some epidemiological studies, a few studies failed to show the same beneficial effects. The molecular and cellular mechanisms for these conflicting observations are not clear. In this study, we compared the inhibitory effects of cell growth by metformin on several hepatic tumor cell lines: SMMC-7721, HCC-97L, HCC-LM3 and HepG2. While metformin inhibited cell growth in all these cells, we found that SMMC-7721, HCC-97L and HCC-LM3 cells were more resistant than HepG2 cells. Mechanistically, we found that metformin inhibited mTOR in all these hepatic tumor cells. However, SMMC-7721 cells had higher levels of basal autophagy and mTORC2-mediated feedback activation of Akt than HepG2 cells, which may render SMMC-7721 cells to be more resistant to metformin-induced inhibition of cell growth. Similarly, HCC-97L and HCC-LM3 cells also had higher feedback activation of AKT than HepG2 cells, which may also account for their resistance to metformin-induced inhibition of cell growth. Therefore, the various basal autophagy and mTOR activity in different cancer cells may contribute to the controversial findings on the use of metformin in inhibition of cancers in humans.