Yuan-Fei Peng

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.

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.

Autophagy inhibition suppresses pulmonary metastasis of HCC in mice via impairing anoikis resistance and colonization of HCC cells.

Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma (HCC), which has been linked to cell-death resistance. Autophagy is an important survival mechanism under conditions of cell stress. We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect. Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes. Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition. The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo. Further invasion, migration, detachment, lung colonization, and epithelial-mesenchymal transition (EMT) assays indicated that autophagy inhibition did not affect cell invasiveness, migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells. Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling. As autophagy inhibition was shown to be able to suppress HCC metastasis, an autophagy-based HCC tissue-specific target therapy system (AFP-Cre/LoxP-shRNA) was constructed. In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner. Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis. Together, our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells. Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.

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.

Promoting colonization in metastatic HCC cells by modulation of autophagy.

Background Autophagy is an important adaptive survival mechanism, which has been postulated to be involved in cancer metastasis. The purpose of this study was to investigate autophagy in metastasis of hepatocellular carcinoma (HCC). Methods Immunohistochemical analysis of autophagic activity in metastatic and paired primary HCC tissues using LC3 as autophagosome marker was performed in samples from 216 HCC patients diagnosed with metastasis (including 158 intravascular, 42 intrabiliary, 8 lymph node, 4 bone and 4 lung metastases). Then a mouse model of pulmonary metastasis was established using a highly metastatic HCC cell line (HCCLM3). Autophagy in pulmonary metastases and paired primary tumors were analyzed by LC3 immunohistochemistry, transmission electron microscopy (TEM) and western blot analysis. Further, mouse model of pulmonary metastasis and in vitro cell migration, invasion and detachment models were established using a stable GFP-LC3-expressing HCCLM3 cell line (HCCLM3-GFP-LC3). Autophagic alterations during metastatic colonization, migration, invasion and detachment were determined by GFP-LC3 analysis and western blot analysis. Results LC3 immunohistochemistry of metastases and primary tumors from HCC patients revealed significantly higher LC3 expression in metastases than primary HCC, which suggested a higher level of autophagy in HCC metastases. Further immunohistochemical, TEM, western blot and in vivo GFP-LC3 analyses of lung metastases and primary tumors in mouse model of pulmonary metastasis confirmed that metastatic colonies displayed higher level of autophagy than primary tumors and the early metastatic colonies displayed highest level. The dynamic monitoring of autophagy in cell migration, invasion and detachment showed that autophagy did not significantly alter in those processes. Conclusions Autophagy is activated in metastatic colonization but not in invasion, migration and detachment of HCC cells. Autophagy may play a role in HCC metastasis via promoting metastatic colonization of HCC cells.

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.

High expression of 5-hydroxymethylcytosine and isocitrate dehydrogenase 2 is associated with favorable prognosis after curative resection of hepatocellular carcinoma

BackgroundThe expression of 5-hydroxymethylcytosine (5-hmC) and isocitrate dehydrogenase 2 (IDH2) is frequently downregulated in numerous cancers. 5-hmC and IDH2 expression in hepatocellular carcinoma (HCC) has yet to be determined.MethodsThe immunohistochemical expression of 5-hmC and IDH2 were analyzed in tissue microarrays containing samples from 646 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of 5-hmC and IDH2 were evaluated by Cox regression and Kaplan-Meier analyses.ResultsWe discovered that low 5-hmC and IDH2 expression was associated with malignant behaviors. Low 5-hmC or IDH2 expression alone and combined 5-hmC and IDH2 expression were associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that 5-hmC or IDH2 and 5-hmC/IDH2 were independent prognostic indicators for OS and time to recurrence (TTR), which was confirmed in an independent validation cohort.Conclusions5-hmC and IDH2 correlate with less aggressive tumor behavior in HCC. When 5-hmC and IDH2 are considered together, they serve as a prognostic marker in patients with surgically resected HCCs.

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.

CCL24 contributes to HCC malignancy via RhoB- VEGFA-VEGFR2 angiogenesis pathway and indicates poor prognosis

CCL24 is one chemotactic factor extensively studied in airway inflammation and colorectal cancer but less studied in hepatocellular carcinoma (HCC) retrospectively. So HCC tissue microarray (TMA) was used to estimate relationship between CCL24 and prognosis, cell experiments were conducted to study its influence for HCC cell biological behavior. CCL24 was injected to nude mice to monitor tumor formation and pulmonary metastasis; qRT-PCR, western blot and Immunohistochemistry were used to explore potential mechanism. CCL24 plays roles in target cells via its downstream CCR3, or it is regulated by Type 2 helper T cells (Th2 cell) factors, so immune related experiments were conducted. Meanwhile, Rho GTPase family have close relation not only with T cell priming, but with neovascularization; CCL24 contributes to neovascularization in age-related macular degeneration via CCR3, so Rho GTPase family, Th2 cell factors, Human Umbilical Vein Endothelial Cells were used to uncover their trafficking. Ultimate validation was confirmed by small interfering RNA. Results showed CCL24 expression was higher in caner tissues than adjacent normal tissues, it could contribute to proliferation, migration, and invasion in HCCs, could accelerate pulmonary metastasis, promote HUVECs tube formation. Th2 cell factors were irrelevant with CCL24 in HCCs; and RhoB, VEGFA, and VEGFR2 correlated with CCL24 in both mRNA and protein level. Downstream RhoB-VEGFA signaling pathway was validated by siRhoB and siVEGFA inhibition. In a word, CCL24 contributes to HCC malignancy via RhoB-VEGFA-VEGFR2 angiogenesis pathway and indicates poor prognosis, which urges us to study further CCL24 effects on diagnosis and potential therapy for HCC.

Inhibition of peritoneal dissemination of colon cancer by hyperthermic CO2 insufflation: A novel approach to prevent intraperitoneal tumor spread.

Background The increasing use of laparoscopic surgery for advanced gastrointestinal cancer raises concerns about intra-peritoneal tumor spread. Prevention of peritoneal dissemination is extremely important but a preventive modality is lacking. The aim of this study was to examine a novel approach (hyperthermic CO2 insufflation, HT-CO2) for preventing peritoneal dissemination during laparoscopic surgery. Methods A peritoneal dissemination model was established in Balb/c nu/nu mice by intraperitoneal injection of human colon cancer cells (SW1116, 1×106). The mice (n = 48) were subsequently randomized into two groups and subjected to hyperthermic CO2 (43°C, >95% humidity, HT-CO2 group) or standard normothermic CO2 (21°C, <1% relative humidity, NT-CO2 group) insufflation for 3 hours. The mice were sacrificed 28 days later. The peritoneal dissemination was quantitatively analyzed by counting and weighing the peritoneal nodules. The port sites and ascites volume were measured. The peritoneal damage of HT-CO2 was histologically examined with light microscopy and scanning electron microscopy. Intra-abdominal adhesions were evaluated 4 weeks later. Results The number of peritoneal nodules in the HT-CO2 group was significantly less than that in the NT-CO2 group (10.21±3.72 vs. 67.12±5.49, P<0.01). The mean weight of metastatic tumors in the HT-CO2 group was significantly lower than that in the NT-CO2 group (0.31±0.10g vs. 2.16±0.31g, P<0.01). Massive ascites were found in the NT-CO2 group while significantly less ascites developed in HT-CO2- treated mice (8.26±0.31ml vs. 1.27±0.28ml, P<0.01). No port-site metastases were detected in the HT-CO2 group while the incidence of the NT-CO2 group was 12.5% (3/24). HT-CO2 subjection resulted in slight peritoneal damage; the peritoneum returned to normal within five days. No adhesions formed after HT-CO2 treatment. Conclusions HT-CO2 can suppress peritoneal dissemination of colon cancer cells and only causes slight and transient peritoneal damage. HT-CO2 may serve as a promising adjuvant treatment for preventing peritoneal dissemination in laparoscopic resection of advanced colorectal cancer.