[Cholesterol Metabolism and Tumor Immunity].

Cholesterol, an important lipid molecule of organisms, is involved in the formation of cell membrane structure, bile acid metabolism and steroid hormone synthesis, playing an important role in the regulation of cell structure and functions. In recent years, a large number of studies have shown that cholesterol metabolism is reprogrammed during tumor formation and development. In addition to directly affecting the biological behavior of tumor cells, cholesterol metabolic reprogramming also regulates the antitumor activity of immune cells in the tumor microenvironment. We reviewed herein the cholesterol metabolism reprogramming of and interactions among immune cells including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), dendritic cells (DCs), and T cells in the tumor microenvironment. However, the relationship between cholesterol metabolism and tumor immunity in tumor microenvironment is complex and diversified. The differences and similarities of cholesterol metabolism reprogramming in tumor microenvironment in regulating immune cell activity and the specific regulatory mechanism are still unresolved issues. Targeted intervention of the cholesterol metabolism pathway of immune cells is expected to become a new strategy of cholesterol metabolism in tumor immunotherapy.

[Phosphorylation of eukaryotic initiation factor 2-alpha inhibits cisplatin-mediated apoptosis of hepatocellular carcinoma cells].

OBJECTIVE: To investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC). METHODS: The human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test. RESULTS: Cisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05). CONCLUSION: Phosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.

The suppressive role of p38 MAPK in cellular vacuole formation.

Vacuolization of the cytoplasm is one of the dramatic and frequently observed phenomena in various cell types. Cellular vacuoles occur spontaneously or via a wide range of inductive stimuli, but the molecular mechanism involved in this process remains largely unknown. In this study, we investigated the role of the p38 and JNK pathways in the formation of cytoplasmic vacuoles. We found that p38 and JNK agonist anisomycin abolishes spontaneous cytoplasmic vacuolization of HepG2 cells through p38 activation, but not through JNK activation. Importantly, blocking the activity of p38 or suppression the expression of p38 elicits cytoplasmic vacuoles formation in various cancer cells. Furthermore, cytoplasmic vacuoles induced by p38 blocking are derived from the perinuclear region. These observations provide direct evidence for a role of p38 signaling in regulating the formation of cytoplasmic vacuoles.

High expression of proline-rich tyrosine kinase 2 is associated with poor survival of hepatocellular carcinoma via regulating phosphatidylinositol 3-kinase/AKT pathway.

BACKGROUND: The peritumoral environment has been implicated to be important in the process of metastasis and recurrence in hepatocellular carcinoma (HCC). Our aims were to assess the prognostic value of proline-rich tyrosine kinase 2 (Pyk2) in HCC and investigate related molecular mechanism. METHODS: Expression of Pyk2 was tested by immunohistochemistry in tissue microarrays containing 141 paired HCC samples. Correlation between Pyk2 and vascular endothelial growth factor (VEGF) expression in clinical samples was analyzed by Spearman rank correlation. Matrigel invasion, anchorage-independent growth assay and immunoblotting were performed to study the effect of Pyk2 on the invasion and progression of HCC cells and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. RESULTS: Higher Pyk2 density in both tumor and peritumor was associated with lower overall survival (P = 0.044; P = 0.041, respectively), serum AFP levels > 1,000 ng/ml (P = 0.013; P = 0.032, respectively) and postoperative distant metastasis (both P < 0.001). However, only higher peritumoral Pyk2 density was related to lower disease-free survival (P = 0.014) and vascular invasion (P = 0.035). A significant correlation between Pyk2 and VEGF density in tumor or peritumoral liver tissue was observed (r = 0. 3133, P = 0.0002; r = 0.5176, P < 0.0001, respectively). Immunoblotting showed that Pyk2 activated PI3K-AKT pathway to upregulate VEGF expression in HL-7702, SMMC-7721 and HepG2 cells. CONCLUSIONS: High Pyk2, especially peritumoral Pyk2 was associated with poor survival, disease recurrence, and metastasis in HCC. PI3K-AKT pathway was involved in Pyk2-mediated VEGF expression during HCC progression and invasion.

c-Met function requires N-linked glycosylation modification of pro-Met.

c-Met, the receptor for hepatocyte growth factor (HGF), is cell surface tyrosine kinase that controls cancer cell growth, survival, invasion, and metastasis. Post-translational modification, such as glycosylation, plays an essential role in regulating the function of cell surface molecules. Whether glycosylation modification regulates the enzymatic properties of c-Met is unknown. In this study, we investigated the effect of glycosylation on the function of c-Met. We found that c-Met is an N-linked glycosylated protein. Both pro-Met and p145Met (the ß subunit of mature c-Met) have N-linked glycosylation. Glycosylation inhibitor studies revealed that the N-glycosylation modification of p145Met is from pro-Met, but not due to the further modification of pro-Met. Importantly, blocking the N-glycosylation targets pro-Met to cytoplasm and initiates its phosphorylation independent of HGF engagement. Nonglycosylated pro-Met activates c-Met downstream pathways to a certain extent to compensate for the degradation of p145Met induced by glycosylation blocking-mediated endoplasmic reticulum (ER) stress.

Profound impact of gut homeostasis on chemically-induced pro-tumorigenic inflammation and hepatocarcinogenesis in rats.

BACKGROUND & AIMS: Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC). METHODS: Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. RESULTS: Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver. CONCLUSIONS: The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.

p28(GANK) prevents degradation of Oct4 and promotes expansion of tumor-initiating cells in hepatocarcinogenesis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), although little is known about their stem cell-like properties. METHODS: We quantified levels of p28(GANK) (Gankyrin), OV6, and Oct4 in 130 human HCC samples using immunohistochemistry. Magnetic-activated cell sorting was used to isolate OV6+ HCC cells. T-IC properties were evaluated by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and spheroid formation. We used a coimmunoprecipitation assay to study interactions among p28(GANK), Oct4, and WWP2. Tumorigenicity and pulmonary metastasis were examined in nonobese diabetic and severe combined immunodeficient mice. RESULTS: In HCC samples, high levels of p28(GANK) correlated with expansion of OV6+ tumor cells; the combination of high levels of p28(GANK) and OV6 was associated with progression of HCC. p28(GANK) was predominantly expressed in liver T-ICs, isolated by magnetic sorting, and undifferentiated primary HCC spheroids. Increased levels of p28(GANK) in T-ICs increased their percentages in HCC samples, expression of stem cell genes, self-renewal potential, chemoresistance in vitro, and tumorigenicity and ability to develop into pulmonary metastases in mice. Conversely, knockdown of p28(GANK) reduced their T-IC properties. p28(GANK) likely activates liver T-ICs by impeding ubiquitination and degradation of the transcription factor Oct4 by WWP2. In support of this concept, levels of p28(GANK) correlated with those of Oct4 in HCC samples. CONCLUSIONS: p28(GANK) activates and maintains liver T-ICs in HCCs by preventing degradation of Oct4. Inhibitors of p28(GANK) might therefore be developed to inactivate T-ICs and slow tumor progression.

The oncoprotein p28GANK establishes a positive feedback loop in ß-catenin signaling.

p28(GANK) (also known as PSMD10 or gankyrin) is a novel oncoprotein that is highly expressed in hepatocellular carcinoma (HCC). Through its interaction with various proteins, p28(GANK) mediates the degradation of the tumor suppressor proteins Rb and p53. Although p53 was reported to downregulate ß-catenin, whether p28(GANK) is involved in the regulation of ß-catenin remains uncertain. Here we report that both growth factors and Ras upregulate p28(GANK) expression through the activation of the phosphoinositide 3-kinase-AKT pathway. Upregulation of p28(GANK) expression subsequently enhanced the transcription activity of ß-catenin. This effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for the p28(GANK)-mediated activation of ß-catenin. p28(GANK) overexpression also reduced E-cadherin protein levels, leading to increased release of free ß-catenin into the cytoplasm from the cadherin-bound pool. Interestingly, exogenous expression of p28(GANK) resulted in elevated expression of the endogenous protein. We also observed that both ß-catenin and c-Myc were transcriptional activators of p28(GANK), and a correlation between p28(GANK) overexpression and c-Myc, cyclin D1 and ß-catenin activation in primary human HCC. Together, these results suggest that p28(GANK) expression is regulated by a positive feedback loop involving ß-catenin, which may play a critical role in tumorigenesis and the progression of HCC.

[The role of mir-221/222 in inhibiting endoplasmic reticulum stress-induced human hepatocarcinoma cell apoptosis].

OBJECTIVE: To investigate the role of miR-221/222 in inhibiting endoplasmic reticulum stress-induced human hepatocarcinoma cells apoptosis. METHOD: miR-221/222 mimics and inhibitors were used to mimic or block the function of endogenous miR-221/222 respectively. Western blot and flow cytometry were used to test the effects of miR-221/222 on cell cycle and apoptosis under endoplasmic reticulum stress in human hepatocellular carcinoma cells. RESULTS: Endoplasmic reticulum stress resulted in miR-221/222 down-regulation in human hepatocellular carcinoma cells. miR-221/222 mimics and inhibitors inhibited and promoted respectively endoplasmic reticulum stress-mediated p27Kip1 induction. Moreover, p27Kip1 suppression not only resulted in reduction in the fraction of G1 phase cells, but also promoted the endoplasmic reticulum stress-mediated apoptosis in human hepatocellular carcinoma cells. CONCLUSION: miR-221/222 were downregulated by endoplasmic reticulum stress in human hepatocellular carcinoma cells, which subsequently protected human hepatocellular carcinoma cells against endoplasmic reticulum stress-induced apoptosis through p27Kip1 regulation.

[Cross-talk between PI3K/Akt and MEK/ERK pathways regulates human hepatocellular carcinoma cell cycle progression under endoplasmic reticulum stress].

OBJECTIVE: To investigate the cross-talk between the PI3K/Akt and MEK/ERK pathways and its role in cell cycle regulation under endoplasmic reticulum stress in human hepatocellular carcinoma cells. METHODS: PI3K inhibitor LY294002 and MEK inhibitor U0126 were used to block the PI3K/Akt and MEK/ERK pathways respectively, and constitutively activated Akt mutant construct was used to activate the PI3K/Akt pathway. Western blot was used to study the potential cross-talk between the PI3K/Akt and MEK/ERK pathways under endoplasmic reticulum stress in human hepatocellular carcinoma cells. the role of the cross-talk between the PI3K/Akt and MEK/ERK pathways in cell cycle regulation was investigated by using propidium iodide staining. RESULTS: LY294002 not only blocked Akt activation efficiently but also increased ERK phosphorylation markedly under endoplasmic reticulum stress in SMMC-7721 and Hep3B cells. Furthermore, myr-Akt inhibited endoplasmic reticulum stress-mediated ERK phosphorylation. In contrast, MEK inhibitor U0126 had no effect on endoplasmic reticulum stress-induced Akt activation. It is notable that both myr-Akt overexpression and MEK inhibitor U0126 inhibited endoplasmic reticulum stress-induced G0/G1 phase arrest in SMMC-7721 cells. CONCLUSION: Endoplasmic reticulum stress-induced Akt activation is mediated through PI3K and the PI3K/Akt pathway inactivation is involved in increased ERK activity in human hepatocellular carcinoma cells. The cross-talk between the PI3K/Akt and MEK/ERK cascades plays an important role in endoplasmic reticulum stress-induced human hepatocellular carcinoma cell cycle arrest.

p28GANK inhibits endoplasmic reticulum stress-induced cell death via enhancement of the endoplasmic reticulum adaptive capacity.

It has been shown that oncoprotein p28(GANK), which is consistently overexpressed in human hepatocellular carcinoma (HCC), plays a critical role in tumorigenesis of HCC. However, the underlying mechanism remains unclear. Here, we demonstrated that p28(GANK) inhibits apoptosis in HCC cells induced by the endoplasmic reticulum (ER) stress. During ER stress, p28(GANK) enhances the unfolded protein response, promotes ER recovery from translational repression, and thereby facilitates cell's ability to cope with the stress conditions. Furthermore, p28(GANK) upregulates glucose-regulated protein 78 (GRP78), a key ER chaperone protein, which subsequently enhances the ER folding capacity and promotes recovery from ER stress. We also demonstrated that p28(GANK) increases p38 mitogen-activated protein kinase and Akt phosphorylation, and inhibits nuclear factor kappa B (NF-kappaB) activation under ER stress, which in turn contributes to GRP78 upregulation. Taken together, our results indicate that p28(GANK) inhibits ER stress-induced apoptosis in HCC cells, at least in part, by enhancing the adaptive response and GRP78 expression. We propose that p28(GANK) has potential implications for HCC progression under the ER stress conditions.