Sirtuin 4 Depletion Promotes Hepatocellular Carcinoma Tumorigenesis Through Regulating Adenosine-Monophosphate-Activated Protein Kinase Alpha/Mammalian Target of Rapamycin Axis in Mice.

Sirtuin 4 (SIRT4) has been reported to play a vital role in the maintenance of glutamine catabolism and adenosine triphosphate (ATP) homeostasis, but its character in hepatocellular carcinomas (HCCs) remains obscure. In this study, we observed low expression of SIRT4 in both HCC cell lines and HCCs from patients. Decreased disease-free survival time is associated with low tumor levels of SIRT4 in patients. Deficiency of SIRT4 facilitated liver tumor development and lung metastasis in xenografts and knockout (KO) mice by promoting colony formation and migration of hepatoma cells and enhancing sphere formation of HCCs. Mechanistically, SIRT4 deletion augmented mammalian target of rapamycin (mTOR) signaling by inactivating adenosine-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through regulation of glutamine catabolism and subsequent AM)/liver kinase B1 (LKB1) axis. Blockage of mTOR by rapamycin or inhibition of glutaminolysis abolished the discrepancy in tumorigenic capacity between SIRT4-depleted hepatoma cells and control cells. Suppression of LKB1 or promotion of AMP by metformin also abrogated the hyperproliferative phenotype caused by SIRT4 loss, which further confirmed that the LKB1/AMPKα/mTOR axis is required in SIRT4-deficiency-promoted HCC tumorigenesis. Conclusion: SIRT4 could exert its tumor suppressive function in HCC by inhibiting glutamine metabolism and thereby increasing the adenosine diphosphate (ADP)/AMP levels to phosphorylate AMPKα by LKB1, which blocks the mTOR signaling pathway.

HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail.

BACKGROUND: Liver cancer is one of the main causes of cancer-related death in human. HOXA7 has been proved to be related with several cancers. METHOD: The expression levels of HOXA7 were examined by Western blot, qRT-PCR or ICH. MTT was used to detect the proliferative rate of liver cancer cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of liver cancer cells were revealed in BALB/c nude mice. RESULTS: In this report, we revealed that HOXA7 promoted metastasis of HCC patients. First, increased levels of HOXA7 were examined in liver cancer especially in metastatic liver cancer. Moreover, higher expression level of HOXA7 was associated with poorer prognosis of liver cancer patients. Overexpression of HOXA7 significantly enhanced proliferation, migration, invasion in vitro and tumor growth and metastasis in vivo meanwhile silencing HOXA7 significantly inhibited the aboves abilities of liver cancer cells. In this research, we identified that HOXA7 performed its oncogenic characteristics through activating Snail. CONCLUSION: Collectively, we identify the critical role and possible mechanism of HOXA7 in metastasis of liver cancer which suggest that HOXA7 may be a potential therapeutic target of liver cancer patients.

[Retracted] Expression of USP22 and Survivin is an indicator of malignant behavior in hepatocellular carcinoma.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that a pair of data panels showing the results of immunohistochemical staining experiments in Fig. 1 on p. 2210 had already appeared in a previously published paper (Zhang Y, Li Y, Lin C, Ding J, Liao G and Tang B: Aberrant upregulation of 14­3­3σ and EZH2 expression serves as an inferior prognostic biomarker for hepatocellular carcinoma. PLos ONE 9: e107251, 2014).  Owing to the fact that the contentious data in the above article had already been published prior to its submission to International Journal of Oncology, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 47: 2208­2216, 2015; DOI: 10.3892/ijo.2015.3214].

Correction: JARID1B promotes metastasis and epithelial-mesenchymal transition via PTEN/AKT signaling in hepatocellular carcinoma cells.

[This corrects the article DOI: 10.18632/oncotarget.3713.].

High expression of ubiquitin carboxyl-terminal hydrolase 22 is associated with poor prognosis in hepatitis B virus-associated liver cancer.

Deubiquitinating enzymes regulate protein activity and cell homeostasis by removing ubiquitin moieties from various substrates. Ubiquitin carboxyl-terminal hydrolase 22 (USP22) is a member of the deubiquitinating protease family and is associated with the development of several tumor types. A previous study demonstrated that USP22 is highly expressed in liver cancer, and its high expression is associated with resistance to chemotherapy. However, the role of USP22 in hepatitis B virus (HBV)-associated liver cancer has not yet been elucidated. The current study demonstrated that USP22 was highly expressed in the tissues of patients with HBV-associated liver cancer, and its high expression was associated with clinicopathological characteristics, including tumor size, clinical stage and prognosis. Further results indicated that USP22 may regulate the proliferative and apoptotic abilities of HepG2.2.15 cells. Additionally, investigation into the underlying mechanism, using small interfering RNA, revealed that the downregulation of USP22 inhibited proliferation and promoted apoptosis though the phosphoinositide 3-kinase/protein kinase B signaling pathway. Therefore, USP22 has the potential to be used as an independent predictor of patient prognosis, as well as a therapeutic target for the treatment of HBV-associated liver cancer.

microRNA-874 suppresses tumor proliferation and metastasis in hepatocellular carcinoma by targeting the DOR/EGFR/ERK pathway.

The δ opioid receptor (DOR) is involved in the regulation of malignant transformation and tumor progression of hepatocellular carcinoma (HCC). However, regulation of the DOR in HCC remains poorly defined. We found that miR-874 was identified as a negative regulator of the DOR, which is a direct and functional target of miR-874 via its 3' untranslated region (UTR). Moreover, miR-874 was downregulated in HCC and its expression was inversely correlated with DOR expression. Downregulation of miR-874 was also associated with larger tumor size, more vascular invasion, a poor TNM stage, poor tumor differentiation, and inferior patient outcomes. Functionally, overexpression of miR-874 in the HCC cell line SK-hep-1 inhibited cell growth, migration, in vitro invasion, and in vivo tumorigenicity. Furthermore, miR-874 overexpression suppressed the DOR, resulting in a downregulated epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation. The EGFR activator-epidermal growth factor (EGF)-can rescue the proliferation and migration suppression induced by miR-874 overexpression, and the rescue effects of the EGF were blocked by an ERK inhibitor. Our study results suggest that miRNA-874 is a negative regulator of the DOR that can suppress tumor proliferation and metastasis in HCC by targeting the DOR/EGFR/ERK pathway, which may be a potential target for HCC treatment.

Poly(γ-glutamic acid)-coated lipoplexes loaded with Doxorubicin for enhancing the antitumor activity against liver tumors.

The study was to develop poly-γ-glutamic acid (γ-PGA)-coated Doxorubicin (Dox) lipoplexes that enhance the antitumor activity against liver tumors. γ-PGA-coated lipoplexes were performed by electrostatistically attracting to the surface of cationic charge liposomes with anionic γ-PGA. With the increasing of γ-PGA concentration, the particle size of γ-PGA-coated Dox lipoplexes slightly increased, the zeta potential from positive shifted to negative, and the entrapment efficiency (EE) were no significant change. The release rate of γ-PGA-coated Dox lipoplexes slightly increased at acidic pH, the accelerated Dox release might be attributed to greater drug delivery to tumor cells, resulting in a higher antitumor activity. Especially, γ-PGA-coated Dox lipoplexes exhibited higher cellular uptake, significant in vitro cytotoxicity in HepG2 cells, and improved in vivo antitumor efficacy toward HepG2 hepatoma-xenografted nude models in comparison with Dox liposomes and free Dox solution. In addition, the analysis results via flow cytometry showed that γ-PGA-coated Dox lipoplexes induce S phase cell cycle arrest and significantly increased apoptosis rate of HepG2 cells. In conclusion, the presence of γ-PGA on the surface of Dox lipoplexes enhanced antitumor effects of liver tumors.

Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle.

Primary liver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death and its incidence is increasing in many countries, becoming a serious threat to human health. Many researches focused on the treatment and prevention of liver cancer. However, due to the underlying molecular mechanism of liver cancer still not fully understood, the studies and development of treatments were forced to be delayed. Akt has been suggested to play an essential role in the progression of inflammation response and apoptosis. Hence, in this study, Akt-knockout mice and cells of liver cancer were used as a model to investigate the molecular mechanism of Akt-associated inflammatory and apoptotic signaling pathway linked with NF-κB and Bcl-2-associated death promoter (Bad) for the progression of liver cancer. Carnosic acid (CA), as a phenolic diterpene with anticancer, antibacterial, antidiabetic, as well as neuroprotective properties, is produced by many species from Lamiaceae family. Administration of CA nanoparticles was sufficient to lead to considerable inhibition of liver cancer progression. The results indicated that, compared to the normal liver cells, the expression of Akt was significantly higher in liver cancer cell lines. Also, we found that Akt-knockout cancer cell lines modulated inflammation response and apoptosis via inhibiting NF-κB activation and inducing apoptotic reaction. Our results indicated that the downstream signals, including cytokines regulated by NF-κB and caspase-3-activated apoptosis affected by Bad, were re-modulated for knockout of Akt. And CA nanoparticles, acting as Akt-knockout, could inhibit inflammation and accelerate apoptosis in liver cancer by altering NF-κB activation and activating caspase-3 through Bad pathway. These findings demonstrated that the nanoparticulate drug CA performed its effective role owing to its ability to reduce inflammatory action and enhance apoptosis for the overexpression of NF-κB and Bad via Akt signaling pathway, playing a direct role in liver cancer progression. Thus, nanoparticle CA might be an important and potential choice for the clinical treatment in the future.

The relationship between the expression of USP22, BMI1, and EZH2 in hepatocellular carcinoma and their impacts on prognosis.

Recent studies have shown that deubiquitination plays a key role in tumor progression, metastasis, resistance to chemotherapy drugs, and prognosis. In this study, we investigated the effects of the deubiquitinating enzyme USP22 on the expression of the drug-resistance genes BMI1 and EZH2 in hepatocellular carcinoma (HCC) and on prognosis. Downregulation of USP22 expression with interference ribonucleic acid in resistant HCC cell lines with high USP22 expression resulted in decreased BMI1 expression, but had no effect on EZH2 expression. USP22, BMI1, and EZH2 were highly expressed in HCC tissue, and the expression levels were positively correlated with tumor grade and clinical stage. Correlation analysis showed that USP22 expression was positively correlated with that of BMI1. Kaplan-Meier analysis showed that high levels of USP22 and BMI1 expression were associated with poor overall survival and relapse-free survival in all of the cases and in patients treated with transcatheter arterial chemoembolization. These results suggested that high levels of USP22 expression played an important role in drug resistance to chemotherapeutic drugs in HCC patients by upregulating the expression of BMI1; therefore, coexpression of USP22 and BMI1 may become a new predictor for HCC prognosis and may help guide clinical treatment.

Overexpression of CTNND1 in hepatocellular carcinoma promotes carcinous characters through activation of Wnt/ß-catenin signaling.

BACKGROUND: Increasing evidence supports the association of CTNND1 with tumor development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in hepatocellular carcinoma (HCC) remains unknown. In this study, we aim to investigate the role of CTNND1 in HCC. METHODS: qRT-PCR and immunohistochemical analyses were used to measure the levels of CTNND1 in HCC specimens and HCC cell lines. CTNND1 and shCTNND1 were transfected into HCC cell lines to investigate its role in HCC. Cell migration and invasion were measured by Transwell and Matrigel analyses in vitro. In vivo metastasis assays were performed in SCID mice. RESULTS: In clinical HCC samples, we found that CTNND1 expression was significantly up-regulated in cancer lesions compared with paired normal liver tissues. By silencing or overexpressing CTNND1 in HCC cells, we found that CTNND1 could promote cell proliferation, migration, and invasion in vitro. An in-vivo assay showed that CTNND1 dramatically promoted HCC cell tumor formation and metastasis. Moreover, CTNND1 promoted HCC metastasis, at least in part, by indirectly enhancing Wnt/ß-catenin signaling. Consistent with these results, the expression of CTNND1 was positively correlated with ß-catenin, WNT11, Cyclin D1, and BMP7 expression in human HCC specimens. CONCLUSIONS: Our study provides evidence that CTNND1 functions as a novel tumor oncogene in HCC, and may be a potential therapeutic target for HCC management.

MicroRNA-155-3p promotes hepatocellular carcinoma formation by suppressing FBXW7 expression.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignant tumors. In the present study, we analyzed the role miR-155-3p plays in Hepatocellular carcinoma (HCC), which has been reported participation in some other types of cancer. METHODS: qRT-PCR was used to measure the levels of miR-155-3p in HCC specimens and HCC cell lines. Overexpression of miR-155-3p and miR-155-3p inhibitor were transfected into HCC cell lines to investigate its role in HCC. Colony formation assay and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays were used to analyses cell proliferation in vitro. In vivo tumor formation assays were performed in BALB/c nude mice. Luciferase reporter assay was carried out to measure the translation of F-Box and WD repeat romain containing 7 (FBXW7). RESULTS: We found that miR-155-3p was remarkably upregulated both in HCC tissue and cell lines. Overexpression of miR-155-3p enhanced HCC cell proliferation in vitro and tumorigenesis in vivo. In addition, overexpression of miR-155-3p is correlated with decreased levels FBXW7 mainly through inhibiting the expression of FBXW7. CONCLUSIONS: Our studies suggest that miR-155-3p plays an important role in the pathogenesis of HCC and implicates its potential applications in the treatment of HCC cancer.

The mechanism underlying alpinetin-mediated alleviation of pancreatitis-associated lung injury through upregulating aquaporin-1.

Characterized by its acute onset, critical condition, poor prognosis, and high mortality rate, severe acute pancreatitis (SAP) can cause multiple organ failure at its early stage, particularly acute lung injury (ALI). The pathogenesis of ALI is diffuse alveolar damage, including an increase in pulmonary microvascular permeability, a decrease in compliance, and invasion of many inflammatory cells. Corticosteroids are the main treatment method for ALI; however, the associated high toxicity and side effects induce pain in patients. Recent studies show that the effective components in many traditional Chinese medicines can effectively inhibit inflammation with few side effects, which can decrease the complications caused by steroid consumption. Based on these observations, the main objective of the current study is to investigate the effect of alpinetin, which is a flavonoid extracted from Alpinia katsumadai Hayata, on treating lung injury induced by SAP and to explore the mechanism underlying the alpinetin-mediated decrease in the extent of ALI. In this study, we have shown through in vitro experiments that a therapeutic dose of alpinetin can promote human pulmonary microvascular endothelial cell proliferation. We have also shown via in vitro and in vivo experiments that alpinetin upregulates aquaporin-1 and, thereby, inhibits tumor necrosis factor-α expression as well as reduces the degree of lung injury. Overall, our study shows that alpinetin alleviates SAP-induced ALI. The likely molecular mechanism includes upregulated aquaporin expression, which inhibits tumor necrosis factor-α and, thus, alleviates SAP-induced ALI.

Aberrant JMJD3 Expression Upregulates Slug to Promote Migration, Invasion, and Stem Cell-Like Behaviors in Hepatocellular Carcinoma.

The Jumonji domain-containing chromatin remodeling factor JMJD3 has important roles in development and cancer. Here, we report a pivotal role for JMJD3 in sustaining the phenotype of aggressive hepatocellular carcinomas. Expression levels of JMJD3 in clinical specimens of hepatocellular carcinoma correlated inversely with patient survival. In hepatocellular carcinoma cells, we found that enforcing its overexpression induced epithelial-mesenchymal transition (EMT), invasive migration, stem cell-like traits, and metastatic properties. Conversely, silencing JMJD3 in hepatocellular carcinoma cells overexpressing it inhibited these aggressive phenotypes. Mechanistically, JMJD3 modulated H3K27me3 in the SLUG gene promoter, a histone mark associated with active SLUG transcription. SLUG silencing blocked JMJD3-induced EMT, stemness, and metastasis. Furthermore, SLUG expression in hepatocellular carcinoma clinical specimens correlated positively with JMJD3 expression. Our results establish JMJD3 as a critical driver of hepatocellular carcinoma stem cell-like and metastatic behaviors, with implications for prognosis and treatment. Cancer Res; 76(22); 6520-32. ©2016 AACR.

MicroRNA-506 suppresses tumor proliferation and metastasis in colon cancer by directly targeting the oncogene EZH2.

Increasing evidence reveals that aberrant expression of microRNA contributes to the development and progression of colon cancer, but the roles of microRNA-506 (miR-506) in colon cancer remain elusive. Here, we demonstrated that miR-506 was down-regulated in colon cancer tissue and cells and that miR-506 expression was inversely correlated with EZH2 expression, tumor size, lymph node invasion, TNM stage and metastasis. A high level of miR-506 identified patients with a favorable prognosis. In vitro and in vivo experiments confirmed that miR-506 inhibits the proliferation and metastasis of colon cancer, and a luciferase reporter assay confirmed that EZH2 is a direct and functional target of miR-506 via the 3'UTR of EZH2. The restoration of EZH2 expression partially reversed the proliferation and invasion of miR-506-overexpressing colon cancer cells. Moreover, we confirmed that the miR-506-EZH2 axis inhibits proliferation and metastasis by activating/suppressing specific downstream tumor-associated genes and the Wnt/ß-catenin signaling pathway. Taking together, our study sheds light on the role of miR-506 as a suppressor for tumor growth and metastasis and raises the intriguing possibility that miR-506 may serve as a new potential marker for monitoring and treating colon cancer.

Inhibition of tribbles protein-1 attenuates radioresistance in human glioma cells.

Radiotherapy is one of the remedies in the treatment of glioma. The radioresistance is a major drawback, of which the mechanism is unclear. Tribble protein and histone deacetylase are involved in the cancer pathogenesis. This study aims to test a hypothesis that the histone deacetylase inhibitors attenuate the radioresistance in human glioma cells. In this study, human glioma cells were cultured. The cells were treated with irradiation with or without a histone deacetylase inhibitor, butyrate. Apoptosis of the glioma cells was assessed by flow cytometry. The results showed that human glioma cells expressed a low level of Trib1, which was significantly up regulated by exposure to small doses (2 Gy/day for 4 days) of irradiation. Trib1-deficient glioma cells showed an enhanced response to irradiation-induced apoptosis. Exposure to small doses of irradiation, Trib1 formed a complex with pHDAC1 (phosphor histone deacetylase-1) to inhibit p53 expression in glioma cells. The presence of HDAC1 inhibitor, butyrate or parthenolide, significantly enforced irradiation-induced glioma cell apoptosis. In conclusion, the Trib1 plays a critical role in the development of radioresistance of glioma cells. The data suggest that inhibition of Trib1 or HDAC1 has the potential to prevent or attenuate the radioresistance.

Expression of USP22 and Survivin is an indicator of malignant behavior in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor type, ranking as the third leading cause of all cancer-related deaths in the world. The post-surgical 5-year survival rate is low, largely due to the high recurrence rate. Therefore, the identification of target molecules that control the biological characteristics of HCC is of great importance. Ubiquitin-specific protease 22 (USP22) is a newly discovered deubiquitinating enzyme and is a cancer stem cell marker that plays a role in tumorigenesis, therapy resistance and cell cycle progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is known to function either as an inhibitor for apoptosis or as a regulator of cell division. Levels of survivin are correlated with the aggressiveness of tumors and a poor prognosis in various cancers including HCC. In the present study, we examined the USP22 expression and its association with survivin expression and clinicopathological features in HCC. First, we examined the expression of USP22 and survivin in 151 HCC cases by immunohistochemistry. High expression of USP22 and survivin was frequently observed in HCC cases, in comparison with normal adjacent liver tissues. Expression of USP22 and survivin was well correlated with malignant behavior including tumor size, stage and differentiation in HCC cases. Importantly, HCC patients with high expression of USP22 and survivin showed poor prognosis. USP22 expression was well correlated with survivin expression in HCC cases. This correlation was confirmed in HCC cell lines and tissues by RT-PCR and western blot analysis. Next, to investigate the biological role of USP22 in HCC, we examined the effect of USP22 knockdown on the cell growth and the expression of cell cycle-related protein including survivin in HCC cells. USP22 siRNA suppressed cell growth. Moreover, USP22 siRNA decreased survivin expression together with upregulation of CDK inhibitor, p21 and downregulation of cyclin B. These findings suggest that USP22 may be involved in HCC progression in cooperation with survivin. We suggest that USP22 can be useful as a new prognostic marker and therapeutic target in HCC patients.

JARID1B promotes metastasis and epithelial-mesenchymal transition via PTEN/AKT signaling in hepatocellular carcinoma cells.

JARID1B is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). JARID1B has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in hepatocellular carcinoma (HCC) remain unknown. Here we show that JARID1B is elevated in HCC and its expression level is positively correlated with metastasis. In addition Kaplan-Meier survival analysis showed that high expression of JARID1B was associated with decreased overall survival of HCC patients. Overexpression of JARID1B in HCC cells increased proliferation, epithelial-mesenchymal transition, migration and invasion in vitro, and enhanced tumorigenic and metastatic capacities in vivo. In contrast, silencing JARID1B in aggressive and invasive HCC cells inhibited these processes. Mechanistically, we found JARID1B exerts its function through modulation of H3K4me3 at the PTEN gene promoter, which was associated with inactive PTEN transcription. PTEN overexpression blocked JARID1B-driven proliferation, EMT, and metastasis. Our results, for the first time, portray a pivotal role of JARID1B in stimulating metastatic behaviors of HCC cells. Targeting JARID1B may thus be a useful strategy to impede HCC cell invasion and metastasis.