Epstein-Barr virus-driven metabolic alterations contribute to the viral lytic reactivation and tumor progression in nasopharyngeal carcinoma.

Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.

Significantly Enhanced Nitrate and Phosphorus Removal by Pyrite/Sawdust Composite-Driven Mixotrophic Denitrification with Boosted Electron Transfer: Comprehensive Evaluation of Water-Gas-Biofilm Phases during a Long-Term Study.

Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent's DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Integrated treatment of suburb diffuse pollution using large-scale multistage constructed wetlands based on novel solid carbon: Nutrients removal and microbial interactions.

In this study, an integrated treatment system was proposed and applied in situ, including detention tank, multistage constructed wetlands (CWs) and wastewater treatment plants (WWTPs), preventing nutrients flowing into Dianchi Lake, in which the treatment performance of multistage CWs were evaluated principally. Results skillfully realized the bypass purification of upstream river at dry reasons, as well as the effective management and treatment of the collected diffuse pollution at rainy reasons. The purified water flowing into water bodies could satisfy the Grade III of environmental quality standards for surface water in China with the average effluent concentrations of COD, NH4+-N, TN and TP decreased to 10 (51.2-72.7%), 0.5 (67.2-83.0%), 1.0 (71.2-79.6%) and 0.15 (72.3-89.4%) mg L-1, respectively. High-throughput sequencing results indicated that the application of poly-3-hydroxybutyrate-cohyroxyvelate-sawdust (PS) blends could enrich norank_f_Anaerolineaceae (7.95%) and Bradyrhizobium (10.2%), which were distinct from the dominant genera of Pleurocapsa (13.0%) in gravel-based CWs. Functional genes and metabolism analysis uncovered that the heterotrophic denitrification was the main pathway of nitrogen removal with the abundance of genes encoding TCA cycle, glycolysis and denitrification process up-regulated. In addition, molecular ecological network (MEN) analysis suggested the denitrification genes were positively correlated with the predominant microbes in PS-based CWs, favorable for denitrifiers to transfer and utilize electron donors during denitrification process. This study proved that the developed PS blends as carbon supplies in CWs and the proposed integrated treatment system are effective methods for watershed management, providing valuable reference to low-pollution wastewater treatment in practical engineering projects.

Pilot-scale two-stage constructed wetlands based on novel solid carbon for rural wastewater treatment in southern China: Enhanced nitrogen removal and mechanism.

Constructed wetlands (CWs) have been proved to be an alternative to the treatment of various wastewater. However, there are few studies focused on the removal performance and mechanisms of pollutants in pilot-scale CWs packed with novel solid carbon. In this study, we investigated the effect of poly-3-hydroxybutyrate-co-3-hydroxyvalerate/polyacetic acid (PHBV/PLA) blends as carbon source on pollutant's transformation, microbial communities and functional genes in pilot-scale aeration-anoxic two-stage CWs for polishing rural runoff in southern China. Results showed a striking improvement of TN removal in CWs with PHBV/PLA blends (64.5%) compared to that in CWs with ceramsite (52.9%). NH4+-N (61.3-64.6%), COD (40.4-53.8%) and TP (43.6-47.1%) were also removed effectively in both two CWs. In addition, the strains of Rhodocyclaceae and Bacteroidetes were the primary denitrifiers on the surface of PHBV/PLA blends. Further, the aerobic stage induced gathering of 16 S and amoA genes and the anoxic zone with PHBV/PLA blends increased the nirS genes, which fundamentally explained the better denitrification performance in CW based on PHBV/PLA blends. Consequently, this study will provide straightforward guidance for the operation of engineering CWs packed with polymers to govern the low-C/N rural wastewater.

HNRNPAB-regulated lncRNA-ELF209 inhibits the malignancy of hepatocellular carcinoma.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

Functional Analysis of Peptidyl-prolyl cis-trans Isomerase from Aspergillus flavus.

Aspergillus flavus, a ubiquitous filamentous fungus found in soil, plants and other substrates has been reported not only as a pathogen for plants, but also a carcinogen producing fungus for human. Peptidyl-Prolyl Isomerase (PPIases) plays an important role in cell process such as protein secretion cell cycle control and RNA processing. However, the function of PPIase has not yet been identified in A. flavus. In this study, the PPIases gene from A. flavus named ppci1 was cloned into expression vector and the protein was expressed in prokaryotic expression system. Activity of recombinant ppci1 protein was particularly inhibited by FK506, CsA and rapamycin. 3D-Homology model of ppci1 has been constructed with the template, based on 59.7% amino acid similarity. The homologous recombination method was used to construct the single ppci1 gene deletion strain Δppci1. We found that, the ppci1 gene plays important roles in A. flavus growth, conidiation, and sclerotia formation, all of which showed reduction in Δppci1 and increased in conidiation compared with the wild-type and complementary strains in A. flavus. Furthermore, aflatoxin and peanut seeds infection assays indicated that ppci1 contributes to virulence of A. flavus. Furthermore, we evaluated the effect of PPIase inhibitors on A. flavus growth, whereby these were used to treat wild-type strains. We found that the growths were inhibited under every inhibitor. All, these results may provide valuable information for designing inhibitors in the controlling infections of A. flavus.

Differential responses of MET activations to MET kinase inhibitor and neutralizing antibody.

BACKGROUND: Aberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive. METHODS: With the goal to investigate the molecular basis of MET amplification (METamp) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring METamp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo. RESULTS: We find that cancer cells driven by METamp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit METamp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors. CONCLUSIONS: Our results demonstrate that METamp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring METamp, a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

Anti-Tumor Effects of Paeoniflorin on Epithelial-To-Mesenchymal Transition in Human Colorectal Cancer Cells.

BACKGROUND Colorectal cancer is one of the leading causes of death in China, and the development of effective drugs is urgently needed. Here, we report on Paeoniflorin (PF), a product isolated from the roots of the peony plant, as a possible candidate because of its anti-tumor effects on epithelial-to-mesenchymal transition (EMT) of PF in human colorectal cancer (CRC). MATERIAL AND METHODS Cell proliferation, wound healing, and Transwell assays were used to analyze the effects of PF on in vitro cell migration and invasion of HCT116 and SW480, 2 colorectal cancer cell lines. The tumor xenograft model was used to verify the anti-metastasis effects of PF in vivo. The RNA and protein levels of epithelia-cadherin (E-cadherin), Vimentin, and histone deacetylase2 (HDAC2) were measured by qPCR and Western blot analysis to explore the mechanism involved. RESULTS Our results showed that PF inhibited colorectal cancer cell migration and invasion and suppressed the metastatic potential of the cancer cells in vivo. Moreover, PF significantly decreased the expression of HDAC2 and Vimentin, while increasing the expression of E-cadherin. CONCLUSIONS These results suggest that PF inhibits colorectal cancer cell migration and invasion ability and reverses the EMT process, through inhibiting the expression of HDAC2, and then affects the expression level of E-cadherin and Vimentin at the cell level. Our results were also verified in the tumor xenograft model. This indicates that PF may be a candidate for colorectal cancer treatment.

MiR-7 inhibits progression of hepatocarcinoma by targeting KLF-4 and promises a novel diagnostic biomarker.

BACKGROUND: MicroRNAs are 22-24 nt non-coding RNAs that bind to the 3' UTR of target mRNAs, thereby inducing mRNA degradation or inhibiting mRNA translation. Due to their implication in the regulation of post-transcriptional processes, the role of miRNAs in hepatocellular carcinoma (HCC) has been extensively studied. However, the function of miR-7 in HCC remains to be demonstrated. METHODS: 50 paired HCC tissues and matched peritumor tissues from patients were collected. The mRNA level of miR-7 was detected by qRT-PCR. The protein level of Kruppel-like factor 4 (KLF-4) was determined by western blot. Cell proliferation and invasive ability were measured using MTT and transwell invasion assay, respectively. RESULTS: We demonstrated that miR-7 was downregulated in 50 HCC tissues and the low expression of miR-7 was significantly correlate with tumour size. Moreover, overexpression of miR-7 significantly inhibited the proliferation and invasion of HCC cells. Over 100 target genes of miR-7 were predicted by Targetscan, and KLF-4 was indicated as the most promising candidate. Luciferase report assay showed that KLF-4 could be silenced by miR-7, so as to restore the impairment of cell proliferation and invasion in HCC cells. CONCLUSIONS: In summary, we revealed a role of miR-7-KLF-4 axis in HCC cells, and the combination of both biomarkers might improve HCC diagnosis.

Neovascularization of hepatocellular carcinoma in a nude mouse orthotopic liver cancer model: a morphological study using X-ray in-line phase-contrast imaging.

BACKGROUND: This study aimed to determine whether synchrotron radiation (SR)-based X-ray in-line phase-contrast imaging (IL-PCI) can be used to investigate the morphological characteristics of tumor neovascularization in a liver xenograft animal model. METHODS: A human hepatocellular carcinoma HCCLM3 xenograft model was established in nude mice. Xenografts were sampled each week for 4 weeks and fixed to analyze tissue characteristics and neovascularization using SR-based X-ray in-line phase contrast computed tomography (IL-XPCT) without any contrast agent. RESULTS: The effect of the energy level and object-to-detector distance on phase-contrast difference was in good agreement with the theory of IL-PCI. Boundaries between the tumor and adjacent normal tissues at week 1 were clearly observed in two-dimensional phase contrast projection imaging. A quantitative contrast difference was observed from weeks 1 to 4. Moreover, 3D image reconstruction of hepatocellular carcinoma (HCC) samples showed blood vessels inside the tumor were abnormal. The smallest blood vessels measured approximately 20 µm in diameter. The tumor vascular density initially increased and then decreased gradually over time. The maximum tumor vascular density was 4.29% at week 2. CONCLUSION: IL-XPCT successfully acquired images of neovascularization in HCC xenografts in nude mice.

Stress Hormone Cortisol Enhances Bcl2 Like-12 Expression to Inhibit p53 in Hepatocellular Carcinoma Cells.

BACKGROUND AND AIMS: The pathogenesis of hepatocellular carcinoma (HC) is unclear. It is suggested that psychological stress associates with the pathogenesis of liver cancer. Bcl2-like protein 12 (Bcl2L12) suppresses p53 protein. This study tests a hypothesis that the major stress hormone, cortisol, inhibits the expression of p53 in HC cells (HCC) via up regulating the expression of Bcl2L12. METHODS: Peripheral blood samples were collected from patients with HC to be analyzed for the levels of cortisol. HCC were cultured to assess the role of cortisol in the regulation of the expression of Bcl2L12 and p53 in HCC. RESULTS: We observed that the serum cortisol levels were higher in HC patients. Expression of Bcl2L12 in HCC was correlated with serum cortisol. Cortisol enhanced the Bcl2L12 expression in HCC. Bcl2L12 binding to the TP53 promoter was correlated with p53 expression in HCC. Cortisol increased the Bcl2L12 expression in HCC to inhibit p53 expression. CONCLUSIONS: Stress hormone cortisol suppresses p53 in HCC via enhancing Bcl2L12 expression in HCC. The results suggest that cortisol may be a therapeutic target for the treatment of HC.

Recovery of Oil with Unsaturated Fatty Acids and Polyphenols from Chaenomelessinensis (Thouin) Koehne: Process Optimization of Pilot-Scale Subcritical Fluid Assisted Extraction.

The potential effects of three modern extraction technologies (cold-pressing, microwaves and subcritical fluids) on the recovery of oil from Chaenomelessinensis (Thouin) Koehne seeds have been evaluated and compared to those of conventional chemical extraction methods (Soxhlet extraction). This oil contains unsaturated fatty acids and polyphenols. Subcritical fluid extraction (SbFE) provided the highest yield-25.79 g oil/100 g dry seeds-of the three methods. Moreover, the fatty acid composition in the oil samples was analysed using gas chromatography-mass spectrometry. This analysis showed that the percentages of monounsaturated (46.61%), and polyunsaturated fatty acids (42.14%), after applying SbFE were higher than those obtained by Soxhlet, cold-pressing or microwave-assisted extraction. In addition, the oil obtained under optimized SbFE conditions (35 min extraction at 35 °C with four extraction cycles), showed significant polyphenol (527.36 mg GAE/kg oil), and flavonoid (15.32 mg RE/kg oil), content, had a good appearance and was of high quality.

A c-Myc/miR-17-5p feedback loop regulates metastasis and invasion of hepatocellular carcinoma.

The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.

Circulating tumor cells are correlated with disease progression and treatment response in an orthotopic hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent hematogenous metastasis. A minimally invasive diagnostic biomarker that can predict disease progression and treatment response would be of extraordinary benefit. Therefore, we have investigated whether the number of circulating tumor cells (CTCs) is correlated with disease progression and treatment response in HCC. Here we report that the number of CTCs, monitored by in vivo flow cytometry (IVFC), is strongly correlated with disease progression and treatment response in a highly metastatic orthotopic nude mouse model of green fluorescent protein (GFP)-labeled HCC. Sorafenib treatment reduces the number of CTCs significantly. The decreased number of CTCs is consistent with low lung metastasis. This study has demonstrated a considerable clinical value of CTCs as a biomarker in predicting disease progression and monitoring therapeutic efficacy in patients with HCC.

Malic enzyme 1 induces epithelial-mesenchymal transition and indicates poor prognosis in hepatocellular carcinoma.

Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.

Establishment of monoclonal HCC cell lines with organ site-specific tropisms.

BACKGROUND: Organ site-specific metastasis is an ominous feature for most poor-prognostic hepatocellular carcinoma (HCC) patients. Cancer cell lines and animal models are indispensable for investigating the molecular mechanisms of organ specific tropism. However, till now, little is known about the drivers in HCC metastatic tropism, and also no effective way has been developed to block the process of tropistic metastasis. METHODS: In this study, we established several monoclonal HCC cell lines from HCCLM3-RFP together with their xenograft models, and then analyzed their metastatic potentials and tropisms using in-vitro and in-vivo assays, and finally elucidated the driving forces of HCC tropistic metastases. RESULTS: Six monoclonal cell lines with different organ site-specific tropism were established successfully. SPARC, VCAM1 and ANGPTL4 were found positively correlated with the potentials of lung metastasis, while ITGA1 had a positive relation to lymph node metastasis of enterocoelia. CONCLUSIONS: By our powerful platforms, HCC metastatic tropisms in clinic could be easily mimicked and recapitulated for exploring the bilateral interactions between tumor and its microenvironment, elucidating the drivers of HCC metastatic tropisms, and testing anti-cancer effects of newly developed agent in pre-clinical stage.

Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK-Src signalling pathways.

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.

Downregulation of ARNT2 promotes tumor growth and predicts poor prognosis in human hepatocellular carcinoma.

BACKGROUND AND AIM: Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is a transcriptional regulator and member of the basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) superfamily. Recently, evidence of that ARNT is involved in carcinogenesis and cancer progression has emerged. The aim of current study was to investigate the role of ARNT2, a homolog of ARNT, in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC). METHODS: Tissue microarray and immunohistochemical staining were used to examine the expression of ARNT2 in 195 HCC tissues. Factors associated with ARNT2 levels were assessed by univariate and multivariate Cox regression analyses. Cell proliferation, migration, and invasion assays were performed by using ARNT2 silencing and overexpressing HCCLM6 cell line. Orthotopic xenograft HCC model was used to elucidate the effects of ARNT2 on HCC progression in vivo. RESULTS: High intratumoral of ARNT2 level was well correlated with longer overall survival (OS) and lower tumor to recurrence (TTR) of HCC patients after resection. Multivariate analysis revealed that intratumoral ARNT2 overexpression was an independent prognostic factor for both OS and TTR. Knockdown of ARNT2 in HCCLM6 cells was significantly enhanced while overexpression of ARNT2 significantly inhibited the ability of cell proliferation, invasion, and migration. In animal studies, downregulation of ARNT2 in HCCLM6 cells promoted, whereas upregulation of ARNT2 in HCCLM6 cells reduced HCCLM6 growth in vivo. CONCLUSIONS: Our data demonstrate that ARNT2 plays an inhibitory role in HCC progression and suggest that ARNT2 may be a potential prognostic predictor and therapeutic target for HCC.

Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin ß1.

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin ß1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin ß1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin ß1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.