HCC Metastasis Research Articles

MicroRNA-20a Suppresses Tumor Proliferation and Metastasis in Hepatocellular Carcinoma by Directly Targeting EZH1.

PurposeHepatocellular carcinoma (HCC), a worldwide leading cause of morbidity and mortality, is the most frequent primary liver tumor. Most HCC patients are diagnosed with advanced liver cancer, resulting in a very low 5-year survival rate. Thus, there is an urgent need for the development of targeted therapies. In this study, we aimed to investigate the effect and mechanism of the miR-20a/EZH1 axis on the proliferation and metastasis of HCC and the inhibitory effect of the EZH1/EZH2 inhibitor UNC1999 on HCC.Materials and MethodsThe expression of miR-20a in human HCC tissues and cell lines was detected using quantitative real-time PCR (qRT-PCR). The expressions of proteins were analyzed with immunohistochemistry and Western blotting. Luciferase assay was used to verify whether miR-20a targets EZH1 or EZH2. The effect of miR-20a on HCC progression was studied in vivo and in vitro. The tumor inhibitory effect of UNC1999 was confirmed in vivo. CCK8 assay, wound healing assay, cell migration and invasion assay were used to evaluate the synergistic effect of UNC1999 with sorafenib. RNA sequencing (RNA-seq) was performed to screen the differentially expressed genes in the Huh7 and SMMC7721 cell lines after UNC1999, sorafenib, and combination treatments.ResultsIn this study, miR-20a showed a lower expression in both HCC tissues and cell lines. MiR-20a inhibited the proliferation and migration of SMMC7721 and Huh7 cells. The results of the luciferase assay and Western blot analysis revealed that miR-20a directly targeted EZH1, a histone methyltransferase. We demonstrated that miR-20a negatively regulated the expression of EZH1 and inhibited the proliferation and metastasis of HCC by reducing H3K27 methylation. We found UNC1999 inhibited tumor cells proliferation and enhanced the inhibitory effect of sorafenib.ConclusionWe demonstrated that miR-20a suppresses the tumor proliferation and metastasis in HCC by directly targeting EZH1. UNC1999 can inhibit tumor proliferation in vivo and increase the sensitivity of hepatoma cell lines to sorafenib.

Retracted: M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring αMβ2 Integrin to Tumor Cells.

Retracted: M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring αMβ2 Integrin to Tumor Cells.

Circular RNA circ_GLIS2 suppresses hepatocellular carcinoma growth and metastasis.

Hepatocellular carcinoma (HCC) is one of the leading causes of tumour-related death. Here, we investigated the molecular mechanism of HCC by studying the function of circ_GLIS2. Human HCC specimens and cell lines were used. Sanger sequencing, actinomycin D and RNase R treatment were performed to validate circular RNA features of circ_GLIS2. qRT-PCR, western blotting, immunostaining, and IHC were employed to examine levels of circ_GLIS2, GLIS2 mRNA, and EMT-related markers. CCK-8, colony formation, flow cytometry, wound healing assay, and transwell assays were performed to evaluate cancer cell proliferation, apoptosis, migration, and invasion. RIP and RNA pull-down assay were used to validate EIF4A3/GLIS2 mRNA interaction. MSP was performed to measure the methylation status of GLIS2 promoter. Nude mouse xenograft model was used to examine tumour growth and metastasis in vivo. Circ_GLIS2 and linear GLIS2 mRNA were reduced in human HCC tissues and cells. Their low levels correlated with a poor survival rate of HCC patients. Overexpression of circ_GLIS2 and GLIS2 suppressed HCC cell proliferation, migration, and invasion but promoted cell apoptosis. GLIS2 promoter region was hypermethylated in HCC cells. EIF4A3 was directly bound with GLIS2 mRNA and promoted circ_GLIS2/GLIS2 expression. Moreover, overexpression of circ_GLIS2 restrained HCC tumour growth and metastasis in vivo. Circ_GLIS2 suppresses HCC growth and metastasis by inhibiting cell proliferation, migration, and invasion, but promoting cell apoptosis. These findings provide molecular insights into the mechanism of HCC and indicate that circ_GLIS2 could serve as a diagnosis marker or therapeutic target for HCC.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Research on Mechanism of miRNA-22 Related with Hepatocellular Carcinoma Metastasis for Regulating Process of Tumor Protein P53

The action of miRNA-22 related with HCC metastasis was analyzed in our study and the mechanism of miRNA-22 related with HCC metastasis was discussed. The HCC hep2 cell was transfected with miRNA-22 mimics and miRNA-22 NC instantaneously followed by analysis of cell migration by Transwell assay, cell viability by MTT and clone formation and cell apoptosis by flow cytometry. The action of miRNA-22 mimics and miRNA-22 on the expression of P53 mRNA in HCC Hep2 cell was detected by RT-PCR. The cell activity in miRNA-22 mimics group was significantly elevated compared with miRNA-22 NC group (P < 0.01). Meanwhile, the apoptotic rate, migrated and invaded capacity of HCC cell was significantly elevated (P < 0.01). The expression level of P53 mRNA was reduced (P < 0.01). In conclusion, overexpression of miRNA-22 could restrain the apoptosis of HCC hep2 cell and down-regulated the expression of P53 so as to prompt cell invasion capacity.

CHD1L augments autophagy-mediated migration of hepatocellular carcinoma through targeting ZKSCAN3

Autophagy is an important biological process in normal cells. However, how it affects tumor progression still remains poorly understood. Herein, we demonstrated that the oncogenic protein Chromodomain-helicase-DNA-binding-protein 1-like gene (CHD1L) might promote HCC cells migration and metastasis through autophagy. CHD1L could bind to the promotor region of Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3), a pivotal autophagy suppressor, and inhibit its transcription. We established inducible CHD1L conditional knockout cell line (CHD1L-iKO cell) and found that the deletion of CHD1L significantly increased ZKSCAN3 expression both at mRNA and protein level. Deletion of CHD1L impaired the autophagic flux and migration of HCC cells, while specifically inhibiting ZKSCAN3 blocked these effects. Further exploration demonstrated that the enhanced tumor cell migration and metastasis induced by CHD1L was mediated through ZKSCAN3-induced autophagic degradation of Paxillin. In summary, we have characterized a previously unknown function of CHD1L in regulating tumor migration via ZKSCAN3-mediated autophagy in HCC. Further inhibition of CHD1L and its downstream autophagy signaling might shed new light on cancer therapeutics.

Compound Kushen Injection intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling

BackgroundHepatocellular carcinoma (HCC) is one of the most extensive and most deadly cancers worldwide. The invasion and metastasis characteristics of HCC dramatically affect the prognosis and survival of HCC patients. Compound Kushen Injection (CKI) is a GMP produced, proverbially applied traditional Chinese medicine formula in China to treat cancer-associated pains, and used as an adjunctive therapy for HCC. Until so far, whether CKI could suppress the metastasis of HCC through regulation of epithelial-mesenchymal transition or metabolic reprogramming is still ambiguous. PurposeIn this study, the anti-metastasis effects of CKI were clarified and its pharmacological mechanisms were systematically explored. MethodsCell invasion and cell adhesion assay were performed in SMMC-7721 cells to assess the anti-metastasis role of CKI, and the histopathological evaluation and biochemical detection were utilized in DEN-induced HCC rats to verify the anti-HCC effect of CKI. Serum and liver samples were analyzed with 1H NMR metabolomics approach to screen the differential metabolites and further target quantification the content of key metabolites. Finally, western blotting and immunofluorescence assay were applied to verify the crucial signaling pathway involved in metabolites. ResultsCKI markedly repressed the invasion and adhesion in SMMC-7721 cells and significantly improved the liver function of DEN-induced HCC rats. CKI significantly regulated the expression of epithelial-mesenchymal transition (EMT) markers (Vimentin and E-cadherin). Metabolomics results showed that CKI regulated the metabolic reprogramming of HCC by inhibiting the key metabolites (citrate and lactate) and enzymes (HK and PK) in glycolysis process. Importantly, we found that c-Myc mediates the inhibitory effect of CKI on glycolysis. We further demonstrated that CKI inhibits c-Myc expression through modulating Wnt/β-catenin pathway in SMMC-7721 cells and DEN-induced HCC rats. Furthermore, through activating Wnt/β-catenin pathway with LiCl, the inhibitory effects of CKI on HCC were diminished. ConclusionTogether, this study reveals that CKI intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling pathway. Our research provides a new understanding of the mechanism of CKI against invasion and metastasis of HCC from the perspective of metabolic reprogramming.

Insufficient Radiofrequency Ablation Promotes Hepatocellular Carcinoma Metastasis Through N6-Methyladenosine mRNA Methylation-Dependent Mechanism.

The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

IL-1β-Induced Elevation of Solute Carrier Family 7 Member 11 Promotes Hepatocellular Carcinoma Metastasis Through Up-regulating Programmed Death Ligand 1 and Colony-Stimulating Factor 1.

Because of a paucity of effective treatment options, metastasis is still a major cause for HCC-associated mortality. The molecular mechanism of inflammation-induced HCC metastasis is open for study. Here, we characterized the function of solute carrier family 7 member 11 (SLC7A11) in inflammation-related HCC metastasis and probed therapy strategies for this subpopulation of patients. Elevated expression of SLC7A11 was positively correlated with poor tumor differentiation, and higher tumor-nodule-metastasis stage, and indicated poor prognosis in human HCC. SLC7A11 increased HIF1α expression through reducing α-ketoglutarate (αKG) level by exporting glutamate. SLC7A11 up-regulated programmed death ligand 1 (PD-L1) and colony-stimulating factor 1 (CSF1) expression through αKG-HIF1α cascade. SLC7A11 overexpression in HCC cells promoted intratumoral tumor-associated macrophage (TAM) and myeloid-derived suppressor cell (MDSC) infiltration through the CSF1/colony-stimulating factor 1 receptor (CSF1R) axis, whereas knockdown of CSF1 attenuated SLC7A11-mediated intratumoral TAM and MDSC infiltration and HCC metastasis. Depletion of either TAMs or MDSCs decreased SLC7A11-mediated HCC metastasis. Furthermore, the combination of CSF1R inhibitor BZL945 and anti-PD-L1 antibody blocked SLC7A11-induced HCC metastasis. In addition, IL-1β up-regulated SLC7A11 expression through the interleukin-1 receptor type 1 (IL-1R1)/extracellular signal-regulated kinase/specificity protein 1 pathway. SLC7A11 knockdown impaired IL-1β-promoted HCC metastasis. Anakinra, an IL-1R1 antagonist, reversed IL-1β-promoted HCC metastasis. In human HCC tissues, SLC7A11 expression was positively associated with HIF1α, PD-L1, and CSF1 expression and intratumoral TAM and MDSC infiltration. IL-1β-induced SLC7A11 overexpression up-regulated PD-L1 and CSF1 through the αKG/HIF1α axis, which promoted TAM and MDSC infiltration. Interruption of this oncogenic loop may provide a promising therapy strategy for the inhibition of SLC7A11-mediated HCC metastasis.

FNDC4 acts as an extracellular factor to promote the invasiveness of hepatocellular carcinoma partly via the PI3K/Akt signalling pathway.

FNDC4 is highly homologous to the exercise‐associated myokine FNDC5/irisin, which is highly expressed and promotes the invasion and metastasis of HCC cells. However, the function of FNDC4 remains unknown. Here, we report that FNDC4, an extracellular factor, plays important roles in the invasion and metastasis of HCC. We found that high FNDC4 expression is associated with poor survival in HCC patients and FNDC4 promotes the migration and invasion of HCC cells. Mechanistically, we found that FNDC4 is related to the PI3K/Akt signalling pathway to a certain extent. Specifically, the extracellular domain of FNDC4 acts as an extracellular factor to promote Akt phosphorylation levels in this pathway. These findings reveal that FNDC4 promotes the invasion and metastasis of HCC partly via the PI3K/Akt signalling pathway.

SOX2 and Bcl-2 as a Novel Prognostic Value in Hepatocellular Carcinoma Progression.

Sex-determining region Y-box 2 (SOX2) is a stem cell transcription factor and a major regulator of self-renewal and pluripotency of cancer stem cells (CSCs). In many types of cancer, SOX2 is dysregulated due to overexpression associated with tumor progression and low survival rate. Many HCC cases encounter recurrence and metastasis which might be due to CSCs and also apoptosis. Since little is known about the expression pattern of SOX2 and apoptotic genes in HCC, we aimed to determine the prognostic significance of SOX2, Bax, and Bcl-2 in clinicopathological features, tumor progression, and survival rate of the HCC patients. The expression of SOX2, Bax, and Bcl-2 were evaluated using qRT-PCR in 53 formalin-fixed, paraffin-embedded tissues (FFPE) of patients and 44 controls. Correlation of these genes was analyzed with clinicopathological features and tumor progression. The correlationship between SOX2 expression and ALBI grade as prognostic indicators were calculated. Survival rates were determined by Kaplan–Meier survival curves. SOX2 and Bcl-2 were remarkably overexpressed in HCC patients compared to controls (p = 0.04 and p = 0.003, respectively). A significant association was found for both SOX2 and Bcl-2 overexpression with TNM staging (p = 0.02, p = 0.04) and tumor grading (p = 0.01, p = 0.003), respectively. A significant correlation was observed: patients with SOX2 overexpression had a lower 5-year overall survival rate (p = 0.04); however, there was no significant association between Bcl-2 and survival (p = 0.5). Collectively, overexpression of SOX2 and Bcl-2, alone or combined, may be a potential marker to evaluate prognosis and response to HCC treatment.

Implication of human endogenous retrovirus W family envelope in hepatocellular carcinoma promotes MEK/ERK-mediated metastatic invasiveness and doxorubicin resistance

Human endogenous retrovirus (HERVs), originating from exogenous retroviral infections of germ cells millions of years ago, have the potential for human diseases. Syncytin-1, an envelope protein encoded by the HERV W family, participates in the contexts of schizophrenia, multiple sclerosis, diabetes, and several types of cancers. Nevertheless, there is no report on the expression pattern and potential mechanism of Syncytin-1 in HCC. Here we found Syncytin-1 expression was up-regulated in HCC compared to adjacent non-tumorous tissues, especially in advanced HCC. Syncytin-1 was an independent risk factor to predict vascular invasion, metastasis, larger tumor size, and poor prognosis in HCC patients. Further analysis discovered that Syncytin-1 overexpression positively associated with HCC patients with serum HBsAg positive. Functional experiments in vitro and in vivo demonstrated that Syncytin-1 enhanced cell proliferation, metastasis, and tumorigenicity in HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated protein kinase (ERK) pathway was involved in HCC. Our clinical data indicated that the levels of phosphorylation MEK1/2 and ERK1/2 were increased in HCC comparing with adjacent non-tumorous tissues. It showed the linear correlation between Syncytin-1 expression and upregulated MEK1/2 and ERK1/2 phosphorylation levels in HCC. Furthermore, Syncytin-1 activated MEK/ERK pathway in HCC cells. In-depth research showed that the inflammation-activated MEK/ERK pathway was essential in Syncytin-1 promoted hepatocarcinogenesis. Syncytin-1 suppressed doxorubicin-induced apoptosis via MEK/ERK cascade. In conclusion, Syncytin-1 promoted HCC progression and doxorubicin resistance via the inflammation-activated MEK/ERK pathway. Our findings revealed that Syncytin-1 was a potential prognostic biomarker and therapeutic target for HCC.

Myofibroblast-Specific Msi2 Knockout Inhibits HCC Progression in a Mouse Model.

Myofibroblasts play a pivotal role in the development and progression of HCC. Here, we aimed to explore the role and mechanism of myofibroblast Musashi RNA binding protein 2 (MSI2) in HCC progression. Myofibroblast infiltration and collagen deposition were detected and assessed in the tissues from 117 patients with HCC. Transgenic mice (Msi2ΔCol1a1 ) with floxed Msi2 allele and collagen type I alpha 1 chain (Col1a1)-ligand inducible Cre recombinases (CreER) were constructed to generate a myofibroblast-specific Msi2 knockout model. Mouse HCC cells were orthotopically transplanted into the Msi2ΔCol1a1 or the control mice (Msi2F/F ). We found that the deposition of collagen fibers, the main product of myofibroblasts, predicted a poor prognosis for HCC; meanwhile, we detected high MSI2 expression in the peritumoral infiltrated myofibroblasts. Conditional deletion of Msi2 in myofibroblasts significantly inhibited the growth of orthotopically implanted HCC, reduced both intrahepatic and lung metastasis, and prolonged the overall survival of tumor-bearing mice (P=0.002). In vitro analysis demonstrated that myofibroblasts promoted cell proliferation, invasion, and epithelial-mesenchymal transformation of HCC cells, whereas Msi2 deletion in myofibroblasts reversed these effects. Mechanically, Msi2 knockout decreased myofibroblast-derived IL-6 and IL-11 secretion by inhibiting the extracellular signal-regulated kinase 1/2 pathway, and thus attenuated the cancer stem cell-promoting effect of myofibroblasts. Interestingly, we found that the simultaneous knockout of Msi2 in myofibroblasts and knockdown of Msi2 in HCC cells could not further attenuate the implanted HCC progression. Myofibroblast-specific Msi2 knockout abrogated the tumor-promoting function of myofibroblasts and inhibited HCC progression in mouse models. Targeting myofibroblast MSI2 expression may therefore prove to be a therapeutic strategy for HCC treatment in the future.

Abstract 2457: NANOG bindsPRC2 to reprogram metabolismandpromotelabel-retaining populationin experimental mouse and clinical carcinoma

Abstract Background: Alcohol and HCV synergistically promote hepatocarcinogenesis. Our previous studies indicated that this synergistic effect of HCV and alcohol was mediated by TLR4, which increases the expression of NANOG, a transcription factor important for the stemness of TICs. Our recent studies further indicated that NANOG could repress mitochondrial oxidative phosphorylation (OXPHOS) genes to support the self-renewal and drug resistance of tumor-initiating stem cell-like cells (TICs), leading to relapse and metastasis (Chen et al., Cell Metabolism). In addition, approximately 39% of HCV-alcohol-associated HCCs have mutations in ARID1A, a component of the chromatin remodeling complex SWI/SNF. Hypothesis: The efficacy of the combined FAOi and PRC2 inhibition (PRC2i) therapy relies on activation of cell death signaling. Aims: We (1) tested if NANOG together with the PRC2 complex inhibits OXPHOS and generates TICs in the presence of ARID1A mutations; (2) tested if specific genetic alterations and NANOG cooperate to generate different classes of TICs for the development of HCC; (3) tested if ARID1A mutations with NANOG induction generate TICs. Methods: We transduced LPCs with a lentiviral vector that expresses mCherry and used the CRISPR/Cas9 system to target Arid1a or Ctnnb1. EZH2, SUZ12 and EED were silenced in patient-derived TICs (with or without ARID1A mutations). Surviving cells are subjected to the spheroid formation assay on ultra-low attachment plates for analysis of their self-renewal ability. Results: Mutant Arid1a HCCs with Nanog overexpression caused HCC metastasis to the lung. The humanized FRG mice subjected to alcohol feeding and/or HCV infection developed HCCs, especially in the CTNNB1 or ARID1A mutant hepatoblast groups, six months post-HCV infection and/or alcohol feeding. HCV-infected humanized livers in these mice contained HCV RNA, but not in the UV-irradiated HCV infected group. Conclusion: ARID1A gene loss promotes sorafenib-resistance phenotype and self-renewal through PRC2 complex genes. We demonstrated that sorafenib induced cytochrome c release from mitochondria to cause apoptosis of TICs, that restoration of OXPHOS was overexpressed in TICs. The studies established a novel therapy to overcome the chemoresistance of TICs for HCC treatment. Citation Format: Da-Wei Yeh, Juan Carlos Hernandez, Keigo Machida. NANOG bindsPRC2 to reprogram metabolismandpromotelabel-retaining populationin experimental mouse and clinical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2457.

Tumor-Associated Neutrophils in Hepatocellular Carcinoma Pathogenesis, Prognosis, and Therapy.

Simple SummaryHepatocellular carcinoma is the most prevalent primary liver cancer, accounting for >80% of primary liver cancers worldwide. Inflammation has come to light as a hallmark of cancer development, and it has become increasingly apparent over the past decade that tumor-associated inflammation drives the involvement of neutrophils in disease progression and metastasis. Infiltrating TANs exhibit either anti-tumorigenic (N1) or protumorigenic (N2) phenotypes. In the vast majority of solid human tumors, high infiltration with tumor-associated neutrophils (TANs) has been correlated with increased tumor growth, lymph node metastasis, and poor prognosis overall, whereas evidence from other studies advocate that under certain conditions, TANs exert cytotoxic and inhibitory activity towards tumor cells, halting the progression of cancer. In this review, we summarize current evidence on the role of neutrophils in the pathogenesis and progression of HCC and we highlight their potential utilization in HCC prognosis and therapy.Hepatocellular carcinoma represents the most prevalent primary liver cancer worldwide, and it is either caused by intrinsic genetic mutations or by a multitude of extrinsic risk factors. Even though the interplay between chronic inflammatory changes and hepatocarcinogenesis has been at the forefront of clinical investigation for the past few decades, the role of tumor-associated neutrophils (TANs) in HCC development still remains ambiguous. On the one hand, N1 TANs exhibit an anti-tumorigenic activity, mediated by direct or indirect tumor cell lysis, whereas on the other hand, N2 TANs have been correlated with increased HCC growth, invasiveness, and metastasis. The association of an elevated Neutrophil-to-Lymphocyte Ratio (NLR) with poor prognosis in patients with HCC, has been recently brought into spotlight, consolidating its widespread use as a reliable biomarker. Due to the decisive involvement of TANs in HCC pathogenesis and development, the utilization of various neutrophil-centered anticancer treatment modalities has been under clinical experimentation, selectively targeting and modulating the processes of neutrophil recruitment, activation, and migration. This review summarizes current evidence on the role of TANs in HCC pathogenesis and progression, as well as in their potential involvement in tumor therapy, shedding light on emerging anticancer treatment methods targeting neutrophils.

Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of β-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

Compound Phyllanthus urinaria L. (CP) is a traditional Chinese medicine (TCM) formula for cancer treatment in the clinic, particularly during progression of hepatitis B-associated hepatocellular carcinoma (HBV-associated HCC). Nevertheless, its anti-metastatic action and mechanisms are not well elucidated. In this study, CP was found to exert remarkable inhibitory effects on the proliferation, migration and invasion of HBV-associated HCC cells. The following network and biological analyses predicted that CP mainly targeted Caveolin-1 (Cav-1) to induce anti-metastatic effects, and Wnt/β-catenin pathway was one of the core mechanisms of CP action against HBV-associated HCC. Further experimental validation implied that Cav-1 overexpression promoted metastasis of HBV-associated HCC by stabilizing β-catenin, while CP administration induced autophagic degradation of Cav-1, activated the Akt/GSK3β-mediated proteasome degradation of β-catenin via ubiquitination activation, and subsequently attenuated the metastasis-promoting effect of Cav-1. In addition, the anti-cancer and anti-metastatic action of CP was further confirmed by in vivo and ex vivo experiments. It was found that CP inhibited the tumor growth and metastasis of HBV-associated HCC in both mice liver cancer xenograft and zebrafish xenotransplantation models. Taken together, our study not only highlights the novel function of CP formula in suppressing metastasis of HBV-associated HCC, but it also addresses the critical role of Cav-1 in mediating Akt/GSK3β/β-catenin axis to control the late-phase of cancer progression.

RETRACTED ARTICLE: Exosomal DLX6-AS1 from hepatocellular carcinoma cells induces M2 macrophage polarization to promote migration and invasion in hepatocellular carcinoma through microRNA-15a-5p/CXCL17 axis

BackgroundHepatocellular carcinoma (HCC) cells-secreted exosomes (exo) could stimulate M2 macrophage polarization and promote HCC progression, but the related mechanism of long non-coding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) with HCC-exo-mediated M2 macrophage polarization is largely ambiguous. Thereafter, this research was started to unearth the role of DLX6-AS1 in HCC-exo in HCC through M2 macrophage polarization and microRNA (miR)-15a-5p/C-X-C motif chemokine ligand 17 (CXCL17) axis.MethodsDLX6-AS1, miR-15a-5p and CXCL17 expression in HCC tissues and cells were tested. Exosomes were isolated from HCC cells with overexpressed DLX6-AS1 and co-cultured with M2 macrophages. MiR-15a-5p/CXCL17 down-regulation assays were performed in macrophages. The treated M2 macrophages were co-cultured with HCC cells, after which cell migration, invasion and epithelial mesenchymal transition were examined. The targeting relationships between DLX6-AS1 and miR-15a-5p, and between miR-15a-5p and CXCL17 were explored. In vivo experiment was conducted to detect the effect of exosomal DLX6-AS1-induced M2 macrophage polarization on HCC metastasis.ResultsPromoted DLX6-AS1 and CXCL17 and reduced miR-15a-5p exhibited in HCC. HCC-exo induced M2 macrophage polarization to accelerate migration, invasion and epithelial mesenchymal transition in HCC, which was further enhanced by up-regulated DLX6-AS1 but impaired by silenced DLX6-AS1. Inhibition of miR-15a-5p promoted M2 macrophage polarization to stimulate the invasion and metastasis of HCC while that of CXCL17 had the opposite effects. DLX6-AS1 mediated miR-15a-5p to target CXCL17. DLX6-AS1 from HCC-exo promoted metastasis in the lung by inducing M2 macrophage polarization in vivo.ConclusionDLX6-AS1 from HCC-exo regulates CXCL17 by competitively binding to miR-15a-5p to induce M2 macrophage polarization, thus promoting HCC migration, invasion and EMT.

Mechanistic Investigation on the Regulation of FABP1 by the IL-6/miR-603 Signaling in the Pathogenesis of Hepatocellular Carcinoma.

Background Abnormal lipid metabolism is closely associated with the invasiveness and metastasis of cancer. Fatty acid-binding proteins (FABPs) play essential roles in lipid metabolism, and miRNAs can affect lipid metabolism by targeting FABPs. However, the exact mechanism is unknown. Methods FABP1 expression in HCC tissues was analyzed by immunochemistry with tissue microarrays. The lipid content was detected by Oil Red O staining, and the interaction between FABP1 and free fatty acid (FFA) was studied by a labeling and tracking method. miRNA arrays were used to detect the expression of miRNAs in IL-6-stimulated HCC cells. miR-603 expression was verified by qPCR. The proteins were checked by Western blot analysis. Gain and loss function evaluation was assessed by lentivirus and miRNA mimic transfection in Huh-7 cells, while reactive oxygen species (ROS) were detected by fluorescence. Results FABP1 expression was significantly decreased in approximately 90% (81/90) of HCC patients. FABP1 expression in adjacent tissues was closely associated with overall survival. Meanwhile, lipid was abundant in the adjacent tissues, yet significantly reduced in HCC tissues. FABP1 and FFA can promote each other for being uptaken by Huh-7 cells. FABP1 overexpression induced apoptosis and inhibited the proliferation, migration, invasion, and metastasis of Huh-7 cells. IL-6 treatment affected the expression of miRNAs, and miR-603 was overexpressed in HCC tissues. Also, miR-603 overexpression promoted the proliferation, migration, invasion, and metastasis of Huh-7 cells. Bioinformatic analysis predicted that miR-603 targets the 3′-UTR region of FABP1. However, miR-603 overexpression inhibited the expression of the FABP1 but increased the CPT1A, PPAR-α, and SREBP1 expressions. FABP1 overexpression reduced ROS in HCC cells, while miR-603 can reverse these effects. Conclusion Our results indicate that in the pathogenesis of HCC, IL-6 induces miR-603 expression, which subsequently inhibits FABP1 expression, promotes the lipid metabolism- and synthesis-related proteins, and finally increases the cellular oxidative stress level and leads to the metastasis of HCC.

Long-term survival of 11\xa0years with multidisciplinary therapy for hepatocellular carcinoma metastasis to the ovary and peritoneum: a case report

We herein report a rare case of HCC metastases to the ovary and peritoneum in a 61-year-old female patient who has achieved 11-year survival with multidisciplinary therapy. The patient was diagnosed with HCC during balloon angioplasty performed for Budd–Chiari syndrome in 1994 and underwent partial hepatectomy twice. Five years after the second hepatectomy, allochronic recurrence of a single nodule detected in S8 was treated by radiofrequency ablation, followed by percutaneous ethanol injection therapy and stereotactic body radiotherapy. However, her α-fetoprotein level rose to 1862 ng/mL within one year and computed tomography revealed a large pelvic tumor suggesting HCC metastasis to the ovary. The subsequent laparotomy revealed one 11-cm left ovarian tumor, one small right ovarian nodule, and numerous peritoneal nodules. Bilateral salpingo-oophorectomy and peritoneal resection of as many nodules as possible were performed. Combination therapy with intravenous 5-fluorouracil plus cisplatin and ramucirumab monotherapy effectively suppressed tumor progression with maintenance of hepatic functional reserve, and she has achieved long-term survival of 11 years, illustrating that multidisciplinary therapy with favorable hepatic functional reserve maintenance can contribute to long-term survival in HCC with extrahepatic spread.

Identification of SRGAP2 as a potential oncogene and a prognostic biomarker in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the common malignancies worldwide. Slit-Robo GTPase-activating proteins (SRGAPs) have been shown to regulate the occurrence and development of various tumors. However, their specific roles in HCC remain elusive. MethodsThe expression pattern, genetic alteration and prognostic value of SRGAPs in HCC are analyzed by bioinformatics tools. The biological functions of SRGAP2 in HCC cells are demonstrated by in vitro experiments. The high-throughput RNA sequencing is conducted to explore the underlying molecular mechanisms of SRGAP2 in HCC cells. ResultsThe expression levels of SRGAP1 and SRGAP2 are significantly elevated in HCC tissues compared to the normal both in Oncomine and TCGA datasets, and SRGAP2 are dramatically upregulated both in mRNA and protein levels. Moreover, higher SRGAP2 is significantly related to the clinical stages of HCC. Meanwhile, SRGAP2 might be an independent prognostic indicator, as it correlates negatively with the clinical outcomes of HCC patients. Further SRGAP2-silencing experiments imply that SRGAP2 might remarkably promote the migration and invasion of HCC cells in an EMT-independent pattern. Based on the high-throughput RNA sequencing of SRGAP2-knockdown HCC cells, enrichment and network analyses demonstrate that SRGAP2 is closely associated with cellular metabolic signaling. ConclusionsOur study firstly illustrates the crucial role of SRGAP2 in the metastasis of HCC and explores its underlying molecular mechanisms. We identify SRGAP2 as a promising prognostic biomarker and a novel therapeutic target for HCC patients.