Epithelial-to-mesenchymal Transition In Hepatocellular Carcinoma Cells Research Articles

TGF-β-MTA1-SMAD7-SMAD3-SOX4-EZH2 Signaling Axis Promotes Viability, Migration, Invasion and EMT of Hepatocellular Carcinoma Cells.

IntroductionEnhancer of zeste homolog 2 (EZH2) is implicated in hepatocellular carcinoma (HCC), but whether transforming growth factor-β (TGF-β)-metastasis associated 1 (MTA1)-SMAD7-SMAD3-SRY-Box Transcription Factor 4 (SOX4)-EZH2 signaling axis, in which EZH2 participates, is also involved in HCC remained unknown.MethodsData on EZH2 expression in liver hepatocellular carcinoma (LIHC) and its relation with prognosis of HCC patients were predicted and analyzed using online databases. Following transfection with or without TGF-β1, HCC cell viability, migration and invasion were determined with MTT, Scratch and Transwell assays. Relative expressions of epithelial-to-mesenchymal transition (EMT)-related factors (N-Cadherin, Vimentin, and E-Cadherin) and TGF-β-MTA1-SMAD7-SMAD3-SOX4-EZH2 signaling axis factors (TGF-β, MTA1, SMAD7, phosphorylated-SMAD3, SOX4 and EZH2) were calculated via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot.ResultsEZH2 was upregulated in HCC, which was related to poor prognosis. Silencing EZH2 suppressed EZH2 expression and HCC cell viability, migration, and invasion, and increased E-Cadherin expression yet decreased N-Cadherin and Vimentin expression, whereas EZH2 overexpression did conversely. Also, silencing EZH2 reversed the effects of TGF-β1 on promoting viability, migration, and invasion, as well as N-Cadherin and Vimentin expressions, yet suppressing E-Cadherin expression in HCC cells. In addition, TGF-β1 promoted TGF-β, MTA1, SOX4 and EZH2 expressions and p-SMAD3/SMAD3 ratio yet suppressed SMAD7, whereas silencing EZH2 solely reversed the effects of TGF-β1 on EZH2 expression in HCC cells.ConclusionThe present study provides a theoretical basis for TGF-β-MTA1-SMAD7-SMAD3-SOX4-EZH2 signaling cascade in viability, migration, invasion, and EMT of HCC cells. Inhibiting these signals may represent a therapeutic pathway for the treatment of metastatic HCC.

Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment.

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib.

Regorafenib inhibits migration, invasion, and vasculogenic mimicry of hepatocellular carcinoma via targeting ID1-mediated EMT.

Regorafenib is approved for patients with unresectable hepatocellular carcinoma (HCC) following sorafenib. However, the effect of regorafenib on HCC metastasis and its mechanism are poorly understood. Here, our data showed that regorafenib significantly restrained the migration, invasion and vasculogenic mimicry (VM) of HCC cells, and downregulated the expression of epithelial-to-mesenchymal transition (EMT)/VM-related molecules. Using RNA-seq and cellular thermal shift assays, we found that inhibitor of differentiation 1 (ID1) was a key target of regorafenib. In HCC tissues, the protein expression of ID1 was positively correlated with EMT and VM formation (CD34- /PAS+ ). Functionally, ID1 knockdown inhibited HCC cell migration, invasion, metastasis, and VM formation in vitro and in vivo, with upregulation of E-cadherin and downregulation of Snail and VE-cadherin. Moreover, Snail overexpression promoted the migration, invasion, and VM formation of ID1 knockdown cells. Snail knockdown reduced the migration, invasion, and VM formation of ID1 overexpression cells. Finally, regorafenib suppressed VM formation and decreased the expression of ID1, VE-cadherin and Snail in HCC PDX model. In conclusion, we manifested that regorafenib distinctly inhibited EMT in HCC cells via targeting ID1, leading to the suppression of cell migration, invasion and VM formation. These findings suggest that regorafenib may be developed as a suitable therapeutic agent for HCC metastasis.

Ezrin promotes hepatocellular carcinoma progression by modulating glycolytic reprogramming.

Ezrin, one of the ezrin‐radixin‐moesin (ERM) proteins, is involved in the formation of cell membrane processes and has been implicated in the promotion of cancer proliferation and metastasis. However, the possible role of ezrin in hepatocellular carcinoma (HCC) metastasis and glycolysis reprogramming has remained unclear. In this study, we found that ezrin was upregulated in HCC tissues, and its overexpression was linked with HCC patients’ aggressive tumor characteristics and poor prognosis. Functional experiments further revealed that ezrin overexpression promoted HCC cell proliferation, epithelial‐to‐mesenchymal transition (EMT) progression and angiogenesis. In addition, by measuring glucose consumption, lactate production, ATP levels and the expression of glucose metabolism‐related markers in HCC cells, we investigated whether ezrin regulated glucose metabolism. Moreover, 2‐deoxy‐D‐glucose (2‐DG) affected ezrin‐mediated proliferation, migration and EMT of HCC cells, which suggested that ezrin may, at least in part, promote HCC progression by regulating glycolysis reprogramming. Based on our results, we proposed that ezrin was involved in HCC progression and may be a valid prognostic marker.

Inhibition of PI3K/Akt signaling suppresses epithelial-to-mesenchymal transition in hepatocellular carcinoma through the Snail/GSK-3/beta-catenin pathway.

Background/AimsPatients with advanced hepatocellular carcinoma (HCC) have a poor prognosis due to the lack of effective systemic therapies. Epithelial-to-mesenchymal transition (EMT) is a pivotal event in tumor progression, during which cancer cells acquire invasive properties. In this study, we investigated the effects of phosphatidylinositol 3-kinase (PI3K) inhibitors, including LY294002 and idelalisib, on the EMT features of HCC cells in vitro.MethodsHuman HCC cell lines, including Huh-BAT and HepG2, were used in this study. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and cell cycle distributions were evaluated using a flow cytometer by propidium iodide staining. Immunofluorescence staining, quantitative real-time polymerase chain reaction, and immunoblotting were performed to detect EMT-associated changes.ResultsPI3K inhibitors suppressed the proliferation and invasion of HCC cells and deregulated the expression of EMT markers, as indicated by increased expression of E-cadherin, an epithelial marker, and decreased expression of N-cadherin, a mesenchymal marker, and Snail, a transcription factor implicated in EMT regulation. Furthermore, LY294002 and idelalisib inhibited the phosphorylation of GSK-3β and induced the nuclear translocation of GSK-3β, which corresponded to the downregulation of Snail and β-catenin expressions in Huh-BAT and HepG2 cells.ConclusionsThe inhibition of PI3K/Akt signaling decreases Snail expression by enhancing the nuclear translocation of GSK-3β, which suppresses EMT in HCC cells, suggesting the potential clinical application of PI3K inhibitors for HCC treatment.

MicroRNA‑875‑5p inhibits tumor growth and metastasis of hepatocellular carcinoma by targeting eukaryotic translation initiation factor 3 subunit a.

Accumulating evidence has demonstrated that aberrant microRNA (miRNA) expression is involved in hepatocellular carcinoma (HCC) progression. Previous findings suggested that miRNA (miR)-875-5p participates in the development of various types of cancer. However, the expression and function of miR-875-5p in HCC remains largely unclear. The analysis of clinical samples in the present study demonstrated that miR-875-5p expression was downregulated in HCC tissues compared to adjacent non-tumor tissues, which was associated with a large tumor size, venous infiltration, advanced tumor-node-metastasis stage and unfavorable overall survival. In vitro experiments revealed that ectopic expression of miR-875-5p suppressed, whereas inhibition of miR-875-5p promoted HCC cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT) progression. Overexpression of miR-875-5p restrained HCC tumor growth and metastasis in vivo. Mechanistically, eukaryotic translation initiation factor 3 subunit a (eIF3a) was identified as the downstream target of miR-875-5p in HCC. Further experiments demonstrated that the expression of eIF3a was upregulated and negatively correlated with that of miR-875-5p in HCC tissues. In addition, miR-875-5p negatively regulated the luciferase activity of wild-type, but not mutant 3′-untranslated region (3′UTR) of eIF3a mRNA. miR-875-5p suppressed eIF3a expression at the mRNA and protein level in HCC cells. Additionally, eIF3a exerted an oncogenic role, and knockdown of eIF3a inhibited the proliferation, motility and EMT of HCC cells. In addition, eIF3a overexpression abolished the inhibitory effects of miR-875-5p on the proliferation, motility and EMT in HCC cells. In conclusion, miR-875-5p, which was downregulated in HCC, may inhibit tumor growth and metastasis by eIF3a downregulation via targeting its 3′UTR and may be a promising prognostic and therapeutic strategy in HCC.

LncRNA SNHG1 promotes cell progression and metastasis via sponging miR-377-3p in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most famous fatal malignancies in the world. LncRNA SNHG1 has been shown to play roles in the development and progression of various tumors, including HCC. The present study aims to investigate the deeper molecular mechanisms of SNHG1 in HCC. The expression levels of SNHG1 and miR-377-3p were detected by qRT-PCR in HCC tissues and cells. MTT assay was used to examine cell proliferation. Cell apoptosis was evaluated by detecting the apoptotic rate and the protein level of C-caspase 3 using flow cytometry and western blot assays. The protein levels of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin) were measured by western blot. Cell migration and invasion were examined by transwell assay. Xenograft analysis was performed to explore the tumor growth in vivo. The binding sites of SNHG1 and miR-377-3p were predicted by the online software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay. We found that SNHG1 was markedly upregulated in HCC tissues and cells. Knockdown of SNHG1 induced apoptosis and inhibited proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT) of HCC cells. SNHG1 knockdown suppressed the tumor growth of HCC in vivo. SNHG1 directly bound to miR-377-3p. Knockdown of miR-377-3p attenuated the effect of SNHG1 knockdown on proliferation, apoptosis, migration, invasion, and EMT of HCC cells. In conclusion, SNHG1 inhibited apoptosis and induced proliferation, migration, invasion, and EMT by sponging miR-377-3p in HCC, which indicated that SNHG1 may be a potential biomarker and therapeutic target for HCC treatment.

Epithelial Mesenchymal and Endothelial Mesenchymal Transitions in Hepatocellular Carcinoma: A Review.

Purpose To present a comprehensive review of the literature data, published between 2000 and 2019 on the PubMed and Web of Science databases, in the field of the tumor microenvironment in hepatocellular carcinoma (HCC). All the data were combined with the personal experiences of the authors. Design From 1002 representative papers, we selected 86 representative publications which included data on epithelial-to-mesenchymal transition (EMT), angiogenesis, cancer stem-like cells (CSCs), and molecular background of chemoresistance or resistance to radiotherapy. Results Although the central event concerns activation of the Wnt/β-catenin pathway, other signal pathways, such as c-Met/HGF/Snail, Notch-1/NF-κB, TGF-β/SMAD, and basic fibroblast growth factor-related signaling, play a role in the EMT of HCC cells. This pathway is targeted by specific miRNAs and long noncoding RNAs, as explored in this paper. A central player in the tumor microenvironment proved to be the CSCs which can be marked by CD133, CD44, CD90, EpCAM, and CD105. CSCs can induce resistance to cytotoxic therapy or, alternatively, can be synthesized, de novo, after chemo- or radiotherapy, especially after transarterial chemoembolization- or radiofrequency ablation-induced hypoxia. The circulating tumor cells proved to have epithelial, intermediate, or mesenchymal features; their properties have a critical prognostic role. Conclusion The metastatic pathway of HCC seems to be related to the Wnt- or, rather, TGFβ1-mediated inflammation-angiogenesis-EMT-CSCs crosstalk link. Molecular therapy should target this molecular axis controlling the HCC microenvironment.

CASC15 promotes epithelial to mesenchymal transition and facilitates malignancy of hepatocellular carcinoma cells by increasing TWIST1 gene expression via miR-33a-5p sponging

Long noncoding RNA cancer susceptibility candidate 15 (CASC15) facilitates progression of hepatocellular carcinoma (HCC) cells, but the molecular mechanisms remain unknown. CASC15 co-expressing genes were explored in the Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset. Putative co-expressing genes were analyzed by Gene Ontology and biological pathway enrichment analysis. CASC15 overexpression or knockdown and TWIST1 overexpression or knockdown in HCC cells was achieved by lentiviral transduction or plasmid transfection. Interaction between CASC15 and microRNA-33a-5p (miR-33a-5p) was verified by argonaute 2-RNA Immunoprecipitation (AGO2-RIP) and luciferase reporter assays. HCC cell malignancy was determined by cell proliferation, apoptosis, migration and invasion assays. Tumorigenicity was evaluated by xenograft assay. Epithelial-to-mesenchymal transition (EMT) of HCC cells was assessed by Western blot. TWIST1, sex-determining region Y-related high-mobility group box 4 (Sox4) and Versican were found as putative CASC15 co-expressing genes. CASC15 positively regulated TWIST1 gene expression as well as protein level of Sox4 and Versican in HCC cells. Positive correlation in expression between CASC15 and TWIST1 mRNA was verified in 42 pairs of HCC pathologic and adjacent tissue specimens. CASC15 upregulated TWIST1 gene expression in HCC cells by sponging miR-33a-5p. CASC15 promoted EMT in HCC cells by increasing N-cadherin and Vimentin protein level while decreasing that of E-cadherin through TWIST1. CASC15 facilitated HCC cell proliferation, migration and invasion while reducing cell apoptosis through TWIST1. CASC15 facilitated the tumorigenicity of HCC cells in vivo. a CASC15 could promote EMT and facilitate malignancy of HCC cells by increasing TWIST1 gene expression via miR-33a-5p sponging.

Metformin enhances radiosensitivity in hepatocellular carcinoma by inhibition of specificity protein 1 and epithelial-to-mesenchymal transition.

Radiotherapy becomes more and more important in hepatocellular carcinoma (HCC) due to the development of technology, especially in unresectable cases. Metformin has a synergistic benefit with radiotherapy in some cancers, but remains unclear in HCC. This study aims to investigate the effect of metformin on radiosensitivity of HCC cells and the roles of specificity protein 1 (Sp1) as a target of metformin. The SMMC-7721 cell line was exposed to various doses of γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and with or without different concentrations of metformin (0, 1, 5, 10, and 20 mM) to measure the radiosensitivity using MTT assay. Flow cytometry was used to determine cell cycle by propidium iodide (PI) staining and apoptosis by Hoechst 33342/PI staining and Annexin V-FITC/PI staining. Real-time polymerase chain reaction and Western blotting were performed to analyze the Sp1 mRNA and protein expressions of Sp1 and epithelial-to-mesenchymal transition (EMT) marker E-cadherin and Vimentin. The invasion capability was measured by the Boyden chamber assay. In SMMC-7721 cells exposed to irradiation, metformin reduced proliferation and survival cells at various concentrations (0, 1, 5, 10, and 20 mM) and induced cell cycle arrest, apoptosis, and inhibited invasion. In SMMC-7721 cells with irradiation, the mRNA and protein expressions of Sp1 were significantly decreased by metformin as well as a selective Sp1 inhibitor. Metformin attenuated transforming growth factor-β1 induced decrease of E-cadherin and increase of Vimentin proteins. Metformin demonstrated enhanced radiosensitivity and inhibition of EMT in HCC cells. Sp1 might be a target of metformin in radiosensitization.

MiR-HCC2 Up-regulates BAMBI and ELMO1 Expression to Facilitate the Proliferation and EMT of Hepatocellular Carcinoma Cells.

MicroRNAs (miRNAs) are a class of gene expression regulators that participate in the occurrence and development of hepatocellular carcinoma (HCC), although the underlying mechanism by which they function in HCC has not been fully elucidated. Here, small RNA deep sequencing was used to identify aberrantly expressed miRNAs in HCC tissues, and a novel miRNA named miR-HCC2 was identified. RT-qPCR analysis demonstrated that miR-HCC2 displayed higher expression in HCC tissues than in adjacent non-tumor tissues. We documented that miR-HCC2 facilitated the growth, migration and invasion of HCC cells by accelerating cell cycle progression, incressing the expression of epithelial-to-mesenchymal transition (EMT)-associated marker vimentin but decreasing the expression of E-cadherin. MiR-HCC2 directly targeted the 3′ UTR of BAMBI and ELMO1 and up-regulated their expression. Both BAMBI and ELMO1 had the same patterns of expression with miR-HCC2 in HCC tissues. Additionally, blocking BAMBI or ELMO1 counteracted the phenotypic alterations elicited by miR-HCC2. Collectively, our investigation identified miR-HCC2 as a new positive modulator of HCC aggressiveness that may serve as a potential biomarker for the development of diagnostic and therapeutic approaches for HCC.

Delphinidin inhibits epidermal growth factor‐induced epithelial‐to‐mesenchymal transition in hepatocellular carcinoma cells

Epithelial-to-mesenchymal transition (EMT), important cellular process in metastasis of primary tumors, is characterized by loss of their cell polarity, disruption of cell-cell adhesion, and gain certain properties of mesenchymal phenotype that enable migration and invasion. Delphinidin is a member of anthocyanidin belong to flavonoid groups, known as having pharmacological and physiological effects including anti-tumorigenic, antioxidative, anti-inflammatory, and antiangiogenic effects. However, the effects of delphinidin on EMT is rarely investigated. Epidermal growth factor (EGF) is known as a crucial inducer of EMT in various cancer including hepatocellular carcinoma (HCC). To determine whether delphinidin inhibits EGF-induced EMT in HCC cells, antiproliferative effect of delphinidin on Huh7 and PLC/PRF/5 cells were measured by Cell Counting Kit-8 assay. As a result, delphinidin inhibited cell proliferation in a dose-dependent manner. Based on the result of proliferation, to measure the effects of delphinidin on EGF-induced EMT, we designated a proper concentration of delphinidin, which is not affected to cell proliferation. We found that delphinidin inhibits morphological changes from epithelial to mesenchymal phenotype by EGF. Moreover, delphinidin increased the messenger RNA and protein expression of E-cadherin and decreased those of Vimentin and Snail in EGF-induced HCC cells. Also, delphinidin prevented motility and invasiveness of EGF-induced HCC cells through suppressing activation of matrix metalloproteinase 2, EGF receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK). Taken together, our findings demonstrate that delphinidin inhibits EGF-induced EMT by inhibiting EGFR/AKT/ERK signaling pathway in HCC cells.

TGF-β2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment.

Hepatocellular carcinoma (HCC) typically develops in a chronic inflammatory setting causal to release of a plethora of growth factors and cytokines. However, the molecular effect of these cytokines on HCC progression is poorly understood. In this study, we exposed HCC cells to TGF-β2 (Transforming Growth Factor-β2), which resulted in a significant elevation of EMT (Epithelial to Mesenchymal Transition) like features. Molecular analysis of EMT markers showed an increase at both RNA and protein levels upon TGF-β2 administration along with up-regulation of TGF-β-induced Smad signaling. Induction of EMT was associated with a simultaneous increase in reactive oxygen species (ROS) and cytostasis of TGF-β2-treated cells. Importantly, quenching of ROS resulted in a significant promotion of TGF-β2-induced EMT. Furthermore, cells treated with TGF-β2 also showed an enhanced autophagic flux. Interestingly, inhibition of autophagy by chloroquine-di-phosphate (CQDP) or siRNA-mediated ablation of ATG5 drastically inhibited TGF-β2-induced EMT. Autophagy inhibition significantly increased ROS levels promoting apoptosis. It was further observed that pro-inflammatory cytokine like, TNF-α (Tumor Necrosis Factor-α) can antagonize TGF-β2-induced response by down-regulating autophagy, increasing ROS levels and thus inhibiting EMT in HCC cells. This inhibitory effect of TNF-α is serum-independent. Transcriptomic analysis through RNA sequencing was further performed which validated that TGF-β2-induced autophagic genes are inhibited by TNF-α treatment suppressing EMT. Our study suggests that autophagy plays a pro-metastatic role facilitating EMT by regulating ROS levels in HCC cells and TNF-α can suppress EMT by inhibiting autophagy. We provide unique mechanistic insights into the role of TGF-β2 in HCC cells, along with appropriate cues to effectively control the disease.

MicroRNA-1296 inhibits metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting SRPK1-mediated PI3K/AKT pathway

BackgroundIncreasing evidences demonstrate that miRNAs contribute to development and progression of hepatocellular carcinoma (HCC). Underexpression of miR-1296 is recently reported to promote growth and metastasis of human cancers. However, the expression and role of miR-1296 in HCC remain unknown.MethodsThe levels of miR-1296 in HCC tissues and cells were detected by qRT-PCR. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. Transwell assays were performed to determine migration and invasion of HCC cells. A lung metastasis mouse model was used to evaluated metastasis of HCC in vivo. The putative targets of miR-1296 were disclosed by public databases and a dual-luciferase reporter assay.ResultsWe found that the expression of miR-1296 was reduced in HCC tissues and cell lines, and it was associated with metastasis and recurrence of HCC. Notably, miR-1296 overexpression inhibited migration, invasion and EMT progress of HCCLM3 cells, while miR-1296 loss facilitated these biological behaviors of Hep3B cells in vitro and in vivo. In addition, miR-1296 inversely regulated SRPK1 abundance by directly binding to its 3′-UTR, which subsequently resulted in suppression of p-AKT. Either SRPK1 re-expression or PI3K/AKT pathway activation, at least partially, abolished the effects of miR-1296 on migration, invasion and EMT progress of HCC cells. Furthermore, miR-1296 and SRPK1 expression were markedly correlated with adverse clinical features and poor prognosis of HCC patients. We showed that hypoxia was responsible for the underexpression of miR-1296 in HCC. And the promoting effects of hypoxia on metastasis and EMT of HCC cells were reversed by miR-1296.ConclusionsUnderexpression of miR-1296 potentially serves as a prognostic biomarker in HCC. Hypoxia-induced miR-1296 loss promotes metastasis and EMT of HCC cells probably by targeting SRPK1/AKT pathway.

Knockdown of FRAT1 inhibits hypoxia-induced epithelial-to-mesenchymal transition via suppression of the Wnt/β-catenin pathway in hepatocellular carcinoma cells.

Hypoxia-induced epithelial-to-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) was investigated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a positive regulator of the Wnt/β-catenin signaling pathway and is overexpressed in many human tumors. However, the expression and role of FRAT1 in HCC has not been elucidated. In this study, we investigated the effect of FRAT1 on EMT process in HCC cells induced by hypoxia. Our results showed that FRAT1 is highly expressed in HCC tissues and cell lines. Hypoxia significantly induced FRAT1 expression in HCC cells. FRAT1 knockdown inhibited hypoxia-induced cell migration/invasion, downregulation of epithelial markers and upregulation of mesenchymal markers. Moreover, FRAT1 knockdown suppressed the expression levels of β-catenin, cyclinD1 and c-myc in HCC cells under the same hypoxic condition. Our results revealed that FRAT1 is a hypoxia factor that is critical for the induction of EMT in HCC cells. These data suggest a potential role for targeting FRAT1 in the prevention of hypoxia-induced HCC cancer progression and metastasis mediated by EMT.

MicroRNA-379-5p inhibits tumor invasion and metastasis by targeting FAK/AKT signaling in hepatocellular carcinoma

Some microRNAs (miRNAs) have been implicated in hepatocellular carcinoma (HCC) development and progression. However, the roles and mechanisms of several miRNAs in HCC remain poorly understood. Here, we report that miR-379-5p, which is down-regulated in HCC tissues and cell lines, is associated with advanced TNM stage and metastasis in HCC. The ectopic overexpression of miR-379-5p inhibited HCC cell migration, invasion, epithelial-to-mesenchymal transition (EMT) and metastasis both in vitro and in vivo. Conversely, miR-379 knockdown increased migration, invasion and EMT in HCC cells. Moreover, miR-379-5p exerted this function by directly targeting focal adhesion kinase (FAK) 3′-UTR and repressing FAK expression, thus leading to suppression of AKT signaling. Furthermore, the tumor suppressive effects of miR-379-5p in HCC cells were reversed by activating AKT signaling or restoring FAK expression. In clinical samples of HCC, miR-379-5p negatively correlated with FAK, which was up-regulated in HCC. Taken together, our findings highlight the important role of miR-379-5p in regulating the EMT and metastasis of HCC by targeting FAK/AKT signaling, suggesting that miR-379-5p may represent a novel potential therapeutic target and prognostic marker for HCC.

HOXB9 promotes epithelial-to-mesenchymal transition via transforming growth factor-β1 pathway in hepatocellular carcinoma cells.

The function of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established. However, the regulatory mechanisms modulating this phenomenon remain unclear. Homeobox B9 (HOXB9) has been proposed as an oncogene in many cancer developments, but its function and underlying mechanisms in HCC metastasis remain unknown. HOXB9 modulates EMT through the transforming growth factor-β1 (TGF-β1) pathway, which is a recognized regulator of EMT in HCC cells. The knockdown of HOXB9 decreased the migration and invasion of HCC cells. Conversely, the HOXB9 overexpression led to an increase in the above-mentioned phenotypes in HCC cells. Further analysis of HOXB9-regulated cellular functions showed the ability of this transcription factor to induce EMT. Moreover, we demonstrated that the TGF-β1 pathway is important in HOXB9-induced EMT in HCC cells. These findings define a novel cellular mechanism regulated by HOXB9, which controls EMT phenotype in HCC. This study is the first to illustrate the pivotal function of HOXB9 in regulating the metastatic behavior of HCC cells.

Crosstalk between Beta-Catenin and Snail in the Induction of Epithelial to Mesenchymal Transition in Hepatocarcinoma: Role of the ERK1/2 Pathway

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.

Abstract C29: Free fatty acid induced epithelial to mesenchymal transition (EMT) in hepatocellular carcinoma cells

Abstract Background: The incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in the United States over the past few decades. From an epidemiological perspective, it has been observed that HCC related mortality rates are significantly higher in obese patients indicating a possible correlation between obesity and metastatic progression of HCC. At the physiological level, obesity manifests an elevation in free fatty acid levels (FFA) levels which is thought to be detrimental for liver cells. While most previous studies have focused on cytotoxic effects of elevated FFA, very few studies have investigated their potential role in progression of liver cancer. In a previous study, we found reduced expression of desmoplakin (DSP) in FFA treated HepG2 cells, suggesting a loss of cell adhesion – one of the features of cells undergoing metastatic progression via EMT. Hence, we hypothesized that elevated FFA levels possess the ability to induce EMT in HCC cells. Methodology and Results: We studied the in vitro effects of FFA on HepG2 and Hep3B cells – two human HCC cell lines of different etiologies. We performed all experiments using FFA conjugated to BSA (bovine serum albumin), and used BSA as treatment control. We first evaluated the overall impact of FFA treatment on cell adhesion. We performed a dispase-based dissociation assay and found significant loss of cell adhesion in FFA treated cells. Next, using scratch wound-healing assay and modified Boyden's chamber assay, we found that FFA significantly enhanced the migration and invasiveness of HCC cells. We further confirmed the occurrence of EMT by measuring the change in expression levels of various markers (Keratin 8/18, Vimentin, E-cadherin) in FFA treated cells using Western blots. Additionally, we screened the change in expression levels of several transcription factors that have been described as inducers of EMT using qRT-PCR. Our studies indicated enhanced expression of a number of EMT-related transcription factors including SNAIL, TWIST, ZEB1 and FOXC2, suggesting a potential transcriptional regulatory mechanism by which elevated FFA could induce EMT. Furthermore, we evaluated the possibility of a direct role of DSP loss in inducing EMT. Using siRNA mediated DSP knock-down we found enhanced expression of EMT markers in both HepG2 and Hep3B cells. Conclusions and future perspectives: Our studies provide evidence that elevated FFA levels may be involved in metastatic progression of HCC cells through transcriptional activation of EMT pathways. Future in vivo studies based on mouse xenograft model will provide additional support to confirm the effects of elevated FFA and DSP loss on enhancing aggressiveness and metastatic potential of HCC. These results may impact HCC therapeutic intervention and drug development, as well as future studies on obesity as a risk factor for cancer tumorigenesis and progression. Citation Format: Aritro Nath, Christina Chan. Free fatty acid induced epithelial to mesenchymal transition (EMT) in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C29.