Metastatic Hepatocellular Carcinoma Cell Research Articles

Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line

ABSTRACT The prevalence of alcohol-related hepatocellular carcinoma (HCC) has been increasing during the last decade. Cancer research requires cell lines suitable for both in vitro and in vivo assays. However, there is a lack of cell lines with a high in vivo metastatic capacity for this HCC subtype. Herein, a new HCC cell line was established, named HCC-ZJ, using cells from a patient diagnosed with alcohol-related HCC. The karyotype of HCC-ZJ was 46, XY, del (p11.2). Whole-exome sequencing identified several genetic variations in HCC-Z that occur frequently in alcohol-associated HCC, such as mutations in TERT, CTNNB1, ARID1A, CDKN2A, SMARCA2, and HGF. Cell counting kit-8 assays, colony formation assays, and Transwell assays were performed to evaluate the proliferation, migration, and sensitivity to sorafenib and lenvatinib of HCC-Z in vitro. HCC-ZJ showed a robust proliferation rate, a weak foci-forming ability, a strong migration capacity, and a moderate invasion tendency in vitro. Finally, the tumorigenicity and metastatic capacity of HCC-Z were evaluated using a subcutaneous xenograft model, an orthotopic xenograft model, and a tail-veil injection model. HCCZJ exhibited strong tumorigenicity in the subcutaneous xenograft and orthotopic tumor models. Moreover, HCC-ZJ spontaneously formed pulmonary metastases in the orthotopic tumor model. In summary, a new HCC cell line derived from a patient with alcohol-related HCC was established, which showed a high metastatic capacity and could be applied for in vitro and in vivo experiments during pre-clinical research. Highlights • An alcohol-related HCC cell line, HCC-ZJ, was established • HCC-ZJ was applicable for in vitro functional experiment and gene editing • HCC-ZJ was applicable for in vivo tumor growth and spontaneous metastasis models

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma.

Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC. Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)-knockdown and -overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7. The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC. This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

TrkB inhibition of DJ-1 degradation promotes the growth and maintenance of cancer stem cell characteristics in hepatocellular carcinoma

Although TrkB may be associated with the pathogenesis of various cancer by upregulation, how upregulation of TrkB led to tumor progression in hepatocellular carcinoma (HCC) and the signaling mechanisms by which TrkB induces motility, invasion, metastasis, drug resistance, and acquisition of self-renewal traits has remained unclear. Here, we demonstrated that TrkB was significantly upregulated in highly metastatic HCC cells and HCC patients. Also, the increased TrkB levels were significantly correlated with tumor stages and poor survival of HCC patients. Furthermore, the upregulated TrkB expression enhances the metastatic ability of HCC cells through reduced anoikis sensitivity, induced migration, and colony formation. Most strikingly, TrkB markedly enhances the activation of STAT3 by preventing DJ-1 degradation through the formation of the TrkB/DJ-1 complex. This signaling mechanism is responsible for triggering cellular traits of highly aggressive HCC. The activation of the EMT program of HCC via increasing DJ-1 stability by TrkB induces the gain of cancer stem cell states and chemoresistance via the upregulation of stem cells cell markers and ABC transporters. Also, TrkB-mediated inhibition of DJ-1 degradation promotes tumor formation and metastasizes to other organs in vivo. Our observations illustrate that TrkB is a prognostic and therapeutic targeting in promoting aggressiveness and metastasis of HCC.

Small Extracellular Vesicle-Derived vWF Induces a Positive Feedback Loop between Tumor and Endothelial Cells to Promote Angiogenesis and Metastasis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular malignancy by which its growth and dissemination are largely driven by the modulation of tumor-derived small extracellular vesicles (sEVs). Proteomic profiling of circulating sEVs of control individuals and HCC patients identifies von Willibrand factor (vWF) to be upregulated progressively along HCC stages. Elevated sEV-vWF levels are found in a larger cohort of HCC-sEV samples and metastatic HCC cell lines compared to their respective normal counterparts. Circulating sEVs of late-stage HCC patients markedly augment angiogenesis, tumor-endothelial adhesion, pulmonary vascular leakiness, and metastasis, which are significantly compromised by anti-vWF antibody. The role of vWF is further corroborated by the enhanced promoting effect of sEVs collected from vWF-overexpressing cells. sEV-vWF modulates endothelial cells through an elevated level of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Mechanistically, secreted FGF2 elicits a positive feedback response in HCC via the FGFR4/ERK1 signaling pathway. The co-administration of anti-vWF antibody or FGFR inhibitor significantly improves the treatment outcome of sorafenib in a patient-derived xenograft mouse model. This study reveals mutual stimulation between HCC and endothelial cells by tumor-derived sEVs and endothelial angiogenic factors, facilitating angiogenesis and metastasis. It also provides insights into a new therapeutic strategy involving blocking tumor-endothelial intercellular communication.

Inhibition of TRPV4 remodels single cell polarity and suppresses the metastasis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor, frequently causing both intrahepatic and extrahepatic metastases. The overall prognosis of patients with metastatic HCC is poor. Recently, single-cell (sc) polarity is proved to be an innate feature of some tumor cells in liquid phase, and directly involved in the cell adhesion to blood vessel and tumor metastasis. Here, we characterize the maintained sc polarity of HCC cells in a suspension culture, and investigate its roles and regulatory mechanisms during metastasis. We demonstrate that transient receptor potential vanilloid 4 (TRPV4) is a promoting regulator of sc polarity via activating Ca2+-dependent AMPK/MLC/ERM pathway. This attenuates the adhesion of metastatic HCC cells to vascular endothelial cells. The reduction of cancer metastases can result from TRPV4 inhibition, which not only impacts the migration and invasion of tumor cells, but also prevents the adhesion to vascular endothelial cells. Additionally, we discover a brand-new TRPV4 inhibitor called GL-V9 that modifies the degree of sc polarization and significantly decreases the metastatic capacity of HCC cells. Taken together, our data shows that TRPV4 and calcium signal are significant sc polarity regulators in metastatic HCC, and that the pharmacological intervention that results in HCC cells becoming depolarized suggests a promising treatment for cancer metastasis.

Hepatocellular carcinoma presenting as a chest wall mass - An unusual case diagnosed on cytology with review of literature.

Hepatocellular carcinoma (HCC) is associated with an aggressive behavior and a strong tendency for extrahepatic metastasis. Although 5%-15% patients have metastases at diagnosis, presentation with symptoms exclusively related to extrahepatic metastases is rare. An 82-year-old male presented with an isolated left anterolateral chest wall swelling. Ultrasonography revealed a soft tissue mass involving the anterior chest wall with adjacent rib erosion. Serum protein electrophoresis showed increase in beta-2 region. A clinical diagnosis of multiple myeloma was considered. Fine needle aspiration cytology from the swelling showed loosely cohesive clusters of polygonal cells with traversing blood vessels. Cells showed abundant vacuolated to granular cytoplasm, round nuclei with frequent intranuclear cytoplasmic inclusions. A differential of metastatic HCC and renal cell carcinoma was considered. Subsequent imaging showed a 12 cm mass in the liver. Biopsy from chest wall mass with immunohistochemistry confirmed the diagnosis. Lungs and lymph nodes are the commonest sites for metastatic HCC; presentation as chest wall metastasis is rarely reported. The classical cytomorphology of HCC proved useful in diagnosing metastasis at a rare site. Recent studies have shown that beta-2-globulin is a promising biomarker for early diagnosis of HCC in patients with chronic liver disease.

Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line

Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line

BORIS variant SF2(C2/A4) promotes the malignant development of liver cancer by activating epithelial-mesenchymal transition and hepatic stellate cells.

The underlying mechanisms of metastasis and recurrence of liver cancer remain largely unknown. Here, we found that Brother of the Regulator of Imprinted Sites (BORIS) variant SF2(C2/A4) was highly expressed in high metastatic potential hepatocellular carcinoma (HCC) cells and clinical tumor samples, related to the formation of satellite nodules. Its over expression promoted self-renewal, the expression of tumor stem cell markers, chemoresistance, wound healing rate, invasion and metastasis of HepG2 and Hep3B cells; reinforced epithelial-mesenchymal transition (EMT), decreased the expression of E-cadherin and increased N-cadherin and Vimentin. Subcellular localization experiment showed that BORIS SF2(C2/A4) was localized in nucleus and cytoplasm. Further double luciferase reporter gene experiment confirmed that it bound to TWIST1 gene promoter and significantly increased latter expression. BORIS SF2(C2/A4) knock down induced apoptosis of HCCLM3 and PLC/PRF/5 cells, and increased the protein content of cleaved caspase 3. Additionally, BORIS SF2(C2/A4) over expression increased the expression of fibroblast growth factor 2 (FGF2) in HepG2 and Hep3B cells. FGF2 expressed higher in HCC tumor tissues than in paired peri-tumor tissues, and its expression was positively correlated with BORIS SF2(C2/A4). Interestingly, high expression of FGF2 is also associated with the formation of satellite nodules. Moreover, using the medium from BORIS SF2(C2/A4) overexpressed cell lines to coculture hepatic stellate cell (HSCs) line LX-2, the latter could be activated and increased the expression of CD90 and PIGF, which is consistent with the effect of adding bFGF alone. These results indicate that BORIS SF2(C2/A4) plays a role in deterioration of liver cancer by regulating TWIST1 to induce EMT, and by FGF2 to activate HSCs.

S100A11 Promotes Metastasis via AKT and ERK Signaling Pathways and Has a Diagnostic Role in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common and malignant liver tumor worldwide, although the treatment approaches for HCC continue to evolve, metastasis is the main reason for high mortality rates. S100 calcium-binding protein A11 (S100A11), an important member of the S100 family of small calcium-binding proteins, is overexpressed in various cells and regulates tumor development and metastasis. However, few studies report the role and underlying regulatory mechanisms of S100A11 in HCC development and metastasis. Herein, we discovered that S100A11 is overexpressed and associated with poor clinical outcomes in HCC cohorts, and we provided the first demonstration that S100A11 could serve as a novel diagnostic biomarker used in conjunction with AFP for HCC. Further analysis implied that S100A11 outperforms AFP in determining whether HCC patients have hematogenous metastasis or not. Using in vitro cell culture model, we demonstrated that S100A11 is overexpressed in metastatic hepatoma cells, knockdown of S100A11 decreases hepatoma cells proliferation, migration, invasion, and epithelial-mesenchymal transition process by inhibiting AKT and ERK signaling pathways. Altogether, our study provides new sights into the biological function and mechanisms underlying S100A11 in promoting metastasis of HCC and explores a novel target for HCC diagnosis and treatment.

Transmembrane serine protease 2 cleaves nidogen 1 and inhibits extrahepatic liver cancer cell migration and invasion.

We aimed to confirm whether transmembrane serine protease 2 (TMPRSS2) regulates nidogen 1 (NID1) expression in extracellular vesicles (EVs) and metastatic hepatocellular carcinoma (HCC) cells. HCC cells, HUVEC cells, MRC-5 cells, HLE cells, MHCCLM3 cells, MHCC97L cells, H2P cells, H2M cells, as well as LO2 cells were cultured according to providers' instruction and EV models were established by using BALB/cAnN-nu mice to facilitate the verifications. We found that TMPRSS2 expression was inversely correlated with the metastatic potential of HCC cell lines. The expression of TMPRSS2 decreased in a time-dependent manner in tumor-bearing model mice implanted with MHCCLM3 cells compared with uninoculated mice. TMPRSS2 overexpression in MHCCLM3 and MHCC97L cells led to the significant downregulation of NID1 expression in total cell lysates and isolated EVs. In contrast, TMPRSS2 silencing resulted in the elevation of NID1 expression in cells and EVs. Administration of EVs from MHCCLM3 and MHCC97L cells with overexpressed or silenced TMPRSS2 inhibited or strengthened, respectively, the invasion, proliferation, and migration of LO2 tumor cells. EVs derived from MHCCLM3 and MHCC97L cells with overexpressed or depleted TMPRSS2 also deactivated or activated fibroblasts, respectively. These EVs secrete inflammatory cytokines and phosphorylated p65, facilitate the colonization of fibroblasts, and augment fibroblast growth and motility. These findings provide evidence for a new candidate drug targeting tumorigenic EV-NID1 to treat HCC.

Large Oncosome-Loaded VAPA Promotes Bone-Tropic Metastasis of Hepatocellular Carcinoma Via Formation of Osteoclastic Pre-Metastatic Niche.

Tumor-derived extracellular vesicles (EVs) function as critical mediators in selective modulation of the microenvironment of distant organs to generate a pre-metastatic niche that facilitates organotropic metastasis. Identifying the organ-specific molecular determinants of EVs can develop potential anti-metastatic therapeutic targets. In the current study, large oncosomes (LOs), atypically large cancer-derived EVs, are found to play a crucial role in facilitating bone-tropic metastasis of hepatocellular carcinoma (HCC) cells by engineering an osteoclastic pre-metastatic niche and establishing a vicious cycle between the osteoclasts and HCC cells. Transmembrane protein, VAMP-associated protein A (VAPA), is significantly enriched on LOs surface via direct interaction with LOs marker αV-integrin. VAPA-enriched LOs-induced pre-metastatic education transforms the bone into a fertile milieu, which supports the growth of metastatic HCC cells. Mechanically, LOs-delivered VAPA integrates to plasma membrane of osteoclasts and directly interacts with and activates neural Wiskott-Aldrich syndrome protein (N-WASP) via dual mechanisms, consequently resulting in ARP2/3 complex-mediated reorganization of actin cytoskeleton in osteoclasts and osteoclastogenesis. Importantly, treatment with N-WASP inhibitor 187-1-packaged LOs (LOs/187-1) dramatically abolishes the inductive effect of VAPA-enriched LOs on pre-metastatic niche formation and precludes HCC bone metastasis. These findings reveal a plausible mechanism for bone-tropism of HCC and can represent a potential strategy to prevent HCC bone metastasis.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

TMEM106A transcriptionally regulated by promoter methylation is involved in invasion and metastasis of hepatocellular carcinoma.

Expression of transmembrane protein 106A (TMEM106A) has been reported to be dysregulated in several types of cancers. However, the role of TMEM106A in hepatocellular carcinoma (HCC) is still unknown. In the present study, we demonstrate that TMEM106A is markedly downregulated in HCC compared with normal liver tissue. In particular, tumor-specific DNA methylation of TMEM106A is frequently observed in tumor tissues from HCC patients. Immunohistochemistry and pyrosequencing reveal a significant relationship between TMEM106A methylation and downregulation of protein expression. Receiver operating characteristic (ROC) curve analysis reveals that methylation of TMEM106A in tumor samples is different from that in non-malignant adjacent tissues of HCC patients. Moreover, HCC patients with TMEM106A hypermethylation have a poor clinical prognosis. 5-Aza-2'-deoxycytidin treatment of hypermethylated TMEM106A in highly metastatic HCC cells increases the expression of TMEM106A. Functional assays reveal that overexpression of TMEM106A significantly suppresses the malignant behavior of HCC cells in vitro and decreases tumorigenicity and lung metastasis in vivo. Mechanistically, TMEM106A inhibits epithelial mesenchymal transition (EMT) of HCC cells through inactivation of the Erk1/2/Slug signaling pathway. In conclusion, our findings demonstrate that TMEM106A is an inhibitor of HCC EMT and metastasis, and TMEM106A is often transcriptionally downregulated by promoter methylation, which results in reduced levels of TMEM106A protein and predicts poor survival outcomes for HCC patients.

Actin-related protein 2/3 complex subunit 2-enriched extracellular vesicles drive liver cancer metastasis.

Extracellular vesicles (EVs) play pivotal roles in tumor growth, cancer metastasis and angiogenesis. Here, we aimed to identify proteins that contribute to the functionality of EVs derived from metastatic hepatocellular carcinoma (HCC) cells. Proteins of EVs derived from metastatic HCC cells and normal liver cells were analyzed by mass spectrometry. Proteomic profiling identified actin-related protein 2/3 complex subunit 2 (ARPC2) to be highly expressed in EVs of metastatic HCC cells. The expression of ARPC2 in EVs and HCC tissues was examined using immunoblotting and TCGA database, respectively. The functional roles of EV-ARPC2 were investigated by knockout approach and various in vitro and in vivo assays. ARPC2 was highly expressed in EVs of metastatic cells but barely detected in non-metastatic HCC cells and normal liver cells. Immunogold labeling showed the presence of APRC2 on the surface of EVs. Analysis of TCGA database of liver cancer revealed ARPC2 overexpression was correlated with poor prognosis of patients. ARPC2 was knockout in metastatic HCC cells. EVs derived from knockout cells displayed compromised activity in enhancing cell growth, motility and metastasis compared to EVs of control cells. Pimozide, an inhibitor of APRC2, also inhibited the promoting effect of EVs of metastatic cells in lung colonization of tumor cells in mice. This study reveals previously unreported expression and function of ARPC2 in EVs. EVs with highly expressed ARPC2 enhance cancer cell growth and metastasis. ARPC2 may provide a prospective target for the novel treatment of HCC patients.

CircRPN2 Inhibits Aerobic Glycolysis and Metastasis in Hepatocellular Carcinoma.

The circRNA circRPN2 is a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma that suppresses aerobic glycolysis and metastasis.

[GPCR48 promotes invasion and metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma cells].

Objective: To observe the G protein-coupled receptor 48 (GPCR48) expression in hepatocellular carcinoma (HCC) cell lines with different metastatic potential and its characteristics effect on the invasion and metastasis of Huh7 hepatoma cells via epithelial-mesenchymal transition (EMT). Methods: Western blot was used to detect the protein expression level of GPCR48 in HCC cells with different metastatic potential. The lentivirus vector expressing GPCR48 gene was constructed. GPCR48 was overexpressed in Huh7 hepatoma cells. The GPCR48 overexpression level was detected by real-time PCR and Western blot. Transwell invasion and migration assay was used to detect the Huh7 hepatoma cells invasion and migration ability in the Control, Mock and GPCR48 overexpression group. Real-time PCR and Western blot were used to detect Huh7 hepatoma cells mRNA and protein expression levels of the EMT related markers (E-cadherin, N-cadherin, vimentin, and γ catenin) in the Control, Mock and GPCR48 overexpression groups, respectively. Analysis of variance was used to compare the differences between data sets. Results: GPCR48 protein expression level in metastatic HCC cell lines was significantly higher than non-metastatic HCC cell lines (P < 0.05). The lentivirus vector expressing the GPCR48 gene had effectively transfected the Huh7 hepatoma cells and stably expressed the GPCR48mRNA and protein. Compared with the Mock and the Control group, Huh7 hepatoma cells invasion and migration ability in the GPCR48 overexpression group was significantly enhanced (F≥5.54, P < 0.05), and the mRNA and protein expression levels of epithelial phenotypic markers E-cadherin and γ-catenin were decreased (P < 0.05). The mRNA and protein expression levels of the mesenchymal phenotypic markers N-cadherin and Vimentin were increased (P < 0.05), indicating that EMT changes occurred in Huh7 hepatoma cells had overexpressed GPCR48. Conclusion: GPCR48 expression level is positively correlated with the metastatic potential of HCC cells. GPCR48 overexpression can down-regulate the expression of epithelial phenotypic markers and up-regulate the expression of mesenchymal phenotypic markers, and induce EMT changes in HCC cells, thus promoting HCC cells invasion and migration.

TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis.

Rab GTPases are major mediators that ensure the proper spatiotemporal regulation of intracellular trafficking. Functional impairment and altered expression of Rab proteins have been revealed in various human cancers. There is an emerging evidence about the role of Rab proteins in the biogenesis of extracellular vesicles (EVs). In hepatocellular carcinoma (HCC), using RNA sequencing comparing expression profiles of adjacent non‐tumorous tissues and HCC, Rab20 is identified to be the most frequently downregulated Rab member in HCC. Functionally, restoration of Rab20 in metastatic HCC cells results in the release of EVs with a diminished activity to promote cell growth, motility and metastasis. Conversely, EVs released from normal liver cells with Rab20 knockdown loses suppressive effect on HCC cell growth and motility. Proteomic profiling revealed the level of triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, in EVs to be positively associated with Rab20 expression of the releasing cells. TPI1 targeted to be expressed in EVs released by Rab20 knockdown cells compromises the oncogenic activity of EVs. Besides, EVs released by TPI1 knockdown cells recapitulates the promoting effect of EVs derived from HCC cells with Rab20 underexpression. Aerobic glycolysis is beneficial to the survival and proliferation of tumour cells. Here, we observed that the enhanced cell growth and motility are driven by the enhanced aerobic glycolysis induced by EVs with reduced TPI1. The addition of glycolytic inhibitor blocks the promoting effect of EVs with reduced TPI1. Taken together, our study provides a mechanistic link among tumour cell‐derived EVs and glucose metabolism in HCC with Rab20 deregulation.

Characterization of the small RNA transcriptomes of cell protrusions and cell bodies of highly metastatic hepatocellular carcinoma cells via RNA sequencing.

Increasing evidence suggest that hepatocellular carcinoma (HCC) HCCLM3 cells initially develop pseudopodia when they metastasize, and microRNAs (miRNAs/miRs) and circular RNAs (circRNAs) have been demonstrated to serve important roles in the development, progression and metastasis of cancer. The present study aimed to isolate the cell bodies (CBs) and cell protrusions (CPs) from HCCLM3 cells, and screen the miRNAs and circRNAs associated with HCC infiltration and metastasis in CBs and CPs. The Boyden chamber assay has been confirmed to effectively isolate the CBs and CPs from HCCLM3 cells via observation of microtubule immunofluorescence, DAPI staining and nuclear protein H3 western blotting. Following high-throughput sequencing of the successfully isolated CBs and CPs, 64 pairs of miRNAs, including 23 pairs of upregulated genes and 41 pairs of downregulated genes, and 260 sets of circRNAs, including 127 upregulated genes and 133 downregulated genes, were significantly differentially expressed, using the following criteria: HP/HB ratio, fold change ≥|1.5|, P<0.05). PCR analysis verified that changes in the expression levels of hsa-let-7a-5p, hsa-let-7c-3p, hsa-miR-30c-5p, hsa_circ_0059580, hsa_circ_0067475, hsa_circ_0002100 and hsa_circ_00072309 were consistent with the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze the functions and roles of the differentially expressed miRNAs and circRNAs. The interaction maps between miRNAs and circRNAs were constructed, and signaling pathway maps were analyzed to determine the molecular mechanism and regulation of the differentially expressed miRNAs and circRNAs. Taken together, the results of the present study suggest that the Boyden chamber assay can be used to effectively isolate the somatic CBs and CPs of HCC, which can be used to screen the miRNAs and circRNAs associated with invasion and metastasis of HCC.

Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3

Intercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

FMRP regulates STAT3 mRNA localization to cellular protrusions and local translation to promote hepatocellular carcinoma metastasis

Most hepatocellular carcinoma (HCC)-associated mortalities are related to the metastasis of cancer cells. The localization of mRNAs and their products to cell protrusions has been reported to play a crucial role in the metastasis. Our previous findings demonstrated that STAT3 mRNA accumulated in the protrusions of metastatic HCC cells. However, the underlying mechanism and functional significance of this localization of STAT3 mRNA has remained unexplored. Here we show that fragile X mental retardation protein (FMRP) modulates the localization and translation of STAT3 mRNA, accelerating HCC metastasis. The results of molecular analyses reveal that the 3′UTR of STAT3 mRNA is responsible for the localization of STAT3 mRNA to cell protrusions. FMRP is able to interact with the 3′UTR of STAT3 mRNA and facilitates its localization to protrusions. Importantly, FMRP could promote the IL-6-mediated translation of STAT3, and serine 114 of FMRP is identified as a potential phosphorylation site required for IL-6-mediated STAT3 translation. Furthermore, FMRP is highly expressed in HCC tissues and FMRP knockdown efficiently suppresses HCC metastasis in vitro and in vivo. Collectively, our findings provide further insights into the mechanism of HCC metastasis associated with the regulation of STAT3 mRNA localization and translation.