Clathrin light chain A-enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis.

Small extracellular vesicles (sEVs) play a key role in exchanging cargoes between cells in tumour microenvironment. This study aimed to elucidate the functions and mechanisms of hepatocellular carcinoma (HCC) derived sEV-clathrin light chain A (CLTA) in remodelling microvascular niche. CLTA level in the circulating sEVs of HCC patients was analysed by enzyme-linked immunosorbent assay (ELISA). The functions of sEV-CLTA in affecting HCC cancerous properties were examined by multiple functional assays. Mass spectrometry was used to identify downstream effectors of sEV-CLTA in human umbilical vein endothelial cells (HUVECs). Tube formation, sprouting, trans-endothelial invasion and vascular leakiness assays were performed to determine the functions of sEV-CLTA and its effector, basigin (BSG) in HUVECs. BSG inhibitor, SP-8356, was tested in a mouse model of patient-derived xenografts (PDXs). Circulating sEVs of HCC patients had markedly enhanced CLTA levels than control individuals and were reduced in patients after surgery. HCC derived sEV-CLTA enhanced HCC cancerous properties, disrupted endothelial integrity and induced angiogenesis. Mechanistically, CLTA remodels microvascular niche by stabilizing and upregulating BSG. Last, SP-8356 alone or in combination with sorafenib attenuated PDXs growth. The study reveals the role of HCC derived sEV-CLTA in microvascular niche formation. Inhibition of CLTA and its mediated pathway may illuminate a new therapeutic strategy for HCC patients.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: Extracellular vesicles (EVs) play a pivotal role in connecting tumor cells with their local and distant microenvironments. Herein, we aimed to understand the role (on a molecular basis) patient-derived EVs play in modulating cancer stemness and tumorigenesis in the context of hepatocellular carcinoma (HCC). METHODS: EVs from patient sera were isolated, quantified and characterized. The EVs were vigorously tested, both in vitro and in vivo, through various functional assays. Proteomic analysis was performed to identify the functional components of EVs. The presence and level of polymeric immunoglobulin receptor (pIgR) in circulating EVs and tumor and non-tumorous tissues of patients with HCC were determined by ELISA, immunoblotting, immunohistochemistry and quantitative PCR. The functional role and underlying mechanism of EVs with enhanced pIgR expression were elucidated. Blockade of EV-pIgR with neutralizing antibody was performed in nude mice implanted with patient-derived tumor xenografts (PDTXs). RESULTS: Circulating EVs from patients with late-stage HCC (L-HCC) had significantly elevated pIgR expression compared to the EVs released by control individuals. The augmenting effect of L-HCC-EVs on cancer stemness and tumorigenesis was hindered by an anti-pIgR antibody. EVs enriched with pIgR consistently promoted cancer stemness and cancerous phenotypes in recipient cells. Mechanistically, EV-pIgR-induced cancer aggressiveness was abrogated by Akt and ß-catenin inhibitors, confirming that the role of EV-pIgR depends on the activation of the PDK1/Akt/GSK3ß/ß-catenin signaling axis. Furthermore, an anti-pIgR neutralizing antibody attenuated tumor growth in mice implanted with PDTXs. CONCLUSIONS: This study illustrates a previously unknown role of EV-pIgR in regulating cancer stemness and aggressiveness: EV-pIgR activates PDK1/Akt/GSK3ß/ß-catenin signaling cascades. The blockade of the intercellular communication mediated by EV-pIgR in the tumor microenvironment may provide a new therapeutic strategy for patients with cancer. LAY SUMMARY: The World Health Organization estimates that more than 1 million patients will die from liver cancer, mostly hepatocellular carcinoma (HCC), in 2030. Understanding the underlying mechanism by which HCC acquires aggressive attributes is crucial to improving the diagnosis and treatment of patients. Herein, we demonstrated that nanometer-sized extracellular vesicles released by tumors promote cancer stemness and tumorigenesis. Within these oncogenic vesicles, we identified a key component that functions as a potent modulator of cancer aggressiveness. By inhibiting this functional component of EVs using a neutralizing antibody, tumor growth was profoundly attenuated in mice. This hints at a potentially effective therapeutic alternative for patients with cancer.

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.

Suppression of ACADM-Mediated Fatty Acid Oxidation Promotes Hepatocellular Carcinoma via Aberrant CAV1/SREBP1 Signaling.

Lipid accumulation exacerbates tumor development, as it fuels the proliferative growth of cancer cells. The role of medium-chain acyl-CoA dehydrogenase (ACADM), an enzyme that catalyzes the first step of mitochondrial fatty acid oxidation, in tumor biology remains elusive. Therefore, investigating its mode of dysregulation can shed light on metabolic dependencies in cancer development. In hepatocellular carcinoma (HCC), ACADM was significantly underexpressed, correlating with several aggressive clinicopathologic features observed in patients. Functionally, suppression of ACADM promoted HCC cell motility with elevated triglyceride, phospholipid, and cellular lipid droplet levels, indicating the tumor suppressive ability of ACADM in HCC. Sterol regulatory element-binding protein-1 (SREBP1) was identified as a negative transcriptional regulator of ACADM. Subsequently, high levels of caveolin-1 (CAV1) were observed to inhibit fatty acid oxidation, which revealed its role in regulating lipid metabolism. CAV1 expression negatively correlated with ACADM and its upregulation enhanced nuclear accumulation of SREBP1, resulting in suppressed ACADM activity and contributing to increased HCC cell aggressiveness. Administration of an SREBP1 inhibitor in combination with sorafenib elicited a synergistic antitumor effect and significantly reduced HCC tumor growth in vivo. These findings indicate that deregulation of fatty acid oxidation mediated by the CAV1/SREBP1/ACADM axis results in HCC progression, which implicates targeting fatty acid metabolism to improve HCC treatment. SIGNIFICANCE: This study identifies tumor suppressive effects of ACADM in hepatocellular carcinoma and suggests promotion of ß-oxidation to diminish fatty acid availability to cancer cells could be used as a therapeutic strategy.

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1.

In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.

Tumour extracellular vesicle-derived Complement Factor H promotes tumorigenesis and metastasis by inhibiting complement-dependent cytotoxicity of tumour cells.

The complement system is involved in the immunosurveillance of pathogens and tumour cells. Proteomic profiling revealed that extracellular vesicles (EVs) released by metastatic hepatocellular carcinoma (HCC) cells contained a significant number of complement proteins. Complement Factor H (CFH), an abundant soluble serum protein that inhibits the alternative complement pathway, was found to be highly expressed in EVs of metastatic HCC cell lines. Here, we investigated the functional role of EV-CFH and explored the therapeutic efficacy of targeting EV-CFH with an anti-CFH antibody in HCC. The results showed that EVs that are enriched in CFH promoted HCC cell growth, migration, invasiveness and enhanced liver tumour formation in mice. EV-CFH also promoted metastasis, which was significantly abrogated when treated with an anti-CFH antibody. These findings demonstrate an unexplored function of EV-CFH in protecting HCC cells by evading complement attack, thereby facilitating tumorigenesis and metastasis. Lastly, we demonstrated the therapeutic efficacy of an anti-CFH antibody in suppressing tumour formation in a syngeneic mouse model. This study suggests a new therapeutic strategy for HCC, by inhibiting EV-CFH with a tumour specific anti-CFH antibody.

Galectin-1 promotes hepatocellular carcinoma and the combined therapeutic effect of OTX008 galectin-1 inhibitor and sorafenib in tumor cells.

BACKGROUND: Galectins are beta-galactose specific binding proteins. In human cancers, including hepatocellular carcinoma (HCC), galectin-1 (Gal-1) is often found to be overexpressed. In order to combat the dismal diagnosis and death rates of HCC, gene silencing and targeted inhibition of Gal-1 was investigated for its improved therapeutic potential. METHODS: Cellular and secretory Gal-1 levels were analyzed using HCC clinical samples. The study of Gal-1 was carried by both knockdown and overexpression approaches. The stable clones were tested by in vitro assays and in vivo experiments. Mass spectrometry was used to identify downstream targets of Gal-1. The upstream regulator of Gal-1, microRNA-22 (miR-22) was characterized by functional assays. The therapeutic effect of inhibiting Gal-1 was also analyzed. RESULTS: Gal-1 overexpression was observed in HCC and correlated with aggressive clinicopathological features and poorer survival. The loss of Gal-1 resulted in hindered cell migration, invasion and anchorage independent growth. This was also observed in the animal models, in that when Gal-1 was knocked down, there were fewer lung metastases. Proteomic profiling of control and Gal-1 knockdown cells identified that the level of retention in endoplasmic reticulum 1 (RER1) was suppressed when Gal-1 level was reduced. The cell motility of Gal-1 knockdown cells was enhanced upon the rescue of RER1 expression. In HCC tissues, Gal-1 and RER1 expressions displayed a significant positive correlation. The upstream regulator of Gal-1, miR-22 was observed to be underexpressed in HCC tissues and negatively correlated with Gal-1. Silencing of miR-22 resulted in the upregulation of Gal-1 and enhanced cell growth, migration and invasion. However, such enhancement was abolished in cells treated with OTX008, an inhibitor of Gal-1. Combinational treatment of OTX008 and sorafenib significantly reduced tumor growth and size. CONCLUSIONS: Gal-1 overexpression was detected in HCC and this played a role in promoting tumorigenic processes and metastasis. The function of Gal-1 was found to be mediated through RER1. The correlations between miR-22, Gal-1 and RER1 expressions demonstrated the importance of miR-22 regulation on Gal-1/RER1 oncogenic activity. Lastly, the combinational treatment of OTX008 and sorafenib proved to be an improved therapeutic option compared to when administering sorafenib alone.

C-terminal truncated HBx protein activates caveolin-1/LRP6/ß-catenin/FRMD5 axis in promoting hepatocarcinogenesis.

Hepatitis B virus X protein mutants, particularly truncated at C-terminal (HBxΔC), generated during random viral integration, are frequently detected in hepatocellular carcinoma (HCC) and exert a more potent oncogenic effect than full-length form (FL). Here, we showed that caveolin-1 (Cav1), a robust metastasis promoter, is transcriptionally upregulated by HBxΔC but not by FL HBx. Promoting effect of HBxΔC in HCC cell aggressiveness is abolished when Cav1 is suppressed. Expression profiling identified FERM domain containing 5 (FRMD5) protein as a downstream target of Cav1. In accordance with the regulation of Cav1, HBxΔC upregulates FRMD5. Knockdown of FRMD5 in HBxΔC cells recapitulated the functional effect of Cav1 knockdown in HBxΔC cells. The regulation of FRMD5 by HBxΔC-induced Cav1 is mediated by the protein stablilization of LRP6 leading to the activation of ß-catenin. Expression of a constitutively active ß-catenin in Cav1 knockdown cells rescued FRMD5 expression and HCC tumorigenesis and metastasis. Clinical relevance of HBxΔC/Cav1/LRP6/FRMD5 pathway is demonstrated by the significant correlation of Cav1, LRP6 and FRMD5 expressions in HCC. The findings of this study uncover a novel HBxΔC-regulated molecular pathway which has profound implications in HCC therapeutics.

Nuclear Met promotes hepatocellular carcinoma tumorigenesis and metastasis by upregulation of TAK1 and activation of NF-κB pathway.

Presence of Met receptor tyrosine kinase in the nucleus of cells has been reported. However, the functions of Met which expresses in the nucleus (nMet) remain elusive. In this study, we found that nMet was increased in 89% of HCC tumorous tissues when compared with the corresponding non-tumorous liver tissues. nMet expression increased progressively along HCC development and significantly correlated with cirrhosis, poorer cellular differentiation, venous invasion, late stage HCC and poorer overall survival. Western blot analysis revealed that nMet is a 48-kDa protein comprising the carboxyl terminal of Met receptor. Induced expression of nMet promoted HCC cell growth, migration and invasiveness in vitro and tumorigenesis and pulmonary metastasis in vivo. Luciferase assay showed that nMet activated NF-κB pathway. Indeed, p-IKKα/ß and nuclear p-p65 were higher in nMet stable cells than in the control cells. Perturbation of TAK1/NF-κB axis abrogated the aggressiveness of HCC cells, both in vitro and in vivo. In conclusion, nMet was overexpressed and as a potential prognostic biomarker of HCC. Functionally, nMet accelerated HCC tumorigenesis and metastasis via the activation of TAK1/NF-κB pathway.

Mechanisms through Which Hypoxia-Induced Caveolin-1 Drives Tumorigenesis and Metastasis in Hepatocellular Carcinoma.

In solid tumors, hypoxia triggers an aberrant vasculogenesis, enhances malignant character, and elevates metastatic risk. The plasma membrane organizing protein caveolin-1 (Cav1) is increased in a variety of cancers, including hepatocellular carcinoma (HCC), where it contributes to metastatic capability. However, the reason for elevation of Cav1 in tumor cells and the mechanistic basis for its contributions to metastatic risk are not fully understood. Here, we show that in HCC cells, hypoxia elevates expression of Cav1, which then acts through the calcium-binding protein S100P to promote metastasis. Hypoxic regions of HCC xenografts displayed elevated expression of Cav1. Hypoxia promoted HCC cell migration and invasion and distant pulmonary metastases, whereas Cav1 silencing abolished these effects. Gene expression profiling revealed that hypoxia-induced Cav1 functioned as a positive regulator of S100P via activation of the NF-κB pathway. S100P elevation under hypoxic conditions was abrogated by silencing of Cav1 or NF-κB function. Conversely, restoring S100P in Cav1-silenced cells rescued the migratory potential of HCC cells along with tumor formation and lung metastasis. In clinical specimens of HCC, we observed S100P overexpression to correlate with venous invasion, microsatellites, direct liver invasion, and absence of tumor encapsulation. Collectively, our findings demonstrated how hypoxia-induced expression of Cav1 in HCC cells enhances their invasive and metastatic potential. Cancer Res; 76(24); 7242-53. ©2016 AACR.