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.

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.

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.

Proteomic Analysis of Circulating Extracellular Vesicles Identifies Potential Biomarkers for Lymph Node Metastasis in Oral Tongue Squamous Cell Carcinoma.

Lymph node metastasis is the most reliable indicator of a poor prognosis for patients with oral tongue cancers. Currently, there are no biomarkers to predict whether a cancer will spread in the future if it has not already spread at the time of diagnosis. The aim of this study was to quantitatively profile the proteomes of extracellular vesicles (EVs) isolated from blood samples taken from patients with oral tongue squamous cell carcinoma with and without lymph node involvement and non-cancer controls. EVs were enriched using size exclusion chromatography (SEC) from pooled plasma samples of patients with non-nodal and nodal oral tongue squamous cell carcinoma (OTSCC) and non-cancer controls. Protein cargo was quantitatively profiled using isobaric labelling (iTRAQ) and two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. We identified 208 EV associated proteins and, after filtering, generated a short list of 136 proteins. Over 85% of the EV-associated proteins were associated with the GO cellular compartment term "extracellular exosome". Comparisons between non-cancer controls and oral tongue squamous cell carcinoma with and without lymph node involvement revealed 43 unique candidate EV-associated proteins with deregulated expression patterns. The shortlisted EV associated proteins described here may be useful discriminatory biomarkers for differentiating OTSCC with and without nodal disease or non-cancer controls.

The Role of Arginine Metabolism in Oral Tongue Squamous Cell Carcinoma.

Early diagnosis and treatment do not prevent the high morbidity and poor prognosis of oral tongue squamous cell carcinoma (TSCC). Earlier studies have shown that ARG1 signaling is deregulated in TSCC. Here, we investigated the complexity of ARG1 metabolism in this cancer subsite to appreciate the therapeutic potential of this potential biological vulnerability. Various functional studies show that ARG1 overexpression in oral cancer cells inhibits cell proliferation and invasion compared with controls. Further, RNA-sequencing revealed numerous differentially expressed genes (DEGs) and associated networks were dysregulated by ARG1 overexpression, including hypoxia-inducible factor (HIFα) signaling, the natural killer cell signaling pathway and interferon signaling. Our work provides a foundation for understanding the mechanism of action of disrupted arginine metabolism in oral tongue squamous cell carcinoma. This may impact the community for developing further therapeutic approaches.

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.

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.

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.