S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos

Abstract

Objective: Growing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown.

Design: The functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10.

Results: Functionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial-mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects.

Conclusion: Altogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

Keywords: HEPATOCELLULAR CARCINOMA; MOLECULAR MECHANISMS; MOLECULAR ONCOLOGY.

Figure 1

Clinical significance of S100A10. (A) Human ES cells were induced to differentiate along hepatic lineages into adult hepatocytes. Cells from different developmental stages, including ES cells, DE, LP cells and PHs, as well as normal liver (NL) and HCC tissues, were used for transcriptomic sequencing. The expression pattern of S100A10 was confirmed using qRT-PCR. (B) Relative expression of S100A10 in HCC and corresponding non-tumorous livers from 86 pairs of samples of patients with HCC. Paired t-test. (C) Kaplan-Meier overall survival curve and numbers at risk for these 86 patients with HCC. (D) Relative S100A10 expression and Kaplan-Meier curve for overall survival of TCGA cohort (analysed by GEPIA website). (E) Correlation between S100A10 expression and clinicopathological features in 86 HCC cases. Fisher exact test. (F) CNV of S100A10 in 80 pairs of samples of patients with HCC. (G) S100A10 expression stratified according to S100A10 CNV (left panel), non-parametric Mann-Whitney test. *P<0.05, **P<0.01. Correlation between relative S100A10 expression and relative CNV (right panel). AJCC, American Joint Committee on Cancer; CNV, copy number variation; DE, definitive endoderm; ES, embryonic stem; HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; LP, liver progenitor; NT, non-tumorous liver tissues; TCGA, The Cancer Genome Atlas; TNM, TNM classification of malignant tumours.

Figure 2

S100A10 enhances HCC stemness. (A) Western blot showing efficiency of ectopic S100A10 expression, and S100A10 silencing with shRNA against S100A10 (shS100A10-1: sh1, shS100A10-3: sh3) and with sgRNA against S100A10 (sgS100A10-2: sg2, sgS100A10-4: sg4). S100, S100A10; NTC, non-target control. Tubulin was used as a loading control. Band intensities were quantitated by ImageJ. (B) Relative expression of HCC stemness-related markers with S100A10-OE compared with Vec plasmid by qRT-PCR. (C) Sphere formation assay of PLC-Vec/S100A10, 97L-Vec/S100A10 and 97H-NTC/sgS100A10s, respectively. (D) Limiting dilution assays of PLC-Vec/S100A10. The tumour-initiating frequency is summarised in chart and table form. (E) Chemoresistance assay in nude mice by subcutaneous injection. Representative images of PLC-Vec/S100A10 tumours treated with indicated drugs. Tumour weights expressed as mean±SD of five mice in each group, Mann-Whitney test (lower panel). (F) Western blot showing the expression of p-AKT, AKT, p-ERK and ERK in PLC-Vec/S100A10, 97L-Vec/S100A10 and 97H-NTC/sgS100A10s, respectively. Tubulin was used as a loading control. HCC, hepatocellular carcinoma; NTC, non-target control; p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; Vec, vector control.

Figure 3

S100A10 promotes HCC migratory, invasive and metastatic abilities in vitro and in vivo. (A) The migratory and invasive abilities of PLC-Vec/S100A10, 97L-Vec/S100A10 and Huh7-NTC/shS100A10 were assessed by transwell migration and matrigel invasion assays. (B, C) Representative image of livers from nude mice after intrasplenic injection of indicated cells and representative histology of the corresponding liver sections (lower panel). Scale bar, 100 µm. Bar charts show the numbers of metastatic nodules on the liver surface (yellow arrows) in six mice in each group. (D) Representative image of lungs from NOD SCID mice after tail vein injection of 97L-Vec/S100A10 and representative histology of the corresponding lung sections (lower panel). Scale bar=100 µm. The bioluminescence signals represent mean±SD. *P<0.05, **P<0.01. Mann-Whitney test. (E) Western blot showing the expression of E-cadherin, N-cadherin, vimentin and fibronectin of HCC cells transfected with Vec and S100A10, or NTC and sgS100A10s. HCC, hepatocellular carcinoma; NTC, non-target control; Vec, vector control.

Figure 4

S100A10 is present in HCC-derived EVs and promotes HCC motility and metastasis. (A) Western blot showing detection of S100A10 and EV markers including CD81, CD63, HSP70, CD9, TSG101, Alix, Golgi marker GM130 and nucleoporin p62 in EVs from the plasma of patients with HCC. Each lane represents either a healthy donor or a patient with HCC. (B) Western blot showing detection of S100A10 and EV markers including CD81, CD63, HSP70, CD9, TSG101, Alix and Golgi marker GM130 and nucleoporin p62 in EVs from HCC cells. (C) Representative electron-micrographs of EVs derived from the plasma of patients with HCC, 97H and 97L cells (upper panel). Immunogold labelling of EVs using anti-CD63 and anti-S100A10 antibodies followed by secondary antibodies conjugated to 5 and 10 nm gold particles, respectively (lower panel). (D) Cell migration and invasion assays of PLC cells treated with Vec EVs or S100A10-OE cells (S100 EVs), or with EVs from 97H-NTC (NTC EVs) or -sgS100A10#4 (sgS100 EVs) cells. (E) Schematic diagram of the two EV education mouse models: liver metastasis model (left panel) and lung metastasis model (right panel). (F) Representative images of liver from nude mice of intrasplenic 97L cell injection model educated with PBS or indicated EVs, and bioluminescence signals of lungs from NOD SCID mice after tail vein injection of 97L educated with PBS or indicated EVs (left panel). The numbers of metastatic nodules on liver surface and bioluminescence signals of lung are summarised in the bar charts (right panel). EV, extracellular vesicle; HCC, hepatocellular carcinoma; NTC, non-target control; Vec, vector control; Vec EV, extracellular vesicle from vector control cell.

Figure 5

S100A10-NA abrogates the functions of S100A10 EVs in HCC stemness. (A) Migration and invasion assays on PLC cells treated with PBS or EVs derived from S100A10-OE cells (S100 EVs). The EVs were preincubated and added together with S100A10 NA or IgG control antibody. (B) Intrasplenic injection of HCC cells. EVs were injected together with S100A10 NA or IgG. (C) Bioluminescence signals of lungs from NOD SCID mice after tail vein injection of luciferase labelled 97L, educated with PBS or indicated EVs together with S100A10 NA or IgG. Sphere formation (D) and chemoresistance (E) were assessed on PLC cells treated with PBS, Vec EVs, or EVs from S100A10-OE cells (S100 EVs). The EVs were preincubated and treated together with S100A10 NA or IgG control antibody. (F) Representative images for lung vascular leakiness after tail vein injection of EVs, Texas Red-Dextran and FITC-lectin. Arrowheads indicate the area of endothelial leakiness. Scale bar=20 µm. *P<0.05, **P<0.01. EV, extracellular vesicle; NA, neutralising antibody; ns, not significant; Vec, vector control; Vec EV, extracellular vesicle from vector control cell.

Figure 6

S100A10 alters the protein contents of EVs. (A) Western blot showing the levels of MMP2, EGF, fibronectin and ITGAV in EVs derived from PLC-Vec/S100A10 and 97L-Vec/S100A10. (B) Western blot showing the expression levels of S100A10, MMP2, EGF, fibronectin and ITGAV in EVs derived from 97H-NTC/sgS100A10s or 97 H cells treated with S100A10 NA or IgG 48 hours before EV isolation. The EV markers, including CD63, CD81, HSP70, TSG101 and Alix, were used as loading control. (C) Representative electron micrograph of EVs from the plasma of patients with HCC subjected to immunogold labelling using anti-S100A10 together with anti-MMP2, anti-EGF, anti-fibronectin or anti-ITGAV antibodies, followed by secondary antibodies conjugated to 10 and 5 nm gold particles, respectively. Scale bar=25 nm. (D) Western blot showing the expression of S100A10, MMP2, EGF, fibronectin, surface marker CD81 and intra-EV marker HSP70 in 97H-derived EVs treated with increasing concentrations of proteinase K. Untreated EVs (first lane from left) served as control. (E) Western blot showing MMP2, EGF, fibronectin, S100A10 and ITGAV in EVs. GRADSP peptide (control peptide RAD as control) or GRGDSP peptide (RGD which is integrin-binding) were added to PLC-S100A10 cells for 48 hours before EV isolation. HSP70 was used as a loading control. (F) Coimmunoprecipitation assay showing the interaction between S100A10 and MMP2, EGF, fibronectin and ITGAV, using anti-S100A10 (IP-S100A10) or IgG (IP-IgG) in S100A10-OE PLC cells. Total cell lysate (input) used as positive control. (G) A schematic diagram illustrating the proposed S100A10-mediated binding of MMP2, fibronectin and EGF to the surface of EVs through ITGAV. EV, extracellular vesicle; HCC, hepatocellular carcinoma; ITGAV, integrin αV; NA, neutralising antibody; NTC, non-target control.

Figure 7

EV-S100A10 and S100A10 enhance HCC progression through EGFR activation. (A) Western blot showing the expression levels of EMT-related markers and EGFR, AKT and ERK signalling in cells treated with PBS, Vec EVs, S100 EVs, or S100 EVs, together with S100A10 NA or IgG. (B) Western blot showing the activation of EGFR, AKT and ERK signalling in cells treated with PBS, Vec EVs, S100 EVs, or S100 EVs together with different concentrations of EGFR inhibitor-Gefi (μM). (C, D) Chemoresistance to sorafenib of PLC-Vec, S100A10-OE cells, or S100A10-OE cells treated with Gefi, S100A10 NA or IgG control antibody. (E) Western blot showing the activation of EGFR, AKT and ERK signalling in Vec or S100A10-OE cells treated with S100A10 NA, IgG or different concentrations of Gefi (μM). (F) Nude mice subcutaneously injected with S100A10-OE PLC cells were administered with vehicle and IgG, sorafenib, S100A10 NA, or a combination of sorafenib and S100A10 NA. Sorafenib was administered daily via oral gavage. Control IgG and S100A10 NA were injected peritoneally once every 3 days. the tumour weight and body weight of mice were measured and plotted (right panel). *P<0.05, **P<0.01. EV, extracellular vesicle; Gefi, gefitinib; Gefi-5, gefitinib at 5 µM; p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; Vec, vector control.

Figure 8

EV-S100A10 promotes chemotaxis of HCC cells. (A, B) Cell migration and invasion assays. Vec EVs, or S100A10-OE cells (S100 EVs), or 97H-NTC (NTC EVs) or -sgS100A10#4 (sgS100 EVs) were added to the lower compartment of transwell chambers as indicated. (C) Migratory and invasive abilities of PLC cells assessed with PBS or S100 EVs added to the lower compartment of transwell chambers. EVs were preincubated and added together with S100A10 NA or IgG antibody. (D) Migratory ability of PLC treated with IgG or EGFR NA, with S100A10 EVs and IgG or S100A10 NA were added to the lower compartment of transwell chambers. (E) Migratory ability of PLC treated with IgG or EGFR NA, with CM and IgG or S100A10 NA were added to the lower compartment of transwell chambers. (F) Coimmunoprecipitation assay showing interaction between S100A10 and EGFR using anti-S100A10 (IP-S100A10) or anti-EGFR antibody (IP-EGFR) or IgG (IP-IgG) in S100A10-OE PLC cells. Total cell lysate (input) used as positive control. EV, extracellular vesicle; NTC, non-target control; Vec, vector control; Vec EV, extracellular vesicle from vector control cell.