ARPC2 is highly expressed in EVs of metastatic HCC cells
EVs derived from metastatic HCC cells have been previously demonstrated to promote cancer cell growth, motility and metastasis . To comprehensively investigate the differential biological activity of EVs, proteomic compositions of EVs derived from the immortalized normal liver cell line MIHA, metastatic HCC cell lines MHCC97L and MHCCLM3 were compared (ProteomeXchange Consortium dataset identifier: PXD019566). Expression of proteins with at least 4-fold modulated and p-value less than 0.05 in EVs of metastatic HCC cells compared to MIHA were regarded as significantly different (Fig. 1a). ARPC2 ranked the 4th upregulated EV proteins of MHCC97L cells (Fig. 1b). Other subunits of Arp2/3 complex, ARPC1B and ARPC4, were also upregulated in MHCC97L- and MHCCLM3-EVs compared to MIHA-EVs (Fig. 1c). Elevated expression of ARPC2 in EVs of metastatic MHCC97L and MHCCLM3 cell but not in EVs of non-metastatic HCC cell lines, Huh7 and PLC/PRF/5, and normal MIHA cells was validated by immunoblotting (Fig. 1d). Immunogold labeling of EVs revealed the expression and presence of ARPC2 on the surface of EVs (Fig. 1e). The expression of TSG101 and Alix while absence of GM130 and p62 suggested the tested EVs are small EVs (exosomes) (Fig. 1d). The identity of small EVs was further corroborated by the size range of EVs (Fig. 1f).
Clinical relevance of ARPC2 and other Arp2/3 subunits in HCC
Presence of high levels of various subunits of Arp2/3 complex in EVs of metastatic HCC cells suggest the role of Arp2/3 complex in HCC. To reveal the clinical significance of Arp2/3 complex, gene expressions of various complex subunits were analyzed using TCGA database of liver cancer that comprises 371 HCC samples and 50 normal liver samples. Significant overexpression of ARPC2, ARPC1A, ARPC1B and ARPC4 were all detected in HCC (Fig. 2a). Among the 4 subunits, ARPC2 (p = 0.0151) and ARPC1A (p = 0.00272) expressions were significantly correlated with tumor stage (Fig. 2b). Kaplan-Meier survival analysis revealed the significant correlation between the expressions of all 4 subunits with poorer overall survival but not with disease free survival of HCC patients (Fig. 2c-2d). The upregulation of ARPC2 in EVs of metastatic HCC cells and the clinical relevance of ARPC2 in HCC suggest the crucial role of ARPC2 in HCC.
Pimozide inhibits the promoting effect of ARPC2-enriched EVs of metastatic HCC cells
EVs from metastatic MHCC97L and MHCCLM3 cells have been shown to facilitate pre-metastatic niche, enhance tumor development and augment metastasis in HCC . To study whether APRC2 contributes to the promoting capacity of MHCC97L- and MHCCLM3-EVs, Pimozide, an inhibitor of APRC2, was examined for its effect in EVs in functional assays. As demonstrated by the colony formation, migration and invasion assays, both MHCC97L- and MHCCLM3-EVs significantly enhanced the growth, motility and invasiveness of MIHA and PLC/PRF/5 cells (Fig. 3a-3f). The EV-induced enhancement in cells was hindered when Pimozide was added. Pimozide also suppressed the motility and colony formation ability of MHCC97L and MHCCLM3 cells in which ARPC2 were highly expressed (Supplementary Fig. S1).
The potential role of EV-ARPC2 was further examined in an experimental metastasis assay. Mice were intravenously with murine p53-/-,Myc hepatoblasts alone or with MHCC97L-EVs either with DMSO or Pimozide (Fig. 3g). The findings revealed that the injection of MHCC97L-EVs resulted in a significant enhancement in the colonization of hepatoblasts to lungs of mice as revealed by an increase in bioluminescence signal in whole mice and lung tissues (Fig. 3h-3i). Histological examination confirmed the presence of tumor nodules in lungs (Fig. 3j). Consistent with the effect of Pimozide observed in in vitro functional assays, mice co-injected EVs with Pimozide displayed significant reduction in bioluminescence signals and metastatic lesions in the lungs. These results demonstrate the oncogenic capacity of EVs derived from metastatic HCC cells was inhibited by Pimozide and implicate that ARPC2 is a functional component contributing to the promoting effect of EVs.
EV-ARPC2 augments the cancerous properties of HCC cells
To affirm the role of ARPC2 in EVs of HCC cells, stable ARPC2 knockout (ARPC2-KO1 and ARPC2-KO2) and nontarget knockout control cells (Control-KO) clones were generated in MHCC97L cells (Fig. 4a). Knockout of ARPC2 resulted in a hindered ability of ARPC2-KO cells to grow, migrate, and invade compared to Control-KO cells (Supplementary Fig. S2). EVs were collected from the conditioned medium of stable clones. Knockout of ARPC2 was observed in the validated isolated EVs of stable clones (Fig. 4a-4c). The functional effect of EVs derived from ARPC2-KO and Control-KO clones were subsequently tested on MIHA and PLC/PRF/5 cells, both of which had relatively low level of ARPC2. Recipient cells treated with Control-KO-EVs presented enhanced abilities to form colony, migrate and invade compared to cells treated with PBS (Fig. 4d-4f). Such enhancement in ability was abrogated in cells treated with ARPC2-KO-EVs compared to cells treated with Control-KO-EVs.
The role of EV-ARPC2 in HCC metastasis was further investigated by the experimental metastasis assay in which the lung colonization was compared between mice co-injected with murine p53-/-,Myc hepatoblasts and Control-KO-EVs or ARPC2-KO-EVs (Fig. 5a). CTL-KO-EVs demonstrated a positive effect on metastasis in mice, whereas the EV-induced colonization of hepatoblasts in the lungs was significantly attenuated when ARPC2-KO-EVs were injected (Fig. 5b-5d). Taken together, these findings demonstrate that EV-APRC2 plays an imperative role in HCC metastasis and suggest targeting EV-ARPC2 may play an anti-metastatic effect in HCC.