YAP1 Inhibition in HUVECs Is Associated with Released Exosomes and Increased Hepatocarcinoma Invasion and Metastasis.

Hepatocellular carcinoma is one of the most common gastrointestinal malignancies. Anti-angiogenesis therapies have recently demonstrated promise in the treatment of malignancies, although early treatment benefits may be accompanied by metastasis over time. Additional and more effective anti-angiogenic treatment modalities are therefore needed. We previously found that Yes-associated protein 1 (YAP1) expression is increased in hepatocellular carcinoma (HCC), particularly around tumor-associated blood vessels, suggesting a role in angiogenesis. The YAP1 inhibitor verteporfin is presently in anti-angiogenic clinical trials for the treatment of various cancers. Depleted YAP1 from vascular endothelial cells effectively reduced proliferation and tube formation, validating its utility as an anti-angiogenesis target. We also showed that YAP1 depletion or inhibition in vascular endothelial cells leads to increased release of exosomes containing the long non-coding RNA (lncRNA) MALAT1 into the tumor microenvironment. Direct exosomal transfer of MALAT1 to hepatic cells leads to increased hepatic cell invasion and migration via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. These observations may explain the occurrence of distant tumor metastasis with YAP1-associated anti-angiogenic therapy over time. It provides insight into new pathways and treatment paradigms that may be targeted to increase the long-term success of anti-angiogenic therapies.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and has high annual incidence and mortality. Considering the important role of angiogenesis in tumor progression, anti-angiogenesis therapy has become an effective method for treating tumors. However, some patients achieve significant results with early anti-angiogenesis treatment, but develop distant tumor metastasis over time., Additional and more effective therapeutic targets are needed to improve anti-tumor angiogenesis treatment. The Hippo pathway is a highly conserved signaling pathway. Its core transcriptional regulator, Yes-associated protein 1 (YAP1), regulates multiple pathophysiological processes, including organ size, cell proliferation, and apoptosis. In our previous study, we found that the expression of YAP1 expression is concentrated around blood vessels in HCC, suggesting that YAP1 may be involved in angiogenesis. It is worthy to further study the mechanism of how YAP1 influences blood vessel formation and whether it may be a target for anti-angiogenesis therapy. Previous study has demonstrated that YAP1 plays a critical role in the regulation of long non-coding RNA (lncRNA) expression. It is also a question of whether the role of YAP1 in HCC angiogenesis is related to the regulation of lncRNAs.

Our work is focused on remodeling of the tumor microenvironment by vascular endothelial cells during angiogenesis, and whether this remodeling has an effect on tumor behaviors. Recent studies have shown that exosomes play a critical role in the interaction among different cell types in the tumor microenvironment. The roles of YAP1 in exosomes released from vascular endothelial cells and their effects on tumor cells are questions that need to be addressed. Exosomes are multivesicular bodies (MVBs) derived from invagination of intracellular lysosomal microsomes, and they range in size from 30 to 150 nm., Recent studies have shown that lncRNAs carried by exosomes play an indispensable role in tumor development and therapy., These observations raise the question of whether the anti-angiogenic effect of YAP1 depletion or inhibition in vascular endothelial cells is related to lncRNAs. Can deletion of YAP1 in vascular endothelial cells influence tumor microenvironment remodeling?

In this study, we confirm that YAP1 deletion inhibits angiogenesis, accompanied by exosome release into the tumor microenvironment. These exosomes can transfer lncRNA MALAT1 to HCC cells, activating extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and promoting the expression of MMP2 and MMP9 to promote invasion and metastasis.

Results

YAP1 Contributes to Angiogenesis in HCC

In our previous study, we observed that YAP1 significantly increased and concentrated around blood vessels in HCC, suggesting that YAP1 may be involved in tumor angiogenesis.

To further confirm whether YAP1 is involved in tumor angiogenesis in HCC, we evaluated the correlation between YAP1 expression and expressions of tumor angiogenic factors (CD31, SPHK1, SPHK2, and VEGF) in 82 HCC specimens and by Gene Expression Profiling Interactive Analysis (GEPIA). The results suggested that the YAP1 expression is positively correlated with these angiogenic factors (Figure 1A; Figure S1), and the strongest positive correlation can be seen for VEGFA with an R of 0.7656. Using gene set enrichment analysis (GSEA) to analyze the relationship between YAP1 and angiogenesis in The Cancer Genome Atlas (TCGA) database, we found that YAP1 is closely related to multiple processes of angiogenesis (Figure 1B). Analysis of the microarray datasets (Gene Expression Omnibus [GEO]: GSE73396 and GSE35004) also indicated that YAP1 in HCC cells is closely related to angiogenesis (Figure 1C).

Figure 1

Expression of YAP1 Associated with Angiogenesis

(A) The expression of YAP1 is positively correlated with tumor angiogenic factors (CD31, SPHK1, SPHK2, and VEGF) in 82 HCC samples. (B) Enrichment plot demonstrating the degree of correlation between angiogenesis gene sets and YAP1 expression. (C) Angiogenesis processes are positively correlated with YAP1 expression by analysis of the publicly available datasets GEO: GSE35004 and GSE73396.

YAP1 Deletion Inhibits Proliferation, Migration, and Angiogenesis of Vascular Endothelial Cells

To explore the underlying mechanisms of YAP1 in vascular endothelial cells, we inhibited YAP1 expression in human umbilical vein endothelial cells (HUVECs) using small interfering RNAs (siRNAs) and verteporfin (VP). As shown in Figures 2A–2C, 5 μM VP is most effective at inhibiting YAP1 in vascular endothelial cells. Therefore, this concentration was selected for subsequent experiments.

Figure 2

YAP1 Deletion Inhibits Proliferation, Migration, and Angiogenesis of Vascular Endothelial Cells

(A) A western blot was used to analyze the expression of YAP1 upon different doses of VP simulated for 24 h. (B) Real-time PCR and western blot were used to validate YAP1 knockdown efficiency in HUVECs at 24 h. (C) Western blots were used to validate YAP1 knockdown efficiency in HUVECs at 48 h. NC, negative control. (D and E) Proliferation assay of HUVECs treated with VP (D) or transfected siYAP1 (E) via CCK-8 detection. (F and G) Transwell migration (F) and Matrigel capillary tube formation (G) assays of HUVECs when incubated with VP. (H and I) Transwell migration (H) and Matrigel capillary tube formation (I) assays of HUVECs when transfected with siYAP1. The average number of migrated cells and the number of capillary tubes were counted with ImageJ software. (J and K) Tumors were measured and tumor volume was calculated at the indicated time points. At the end of the experiment, tumors were excised and imaged (n = 8). (L) Tumoral expression of YAP1 and CD31 was detected by IHC. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. #, not significant.

We found that vascular endothelial cell proliferation, migration ability, and angiogenic ability were inhibited after treating with VP or transfecting with siYAP1 (Figures 2D–2I). Moreover, our results also confirmed that activated YAP1 increased vascular endothelial cell proliferation, migration, and angiogenic ability (Figure S2). We further observed the effect of VP on the HCC xenograft model. Tumors from VP-treated mice were significantly smaller (Figures 2J and 2K). Immunohistochemical analysis of tumor sections showed that VP decreased the expression of YAP1 and CD31 (Figure 2L). Also, the expression of YAP1 shifted from the nucleus to the cytoplasm. Taken together, these findings reveal that YAP1 deletion can inhibit angiogenesis in vitro and in vivo.

MALAT1 Is Modulated by YAP1 Deficiency in HUVECs

Tumor angiogenesis is the result of multiple interacting factors., GSEA results showed that there was a positive enrichment of the ncRNA term in the high-risk group (the data came from HUVECs with YAP siRNA knockdown; GEO: GSE61989) (Figure 3A). We examined whether the role of YAP1 in angiogenesis is related to lncRNAs. Using GEO data, we analyzed 100 lncRNAs with the most significant changes (GEO: GSE61989) (Figure 3B). Furthermore, we analyzed the top 20 significantly changed genes by quantitative real-time PCR. Our results identified lncRNA MALAT1 as having the most significant change after transfecting with siYAP1 (Figure 3C). To clarify the role of MALAT1 in vascular endothelial cells, we knocked down MALAT1 expression using siRNAs (Figure 3D). We found that HUVEC proliferation, migration ability, and tube formation were significantly inhibited after MALAT1 knockdown compared to control cells (Figures 3E–3G).

Figure 3

MALAT1 Is Modulated during YAP1 Deficiency in HUVECs

(A) GSEA results show that there was a positive enrichment in ncRNA-related terms in the high-risk group. (B) Heatmap showing the relative expression of 100 lncRNAs with the most significant changes according to YAP1 expression. Genes shown in red are upregulated, and those shown in blue are downregulated. (C) Real-time PCR was used to validate the expression levels of the lncRNAs with YAP1 knockdown in endothelial cells. 18S was used as the internal control. (D) HUVECs were transfected with NC or MALAT1 siRNAs, and real-time PCR was used to validate MALAT1 knockdown efficiency. (E) Cell viability was determined by a CCK-8 assay 24, 48, and 72 h after si-MALAT1 knockdown. (F and G) Transwell migration assay and Matrigel capillary tube formation were used to detect HUVEC migration (F) and tube formation (G) after MALAT1 knockdown. (H and I) Transwell migration assay and Matrigel capillary tube formation were used to detect HUVEC migration (H) and tube formation (I) after MALAT1 knockdown and then transfecting YAP5SA. The average number of migrated cells and number of capillary tubes were counted with ImageJ software. ∗p < 0.05, ∗∗p < 0.01. #, not significant.

To further clarify that the effect of YAP1 on vascular endothelial cells was mediated via MALAT1, we knocked down MALAT1 expression in HUVECs and then performed YAP1 activation by transfection with YAP5SA. Our results revealed that MALAT1-mediated YAP1 activation induced the promotion of proliferation, migration, and tube formation, suggesting that the inhibition of vascular endothelial cells by YAP1 may be mediated by MALAT1 (Figures 3H and 3I).

YAP1 Deficiency Increases the Number of Exosomes Released from HUVECs and Promotes HCC Invasion and Migration

Whether anti-tumor angiotherapy using YAP1 as a therapeutic target may influence hepatoma cells is worthy of further study. A Cell Counting Kit-8 (CCK-8) assay showed that the conditioned medium (CM) (derived from HUVECs treated with VP) had no significant effect on hepatoma cell proliferation (Figures 4A and 4B). However, a transwell assay indicated that the CM could significantly promote cell invasion and migration (Figures 4C and 4D). We also found that the expression of MMP2 and MMP9 was significantly increased (Figure 4E).

Figure 4

YAP1 Deficiency Increases the Number of EVs Released from ECs and Promotes HCC Invasion and Migration

(A and B) The proliferation of Hep3B (A) and Huh-7 cells (B) cultured with CM from HUVECs for 24, 48, and 72 h. (C and D) The migration and invasion of Hep3B (C) and Huh-7 (D) cells cultured with CM from HUVECs were assessed by a transwell assay. (E) Expression of MMP2 and MMP9 of Hep3B (left) and Huh-7 (right) cells cultured with CM. (F) GSEA results showed that there was an enrichment of EXOCYTOSIS_VESICLE terms. (G and H) Representative image of NanoSight analysis of the size distributions and number of exosomes derived from YAP1-deficient HUVECs. (I) YAP1 deficiency-derived exosomes were analyzed for CD63 and CD81 by western blot (left). Transmission electron microscopy images of exosomes (right). (J and K) The migration and invasion of Hep3B (J) and Huh-7 (K) cells cultured with CM from HUVECs treated with VP, CM+RNase A (2 μg/mL), or CM+RNase A combined with Triton X-100 (0.1%) were assessed by a transwell assay. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, not significant.

To find how YAP1 in vascular endothelial cells promotes the invasion and metastasis of hepatoma cells, we used GSEA to find the possible mechanism (GEO: GSE61989). GSEA results showed that there was a positive enrichment of EXOCYTOSIS_VESICLE terms after YAP1 deficiency (Figure 4F). We purified exosomes from the CM treated with VP and analyzed them by nanoparticle tracking analysis (NTA). Exosome-like vesicles were found to be cup-shaped and were 125.2–127.5 nm in diameter, which are characteristic exosome morphologies. NTA results also suggested that exosomes released from vascular endothelial cells are significantly elevated when treating with VP (Figures 4G and 4H). Moreover, western blot (WB) and electron microscopy of purified exosomes further confirmed the release of exosomes (Figure 4I). In order to evaluate whether exosomes released could be internalized to act as paracrine mediators, exosomes labeled with PKH67 dye were assayed. Figure S3 showed that exosomes could be incorporated by Hep3B and Huh-7 cells.

To find the mechanism promoting hepatoma cell invasion by exosomal release, we treated the CM derived from YAP1 deficiency with RNase A or RNase A and Triton X-100, respectively. Triton X-100 lysates the vesicles, and RNase A hydrolyzes the released exosomal RNA. Thus, the RNAs in exosomes would not be able to enter the cells to exert their actions. We found that the effect of the CM on hepatoma cell invasion and migration was not significantly changed after RNase A treatment alone, indicating that extracellular RNAs were mainly wrapped by membrane instead of being directly released. However, when the CM derived from YAP1 deficiency was treated with RNase A and Triton X-100, the capacity to promote invasion and migration was reduced (Figures 4J and 4K). These results suggested that loss of YAP1 in HUVECs could promote the release of exosomes, and the exosomes could significantly promote the invasion and migration of hepatoma cells via RNAs.

YAP1 Deficiency Leads to Exosomal MALAT1 Accumulation and Promotes the Expression of MALAT1 in HCC

We have confirmed that the expression of MALAT1 is significantly reduced in HUVECs after YAP1 depletion. We therefore evaluated whether YAP1 deficiency in vascular endothelial cells promotes the release of exosomes carrying MALAT1. We extracted exosomal RNA and detected MALAT1 expression. Our results confirmed that the expression of MALAT1 was significantly increased in exosomes (Figure 5A). We also confirmed that exosomes released from vascular endothelial cells after YAP1 deficiency could promote the expression of MALAT1 in hepatoma cells (Figures 5B and 5C).

Figure 5

YAP1 Deficiency Leads to Exosomal MALAT1 Accumulation and Promotes the Expression of MALAT1 in Hepatoma Cells

(A) Real-time PCR analysis of MALAT1 expression in exosomes isolated from the CM of YAP1-deficient HUVECs. Cells treated with DMSO acted as the control. (B and C) Real-time PCR analysis of MALAT1 in Hep3B (B) and Huh-7 (C) cells 24 h after incubation with exosomes derived from cells treated with VP. Exosomes extracted from DMSO-treated cells served as controls. (D and E) Nascent RNA was labeled and isolated, and newly synthesized MALAT1 was analyzed using real-time PCR in Hep3B (D) and Huh-7 (E) cells after incubation with exosomes derived from cells treated with VP. Exosomes extracted from DMSO-treated cells served as controls. (F) Real-time PCR analysis of MALAT1 in exosomes and whole CM derived from YAP1-deficient HUVECs. The expression of MALAT1 in CM acted as the control. (G and H) Exosomes isolated from CM of MALAT1-deficient HUVECs+VP or HUVECs+VP were added to Hep3B (G) and Huh-7 (H) cells for 24 h, and a transwell assay was used to detect invasion and migration. (I–K) CCK-8 (I), migration (J), and tube formation (K) assays showed that GW4869 (10 μM) inhibition of the release of exosomes could not be reversed by the inhibitory effect of VP on endothelial cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. #, not significant.

To determine whether YAP1 deficiency-released exosomes promote MALAT1 expression in HCC cells via direct transfer or by promoting new gene expression, we used a Click-iT nascent RNA capture system. We found that exosomes did not affect transcription of MALAT1 in HCC cells (Figures 5D and 5E). We also found that the level of MALAT1 in exosomes is almost equal to that of total CM, further suggesting that exosome-induced elevation of MALAT1 expression in hepatoma cells was mediated by exosomal transfer of MALAT1 (Figure 5F).

We also confirmed that in MALAT1-deficient endothelial cells, exosomes released after YAP1 deficiency had diminished capacity to promote HCC invasion and metastasis (Figures 5G and 5H). These results suggested that exosomes released after YAP1 deficiency promote HCC cell invasion and metastasis via MALAT1. To confirm that the effect of VP on endothelial cells depends on an autocrine exosome, a GW4869 study was performed. Our results found that the inhibitory effect of VP on endothelial cells could not be reversed by inhibiting exosome release (Figures 5I–5K). The results indicated that the effect of VP on endothelial cells was independent of exosomes released.

MALAT1 Levels in Tumor Tissues Correlate with Clinicopathological Parameters

To evaluate the role of MALAT1 in HCC, we analyzed the expression and relationship with clinicopathological parameters. We found that the expression of MALAT1 is significantly associated with TNM (tumor, node, metastasis), differentiation, portal vein infiltration, and lymph node metastasis (Table 1). We confirmed a positive correlation between MALAT1 and YAP1 expression (R = 0.44, p < 0.001). Additionally, the expression of MALAT1 was significantly associated with disease-free survival (DFS) in patients and metastasis, but not with overall survival (OS) (Figures S4A–S4C). By transfecting siMALAT1, we found that MALAT1 deficiency could significantly inhibit cell invasion and migration (Figures S4D and S4E).

Table 1

The Relationship between Expression of MALAT1 and Clinicopathological Parameters in Hepatomas

	n	MALAT1		p
		Low	High
Age (years)				>0.05
>45	37	15	22
<45	45	25	20
Sex				>0.05
Male	50	21	29
Female	32	19	13
Stage of TNM				<0.05
I+II	50	33	17
III+IV	32	7	25
Tumor (cm)				>0.05
>5	27	13	14
<5	55	27	28
Degree of differentiation				<0.05
Well+moderate	50	33	17
Poor	32	7	25
Lymph node metastasis				<0.05
Yes	28	5	23
No	54	35	19
Portal vein invasion				<0.05
Yes	31	6	25
No	51	34	17

VAMP3 Is Required for YAP1-Regulated Exocytosis of MALAT1-Containing Exosomes

Previous studies have identified proteins that mediate regulated exocytosis in endothelial cells, including vesicle-associated membrane protein 3 (VAMP3), VAMP8, syntaxin 4, syntaxin-binding protein 5 (STXBP5), and synaptosomal-associated protein 23 (SNAP23). Most of these factors belong to a superfamily of transmembrane proteins named soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). We therefore examined whether YAP1-mediated exocytic release is mediated by SNAREs. Our results confirmed that VAMP3 protein expression was significantly increased when YAP1 was depleted from vascular endothelial cells (Figures 6A and 6B). The expression of VAMP3 in control cells was mainly concentrated around the nucleus, but became dispersed after YAP1 depletion (Figures 6C and 6D). We knocked down VAMP3 expression in vascular endothelial cells (Figures 6E and 6F). We found that VAMP3 deficiency could significantly inhibit the release of exosomes, but it could not inhibit the expression of MALAT1 in exosomes (Figures 6G and 6H). To determine whether VAMP3 is required for YAP1-regulated exocytosis, we treated the VAMP3-deficient cells with VP. The expression of MALAT1 in exosomes from VAMP3 knockdown cells did not differ significantly (Figure 6H).

Figure 6

VAMP3 Is Required for YAP1-Regulated Exocytosis of MALAT1-Containing Exosomes

(A and B) Western blot was used to assess VAMP3, VAMP8, syntaxin 4, and STXBP5 protein levels in HUVECs treated with VP (A) or transfected siYAP1 (B). The protein levels were quantified by normalizing to β-actin. (C and D) Confocal microscopy confirmed the subcellular localizations of VAMP3 becoming dispersed in HUVECs treated with VP (C) or transfected siYAP1 (D). (E) Real-time PCR was used to validate VAMP3 knockdown efficiency in HUVECs transfected with VAMP3 siRNAs. 18S was used as the internal control. (F) Western blot was used to validate VAMP3 knockdown efficiency in HUVECs transfected with VAMP3 special siRNAs. (G) NanoSight analysis of the size distributions and number of exosomes derived from VAMP3-deficient HUVECs. (H) Real-time PCR analysis of MALAT1 in exosomes derived from VAMP3 knockdown HUVECS and then incubated with VP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. #, not significant.

Exosomes Released by YAP1 Deficiency in HUVECs Promote Invasion and Migration of HCC Cells through ERK1/2

Previous studies have shown that MALAT1 promotes invasion and metastasis through a variety of signaling pathways. By GSEA, we found that the ERK1/2 signaling pathway is a potential target in HCC (Figure 7A). We also found that the expression of phosphorylated (p-)ERK1/2 was rapidly increased, with highest expression levels at 15 min after exosome induction. The expression of p-ERK1/2 gradually decreased over time, while the expression of total ERK1/2 was not significantly changed (Figure 7B). As shown in Figure 7C, PD98059 inhibited p-ERK1/2 in Hep3B and Huh-7 cells. The effect of exosomes on HCC invasion was inhibited when ERK1/2 activity was inhibited by PD98059 (Figure S5; Figure 7D). These results confirm that exosomes released by depletion of YAP1 in HUVECs promote invasion and metastasis of hepatoma cells via ERK1/2 signaling.

Figure 7

Exosomes Released by YAP1 Deficiency in HUVECs Promote Invasion and Migration of Hepatocellular Carcinoma Cells through ERK1/2

(A) GSEA results showing that “ERK pathway” signatures were enriched whereas the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, WNT pathway, cyclic AMP response element binding protein (CREB) pathway, P53 pathway, and nuclear factor κB (NFKB) pathway were not enriched in hepatocellular carcinoma generated from TCGA and categorized into subgroups according to their median YAP1 expression. (B) Western blot analysis of p-ERK1/2 and total-ERK1/2 (T-ERK1/2) protein expression levels after incubation with exosomes derived from cells treated with VP at different times. (C) The ERK1/2 inhibitor PD98059 inhibited ERK1/2 phosphorylation in Hep3B and Huh-7 cells. Hep3B and Huh-7 cells were treated with PD98059 (10 μM) for 60 min. (D) The expression levels of MMP2 and MMP9 of Hep3B and Huh-7 cells cultured with exosomes and exosomes+PD98059 were assessed by western blot. Cells were pretreated with PD98059 for 60 min and then treated with exosomes for 24 h. ∗p < 0.05, ∗∗p < 0.01. #, not significant.

Discussion

YAP1 is a critical nuclear transcription factor downstream of the Hippo pathway, and it has a significant role in tumorigenesis and the tumor microenvironment. In this study, we confirmed that the expression of YAP1 in HCC tissues is significantly higher than in the adjacent tissues, suggesting that YAP1 is involved in the progression of an early event during development. We also found that the expression of YAP1 in HCC is positively correlated with angiogenesis-related factors. By analyzing the GEO database and TCGA database, the results suggested that YAP1 expression is closely related to processes of angiogenesis. Angiogenesis is a complex process, but the most important features are functional changes in endothelial cells, in particular proliferation and migration, which lead to the formation of new blood vessel branches and capillary networks. Our results confirmed that YAP1 deletion can inhibit angiogenesis in in vitro and in vivo. These results suggest that YAP1 may be a target for anti-tumor angiogenesis therapy.

By GSEA, we found that there was a positive enrichment of the ncRNA term. We hypothesized that the mechanism of action of VP against tumor angiogenesis involves lncRNAs. To test this hypothesis, we analyzed the GEO database and used quantitative real-time PCR, and we found that lncRNA MALAT1 expression was most significantly changed. MALAT1 is expressed in a variety of tumor tissues as well as normal tissues, mediating a variety of pathophysiological processes. In particular, study has confirmed the significant role of MALAT1 in angiogenesis. Our results confirmed that the deficiency of MALAT1 could effectively inhibit angiogenesis and the anti-angiogenic effect of VP achieved through MALAT1. Elucidating the molecular basis of angiogenesis could rationally design therapies to block compensatory signaling pathways. Our results demonstrated that a YAP1/MALAT1-targeted therapy could be used as a promising therapeutic strategy for anti-angiogenic therapy.

In previous studies and clinical trials, some patients who received anti-tumor angiogenesis therapy experienced unstable effects in late stages of treatment. To address whether anti-tumor therapy targeting YAP1 is feasible, we administered VP or siYAP1 to vascular endothelial cells and collected CM to examine its effect on hepatocarcinoma cells. Our results showed that targeting YAP1 for anti-angiogenesis treatment promotes HCC invasion and the expression of MMP2 and MMP9. Most MMPs are influenced by chemical agents, physical stress, oncogene products, growth factors, cytokines, and cell-cell or cell-extracellular matrix (ECM) interactions. In our study, we confirmed that exosomes may be one of the new factors that influenced the expression of MMPs. MMPs during cancer progression have been identified, and a correlation among their overexpression with an increase of the disease aggressiveness has been established; these findings identified MMPs as probable prognostic markers and therapeutic targets. Clarifying the mechanism by which YAP1 deficiency in CM promotes the invasion and metastasis of HCC cells may allow effective inhibition of tumor proliferation and angiogenesis while avoiding distant metastasis of tumor cells. By GSEA, we found there was a positive enrichment the EXOCYTOSIS_VESICLE term. Studies have also confirmed that exosomes have become vital mediators of cellular communication between cells within the tumor microenvironment., We hypothesized that the pro-invasive effect of YAP1 deficiency in vascular endothelial cells on HCC cells is mediated by exosomes. Our results found that YAP1-deficient vascular endothelial cell-released exosomes significantly increased. Previous studies have confirmed that lncRNAs are present in exosomes and account for 20.19% of exosomal RNA extracted from the plasma., We confirmed that MALAT1 carried by exosomes can promote the invasion and metastasis of hepatoma cells during anti-angiogenic treatment with YAP1 depletion. Additionally, we confirmed that the effect of VP on endothelial cells was independent of exosome release. Analysis of the relationship between the expression of MALAT1 and clinicopathological parameters suggested that MALAT1 is closely related to TNM, differentiation, lymph node metastasis, and portal vein infiltration. By GEPIA, we confirmed that the expression of MALAT1 is significantly associated with DFS in patients. Thus, our demonstration of YAP1-depleted export of MALAT1 in exosomes might provide a mechanism for eliminating MALAT1, and the expression of MALAT1 may be a prognostic indicator for HCC patients.

We also analyzed HCC samples in TCGA database by GSEA and found enrichment for genes associated with ERK1/2 signaling. We confirmed that exosomes can activate ERK1/2 to promote MMP2 and MMP9 in hepatocarcinoma cells. ERK1/2 plays central roles in cancer development. Also, ERK inhibition has been shown to be effective in cancers. Several ERK inhibitors, including GDC-0994, LTT462, and BVD-523, are in various stages of clinical development.26, 27, 28 Our results reinforce the concept that adopting combined therapies against ERK1/2 may overcome the invasion and metastasis induced by YAP1 deficiency. Certainly, the precision and specificity need to be determined through large-scale clinical trials.

Finally, we examined the mechanism by which YAP1 deficiency promotes exocytic release. Exosomes originated from the endosomal system and released via the fusion of MVBs with the cell membrane. However, the biogenesis mechanisms of exosomes are different. In endothelial cells, the key factors that mediate exocytosis belong to SNAREs.17, 18, 19 During membrane fusion, SNARE can form a complex that mediates granule exocytosis. VAMP3 are localized predominantly to the plasma membrane to participate in exocytotic processes., We confirmed that the process by which YAP1 depletion promotes the release of vascular endothelial cell exosomes is dependent on VAMP3 by promoting MVB fusion with the plasma membrane.

In clinical work, there is no drug that targets YAP1 currently. Therefore, the impact of VP on HCC cells needs to be considered during the treatment of HCC by VP. This will be studied in depth in our future work. In this study, we not only provide targets for antivascular therapy, but we also discovered the important role of exosomes in antivascular therapy, providing new experimental evidence for the development of antivascular drugs.

Collectively, our data demonstrate that vascular endothelial cell YAP1 deficiency promotes the release of MALAT1-containing exosomes and effectively inhibits angiogenesis. Released exosomes promote the distant migration of hepatoma tumor cells by activating the ERK1/2 pathway; accordingly, ERK1/2 inhibitors can reverse the metastasis-promoting effect of YAP1 deficiency. At present, an ERK1/2 small-molecule inhibitor and the YAP1 inhibitor VP have already entered the clinical trial stage. Our results suggest that the combination of VP and ERK1/2 inhibitor may be an effective new approach for anti-angiogenesis therapy.

Materials and Methods

Patients

The RNA sequencing (RNA-seq) data were obtained from TCGA database. GEPIA was used to analyze the correlation between different genes. Eighty-two tumor tissues and adjacent noncancerous tissues from patients were used for quantitative real-time PCR and western blot analysis. All patients had been diagnosed with HCC by pathological examination. No patients had received chemotherapy or radiotherapy prior to surgery. The China Medical University Ethics Committee approved this study.

Cell Culture

HUVECs, Huh-7 cells, and Hep3B cells were obtained from Shanghai Institute of Cell Bank (Shanghai, China). HUVEC and Huh-7 cell lines were cultured in DMEM medium (Biological Industries, Shanghai, China) with 10% fetal bovine serum (FBS) (Biological Industries). Hep3B cell lines were cultured in minimum essential medium (MEM) (Gibco, Shanghai, China) with 10% FBS (Biological Industries). Cells were maintained at 37°C in a 5% CO2 atmosphere. All cell lines were authenticated by short tandem repeat (STR) profiling.

Immunohistochemistry (IHC)

IHC was performed as previously described.

RNA Isolation and Quantitative Real-Time PCR

RNA isolation and quantitative real-time PCR were performed as previously described. Primers are shown in Table S1.

Western Blot

Western blot analysis was performed as previously described. The details of antibodies used in this study are shown in Table S2.

Cell Proliferation Assay

A CCK-8 assay was performed as previously described.

Migration and Invasion

We assessed HCC cell migration and invasive capability using 24-well transwell plates with or without Matrigel (BD Biosciences), respectively.

Tube Formation (In Vitro Angiogenesis Assay)

Briefly, 24-well culture plates were coated with 200 μL of Matrigel (BD Biosciences, USA) at 37°C for 30 min. Then, 8 × 104 HUVECs were plated into the 24-well plates and cultured for 6 h. Five randomly chosen fields were imaged, and the number of tubular structures were quantified using ImageJ. The experiments were repeated at least in triplicate.

Tumorigenicity Assay In Vivo

A tumorigenicity assay was performed as previously described. BALB/c nude mice (4 to 5 weeks old, weighting 21–23 g) were obtained from Beijing Vital River Laboratory Animal Technology. The mice were raised in the Department of Laboratory Animal Science at China Medical University. The study was approved by the Animal Ethics Committee of China Medical University and all experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. To evaluate cancer cell proliferation in vivo, the Huh-7 cells (107 cells in 100 μL of PBS) were inoculated into the axilla of mice after a week of acclimatization. When tumors reached a mean volume of 70–150 mm3, the animals were randomized to two groups. The mice in the VP group were intraperitoneally injected with VP (100 mg/kg body weight) every 2 days whereas the control mice received an intraperitoneal injection with PBS.

CM Collection

HUVECs were plated on dishes at identical densities and in the same volumes of medium. After culture for 24 h, the media were replaced with media containing 5 μM VP. After 12 h, media were replaced with an equal volume of low-serum medium (containing 2% exosome-depleted FBS), and the cells were left to culture for another 24 h. At the end of this time point, the media were collected.

Isolation and Quantification of HUVEC-Derived Exosomes

Exosomes were prepared from culture supernatant after stimulation with VP for 24 h. Culture supernatants were centrifuged (Beckman XPN-100) at 2,000 × g for 10 min and then 1,000 × g for 30 min to remove the debris. Then, the supernatant was centrifuged at 100,000 × g for 70 min. The pellet was re-suspended and washed in PBS, and the supernatant was centrifuged at 100,000 g for 70 min again. Finally, the exosomes were collected.

The number and size of exosomes were directly tracked by the rate of Brownian motion of exosomes using the NanoSight NS 300 system (NanoSight Technology, Malvern, UK). The samples were diluted with Dulbecco’s PBS (DPBS) without any nanoparticles to attain a concentration of 1–20 × 108 particles per milliliter. Exosome numbers and size distribution were explored by the Stokes-Einstein equation. Each sample was measured in triplicate.

Electron Microscopy

Electron microscopy analysis was performed as previously described.

Label and Capture of Nascent RNA

The Click-iT nascent RNA capture kit (Invitrogen) was used to label and isolate newly synthesized RNA. In brief, cells were incubated with 0.2 mM 5-ethymyl uridine for labeling nascent RNA. Total RNA was isolated using TRIzol reagent, and the 5-ethymyl uridine-labeled nascent RNA was biotinylated in Click-iT reaction buffer with 0.5 mM of biotin azide.

Online Microarray Data Availability and Bioinformatics Analysis

The online available microarray datasets GEO: GSE61989, GSE73396, and GSE35004 were downloaded from the NCBI GEO database. The Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 (http://david.abcc.ncifcrf.gov/) and enriched gene ontology (GO) terms were used to analyze the biological functions. GSEA (https://www.broadinstitute.org/gsea/index.jsp) was performed to explore the enrichment of specific gene sets.

Statistical Analysis

All data were analyzed using SPSS 17.0. Comparisons between two groups were conducted using a two-tailed Student’s t test or Mann-Whitney test, and ANOVA with Tukey’s post hoc tests were performed for statistical significance among multiple groups. p < 0.05 was defined as statistically significant.

Author Contributions

Y.L. performed the experiments. X.Z. wrote the manuscript. Y.M. and Q.W. cultured the cells. L.Y. collected the HCC tissues and adjacent normal tissues. Y.Z. performed the IHC and immunofluorescence analysis (IFC). C.Z. performed the bioinformatics analysis. X.P. performed electron microscopy and isolation and quantification of exosomes. Q.Z., B.W., X.M., H.L., and J.L. conceived the study and participated in its design. All authors read and approved the final manuscript.

Conflicts of Interest

No conflicts of interest are reported.