3.1 ST2 is highly expressed in gastric cancer tissues, which is associated with poor prognosis
As shown in Fig. 1A, the immunohistochemical staining of gastric cancer tissue and normal gastric epithelial tissue from the HPA database revealed a significantly higher expression of ST2L in gastric tumor samples compared to normal samples. Analysis of TCGA data demonstrated a significant correlation between ST2 expression levels and gastric cancer progression, with an increasing trend observed as the cancer stage advanced (Fig. 1B). Furthermore, Kaplan-Meier analysis indicated a significant association between high IL1RL1 mRNA expression levels and shorter overall survival in patients with gastric cancer (Fig. 1C). Based on the data in the TCGA database, we found that IL1RL1 expression was significantly correlated with Lymph Node Stage, Cancer Metastasis Stage Code, and TNM stage (Table 1). Thus, we speculated that the high expression of IL1RL1 in patients with gastric cancer may be associated with poor prognosis. Given the crucial role of angiogenesis in tumor development, we further investigated the relationship between IL1RL1 expression and angiogenic markers PCAM1 and CD34. The result revealed a significant positive correlation between IL1RL1 expression and these angiogenic markers (Fig. 1D). Our results suggest that elevated ST2 expression in gastric cancer tissues may promote angiogenesis thereby facilitating disease progression.
Table 1
Pearson correlation analysis was used to analyze the correlation between IL1RL1 gene expression level and clinicopathological features. *P < 0.05, **P < 0.01, ***P < 0.001.
Variables | Number of cases | IL1RL1 mRNA expression | X² | P |
low | high |
Sex | Male | 247 | 127 | 120 | 0.046 | 0.831 |
Female | 137 | 72 | 65 |
Diagnosis Age | ≥ 65 | 220 | 117 | 103 | 0.381 | 0.537 |
<65 | 164 | 82 | 82 |
Local Invasion | T2 | 79 | 45 | 34 | 5.933 | 0.115 |
T3 | 174 | 92 | 82 |
T4 | 112 | 49 | 63 |
T1 | 19 | 13 | 6 |
Lymph Node Stage | N0 | 121 | 80 | 41 | 14.98 | 0.005*** |
N2 | 77 | 34 | 43 |
N3 | 80 | 37 | 43 |
NX | 5 | 3 | 2 |
N1 | 101 | 45 | 56 |
Cancer Metastasis Stage Code | MX | 17 | 4 | 13 | 9.547 | 0.008*** |
M0 | 341 | 186 | 155 |
M1 | 26 | 9 | 17 |
TNM stage | Stage I | 55 | 35 | 20 | 11.888 | 0.008*** |
Stage III | 167 | 78 | 89 |
Stage II | 122 | 72 | 50 |
Stage IV | 40 | 14 | 26 |
3.2 ST2L expression is associated with proliferation, migration, and VEGFA-dependent angiogenesis in vitro
ST2 belongs to the interleukin-1 receptor family and mainly presents two isoforms in vivo: transmembrane ST2 (ST2L) and soluble ST2 (sST2). To further explore the role of ST2 in gastric cancer development, we first attempted to detect sST2 secreted by tumor cells by ELISA. However, the amount of sST2 in the cell culture supernatant was too small to reach the detection limit. Therefore, we hypothesized that among the two subtypes of ST2 in gastric cancer cells, the membrane protein ST2L plays a more important role in the progression of gastric cancer. To further investigate the impact of ST2L expression on the biological behavior of tumor cells, gastric cancer cells (BGC-823 and HGC-27) were transfected with scramble/ST2L KD siRNA/pcDNA/ST2L OE plasmid (Fig. 2A and B). The MTT assay revealed that upregulation of ST2L enhanced the proliferation of gastric cancer cells, whereas downregulation of ST2L led to an inhibition in tumor cell viability (Fig. S1A and B). Both wound healing and transwell assays demonstrated that ST2L overexpression significantly increased the migratory capacity of gastric cancer cells, whereas knockdown of ST2L had an inhibitory effect on cell migration (Fig. 2C, D, and S1C). As angiogenesis is a crucial requisite for tumor metastasis, and our findings indicate a significant positive correlation between the expression of ST2L and angiogenesis markers in gastric cancer patients, we aim to investigate the potential impact of ST2L expressed by gastric cancer cells on tumor angiogenesis. Transwell assay was used to examine the effect of different conditioned mediums (CMs) on the migration ability of HUVECs. The results showed that CM prepared by ST2L overexpressed GC cells significantly promoted HUVEC migration, while CM prepared by ST2LKD GC cells inhibited HUVEC migration (Fig. 2E and S1D). Tube formation assays demonstrated that compared with the respective control group, HUVECs cocultured with ST2LOE cell CM had a significant increase in vascular junctions, while the number of junctions in HUVECs cocultured with ST2L KD cell CM decreased (Fig. 2F and S1E). Since VEGFA is a vital factor in tumor angiogenesis, we wondered whether ST2L expression could regulate VEGFA expression and thus affect tumor angiogenesis. Here, we noted that overexpression of ST2L significantly facilitated VEGFA protein levels in both BGC-823 and HGC-27 cells, while ST2L knockdown inhibited VEGFA expression in GC cells (Fig. 2G, H). In summary, these findings suggest that ST2L expressed by gastric cancer cells can regulate angiogenesis through VEGFA.
3.3 ST2L induces gastric cancer angiogenesis in vitro by binding to its ligand IL-33, and this effect can be blocked by sST2
As the sole ligand of ST2L in vivo, IL-33 shows an important effect on inflammatory response and angiogenesis. The STRING database demonstrated the interaction between IL-33 and ST2 (IL1RL1) (Fig. 3A). Consistent with this, analysis of the TCGA database revealed that IL-33 expression was positively correlated with IL1RL1 expression in gastric cancer patients (Fig. 3B). Additionally, survival analysis indicated that gastric cancer patients with higher expression of IL-33 had significantly worse overall survival (Fig. 3C).
To further demonstrate the interaction between IL-33 and ST2L on the surface of gastric cancer cells to mediate gastric cancer angiogenesis, we treated gastric cancer cells with 30ng/ml human recombinant IL-33 and then the CM was collected. HUVECs treated with the above conditioned medium had a significant increase in migration and tube formation ability (Fig. 3D, E and Fig. S2A, B). However, when we knocked down the expression of ST2L 48 hours before adding IL-33, it completely reversed the aforementioned effects (Fig. 3F, G and Fig. S2C, D). Similarly, knockdown of ST2L reversed the IL-33-mediated increase in VEGFA expression in GC cells (Fig. 3J and S2G). This suggests that ST2L receptors on the membrane of gastric cancer cells are able to interact with IL-33 to promote VEGFA-mediated angiogenesis.
Furthermore, soluble ST2 (sST2), acting as an extracellular decoy receptor, can bind to IL-33 and competitively block its interaction with ST2L. We then examined whether the application of sST2 could inhibit IL-33/ST2L axis activation-induced angiogenesis in GC cells. Gastric cancer cells were pretreated with 200ng/ml sST2 for 2 hours before adding 10ng/ml IL-33 to six-well plates for culturing another 24 hours. The addition of sST2 also partially reversed the IL-33-induced HUVECs migration and increased the number of junctions (Fig. 3H, I and S2E, F). sST2 also reversed IL-33-mediated upregulation of VEGFA protein expression in GC cells (Fig. 3K and S2H).
Taken together, the results suggest that ST2L on the GC cell membrane interacts with IL-33 to induce VEGFA-mediated angiogenesis, while sST2 acts as a decoy receptor to block this effect.
3.4 IL-33/ST2L axis regulates the TRAF6-mediated PI3K/Akt/ NF-κB signaling pathway to promote VEGFA-dependent vascular growth in gastric cancer cells
Next, we aimed to investigate which intracellular signaling pathway is activated by the IL-33/ST2L axis to mediate VEGFA-related angiogenesis. Figure 3A displayed a protein-protein interaction between ST2 and TRAF6. Moreover, the IL-33/ST2 axis can activate the PI3K/AKT pathway through TRAF6 to promote inflammation-induced lymphangiogenesis 24. Numerous studies have shown that the PI3K/Akt signaling pathway is involved in VEGF-dependent tumor angiogenesis. Therefore, we hypothesized that the IL-33/ST2L axis may activate the PI3K/AKT pathway through the adaptor molecule TRAF6 to mediate VEGFA-dependent tumor angiogenesis.
Western blot showed that IL-33 could increase the protein level of TRAF6 in GC cells and activate the PI3K/Akt signaling pathway, leading to elevated expression levels of p-PI3K and p-Akt, while knockdown of ST2L or addition of sST2 significantly inhibited TRAF6 expression and phosphorylation levels of PI3K and Akt (Fig. 4A, B and Fig. S3A, B). Akt activation is a key regulator of Iκ-B degradation and NF-κB activation in NF-κB-dependent gene transcription. In addition, studies have found that NF-κB also promotes tumor angiogenesis by upregulating VEGFA, ultimately leading to tumor hematologic metastasis and growth 25. Therefore, we further examined the phosphorylation levels of IκBα and NF-κB. As expected, p-IκBα and p-NF-κB protein levels increased significantly after incubation with IL-33, and this effect could be blocked by ST2L knockdown or sST2 (Fig. 4A, B and Fig. S3A, B). These results suggest that IL-33 activates the PI3K/AKT pathway through ST2L/TRAF6, which further induces phosphorylation of IκBα to promote the activation of NF-κB.
In order to clarify the key role of TRAF6 in connecting the IL-33/ST2L axis with the downstream pathway, we successfully constructed siRNA to knock down TRAF6 expression in gastric cancer cells (Fig. S3C, D). Transwell and tube formation assay showed that compared with the control group (IL-33 + scramble), CM prepared after 48h IL-33 treatment of TRAF6 KD cells could partially reverse the increase of HUVECs’ migration and angiogenesis capacity (Fig. 4C, D and Fig. S3E, F). Similarly, IL-33-induced upregulation of VEGFA expression in GC cells was reversed by TRAF6 knockdown (Fig. 4E and S3G).
To further demonstrate the above signaling pathways in regulating gastric cancer angiogenesis, we conducted transwell and HUVECs tube formation assays. Compared with the group added with CM from ST2L OE cells, the migration rate (Fig. 4F and S3H) and the tube formation (Fig. 4G and S3I) of HUVECs decreased noticeably adding the CM from ST2L OE cells treated with 10mg/ml Triciribine (Akt inhibitor). Similarly, the up-regulation of VEGFA protein induced by ST2LOE can also be reversed by Triciribine (Fig. 4H and S3J).
Taken together, we can conclude that the IL-33/ST2L axis regulates the activation of IκBα and NF-κB through the TRAF6/PI3K/Akt signaling pathway to promote VEGFA-dependent angiogenesis in gastric cancer, while sST2 can competitively bind to IL-33 and reverse the above effects.
3.5 Overexpression of ST2L promotes GC progression in vivo, while sST2 or blocking Akt pathway can slow GC progression in vivo
It has been demonstrated that the IL-33/ST2L axis promotes VEGFA-dependent angiogenesis in GC cells by regulating TRAF6/PI3K/Akt/NF-κB signaling pathway. Next, to further elucidate its role in tumor development in vivo, we established a xenograft tumor model, and the experimental design is shown in Fig. 5A. When tumor volume reached 100mm3, Triciribine(15mg/kg) was injected intraperitoneally twice a week, or sST2(0.1mg/kg) was injected intratumorally twice a week. Tumor volumes were recorded every five days and tumor growth curves were plotted. After 21 days of modeling, the mice were euthanized, and the solid tumors were dissected. The results showed that subcutaneous inoculation of ST2L OE tumor cells resulted in faster tumor growth and larger and heavier solid tumors compared with the control group, which were partially reversed by Triciribine or sST2 (Fig. 5B-D). Intratumoral injection of sST2 slowed tumor growth and reduced tumor burden compared with the control group (Fig. 5B-D). Immunohistochemical staining of tumor tissue also showed that overexpression of ST2L significantly promoted CD34 expression, and administration of Triciribine or sST2 reduced vascular density (Fig. 5E).
3.6 Transcription factors YY1 and GATA2 participate in IL-33-mediated upregulation of ST2L transcription level
Since IL-33 exerts its biological effects mainly by interacting with ST2L and activating downstream signaling pathways, we wondered whether activation of the IL-33/ST2L axis could feedback regulate ST2L expression to further amplify the malignant progression of IL-33/ST2L-mediated angiogenesis in gastric cancer. We detected the effects of IL-33 and downstream Akt signaling on ST2L expression. The result showed that 30ng/ml IL-33 significantly upregulated the mRNA and protein levels of ST2L in BGC-823 and HGC-27 cells, and this effect was partially inhibited by Triciribine (Fig. 6A, B and S4 A). Huang et al. found a dose-dependent increase in ST2L protein levels after IL-33 treatment of AGS and MKN-45 cells, which partially supports our findings 10. To further explore the mechanism of how IL-33 up-regulates the expression of ST2L in GC cells, we identified five transcription factors that may bind to the ST2L promoter site, namely YY1, GATA2, GATA3, GATA4, and TCF4 (Fig. 6C). Here, we noted that YY1 and GATA2 mRNA levels were increased upon IL-33 treatment and downregulated in the presence of Triciribine. (Fig. 6D, E). Further, we verified the YY1 and GATA2 protein levels after being treated with IL-33 and Triciribine, which also showed the same trend (Fig. 6F and S4 B). Consequently, the binding motifs of YY1 and GATA2 were predicted, and JASPAR online tool was used to identify one YY1 binding site between − 1459 bp and − 1448bp, and three GATA2 binding sites located at -1745 ~ -1739bp, -115 ~ -109bp and + 94 ~ + 100bp, respectively (Fig. 6G). CHIP assay was performed and qPCR primers were designed to amplify the ST2L promoter sequence. The result proved that the occupancy of transcription factors YY1 and GATA2 with ST2L promoter sequences was significantly increased in IL-33-treated gastric cancer cells (Fig. 6H and Fig.S4 C, D). Therefore, we can conclude that IL-33 promotes the expression of YY1 and GATA2 through the Akt pathway, which induces increased occupancy of the promoter region of ST2L, thereby promoting the expression of ST2L. We synthesized siRNA to knock down the expression of YY1 and GATA2 respectively (Fig. 6I and S4E). Moreover, knockdown of YY1 and GATA2 in BGC-823 and HGC-27 cells significantly suppressed ST2L protein expression, while simultaneous knockdown of YY1 and GATA2 had a more significant effect on inhibiting ST2L protein expression (Fig. 6J and S4F). Thus, IL-33 can promote ST2L transcription by improving the expression of transcription factors YY1 and GATA2 through the Akt pathway.