Identification of SFRP2 in ovarian cancer peritoneal metastasis
In order to study the biological function differences between primary ovarian cancer and peritoneal metastases, we analysed the high-throughput RNAseq data set GSE98281 of primary ovarian cancer and metastases of the greater omentum. In 10 omental metastatic tissues and 10 matched primary tumour tissues, we found that genes were upregulated and genes down-regulated in peritoneal metastases. In Figure1A and 1B, we found that the SFRP2 gene was highly expressed in tissues that were metastasised to the greater omentum, and we performed a paired test analysis on the expression of SFRP2. In Figure 1C, the expression of SFRP2 in the metastatic tissues of the greater omentum was significantly higher than that of the primary tissue (P=0.0166). We then performed GO (Figure 1D) and KEGG (Figure 1E) analysis on the highly expressed genes in the tissues of the omentum metastasis. We found that the gene enrichment analysis of omentum transfer shows that the Wnt signalling pathway was significantly activated. Moreover, we conducted a GSEA (Gene Set Enrichment Analysis) analysis of the Wnt signalling pathway and found that omental metastasis was positively correlated with the Wnt signalling pathway (Figure 1F).
SFRP2 activates the wnt signalling pathway through phosphorylation of GSK3β
Ovarian cancer cell lines were selected for the molecular experiments in order to study the mechanism of SFRP2 upregulating the Wnt signalling pathway. The expression level of SFRP2 was analysed in four typical ovarian cancer cell lines. Figure 2A shows that the OVCAR8 cell line expression level of SFRP2 was the lowest; and in A2780, it was the highest. In subsequent experiments, we chose OVCAR8 and A2780 cell lines for corresponding molecular biology experiments.
We constructed siRNA for A2780 to interfere with expression and transferred the SFRP2 vector to the OVCAR8 cell line with low baseline expression, to see if it increased. siNC and empty vectors were treated as controls, respectively. Based on the results of the previous KEGG analysis, we detected protein expression levels of GSK3β, pGSK3β and β-Catenin in ovarian cell lines. We found that after knocking down SFRP2 in A2780 when the expression level of GSK3β was stable, the level of pGSK3β increased, while the expression of β-Catenin protein decreased (p<0.05). Conversely, after upregulating SFRP2 in OVCAR8, when the expression level of GSK3β was stable, the level of pGSK3β decreased, while the expression of β-Catenin protein increased (p<0.05).
SFRP2 affects the invasion and migration of ovarian cancer cells
Based on the results of this study, we aim to verify that SFRP2 affects the metastasis of ovarian cancer through the β-Catenin/Wnt signalling pathway. We tested the invasion and migration ability of A2780 and OVCAR8 cells. In Figure 2C, the invasion ability of A2780-siSFRP2 was lower than that of A2780-siNC (p<0.01). After OVCAR8 was transfected with SFRP2, the invasion ability was significantly enhanced (p<0.01), Figure 2D. In terms of migration ability, the migration ability of A2780-siSFRP2 was lower than that of A2780-siNC (p<0.05), as shown in Figure 2E. The migration ability of OVCAR8 transfected with SFRP2 was significantly enhanced (p<0.05); this can be seen in Figure 2F.
Analysis of SFRP2 protein expression and survival in tumour tissues
We used immunohistochemistry to detect the expression of SFRP2 and β-Catenin protein in 115 cases of ovarian cancer tissues. As shown in Figure 3A, in the primary samples of ovarian cancer, SFRP2 was mainly expressed in the cytoplasm, and the intensity was low. β-Catenin was mainly expressed in the nucleus. In samples with metastatic ovarian cancer, SFRP2 was expressed more strongly in the cytoplasm, while β-Catenin was more strongly expressed in the nucleus than in the primary samples.
Through the follow-up of these 115 patients with ovarian cancer, we found that the prognosis of patients with high SFRP2 expression was worse than that of patients with low SFRP2 expression, in Figure 3B, log-rank P value = 0.0063.
TCGA database explores SFRP2 and prognostic analysis
In order to verify the results of our study with an external cohort, we used the ovarian cancer data set of TCGA-OV to analyse the survival of patients. We have obtained similar results. Patients with high SFRP2 expression have a worse prognosis than patients with low SFRP2 expression, log-rank P value = 0.019 (Figure 3C).
The relationship between SFRP2 and ovarian cancer metastasis also indicated a poor prognosis. We analysed the infiltration of SFRP2 and various immune cells in the microenvironment of ovarian cancer. We found that SFRP2 expression was negatively correlated with B cell infiltration (P=0.0117, Figure 3D), suggesting that SFRP was related to immunosuppression. SFRP2 correlated positively with tumour-associated macrophages (P=0.220, Figure 3E), and also with neutrophils (P=0.440, Figure 3F).