1. PFKFB3 is up-regulated in gastric cancer tissues
We used RNA-seq data of tumors in the TCGA database, and Timer 2.0 analysis showed that PFKFB3 is differentially expressed in various tumors. PFKFB3 was highly expressed in STAD (gastric adenocarcinoma), LUSC (lung squamous cell carcinoma) tumors and lowly expressed in BLCA, BRCA, KIRP, LIHC, LUAD tumors compared with adjacent normal tissues (Figure 1A).
The UALCAN database was used to examined the expression level of PFKFB3, and the results are shown in (Fig.1B), in contrast to adjacent normal tissues, PFKFB3 is highly expressed in STAD (gastric adenocarcinoma). In addition, PFKFB3 expression levels were analyzed in gastric cancer patients with clinicopathological features (gender, clinical stage, histological type, cancer stage, etc.) (Figure 2A-I). In addition, from HPA (Human Protein Profile) (file://localhost/Fig. http/::www.proteinatlas.org:), the level of PFKFB3 protein expression in gastric cancer pathological section examined was downloaded and analyzed (Figure 1C-E). PFKFB3 was expressed at significantly higher levels in gastric tumor tissues than in paired nonadjacent normal tissues (Figure 1C-E).
2. PFKFB3 promotes the proliferation, migration and invasion of gastric cancer cells
We analyzed the expression levels of PFKFB3 protein in four gastric cancer cells using Western blot (Figure 1F). CCK8 assay was used to detect the proliferation ability of gastric cancer cells, and the results showed that PFKFB3 overexpression enhanced the proliferation ability and colony formation ability of SGC-7901 cells (P < 0.01, Figure 2a,b). PFKFB3 knockdown expression inhibited the proliferation ability and colony formation ability of AGS cells (P < 0.001, Figure 3d).3d).Wound healing assays showed that SGC-7901 cells overexpressing PFKFB3 migrated more cells within 24 h than untreated cells (P < 0.001; Figure 2c),Whereas PFKFB3 knockdown expression showed opposite results in AGS cells (P < 0.01; Figure 2d).In addition, transwell invasion assays showed that when PFKFB3 was overexpressed, the number of cells in SGC-7901 cells passing through the transwell membrane was significantly increased compared with AGS cells with PFKFB3 knockdown expression compared with the control group (P < 0.001; Figure2e,f). These results showed that PFKFB3 overexpression promoted the proliferation, migration and invasion of gastric cancer cells. To observe the effect of PFK15 combined with DDP (cisplatin) on AGS tumor in nude mice. Nude mice were randomly divided into 4 groups, 4 mice in each group: control group, PFK15 treatment group, DDP group, PFK15 combined with DDP group. After 30 days of treatment, mice were sacrificed and subcutaneous tumors were collected, the weight and volume were recorded as shown in Figure 3.The results showed that PFK15/DDP combined with PFKFB3 inhibitor could significantly inhibit tumor growth in nude mice.
3. Prognostic analysis of PFKFB3 expression in gastric cancer patients
We performed a pan-cancer analysis based on the Kaplan-Meier plotter database (Supplementary Table 1) and found that PFKFB3 expression significantly affected the prognosis of cancer patients. The results showed that high PFKFB3 expression was associated with poor prognosis in gastric cancer (Figure 4) (OS HR = 0.78, 95% CI = 0.65 to 0.94, P = 0.0098; PFS HR = 0.75, 95% CI = 0.6 to 0.93, P = 0.0093).In breast cancer, low expression of PFKFB3 was associated with worse breast cancer prognosis (OS HR = 1.28, 95% CI = 1.06 to 1.54, P = 0.011; RFS HR = 1.15, 95% CI = 1.04 to 1.27, P = 0.0074) (Figure 3).The RNA sequencing data of TCGA in GEPIA2 database were also validated, and the relationship between PFKFB3 expression and prognostic value in 33 cases of cancer was analyzed, and it was found that high PFKFB3 expression significantly shortened the prognosis of gastric cancer patients. (Supplementary Figure 1).The relationship of clinical characteristics of gastric cancer patients was analyzed in this database. Overexpression of PFKFB3 was associated with tumor differentiation in gastric cancer patients (P < 0.05).PFKFB3 overexpression was associated with OS and differentiation in gastric cancer patients of different genders (P < 0.05), and high PFKFB3 mRNA expression was associated with poor OS in gastric cancer stage 1-3 patients, but not in stage 4 patients showing a significant correlation (OS HR = 0.72, P = 0.13; PFS HR = 1.33, P = 0.32) (Table 1).This is consistent with our previous study finding that high PFKFB3 expression is associated with poor stage and poor prognosis in patients with lymph node metastasis of gastric cancer[20].We used prognostic protein repertoire to analyze the impact of PFKFB3 expression levels on the prognosis of cancer patients. The relationship between PFKFB3 expression levels and different tumor prognosis is shown in (Supplementary Table 1).
4. The expression level of PFKFB3 is closely related to the level of tumor immune cell infiltration
Tumor infiltrating immune cells are closely related to tumorigenesis, invasion and metastasis, and acted as an important part of the tumor microenvironment [21].TIMER2.0 database was used to investigate the relationship between PFKFB3 expression and infiltrating immune cells in different tumor types. The results showed that PFKFB3 expression was significantly correlated with purity in 33 different tumors, and we found that PFKFB3 expression was significantly correlated with infiltration levels of CD4 + T cells, B cells, CD8 + T cells, neutrophils, and macrophages in different types of tumors (Figure 5 and Supplementary Figure 2). PFKFB3 expression is associated with high infiltration of most immune cell types in gastric cancer.PFKFB3 expression levels were associated with B cells (r = 0.119, P = 2.03e-02), CD4 + T cells (r = 0.25, P = 8.35e-07), CD8 + T cells (r = 0.298, P = 3.26e-09), macrophages (r = 0.244, P = 1.51e-6), and neutrophils (r = 0.407, P = 1.53e-16) in STAD. The level of infiltration was positively correlated. However, in BRCA, PFKFB3 expression was associated with poor prognosis but was statistically negatively correlated with the infiltration levels of B cells (r = -0.12, P = 1.53e-04), CD8 + T cells (r = 0.184, P = 5.08e-09), CD4 + T cells (r = 0.192, P = 9.72e-10), macrophages (r = 0.215, P = 7.70e-12), and neutrophils (r = 0.206, P = 5.40e-11).In LUAD, PFKFB3 expression correlated with B cells (r = 0.148, P = 9.92e-04), CD8 + T cells (r = 0.212, P = 2.13e-06), CD4 + T cells (r = 0.261, P = 3.93e-09), macrophages (r = 0.192, P = 1.80e-05), and neutrophils (r = 0.322, P = 2.26e-13) (Figure 4).These findings not only indicate differences between tumor infiltration status, PFKFB3 expression levels, and prognosis of immune cells in different cancers, but also suggest that aberrant PFKFB3 expression related with immune cell infiltration into tumor tissue.
5.Correlation analysis between PFKFB3 mRNA levels and markers of different immune cell subsets
The correlation between PFKFB3 expression and immune cell infiltration was analyzed using TIMER2.0 and GEPIA2 databases, and the correlation between PFKFB3 expression and immune cell status of gastric cancer infiltration was investigated according to the expression levels of immune marker genes in STAD tissues. Immune cells analyzed in STAD tissues included CD8 + T cells, CD4 + T cells, B cells, tumor-associated macrophages (TAM), monocytes, M1 and M2 macrophages, and neutrophils. In addition, different subsets of T cells, namely T helper 1 (Th1), Th2, T follicular helper (Tfh), Th17, regulatory T cells (Tregs), and depleted T cells, were analyzed. Notably, we found that TAM CD68, IL10, macrophage CD163, NRP1, ITGAM, Treg cell TGFB1, FOXP3, CCR8, STAT5B, and T-cell depletion of HAVCR2 were moderately associated with gastric cancer PFKFB3 (P < 0.001; 0.40 > Cor value ≥ 0.30).Th1 ITGAX, Th2 STAT6 and STAT5A were strongly associated with PFKFB3 in gastric cancer (P < 0.001; Cor value ≥ 0.40) (Table 2, Table 3).These findings suggest that overexpression of PFKFB3 is associated with prognosis in gastric cancer patients and is associated with increased immune infiltration of TAMs, Th1 cells, macrophages, and Treg and T-cell depletion in gastric cancer. Therefore, PFKFB3 may play an important role in immune cell infiltration in gastric cancer patients and may serve as a prognostic biomarker in gastric cancer patients. The specific immune infiltrating cells found in gastric cancer suggest that PFKFB3 plays a crucial role in immune escape from the gastric cancer microenvironment.