Low FBXW7 expression reflected poor prognosis in ESCC patients.
Previously, we performed WGS/WES in 104 ESCC cases, and identified 8 significant mutated genes (SMGs) (FDR < 0.178, p < 0.0001). Of them, the mutation frequency of FBXW7 was 7.7% (8/104), covering nonsense mutations 2/8 (25%), insertion/deletion 1/8 (12.5%), and missense mutations 5/8 (62.5%)(Fig. 1A)[18, 19]. In addition, the RNA-seq results from 154 ESCC cases showed that the FBXW7 mRNA level in tumor tissues were significantly lower than those in adjacent normal tissues (Fig. 1B).
In order to clarify the clinical significance of FBXW7 in ESCC, rank sum and Chi-square (χ2) tests were applied in the ESCC cohort (95 cases) from TCGA database. According to the expression of FBXW7 and the survival information of ESCC patients, we drew the ROC curve and determined the critical value of FBXW7 expression (cutoff = 324.78), and divided ESCC patients into low expression group (≤ 324.78) and high expression group (> 324.78). Then the correlation between FBXW7 expression and ESCC clinical variables was analyzed, and it was found that FBXW7 expression was correlated with survival status (p = 0.031) (Table 1). And survival analysis showed that patients with low FBXW7 expression in ESCC had a worse prognosis than those with high FBXW7 expression (p < 0.05, Fig. 1C). In addition, further stratified analysis showed that the prognosis of ESCC patients with low FBXW7 expression was poor in patients ≤ 55 years old, without drinking history and T3 stage (p < 0.05, Fig. 1D). These results suggested that the low expression of FBXW7 may be an independent prognostic factor for poor prognosis of ESCC patients.
Table 1
Correlation analysis between FBXW7 mRNA levels in ESCC and clinicopathological variables
Characteristic | Total | FBXW7 expression [n%] | Chi-square | test |
| (n = 95) | Low(T ≤ 324.7751) n = 23 | Hight(T > 324.7755) n = 72 | χ2 | P-value |
Age | | | | | |
< 55 | 40 | 11(27.500) | 29(72.500) | 0.407 | 0.523 |
≥ 55 | 55 | 12(21.818) | 43(78.182) | | |
Gender | | | | | |
male | 81 | 21(25.926) | 60(74.074) | 0.361 | 0.548 |
female | 14 | 2(14.286) | 12(85.714) | | |
Smoking | | | | | |
No | 11 | 6(54.545) | 5(45.455) | 0.451 | 0.798 |
Yes | 84 | 57(67.857) | 27(32.143) | |
Drinking | | | | | |
No | 69 | 16(23.188) | 53(76.812) | 0.342 | 0.559 |
Yes | 26 | 9(34.615) | 17(65.385) | |
TNM Stage | | | | | |
Ⅰ+Ⅱ | 62 | 16(23.188) | 46(74.194) | 0.116 | 0.734 |
Ⅲ+Ⅳ | 33 | 9(27.273) | 24(72.727) | |
T stage | | | | | |
1 + 2 | 39 | 10(25.641) | 29(74.359) | 0.030 | 0.863 |
3 | 56 | 15(26.786) | 41(73.214) | |
Prognosis | | | | | |
survival | 63 | 11(17.460) | 52(82.540) | 4.644 | 0.031 |
dead | 32 | 2(37.500) | 20(62.500) | | |
*p < 0.05 |
FBXW7 inhibited cell proliferation, cell migration and cell invasion, as well as promoted apoptosis in ESCC
We detected FBXW7 mRNA expression level in nine ESCC cell lines (Fig. 2A) and found that KYSE450 and TE5 with low endogenous FBXW7 mRNA expression. Therefore, we transiently transfected the pcDNA3-FBXW7wt plasmid into KYSE450 and TE5 cells respectively to explore the gene function. The over-expression efficiency was confirmed by qPCR(p < 0.05, Fig. 2B). We observed that FBXW7 overexpression significantly inhibited the proliferation of KYSE450 and TE5 cells by MTT and colony formation assays(p < 0.05, Fig. 2C,D). Moreover, the results of transwell experiments showed that FBXW7 overexpression was able to inhibit the invasion and migration ability of ESCC cells (p < 0.05, Fig. 2E,F). And, the apoptosis of FBXW7 overexpressed cells was detected by Annexin V-FITC/PI double staining using flow cytometry, and it was found that the number of apoptosis cells significantly increased (Fig. 2G). These results indicated that FBXW7 may be a tumor suppressor gene in ESCC development.
Altered pathways and gene-interaction networks affected by FBXW7 over-expressed in ESCC cells
On the basis of clarifying the function of FBXW7, we performed PCR array experiments with FBXW7wt and control cells to explore the mechanism of FBXW7 in ESCC. The results showed that there were 16 genes whose expression changes were more than two times, namely ANGPT1, SERPINB2, SOD1, DDB2, DDIT3, ERCC3, ERCC5, POLB and PPP1R15A, FLT1, KDR, PGF, TEK, BMI1, IGFBP5 and TBX2. And they were mainly concentrated in angiogenesis, DNA damage repair and cell senescence related signaling pathways (Fig. 3A,B), suggesting that FBXW7 might be related to these pathways in ESCC. And this result was also verified by qPCR experiment (Fig. 3C).
Additionally, we also analyzed the gene interaction network between FBXW7 and these genes using cystoscope software. As shown in Fig. 4A, in the angiogenesis network, in addition to the central FBXW7 gene, there were other core genes, including up-regulated ANGPT1 and down-regulated FLT1, KDR, PGF, and TEK. In the cell senescence network, core genes also include up-regulated SOD1, SERPINB2, and down-regulated IGFBP5(Fig. 4B). In the DNA damage repair network, the core genes also include up-regulated DDIT3, PPP1R15A, POLB, ERCC5, and DDB2(Fig. 4C). Overall, above results showed that FBXW7 may play a tumor suppressor role via angiogenesis, cell senescence, DNA damage and repair signaling pathways in ESCC .
FBXW7 regulated angiogenesis, cell senescence and DNA damage repair pathways by inhibiting tumor stemness in ESCC
It has been reported that cancer stem cells (CSCs)play a crucial role in the regulation of tumor angiogenesis and cell senescence, and they coordinate pathological angiogenesis by secreting angiogenic factors during tumor progression[20]. The stemness marker ALDH1A1 promotes tumor angiogenesis through retinoic acid/HIF-1 α/VEGF signaling in MCF-7 breast cancer cells[21, 22]. Hydrogen reduces cellular senescence by regulating ROS/p53/p21 pathways in the mesenchymal stem cell of the bone marrow[23, 24]. Therefore, we propose the hypothesis that whether the effect of FBXW7 on angiogenesis and cell senescence-related pathways in ESCC cells was related to tumor stemness. We performed vascular mimicry assay to examine the effect of FBXW7 on angiogenesis in FBXW7wt and control cells. The results also showed that compared with the control group, the number of tubular structures in FBXW7wt group was significantly reduced, suggesting that FBXW7 could inhibit angiogenesis in ESCC (Fig. 5A). Furthermore, we collected KYSE450 and TE5 stem cells, and found that compared with the parental cells, the FBXW7 expression was decreased in ESCC stem cells, while the stemness markers ALDH1A and LLF4 were significantly increased (Fig. 5B). In addition, FBXW7wt and control group ESCC cells were also suspended and cultured. The number and size of floating cell sphere were counted under the microscope (more than 50µm was counted as tumor stem cell balls). It was found that compared with the control group, the volume of cell spheres formed in the KYSE450-FBXW7wt group was smaller, and the number of cell spheres formed in the TE5-FBXW7wt group was less(p < 0.05) (Fig. 5C). These results suggested that the negative regulation of FBXW7 on stemness might be a universal mechanism in ESCC.