3.1 The expression of TNFRSF9 in breast cancer is significantly decreased, and related to metastasis and malignant
We conducted qRT-PCR assays to detect the mRNA expression of TNFRSF9 in breast cancer tissues and paracancerous normal tissues collected from 30 patients. As shown in Fig. 1A, the expression of TNFRSF9 in breast cancer tissues was remarkably lower than their nearby non-tumor tissues (***p<0.001). The western blot assay result (Fig. 1B) indicates the TNFRSF9 protein expression’s downregulation in eight matched breast cancer tissues and normal tissues. Low expression of TNFRSF9 mRNA and TNFRSF9 protein was observed in five breast cancer cell lines (Fig. 1C-D, ***p<0.001). We also compared the clinicopathological features between low TNFRSF9 expression patients and high TNFRSF9 expression patients (Table 1). Patients with high TNFRSF9 were less likely to have lymphoid metastasis (p = 0.0007) and lower Malignant Tumors (TNM) stage (p = 0.0016). The above results indicate that dysregulation of TNFRSF9 may have an essential role in breast cancer progression.
Table 1
Association analysis of TNFRSF9 expression and the clinicopathological features in 30 breast cancer patients
Characteristics
|
Case number
|
TNFRSF9 expression
|
P-value
|
Low
(n=20)
|
High
(n=10)
|
Number
|
30
|
20
|
10
|
|
Ages (years)
|
|
|
|
0.5921a
|
≤55
|
19
|
12
|
7
|
|
>55
|
11
|
8
|
3
|
|
Tumor size
|
|
|
|
0.5839a
|
≤2 cm
|
10
|
6
|
4
|
|
>2 cm
|
20
|
14
|
6
|
|
HR (estrogen receptor) status
|
|
|
|
0.6048a
|
Negative
|
14
|
10
|
4
|
|
Positive
|
16
|
10
|
6
|
|
HER2 status
|
|
|
|
0.7945 a
|
Negative
|
17
|
11
|
6
|
|
Positive
|
13
|
9
|
4
|
|
Lymph node metastasis
|
|
|
|
0.0007 a
|
No
|
9
|
2
|
7
|
|
Yes
|
21
|
18
|
3
|
|
TNM stage
|
|
|
|
0.0016a
|
I - II
|
12
|
4
|
8
|
|
III
|
18
|
16
|
2
|
|
a Two-sided chi-squared test. |
3.2 Regulating TNFRSF9 expression can mediate p38 phosphorylation and further affect the expression of PAX6
To further investigate the role of TNFRSF9 in breast cancer, we knockdowned TNFRSF9 through transfecting MCF-7 cells and ZR-75-30 with si-TNFRSF9, as these two cell lines have the lowest TNFRSF9 expression in all the breast cancer cell lines. We also overexpressed TNFRSF9 by transfecting MCF-7 cells and ZR-75-30 with pcDNA3.1-TNFRSF9. qRT-PCR assays were used to determine expression regulation efficiency (Fig. 2A left panel & Fig. 2C left panel, **p<0.01, ***p<0.001).
We first confirmed the relationship between TNFRSF9, p38, and PAX6. The mRNA expression of PAX6 was detected using qRT-PCR assays. In both the MCF-7 cell line (Fig. 2A right panel) and ZR-75-30 cell line (Fig. 2C right panel), the mRNA expression of PAX6 was increased with TNFRSF9 downregulation (**p<0.01, ***p<0.001) and decreased with TNFRSF9 upregulation (**p<0.01). As the phosphorylation status of p38 cannot be determined by mRNA, western blot assays were used to assess p-p38 protein expression. In both the MCF-7 cell line (Fig. 2B) and ZR-75-30 cell line (Fig. 2D), with TNFRSF9 knockdown, TNFRSR9 protein expression decreased, p-p38 protein expression increased, PAX6 protein expression increased. In contrast, p38 protein expression remained not significantly changed compared with the knockdown negative controls (*p<0.05, **p<0.01). In contrast, with TNFRSF9 overexpression, TNFRSR9 protein expression increased, p-P38 protein expression decreased remarkably, PAX6 protein expression decreased while p38 protein expression remained not significantly changed when compared with the overexpression negative controls (*p<0.05, **p<0.01, ***p<0.001). The above results indicate that TNFRSR9 mediates p38 phosphorylation and PAX6 protein expression.
3.3 Dysregulated TNFRSF9 expression affected cell proliferation, invasion, and apoptosis
Next, we investigated the dysregulation effect of TNFRSF9 in tumor development. The CCK-8 assays were performed to exam the cell proliferation. In both the MCF-7 cell line (Fig. 3A) and ZR-75-30 cell line (Fig. 3C), when compared with the negative controls, cell proliferation increased with TNFRSF9 knockdown and decreased with TNFRSF9 overexpression. BrdU assays were also performed to exam the cell proliferation. Similarly, comparing with the negative controls, in both MCF-7 cell line (Fig. 3B) and ZR-75-30 cell line (Fig. 3D), cell proliferation increased with TNFRSF9 knockdown (*p < 0.05) and decreased with TNFRSF9 overexpression (*p < 0.05, **p < 0.01). Transwell assays were performed to investigate the cell invasion ability (Fig. 4A & Fig. 4B). Comparing with the negative control, in both cell lines, knockdown TNFRSF9 enhanced cell invasion ability (***p < 0.001) and overexpress TNFRSF9 impaired cell invasion ability (**p < 0.01). ). The cell apoptosis was examed through flow cytometry in both cell lines (Fig. 4C & Fig. 4D). The apoptotic rate decreased when knockdowned TNFRSF9 (*p < 0.05) and increased when overexpressed TNFRSF9 (***p < 0.001). These results suggest that TNFRSF9 is a suppresser of breast cancer malignancy, and activate TNFRSF9 could suppress breast cancer progression.
3.4 TNFRSF9 mediated PAX6 through p-p38 that inhibiting p38 phosphorylation could restore PAX6 expression from TNFRSF9 knockdown
We found that TNFRSF9 dysregulation affects p38 phosphorylation but not p38 expression previously, so we inhibited p38 phosphorylation to determine the regulation axis. qRT-PCR assays and western blot assays were used to determine the efficiency of the p38 phosphorylation inhibitor (p38 MAPK-IN-1). In both the MCF-7 cell line (Fig. 5A) and ZR-75-30 cell line (Fig. 5C), when knockdowned TNFRSF9 with p38 phosphorylation inhibitor, the downregulated TNFRSF9 mRNA expression was not affected, and the mRNA expression of PAX6 was decreased compared to the negative control level (**p < 0.01, ***p < 0.001). The western blot assays (Fig. 5B & fig. 5D) confirmed that the p38 phosphorylation inhibitor inhibited the protein expression of p-p38 without affecting TNFRSF9 protein expression and restored the PAX6 protein expression to low level as the negative control (*p < 0.05, **p < 0.01, ***p < 0.001).
We further investigated the restoration effect of p38 in tumor development. The CCK-8 assays were performed to exam the cell proliferation. In both the MCF-7 cell line (Fig. 6A) and ZR-75-30 cell line (Fig. 6C), comparing with the TNFRSF9 knockdown group, cell proliferation decreased with p38 phosphorylation inhibitor. BrdU assays were also performed to exam the cell proliferation. Similarly, comparing with the TNFRSF9 knockdown group, in both MCF-7 cell line (Fig. 6B) and ZR-75-30 cell line (Fig. 6D) cell proliferation decreased significantly with p38 phosphorylation inhibitor (**p < 0.01, ***p < 0.001). Transwell assays were performed to investigate the cell invasion ability (Fig. 7A & Fig. 7B). Comparing with the TNFRSF9 knockdown group, in both MCF-7 and ZR-75-30 cell lines, inhibiting p38 phosphorylation impaired cell invasion ability compared to the negative control level (**p < 0.01). The cell apoptosis was examed through flow cytometry in both cell lines (Fig. 7C & Fig. 7D). The apoptotic rate decreased when knockdowned TNFRSF9 (*p < 0.05) and increased when p38 MAPK-IN-1 was given to si-TNFRSF9 transfected cells (***p < 0.001). All these findings suggest that TNFRSF9 suppresses breast cell carcinogenesis through inhibiting p38 phosphorylation and inhibit PAX6 protein expression as a result.
3.5 Overexpression of TNFRSF9 can reduce malignancy in breast cancer cell-induced tumor formation
We further gave a subcutaneous injection of MCF-7 cells transfected with or without pLV-TNFRSF9 to mice to induce tumor formation. Tumors induced by pLV-TNFRSF9 injected MCF-7 cells were significantly smaller in size compared to tumors induced by NC injected MCF-7 cells (Fig. 8A and 8B). Furthermore, the tumors’ pathological slides were stained with H&E (Fig. 8C), Bcel-2 antibody, or TNFRSF9 antibody (Fig. 8D). The H&E image indicated that the pLV-TNFRSF9 injected tumor was less malignant. The immunohistochemical image showed less Bcl-2 expression and more TNFRSF9 expression in the pLV-TNFRSF9 injected tumor. qRT-PCR assays were performed to evaluate the mRNA expression of TNFRSF9 and PAX6 in the tumor tissues (Fig. 8E). In all five pLV-TNFRSF9 injected tumors, the mRNA expression of TNFRSF9 was significantly upregulated (upper panel) and the mRNA expression of PAX6 was significantly downregulated (lower panel) (*p < 0.05, **p < 0.01, ***p < 0.001). The western blot assay result (Fig. 8F) indicates that in eight matched tumor tissues, the protein expression of TNFRSF9 was significantly promoted with pLV-TNFRSF9 injection. In contrast, the protein expression of p-p38 and PAX6 were significantly suppressed, and the protein expression of p38 remains unchanged. Therefore, agonistic TNFRSF9 antibody may be a novel therapy for breast cancer patients.