2.1 Cell culture

Prostate cancer cells 22RV1, DU145, and PC3 and the normal prostate epithelial cells RWPE-1 in our study were obtained from the National Collection of Authenticated Cell Cultures, Shanghai, China. The cells were incubated in the corresponding medium supplemented with 10% heat-inactivated fetal bovine serum (PAN, Germany) in a humid environment containing 5% CO₂ at 37℃.

2.2 Tissue samples

We have carried out immunohistochemical detection of the primary prostate tissue samples taken from 84 patients by surgery or biopsy between 2011 and 2017 at the Ningbo First Hospital (Zhejiang Province, China) with the approval of the institutional review board. Clinical data were obtained from the medical record system of the Ningbo First Hospital. Frozen prostate tissues were obtained from the Ningbo Pathology Center with the approval of the institutional review board.

2.3 Immunohistochemical staining and analyses

A total of 84 prostate cancer tissues and 22 precancerous tissues were obtained from the Ningbo Pathology Center of Zhejiang Province. Each case took 2–3 slides for experimentation. Slides were deparaffinized and hydrated at room temperature. Immunohistochemical staining was performed by following the manufacturer's directions of a specific kit (Absin, China). 4E-BP1 (dilution 1:100; Cell Signaling, US) and p4E-BP1 (dilution 1:100; Cell Signaling, US) were applied as the primary antibody at 37℃ for 12 h. For 4EBP1 or p4EBP1 scoring, the cytoplasmic staining intensity (4E-BP1 or p4E-BP1 typically stained the cytoplasmic of cancer cells) was rated as negative (no staining at all), weak (1+ staining intensity), moderate (2+ staining intensity), or strong (3+ staining intensity). In addition, it was stipulated that the product of the area percentage score of positive cells and the cell staining intensity score is regarded as a comprehensive staining score, with a score of ≥3 as positive, and a score of 0–2 as negative.

2.4 TCGA data analysis

Download the gene expression matrix of patients with prostate cancer in the TCGA database. The expression of 4EBP1 in prostate cancer was then evaluated by GraphPad Prism 8.0.

2.5 Western blot

Tumor tissues and cells were extracted with RIPA buffers containing 1% protease inhibitor mix (Solarbio, China) and 1% phosphatase inhibitor mix (Solarbio, China). The SDS-PAGE gel was used to isolate proteins, then the proteins were transferred to a PVDF membrane (Bio-RAD, USA), followed by blocking with 5% skim milk. Subsequently, the membranes were incubated with the corresponding primary antibodies for an overnight at 4℃. The primary antibodies 4E-BP1, p4E-BP1, and β-actin were all purchased from Cell Signaling Technology (USA). Finally, membranes were treated with HRP (horseradish peroxidase) labeled secondary antibody (Cell Signaling Technology, USA) at room temperature for 2–3 h and then detected by chemiluminescence.

2.6 Transwell assay

Cell transwell assays were performed using the 8 μM transwell chambers (Corning, USA) to evaluate the ability of cellular invasion. In brief, about 1 × 10⁵ PC-3 cells in serum-free basal medium were seeded onto the upper compartment. A growth medium containing 10% FBS was filled into the lower chambers. After 48 h of incubation at 37℃, the noninvasive cells in the upper chamber were removed, and then, the invasive cells were fixed with 90% methanol and stained with 0.1% crystal violet (Sigma-Aldrich, Germany).

2.7 Wound healing assay

The ability of cellular migration was tested by performing the wound healing assay. Once the cells achieved 95% confluence in the 6-well plate after transfection, a 200 μl pipette tip was used in a lengthwise stripe was used to create a linear wound, and then the non-adherent cells were rinsed by PBS. In order to allow cells to move into the gap without being affected by cell growth, the medium containing 0.5% fetal bovine serum was added. Three different isometric points in the scratch region were photographed and imaged by a microscope (Olympus, Japan) at 0 h and 24 h, respectively. The migration rate was counted as the proportion of the initial scratch area in each sample, using the ratio of areas remaining cell-free between the two boundaries after cell migration.

2.8 Cell apoptosis assay

Annexin V-fluorescein/propidium iodide double-staining assay was performed to detect the cell apoptosis by using the corresponding kit (Lianke Biotechnology Corporate Limited, China). In short, the cells were harvested and resuspended in 500 μl 1 × binding buffer, and then they were stained with Annexin V-FITC and PI. After incubation in the dark at room temperature for 15 min, the number of apoptotic cells was detected by a FACScan flow cytometer (Becton Dickinson, USA). Data were analyzed with the FlowJo software (BD, USA).

2.9 Statistical analyses

All statistical analyses were done with the SPSS program. Tissues were divided into two groups: (a) prostate cancer, (b) paracancerous tissues. The difference of expression of 4E-BP1 and p4E-BP1 between these two groups was analyzed by using the T test. Correlation analysis between the expression of 4E-BP1/p4E-BP1 and the clinical and pathological data was performed using the χ² test, Fisher's exact probability test, and the Spearman rank correlation analysis. The significant test level was calibrated to p < 0.05 (*p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).

3.1 4E-BP1 is over-expression in prostate cancer tissues

To explore the clinical significance of 4E-BP1 in prostate cancer, 106 pathological samples were detected using immunohistochemistry with a specific 4E-BP1 antibody. According to the staining results, the positive expression rate of 4E-BP1 in 84 prostate cancer samples reached 56.32%, while in 22 precancerous tissues, the positive rate was only 4.76% (p < 0.05, Table 1), which suggested that 4E-BP1 was expressed in higher levels in cancer tissues than in precancerous tissues (Figure 1A). However, there was no obvious correlation between the positive expression of 4E-BP1 and the Gleason score, PSA value, clinical stage, the presence of bone, lymph nodes, or visceral metastases in patients (p > 0.05, Table 2).

3.2 p4E-BP1 is closely related to the malignant degree of prostate cancer

Although we have observed the abnormal expression of 4E-BP1 in prostate cancer tissues, we did not find its relationship with the clinical progression of the tumor. Therefore, we continued to explore the connection between p4E-BP1, the active form of 4E-BP1, and prostate cancer. Similar to the result of 4E-BP1, the positive expression rate of p4E-BP1 in prostate cancer tissue was 55.60%, while it was only 9.09% in precancerous tissues (Figure 1B) (p < 0.05, Table 3). Surprisingly, we found that the expression of p4E-BP1 was conspicuously related with the Gleason score (Figure 1C) and lymph node metastasis (Figure 1D) in patients with prostate cancer (Table 4), which suggested that p4EBP1 plays a key role in the progression of the cancer.

3.3 Inhibition the p4E-BP1 reduces cell cloning-formation, invasion, and migration and effectively promotes the apoptosis in PC3 cells

Using the TCGA and GTEx database search, we found that 4E-BP1 was highly expressed in prostate cancer tissues compared to normal prostate tissues (Figure 2A); however, we did not find the expression and corresponding clinical data analysis of p4E-BP1 in the database. As we found that p4E-BP1 was significantly associated with the Gleason score, lymph node metastasis, and clinical stage in patients with prostate cancer by immunochemistry, we further tested for the expression of 4E-BP1 and p4E-BP1 in various prostate cancer cell lines. We could see that the expression of 4E-BP1 and p4E-BP1 were lower in normal prostate epithelial cells (RWPE-1). While in prostate cancer cells, as the malignant degree of cancer cells increased (cell malignancy rank 2RV1 < DU145 < PC3), the expressions of 4E-BP1 and p4E-BP1 also gradually elevated (Figure 2B). As the PC3 cell line showed the highest expression of p4E-BP1, we chose the PC3 cells for the following experiments.

From the results of the clinical data analysis, we believe that 4E-BP1 played a relevant role in prostate cancer through its phosphorylated form (p4E-BP1). To verify this point, we treated PC3 cells with different concentrations of Everolimus, which is the inhibitor that can suppress the phosphorylation of mTOR, which is the upstream molecule of 4E-BP1, for 24 h. We found that Everolimus could indeed significantly inhibit the phosphorylation level of 4E-BP1 (Figure 2C).

Next, we treated PC3 cells with different concentrations of Everolimus for 24 h and detected the apoptosis level by flow cytometry (Figure 3A–C) and found that inhibition of the phosphorylation level of 4E-BP1 could obviously increase the apoptosis of prostate cancer cells in a dose-dependent manner (Figure 3D).

Then, we estimated the clone-formation ability of prostate cancer cells after Everolimus treatment (Figure 4A), and found that when the phosphorylation level of 4E-BP1 was inhibited, the cloning ability of PC3 cells was significantly reduced.

In addition, we observed that the expression of p4E-BP1 was related to tumor metastasis from the analysis of our clinicopathological data; therefore, we further tested the changes in the invasion and migration ability of prostate cancer cells after treatment with Everolimus (Figure 4B, C). The results showed that the invasion and migration ability of PC3 was significantly reduced after the inhibition of p4E-BP1.