TMED3 promotes prostate cancer via FOXO1a and FOXO3a phosphorylation

Abstract

Background: Transmembrane emp24 trafficking protein 3 (TMED3) is associated with the development of several tumors; however, whether TMED3 regulates the progression of prostate cancer remains unclear.

Materials and methods: Short hairpin RNA was performed to repress TMED3 in prostate cancer cells (DU145 cells) and in a prostate cancer mice model to determine its function in prostate cancer in vitro and in vivo.

Results: In the present study, we found that TMED3 was highly expressed in prostate cancer cells. In vitro, shTMED3 treatment suppressed the proliferation, invasion, and migration and promoted the apoptosis of DU145 cells. Additionally, the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed a strong correlation between TMED3 and forkhead box O transcription factor (FOXO) pathway. Furthermore, TMED3 inhibition efficiently decreased FOXO1a and FOXO3a phosphorylation. In vivo, TMED3 downregulation suppressed the apoptosis, growth, and metastasis of prostate cancer cells via FOXO1a and FOXO3a.

Conclusion: The present findings show that TMED3 participates in the regulation of prostate cancer progression via FOXO1a and FOXO3a phosphorylation, thereby revealing a novel mechanism underlying prostate cancer development and suggesting that TMED3 inhibition may serve as a novel strategy for prostate cancer treatment.

Keywords: Apoptosis; Proliferation; Prostate cancer; Transmembrane emp24 trafficking protein 3 (TMED3); forkhead box O transcription factor (FOXO).

Figure 1. The silencing efficiency of shTMED3. (A) DU145 cells were treated with shTMED3-1, shTMED3-2, and shTMED3-3 for 72 h and subjected to qRT-PCR. Relative TMED3 mRNA expression in DU145 was analyzed. (B) DU145 cells were treated with shTMED3-3 for 72 h and subjected to western blotting. The TMED3 protein level in DU145 was determined. *p < 0.05; **p < 0.01.

Figure 2. TMED3 regulated proliferation and promoted apoptosis in DU145 cells. (A) TMED3 protein levels in normal human prostate cells and prostate cancer cells were determined by western blotting. DU145 cells were infected with a lentivirus to silence TMED3 expression, and shCtrl was used as a scrambled control. (B) Cytotoxicity at different time points was determined by performing the CCK-8 assay. (C) A representative scatter plot of PI⁺Annexin V⁺ and PI⁻Annexin V⁺ DU145 cells by flow cytometry. (D) The proportions of DU145 cells at different stages of apoptosis (early apoptosis, late apoptosis, and total apoptosis) by flow cytometry. (E and F) The cell cycle (G1, S, and G2 phases) of DU145 cells was determined by flow cytometry. **p < 0.01; ***p < 0.001.

Figure 3. TMED3 regulated the invasion and migration of prostate cancer cells. DU145 cells were infected with a lentivirus to silence TMED3 expression, and shCtrl was used as a scrambled control. (A and B) The wound-healing assay was performed to determine the invasion ability of DU145 cells. (C and D) DU145 cell migration was assessed by performing the Transwell assay. ***p < 0.001. Original magnification, 200×.

Figure 4. TMED3 inhibited the FOXO pathway by inducing FOXO1a and FOXO3a phosphorylation during prostate cancer progression. (A) The enrichment analysis of the TCGA data was performed to analyze the correlation of TMED3 with 26 signaling pathways. The sizes of the circles indicate the significance of enrichment, whereas their colors represent the strength and direction of the enrichment (red: positive correlation, blue: negative correlation). (B) The key role of the FOXO family in cancer-related pathways. (C) Western blotting of FOXO1a, p-FOXO1a, FOXO3a, and p-FOXO3a in DU145 cells.

Figure 5. TMED3 downregulation alleviated prostate cancer in vivo. (A and B) The sizes of prostate tumors in mice. (C) Prostate cancer metastasis in mice was evaluated using an in vivo imaging system. (D) Histological alterations in prostate tumors were observed by performing HE staining. (E) Immunohistochemistry of ki67-positive expression in prostate tumors of mice. Original magnification, 400×.

Figure 6. TMED3 downregulation decreased p-FOXO1 and p-FOXO3 levels in a mouse model of prostate cancer. (A) Western blotting of FOXO1a, p-FOXO1a, FOXO3a, and p-FOXO3a in tumor tissues. Immunofluorescence analysis of p-FOXO1a (B) and p-FOXO3a (C) in tumor tissues. Original magnification, 200×.