Downregulation of FOXO6 in breast cancer promotes epithelial-mesenchymal transition and facilitates migration and proliferation of cancer cells

Abstract

Purpose: Increasing evidence indicates that members of forkhead transcription factor family (FOXO) play key roles in cell proliferation and apoptosis in multiple cancers, including prostate cancer. However, the underlying mechanism of FOXO6 was not yet known. The aim of our work is to investigate the function of FOXO6 in breast cancer.

Methods: In the present study, quantitative real-time polymerase chain reaction and Western blotting analyses were used to detect the expression of FOXO6 in breast cancer tissues and cell lines.

Results: The results revealed that FOXO6 was downregulated in breast cancer tissues and cell lines, compared with adjacent normal tissues and MCF-10A cells, respectively. Moreover, the expression of FOXO6 was associated with the expression of epithelial-mesenchymal transition (EMT) indicator proteins, such as E-cadherin and N-cadherin. Additionally, our findings suggested that FOXO6 expression was negatively associated with tumor size (p=0.002), pathological grade (p=0.018) and lymph node metastasis (p=0.003). Sirt6 has been found to promote cell proliferation and metastasis in several cancers, and quantitative chromatin immunoprecipitation and luciferase reporter assays indicated FOXO6 transcriptionally regulated Sirt6 expression. Furthermore, various functional experiments, including wound healing assay, transwell invasion assay, colony formation assay and Cell Counting Kit-8 assay, revealed that FOXO6 suppressed cell migration, invasion, and proliferation of breast cancer cells.

Conclusion: In conclusion, FOXO6 serves as a tumor suppressor in breast cancer, and suppresses EMT through regulation of Sirt6.

Keywords: EMT; FOXO6; Sirt6; breast cancer; migration.

Figure 1

FOXO6 is downregulated in breast cancer tissues and associated with EMT-associated proteins E-cadherin and N-cadherin. Notes: (A) The expression of FOXO6 in tumor tissues and adjacent normal tissues was detected by qRT-PCR and Western blotting analysis, respectively. Tumor tissues vs adjacent normal tissues, *p < 0.05. (B) The expression of FOXO6 in breast cancer cell lines was detected by qRT-PCR and Western blotting analysis, respectively. Human normal breast MCF-10A cells were used as control. MDA-MB-231 or MCF-7 vs MCF-10A, *p < 0.05. (C) Kaplan–Meier analysis was used to analyze overall survival according to the expression status of FOXO6 (log-rank). Abbreviations: EMT, epithelial–mesenchymal transition; qRT-PCR, quantitative real-time polymerase chain reaction; FOXO, forkhead transcription factor family.

Figure 2

FOXO6 suppresses EMT in breast cancer cells. Notes: (A) MCF-7 cells were transfected with vector or FOXO6, or siControl or siFOXO6, respectively. After transfection for 48 h, the expression of FOXO6 was determined by qRT-PCR and Western blotting analyses. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (B) FOXO6 was overexpressed in MCF-7 cells. The mRNA and protein levels of EMT-associated proteins were detected by qRT-PCR and Western blotting analyses. FOXO6 vs vector, *p < 0.05. (C) FOXO6 was knocked down in MCF-7 cells. The mRNA and protein levels of EMT-associated proteins were detected by qRT-PCR and Western blotting analyses. siFOXO6 vs siControl, *p < 0.05. Abbreviations: FOXO, forkhead transcription factor family; EMT, epithelial–mesenchymal transition; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

FOXO6 inhibits breast cancer cells migration and invasion. Notes: (A) MDA-MB-231 cells were transfected with vector or FOXO6, or siControl or siFOXO6, respectively. After transfection for 48 h, the expression of FOXO6 was determined by qRT-PCR and Western blotting analyses. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (B) FOXO6 was overexpressed or knocked down in MDA-MB-231 cells. The effect of FOXO6 on cell invasion was determined by transwell invasion assay. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (C) FOXO6 was overexpressed or knocked down in MDA-MB-231 cells. The effect of FOXO6 on cell migration was determined by wound healing assay. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (D) FOXO6 was overexpressed or knocked down in MDA-MB-231 cells. The secretion of MMP9 was detected by ELISA. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. Abbreviations: FOXO, forkhead transcription factor family; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4

Sirt6 is a direct transcriptional target of FOXO6. Notes: (A) FOXO6 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. The mRNA level of Sirt6 was assessed by qRT-PCR assay. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (B) FOXO6 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. The protein level of Sirt6 was assessed by Western blotting assay. (C) qChIP sassy was performed using anti-FOXO6 antibody. FOXO6 vs IgG, *p < 0.05. (D) FOXO6 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. After transfection for 24 h, relative luciferase activity was detected by dual-luciferase reporter assay kit. *p < 0.05. Abbreviations: FOXO, forkhead transcription factor family; qRT-PCR, quantitative real-time polymerase chain reaction; qChIP, quantitative chromatin immunoprecipitation.

Figure 5

FOXO6 inhibits the proliferation of breast cancer cells. Notes: (A) FOXO6 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. Colony formation assay was used to detect the effect of FOXO6 on cell proliferation. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. (B) FOXO6 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. CCK-8 assay was used to detect the effect of FOXO6 on cell proliferation. FOXO6 vs vector, siFOXO6 vs siControl, *p < 0.05. Abbreviation: CCK-8, Cell Counting Kit-8; FOXO, forkhead transcription factor family.