Transcription factor FOXP4 inversely governs tumor suppressor genes and contributes to thyroid cancer progression

Abstract

Objective: In recent decades, thyroid cancer (TC) has exhibited a rising incidence pattern. Elevated levels of the transcription factor FOXP4 have been strongly linked to the progression of diverse tumors; nevertheless, its specific role in thyroid cancer remains underexplored. The primary objective of this study was to elucidate the functions of FOXP4 and its associated target gene, FBXW7, in the context of thyroid cancer.

Methods: FOXP4 and FBXW7 expression levels in TC tissues and cell lines were assessed through immunohistochemistry and RT-qPCR analyses. The functional aspects of FOXP4, including its effects on cell proliferation, migration capabilities, cell cycle regulation, and epithelial-mesenchymal transition (EMT), were investigated. Furthermore, the interaction between FOXP4 and FBXW7 was confirmed using chromatin immunoprecipitation (ChIP) assays. The impact of FBXW7 on FOXP4-mediated cellular phenotypes was subsequently examined. Additionally, the in vivo role of FOXP4 and FBXW7 in tumor growth was elucidated through the establishment of a murine tumor model.

Results: Elevated levels of FOXP4 were observed in papillary carcinoma tissues, and patients exhibiting high FBXW7 levels showed a more favorable prognosis. KTC-1 cells displayed a concomitant increase in FOXP4 expression and decrease in FBXW7 expression. FOXP4 overexpression in these cells enhanced cell proliferation, migration capabilities, and EMT. The interaction between the FOXP4 protein and the FBXW7 promoter was confirmed, and the effects of FOXP4 were mitigated upon overexpression of FBXW7. Furthermore, knockdown of FOXP4 led to decelerated growth of transplanted tumors and increased FBXW7 levels within the tumors.

Conclusion: The findings of the current study underscore the regulatory role of FOXP4 in the transcription of FBXW7 and establish a clear link between aberrations in FBXW7 expression and the manifestation of malignant phenotypes in highly aggressive TC cells.

Keywords: EMT; FBXW7; FOXP4; Thyroid cancer; Tumor transplant.

Fig. 1

FOXP4 in thyroid tissue (A) The levels of FOXP4 in thyroid cancer and adjacent tissues were determined by immunohistochemistry. (B) Semi-quantitative results of immunohistochemistry. (C) The relationship between the survival rate of patients with FOXP4 and FBXW7 levels was analyzed using the TIMER web server. ***P < 0.001.

Fig. 2

FOXP4 in thyroid cancer cells (A) The levels of FOXP4 and (B) FBXW7 in thyroid cancer cells were determined by RT-qPCR. (C) The effects of FOXP4 knockdown or overexpression on cell viability and (D) colony formation ability were assessed using the CCK8 and colony formation assay. (E) Cell migration was evaluated using Transwell assay. (F) The EMT process was assessed using western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Nthy-ori3-1 or ov-NC; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. sh-NC.

Fig. 3

FOXP4 negatively regulates FBXW7 (A) The distribution of FOXP4 in the nucleus and nucleoplasm was determined by RT-qPCR. (B) The connection between FOXP4 and FBXW7 was verified by the ChIP assay. (C) The levels of FBXW7 in the transfected cells were examined using RT-qPCR and (D) western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Cytoplasm or ov-NC; ^###P < 0.001 vs. sh-NC.

Fig. 4

FBXW7 affects FOXP4 functions (A) The efficacy of FBXW7 knockdown was determined by western blotting. (B) Cells were co-transfected to knock down FBXW7 along with FOXP4 knockdown, and the level of FBXW7 was determined by western blotting. (C) The impacts of FBXW7 on FOXP4 functions were assessed based on cell viability, (D) colony formation ability, (E) migration, (F) cell cycle, and (G) EMT process. **P < 0.01, ***P < 0.001 vs. sh-NC; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. sh-FOXP4 + sh-NC.

Fig. 5

Tumor formation in nude mice (A) Stably transfected cell lines were constructed, MOI = 5 group was used for tumor transplantation. (B) The levels of FOXP4 and FBXW7 in stably transfected KTC-1 cells were examined using western blotting. (C) Photos of mice and tumors. (D) The effects of FOXP4 knockdown on the tumor volume growth and (E) weight were recorded. (F) The expression levels of EMT-related protein in tumors were determined using western blotting. (G) The expression levels of Ki67 protein in tumors were determined using western blotting. ***P < 0.001 vs. sh-NC.

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