ZNF384-ZEB1 feedback loop regulates breast cancer metastasis

Abstract

Background: Breast cancer has become the most frequently diagnosed cancer worldwide. Increasing evidence indicated that zinc finger proteins (ZNFs), the largest family of transcription factors, contribute to cancer development and progression. Although ZNF384 is overexpressed in several types of human cancer, the role of ZNF384 in breast cancer remains unknown. Therefore, our research focused on ZNF384 regulation of the malignant phenotype of breast cancer and the underlying molecular mechanisms.

Methods: CCK-8 and colony formation assays were used to evaluate cell proliferation. Transwell and scratch assays were used to evaluate the cell migration and invasion. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter assays were used to confirm the target relationship between ZNF384 and zinc finger E-box binding homeobox 1 (ZEB1). Xenografts were used to monitor the targets in vivo effects.

Results: We noted that ZNF384 was significantly overexpressed in breast cancer and highlighted the oncogenic mechanism of ZNF384. ZNF384 transactivated ZEB1 expression and induced an epithelial and mesenchymal-like phenotype, resulting in breast cancer metastasis. Furthermore, ZNF384 may be a target of miR-485-5p, and ZEB1 can up-regulate ZNF384 expression by repressing miR-485-5p expression. Together, we unveiled a feedback loop of ZNF384-ZEB1 in breast cancer metastasis.

Conclusions: The findings suggest that ZNF384 can serve as a prognostic factor and a therapeutic target for breast cancer patients.

Keywords: Breast cancer; Epithelial to mesenchymal transition; Metastasis; ZEB1; ZNF384; miR-485.

Fig. 1

ZNF384 is highly expressed in breast cancer and associated with poor prognosis. A ZNF384 expression determined by IHC in 20 cases of primary breast cancer tissues and the paired normal breast tissues. B ZNF384 expression in six breast cancer cells and the normal breast cell line MCF10A determined by western blot. C The Kaplan–Meier analysis of disease-free survival of patients with different ZNF384 expression levels (n = 236)

Fig. 2

ZNF384 overexpression promotes breast cancer migration and invasion in vitro. A ZNF384 expression in T47D and ZR-75-30 cells transfected with a human ZNF384 expression vector, as well as vector control cells determined by western blot. B The ability of cell proliferation of cells as in A determined by CCK-8. C The clonogenic potential of cells as in A determined by colony formation assays. D–F The ability of cell migration and invasion in cells as in A determined by wound-healing (D) and transwell (E, F) assays. *P < 0.05

Fig. 3

Depletion of ZNF384 expression suppresses breast cancer metastasis. A ZNF384 expression in stable ZNF384-depleted cells (shZNF384) and control cells (shControl) determined by western blot. B The ability of cell proliferation of cells as in A determined by CCK-8. C The clonogenic potential of cells as in A determined by colony formation assays. D–F The ability of cell migration and invasion in cells as in A determined by wound-healing (D) and transwell (E–F) assays. G Tumor growth curves for xenograft mice injected with cells as in (A) at the indicated times. H Bioluminescent imaging of metastasis for xenograft mice after tail vein injection of cells as in A. I H&E staining or immunohistochemical staining in primary tumors or metastatic nodules in the lung harvested from mice bearing the indicated xenograft tumors. *P < 0.05

Fig. 4

ZNF384 induces an EMT-like phenotype in breast cancer cells. A Representative images of morphology in ZNF384-overexpressed T47D cells or ZNF384-depleted MDA-MB-231 cells, as well as control cells. B, C The expression of epithelial marker (E-cadherin) and mesenchymal markers (Vimentin and N-cadherin) in cells as in A is determined by immunofluorescence (B) and western blot (C). D The expression of EMT-related transcriptional factors in cells as (A) determined by RT-qPCR. *P < 0.05

Fig. 5

ZNF384 promotes breast cancer progression by transactivation of ZEB1 expression. A Four potential ZNF384-binding sites located in ZEB1 promoter region. B The interaction between ZNF384 and ZEB1 promoter region in 293FT cells verified by ChIP analysis. C The effect of ZNF384 expression on ZEB1 promoter activity determined by dual-luciferase reporter analysis in 293FT cells. A series of fragments containing the predicted ZNF384 binding site is fused upstream of Luc gene. D ZEB1 expression determined by western blot in ZNF384-overexpressed T47D cells or ZNF384-depleted MDA-MB-231 cells, as well as control cells. E–F The ability of cell migration and invasion in ZNF384-depleted MDA-MB-231 with or without ZEB1 overexpression determined by transwell analysis. *P < 0.05

Fig. 6

ZNF384 is a target of miR-485-5p. A ZNF384 identified as a target of miR-485-5p and miR-31-5p using multiple target-predicting programs. B The relationship between ZNF384 and miR-485-5p or miR-31-5p in breast cancer from TCGA data. C The predicted binding site of miR-485-5p in ZNF384 3'-UTR. The mut sequence contains a 4-base mutation at the miR-485-5p target seed region. D The effect of miR-485-5p expression on wild or mutated ZNF384 3'-UTR activity determined by dual-luciferase reporter analysis in 293FT cells. E–F The expression of ZNF384 mRNA levels in miR-485-5p-overexpressed MDA-MB-231 cells or miR-485-5p-depleted T47D cells, as well as control cells, determined by RT-qPCR (E) or western blot (F). *P < 0.05

Fig. 7

ZEB1 represses miR-485-5p expression in a ZNF384–ZEB1 feedback loop. A A potential ZEB1-binding site located in the miR-485 promoter region. B The interaction between ZEB1 and miR-485 promoter region in MDA-MB-231 cells verified by ChIP analysis. C The effect of ZEB1 expression on miR-485 promoter activity determined by dual-luciferase reporter analysis in 293FT cells. A series of fragments containing the predicted ZEB1 binding site is fused upstream of Luc gene. D The expression of miR-485-5p levels in ZEB1-depleted MDA-MB-231 cells or ZEB1-overexpressed T47D cells, as well as control cells, determined by RT-qPCR. E The expression of miR-485-5p levels in ZNF384-depleted MDA-MB-231 cells or ZNF384-overexpressed T47D cells, as well as control cells, determined by RT-qPCR. F Schematic illustration of ZNF384-miR-485-5p feedback loop in regulating breast cancer EMT and metastasis. *P < 0.05