CDCA5 accelerates progression of breast cancer by promoting the binding of E2F1 and FOXM1

Abstract

Background: Breast cancer is one of the most common malignant tumors in women. Cell division cycle associated 5 (CDCA5), a master regulator of sister chromatid cohesion, was reported to be upregulated in several types of cancer. Here, the function and regulation mechanism of CDCA5 in breast cancer were explored.

Methods: CDCA5 expression was identified through immunohistochemistry staining in breast cancer specimens. The correlation between CDCA5 expression with clinicopathological features and prognosis of breast cancer patients was analyzed using a tissue microarray. CDCA5 function in breast cancer was explored in CDCA5-overexpressed/knockdown cells and mice models. Co-IP, ChIP and dual-luciferase reporter assay assays were performed to clarify underlying molecular mechanisms.

Results: We found that CDCA5 was expressed at a higher level in breast cancer tissues and cell lines, and overexpression of CDCA5 was significantly associated with poor prognosis of patients with breast cancer. Moreover, CDCA5 knockdown significantly suppressed the proliferation and migration, while promoted apoptosis in vitro. Mechanistically, we revealed that CDCA5 played an important role in promoting the binding of E2F transcription factor 1 (E2F1) to the forkhead box M1 (FOXM1) promoter. Furthermore, the data of in vitro and in vivo revealed that depletion of FOXM1 alleviated the effect of CDCA5 overexpression on breast cancer. Additionally, we revealed that the Wnt/β-catenin signaling pathway was required for CDCA5 induced progression of breast cancer.

Conclusions: We suggested that CDCA5 promoted progression of breast cancer via CDCA5/FOXM1/Wnt axis, CDCA5 might serve as a novel therapeutic target for breast cancer treatment.

Keywords: Breast cancer; CDCA5; FOXM1; Wnt/β-catenin signaling.

Fig. 1

CDCA5 was upregulated in breast cancer and correlated with poor prognosis. (A) Representative IHC images of CDCA5 staining in human breast cancer tissues and normal para-carcinoma tissues. Scale bar is 50 μm. (B) The IHC staining score of CDCA5 in Fig. 1A. (C) Overall survival curves of breast cancer patients with high/low expression of CDCA5. High expression of CDCA5: 48 samples; Low expression of CDCA5: 48 samples. (D) CDCA5 mRNA and (E) protein expression levels in breast cancer cell lines (BT-549, MDA-MB-231, T47D, MCF-7, MDA-MB-453, MDA-MB-468) and the normal MCF-10 A cell line was detected by qPCR analysis and WB analysis. GAPDH was used as an internal reference. Results were presented as mean ± SD. ^*p < 0.05

Fig. 2

CDCA5 knockdown inhibited proliferation and migration of breast cancer cell in vitro. The BT-549 and MDA-MB-231 cell lines were transfected with shCDCA5 or shCtrl lentivirus. 48 h after lentiviral transfection, (A) the mRNA and protein levels of CDCA5 expression were evaluated by qPCR and WB assays, respectively. GAPDH was used as inner control. (B) Cell viability of BT-549 and MDA-MB-231 cells was determined by Celigo cell counting assay. (C) Capacity of colony formation in BT-549 and MDA-MB-231 cells was assessed by colony formation assays. (D) The apoptotic ratio of BT-549 and MDA-MB-231 cells were determined by flow cytometry. (E) Capacity of cell migration in BT-549 and MDA-MB-231 cells was detected by wound healing and (F) transwell assays. Results were presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 3

CDCA5 promoted the binding of E2F1 to FOXM1 promoter. (A-B) The mRNA levels of AURKB, CREB5, FOXM1, and PRKACB in BT-549 andMDA-MB-231 cells transfected with shCDCA5 or shCtrl lentivirus were determined by qPCR. The dotted line showed the expression of FOXM1 relative to other genes. (C) The protein levels of CDCA5 and FOXM1 in BT-549 and MDA-MB-231 cells transfected with shCDCA5 or shCtrl lentivirus were detected by WB assays. GAPDH was used as an internal reference. (D) The interaction between CDCA5 and E2F1 in MDA-MB-231 cells was validated by Co-IP assays. (E) The combination of E2F1 and FOXM1 promoter was determined by dual-luciferase reporter assays in 293T cells transfected with E2F1 or empty vector and WT or Mut FOXM1 promoter. (F) Enrichment of E2F1 at FOXM1 promoter region was observed by ChIP analysis in MDA-MB-231 cells with or without CDCA5 overexpression. Results were presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 4

FOXM1 depletion attenuated cell proliferation and migration promoted by CDCA5. The BT-549 and MDA-MB-231 cell lines were transfected with CDCA5-overexpressed and FOXM1-depleted scramble vectors (NC group), CDCA5-overexpressed lentivirus and FOXM1-depleted scramble vector (CDCA5 group), CDCA5-overexpressed scramble vector and FOXM1-depleted lentivirus (shFOXM1 group), and CDCA5-overexpressed lentivirus and FOXM1-depleted lentivirus (CDCA5 + shFOXM1 group), respectively. 48 h after lentiviral transfection, (A) Cell viability in four groups of BT-549 and MDA-MB-231 cells was determined by Celigo cell counting assay. (B) The apoptotic ratio in four groups of BT-549 and MDA-MB-231 cells were evaluated by flow cytometry. (C) Cell cycle in four groups of BT-549 and MDA-MB-231 cells was analyzed by flow cytometry. (D-E) Capacity of cell migration in four groups of BT-549 and MDA-MB-231 cells was detected by wound healing and transwell assays. Results were presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 5

FOXM1 is essential for CDCA5-induced tumor growth in vivo. The xenograft model was constructed by subcutaneous injection of MDA-MB-231 cells from NC, shFOXM1, CDCA5 or CDCA5 + shFOXM1 groups. 7 days after inoculation, the tumor volume was calculated based on tumor sizes and (A) tumor growth curves were obtained by tumor volume. (B) Photographs of all mice and tumor tissues in four groups. (C) Tumor weight of mice in four groups. (D) Representative IHC images of Ki-67 staining in tumor tissues from mice in four groups. Scale bar is 50 μm. (E) The protein expression levels of CDCA5, E2F1 and FOXM1 in tumor tissues were determined by WB analysis. GAPDH served as an internal reference. Results were presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 6

CDCA5 facilitated proliferation of breast cancer cells via Wnt/β-catenin signaling pathway. The BT-549 and MDA-MB-231 cell lines were transfected with indicated lentivirus followed by treatment of C59 (Wnt/β-catenin inhibitor, 20 µmol/L) for 24 h or not. (A-C) Wnt3a, β-catenin and c-Myc protein levels in three groups of BT-549 and MDA-MB-231 cells were detected by WB assays. GAPDH was used as an internal reference. (D) Cell viability in three groups of BT-549 and MDA-MB-231 cells were determined by CCK-8 assays. (E) The apoptotic ratio in three groups of BT-549 and MDA-MB-231 cells were analyzed by flow cytometry. (F) Schematic depiction illustrated that CDCA5 promoted Wnt/β-catenin signaling activation and breast cancer progression through upregulating FOXM1 transcription. Results were presented as mean ± SD. ^**p < 0.01, ^***p < 0.001