J Breast Cancer.  2019 Jun;22(2):237-247. 10.4048/jbc.2019.22.e29.

Cell Division Cycle Associated 8 Is a Key Regulator of Tamoxifen Resistance in Breast Cancer

• Yu D
• Shi L
• Bu Y
• Li W

• KMID: 2450118
• DOI: http://doi.org/10.4048/jbc.2019.22.e29

Abstract

PURPOSE
Breast cancer (BC) is one of the most common malignancies globally, and millions of women worldwide are diagnosed with BC every year. Up to 70% of BC patients are estrogen receptor (ER)-positive. Numerous studies have shown that tamoxifen has a significant therapeutic effect on both primary and metastatic ER-positive BC patients. Although tamoxifen is currently one of the most successful therapeutic agents for BC, a significant proportion of patients will eventually become resistant to tamoxifen, leading to tumor recurrence and metastasis. Knowledge about the development of tamoxifen resistance in BC patients is still limited.
METHODS
We applied a loss-and-gain method to study the biological functional role of cell division cycle associated 8 (CDCA8) in tamoxifen resistance in BC cells.
RESULTS
We found that CDCA8 was significantly elevated in tamoxifen-resistant BC cells. Knockdown of CDCA8 expression significantly inhibited the proliferation of tamoxifen-resistant BC cells and reduced their resistance to tamoxifen. In contrast, overexpression of CDCA8 promoted the growth of tamoxifen-sensitive BC cells and induced their resistance to tamoxifen.
CONCLUSION
In this study, we reported that CDCA8 is a key regulator of tamoxifen resistance in BC, suggesting that CDCA8 may serve as a potential therapeutic target for BC treatment.

Keyword

Apoptosis; Breast neoplasms; Cell cycle; CDCA8 protein, human; Tamoxifen

MeSH Terms

Apoptosis
Breast Neoplasms*
Breast*
Cell Cycle*
Cell Division*
Estrogens
Female
Humans
Methods
Neoplasm Metastasis
Recurrence
Tamoxifen*
Estrogens
Tamoxifen

Figure

• 

  Figure 1 Upregulation of CDCA8 in Tam-resistant BC cells. (A, B) MCF-7, MCF-7/TR, T47D, and T47D/TR cell proliferation was determined by the MTT assay after 4OH-Tam treatment for 96 hr. (C) Quantitative reverse transcription-polymerase chain reaction analysis of CDCA8 mRNA levels in BC cells. Glyceraldehyde-3-phosphate dehydrogenase served as loading controls. (D) Western blotting analysis of CDCA8 levels in BC cells. β-actin served as loading controls. Data are presented as mean ± standard deviation from three independent experiments. CDCA8 = cell division cycle associated 8; BC = breast cancer; Tam = tamoxifen. *p < 0.01.

• 

  Figure 2 Reduction of Tam resistance by knockdown of CDCA8 in Tam-resistant BC cells. (A) Western blotting assay for CDCA8 levels in Tam-resistant BC cells stably expressing shCtrl or shCDCA8. β-actin served as loading controls. (B) BC cells were grown in 6-well plates in media containing 10% serum and the cell number was determined at the indicated days with or without knockdown of CDCA8. (C) Cell proliferation of Tam-resistant BC cells was determined by the MTT assay after 4OH-Tam treatment for 5 days. (D) MCF-7/TR and (E) T47D/TR cells were subjected to cell colony formation assay. Tam+ and Tam− represents the culture media with and without 1 μM Tam, respectively. Data are presented as mean ± standard deviation from three independent experiments. CDCA8 = cell division cycle associated 8; BC = breast cancer; shCtrl = shRNA control; shCDCA8 = shRNA targeting CDCA8; Tam = tamoxifen. *p < 0.05; †p < 0.01.

• 

  Figure 3 Induction of cell apoptosis and cell cycle arrest in Tam-resistant BC cells by CDCA8 knockdown. MCF-7/TR-shCtrl, MCF-7/TR-shCDCA8-1, MCF-7/TR-shCDCA8-2, T47D/TR-shCtrl, T47D/TR-shCDCA8-1, and T47D/TR-shCDCA8-2 cells were cultured with or without 4OH-Tam. Total cells were collected and subjected to apoptosis (A, B) and cell cycle (C, D) analysis. Data represent mean ± standard deviation from three independent experiments with each measured in triplicate. BC = breast cancer; CDCA8 = cell division cycle associated 8; shCtrl = shRNA control; shCDCA8 = shRNA targeting CDCA8; Tam = tamoxifen; FITC = fluorescein isothiocyanate. *p < 0.01.

• 

  Figure 4 Induction of Tam resistance in BC cells by forced expression of CDCA8. MCF-7 and T47D cells were infected with retrovirus vector alone or carrying CDCA8. (A) Western blotting analysis of CDCA8 expression in MCF-7 and T47D cells. (B) BC cells were grown in 6-well plates in media containing 10% serum and the cell number was determined at the indicated days with or without forced expression of CDCA8. (C) MCF-7 and T47D cell proliferation was determined by the MTT assay after 4OH-Tam treatment for 5 days. (D) MCF-7 and T47D cells were subjected to cell colony formation assay. Tam+ and Tam− represent the culture media with and without 1 μM Tam, respectively. Data are presented as mean ± standard deviation from three independent experiments. BC = breast cancer; CDCA8 = cell division cycle associated 8; Tam = tamoxifen. *p < 0.05; †p < 0.01.

• 

  Figure 5 Protection of Tam-sensitive BC cells from apoptosis and cell cycle arrest by forced expression of CDCA8. MCF-7-Vec, MCF-7-CDCA8-OE, T47D-Vec, and T47D-CDCA8-OE cells were cultured with or without 4OH-Tam. Total cells were collected and subjected to apoptosis (A, B) and cell cycle (C, D) analysis. Data represent mean ± standard deviation from three independent experiments which each measured in triplicate. BC = breast cancer; CDCA8 = cell division cycle associated 8; Tam = tamoxifen; FITC = fluorescein isothiocyanate. *p < 0.01; †p < 0.01.

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