Combination of palbociclib with enzalutamide shows in vitro activity in RB proficient and androgen receptor positive triple negative breast cancer cells

Abstract

Objectives: Triple negative breast cancer (TNBC) lacks specific drug targets and remains challenging. Palbociclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor is approved for metastatic estrogen receptor (ER)-positive and human epithermal growth factor 2 (HER2)-negative breast cancer. The nature of cell cycle inhibition by palbociclib suggests its potential in TNBC cells. Retinoblastoma (RB, a known substrate of CDK4/6) pathway deregulation is a frequent occurrence in TNBC and studies have revealed that pharmacological CDK4/6 inhibition induces a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models. In addition, recent studies reported that anti-androgen therapy shows preclinical efficacy in androgen-receptor (AR)-positive TNBC cells. Here we examined the effect of palbociclib in combination with an anti-androgen enzalutamide in TNBC cells.

Method: MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Protein expressions were assessed by Western blot analysis. Cytostatic effect was examined by MTT assay. Cell cycle and apoptosis were examined by flow cytometry.

Results: Palbociclib showed inhibitory effect in RB-proficient TNBC cells, and enzalutamide inhibited cell viability in AR-positive TNBC cells. Enzalutamide treatment could enhance the palbociclib-induced cytostatic effect in AR-positive/RB-proficient TNBC cells. In addition, palbociclib-mediated G1 arrest in AR-positive/RB-proficient TNBC cells was attenuated by RB knockdown.

Conclusion: Our study provided a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists.

Fig 1. Effect of palbociclib and enzalutamide on AR and RB protein expressions in TNBC cell lines.

(A) The whole cell extracts of MCF 10A, BT-549, BT-20 MDA-MB-231, MDA-MB-453, MDA-MB-468 and HCC1937 cells were analyzed by Western blot using antibodies against anti-AR, anti-phospho-RB, anti-RB and anti-β-actin. (B-D) The whole cell extracts of MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 were treated with DMSO (-), 1.25 and 2.5 μM palbociclib, 10 and 20 μM enzalutamide and combination of 2.5 μM palbociclib and 20 μM enzalutamide for 48 h which prepared for Western blot analysis using antibodies against anti-AR, anti-phospho-RB, anti-RB and anti-β-actin.

Fig 2. Enzalutamide in combination with palbociclib enhances the cytostatic effect in AR-positive/RB-proficient TNBC cells.

(A) MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 cells were treated with various concentrations of palbociclib, (B) enzalutamide and (C) combination of 0.156 or 2.5 μM palbociclib and 10 or 20 μM enzalutamide for 72 h, the cell viability was determined using MTT assay. The means ± SEM of three independent experiments performed in triplicate are shown. *P < 0.05; **P < 0.01.

Fig 3. Palbociclib-induced G1 arrest is increased in AR-positive/RB-proficient cells.

(A) MDA-MB-453, (B) BT-549, (C) MDA-MB-231 and (D) MDA-MB-468 cells were treated with DMSO (-), 1.25 and 2.5 μM palbociclib, 10 and 20 μM enzalutamide and combination of 2.5 μM palbociclib and 20 μM enzalutamide for 48 h, the cell cycle analysis by DNA content using flow cytometry. The means ± SEM of three independent experiments performed in triplicate are shown.

Fig 4. The effects of combination of palbociclib and enzalutamide are attenuated by RB knockdown.

(A) MDA-MB-453 cells were transfected with siRNA against control (siCtrl) or RB1 (siRB) for 24 h, and the transfected cells were further treated with 2.5 μM palbociclib for 48 h. The treated cells were analyzed by MTT assay. *P < 0.05. (B, C) MDA-MB-453 cells were transfected with siRNA against control (siCtrl) or RB1 (siRB) for 24 h, and the transfected cells were further treated with 2.5 μM palbociclib and 20 μM enzalutamide for 48 h. The treated cells were analyzed by flow cytometry analysis (B) and Western blot (C) using antibodies against anti-phospho-RB, anti-RB, anti-cyclin D1, anti-p21, anti-p16 and anti-β-actin. G0/G1 subpopulation; **P < 0.01.

Fig 5. Palbociclib and enzalutamide do not result in apoptosis in TNBC cells.

(A) MDA-MB-453, (B) BT-549, (C) MDA-MB-231 and (D) MDA-MB-468 cells were treated with DMSO (Ctrl), 1.25 and 2.5 μM palbociclib, 10 and 20 μM enzalutamide, combination of 2.5 μM palbociclib and 10 μM enzalutamide, cisplatin, and doxorubicin for 48 h, the percentage of apoptotic cells were analyzed by flow cytometry. The means ± SEM of three independent experiments performed in triplicate are shown. **P < 0.01; ***P < 0.001.

Fig 6. Clinical significances of AR and RB1 gene expression in patients with TNBC.

(A-D) The level 3 data of mRNA RSEM (RNA-Seq by Expectation Maximization) from patients with TNBC were selected and downloaded from the TCGA and Broad GDAC Firehose data portal. The mean of AR or RB1 gene expressions were the chosen as cut-off value for separating tumors with strong and weak expression. Overall survival rates of TNBC patients were plotted against time in month for different parameters: the level of AR gene (A), the level of RB1 gene (B), and the level of AR/RB1 genes (C). The correlation between AR and RB1 mRNA expressions was analyzed by Pearson correlation analysis (D).