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

ObjectivesTriple 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.MethodMDA-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.ResultsPalbociclib 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.ConclusionOur study provided a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists.### 研究目标 三阴性乳腺癌（Triple negative breast cancer, TNBC）缺乏特异性药物靶点，临床治疗仍颇具挑战。帕博西尼（Palbociclib）作为细胞周期蛋白依赖性激酶4和6（cyclin-dependent kinases 4 and 6, CDK4/6）抑制剂，已获批用于治疗转移性雌激素受体（estrogen receptor, ER）阳性、人表皮生长因子受体2（human epidermal growth factor 2, HER2）阴性乳腺癌。帕博西尼的细胞周期抑制特性提示其在三阴性乳腺癌细胞中的应用潜力。视网膜母细胞瘤蛋白（Retinoblastoma, RB，CDK4/6的已知底物）通路失调在三阴性乳腺癌中较为常见，已有研究表明，在RB功能正常的三阴性乳腺癌模型中，CDK4/6抑制剂的药理学抑制可与多柔比星产生协同细胞生长抑制效应。此外，近期研究报道抗雄激素治疗在雄激素受体（androgen-receptor, AR）阳性的三阴性乳腺癌细胞中展现出临床前疗效。本研究考察了帕博西尼联合抗雄激素药物恩扎卢胺（enzalutamide）在三阴性乳腺癌细胞中的作用效果。 ### 研究方法 本研究采用MDA-MB-453、BT-549、MDA-MB-231及MDA-MB-468三阴性乳腺癌细胞系开展体外实验。通过蛋白质印迹法（Western blot analysis）检测蛋白表达水平，采用MTT比色法检测细胞生长抑制效应，利用流式细胞术分析细胞周期与细胞凋亡情况。 ### 研究结果 帕博西尼对RB功能正常的三阴性乳腺癌细胞具有抑制作用，恩扎卢胺可抑制AR阳性三阴性乳腺癌细胞的活力。在AR阳性/RB功能正常的三阴性乳腺癌细胞中，恩扎卢胺处理可增强帕博西尼诱导的细胞生长抑制效应。此外，RB基因敲低可逆转帕博西尼介导的AR阳性/RB功能正常的三阴性乳腺癌细胞G1期阻滞。 ### 研究结论 本研究为筛选可从CDK4/6抑制剂与AR拮抗剂联合治疗中获益的患者提供了临床前理论依据。