Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, 87036, Italy.
Breast and General Surgery Unit, Regional Hospital Cosenza, Cosenza, 87100, Italy.
J Exp Clin Cancer Res. 2024 Jun 18;43(1):171. doi: 10.1186/s13046-024-03096-7.
The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients.
BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored.
Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs.
Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.
细胞周期蛋白 D1-细胞周期蛋白依赖性激酶（CDK）4/6 抑制剂帕博西尼联合内分泌治疗在管理雌激素受体（ER）阳性和 HER2 阴性晚期乳腺癌（BC）方面显示出显著疗效。然而，帕博西尼的耐药性经常出现，这凸显了需要确定新的靶点，以实现 BC 患者更全面的治疗策略。
生成了对帕博西尼耐药的 BC 细胞系，并将其用作模型系统。基因沉默技术和过表达实验、实时 PCR、免疫印迹和染色质免疫沉淀研究以及细胞活力、集落和 3D 球体形成测定用于评估 G 蛋白偶联雌激素受体（GPER）在 BC 细胞对帕博西尼耐药中的作用。还进行了分子对接模拟，以研究帕博西尼与 GPER 之间的潜在相互作用。此外，还使用从乳腺癌中分离的癌症相关成纤维细胞（CAF）共培养 BC 细胞，以研究 GPER 信号是否有助于肿瘤微环境中功能细胞相互作用对帕博西尼耐药的影响。最后，通过对大量 BC 患者的临床和表达数据进行生物信息学分析和 k-均值聚类，探讨了新的帕博西尼耐药调节剂的临床意义。
剖析了对帕博西尼耐药的 ER 阳性 BC 细胞的分子事件，发现 ERα 下调和 GPER 上调。为了评估 GPER 上调涉及的分子事件，我们确定表皮生长因子受体（EGFR）与 GPER 的启动子区域相互作用并刺激其表达，导致 BC 细胞对帕博西尼治疗产生耐药性。对这些数据的进一步研究表明，帕博西尼通过 GPER 信号诱导 CAF 中的促炎转录事件。值得注意的是，通过进行共培养测定，我们证明 GPER 有助于降低 BC 细胞对帕博西尼的敏感性，并促进 BC 细胞与肿瘤微环境的主要成分 CAFs 之间的功能相互作用。
总之，我们的研究结果提供了关于 GPER 如何促进 BC 细胞对帕博西尼耐药的分子事件的新见解。需要进一步的研究来评估靶向 BC 细胞与 CAFs 之间的 GPER 介导的相互作用是否有助于更全面的治疗策略，以克服对帕博西尼的耐药性。
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