Tavares Inês, Morais Mariana, Dias Francisca, Ferreira Mariana, Martins Gabriela, Fernandes Rui, Bidarra Sílvia Joana, Medeiros Rui, Teixeira Ana Luísa
Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO-Porto), Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), Porto, Portugal; ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal.
Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO-Porto), Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), Porto, Portugal.
Int J Biol Macromol. 2025 Jan;284(Pt 1):137993. doi: 10.1016/j.ijbiomac.2024.137993. Epub 2024 Nov 26.
Enzalutamide (ENZ) has emerged as a major treatment advance in castration-resistant prostate cancer (CRPC) patients; however the development of resistance remains a key challenge. The extracellular vesicles (VEs)-derived miRNAs play crucial roles tumor microenvironment cell communication, thereby influencing resistance mechanisms. Considering the urgent need for molecular biomarkers to monitor ENZ response and predict resistance, we intend to identify an EV-derived miRNA profile associated with ENZ resistance using an innovative 3D-spheroid in vitro model. Through the generation of this model, we provide a comprehensive platform for elucidating the molecular alterations involved in the process. An in vitro model of ENZ resistance was established through continuous exposure of LNCaP to increasing ENZ concentrations. A screening of 799 miRNAs from resistant and normal LNCaP cells were quantified. A bioinformatic analysis was performed using miRTarbase and Cytoscape and top 5 overexpressed miRNAs were selected, that will be analyzed in extracellular vesicles derived from ENZ resistance 3D spheroid models. We identified 12 up- and 13 downregulated miRNAs in LNCaP 30 μM ENZ cells compare to LNCaP·In silico analysis led to the construction of a 76 proteins cluster and functional enrichment revealed terms like PI3K/AKT, TFG-β and FOXO. hsa-miR-22-3p was significantly decreased at 5 and 20 μM ENZ concentration intracellularly, but significantly increased at 20 μM ENZ in EVs. hsa-miR-221-3p and miR-222-3p were upregulated in all concentrations both intracellularly and in EVs. The developed 3D-spheroid model effectively replicated the ENZ resistance to ENZ in an AR-independent manner, underscoring the importance of EVs-derived miRNAs in this adaptive process.
恩杂鲁胺（ENZ）已成为去势抵抗性前列腺癌（CRPC）患者治疗方面的一项重大进展；然而，耐药性的产生仍然是一个关键挑战。细胞外囊泡（EVs）衍生的微小RNA（miRNAs）在肿瘤微环境细胞通讯中发挥着关键作用，从而影响耐药机制。鉴于迫切需要分子生物标志物来监测ENZ反应并预测耐药性，我们打算使用创新的三维球体体外模型来鉴定与ENZ耐药相关的EVs衍生miRNA谱。通过建立这个模型，我们提供了一个全面的平台来阐明该过程中涉及的分子改变。通过将LNCaP细胞持续暴露于不断增加的ENZ浓度，建立了ENZ耐药的体外模型。对耐药和正常LNCaP细胞中的799种miRNAs进行了定量筛选。使用miRTarbase和Cytoscape进行了生物信息学分析，并选择了5种上调最明显的miRNAs，将在ENZ耐药三维球体模型衍生的细胞外囊泡中进行分析。与LNCaP相比，我们在LNCaP 30 μM ENZ细胞中鉴定出12种上调和13种下调的miRNAs。计算机分析导致构建了一个76种蛋白质的簇，功能富集显示出PI3K/AKT、转化生长因子-β（TGF-β）和叉头框蛋白O（FOXO）等术语。在细胞内，hsa-miR-22-3p在5 μM和20 μM ENZ浓度下显著降低，但在20 μM ENZ的细胞外囊泡中显著增加。hsa-miR-221-3p和miR-222-3p在所有浓度下细胞内和细胞外囊泡中均上调。所建立的三维球体模型以雄激素受体（AR）非依赖性方式有效地复制了对ENZ的耐药性，强调了EVs衍生的miRNAs在这一适应性过程中的重要性。
