Associação Portuguesa de Investigação em Cancro
Extracellular vesicles derived-microRNAs predicting enzalutamide-resistance in 3D spheroid Prostate Cancer Model
Extracellular vesicles derived-microRNAs predicting enzalutamide-resistance in 3D spheroid Prostate Cancer Model

Researchers from the Molecular Oncology and Viral Pathology Group (GOMPV) at the Research Center of IPO-Porto (CI-IPOP), in collaboration with researchers from Instituto de Investigação e Inovação em Saúde (i3S), established a 3D in vitro model of castration-resistant prostate cancer, applicable for identifying extracellular vesicle (EV)-derived microRNAs predictive of enzalutamide (ENZ) resistance development. Despite the promising effect of ENZ, a high percentage of patients either present primary resistance or develop resistance to this treatment, which is associated with poorer overall survival and reduced quality of life. Currently, it is known that EVs play a key role in the tumor microenvironment, modulating the acquisition of treatment resistance phenotypes. The project focused on identifying a profile of EV-derived miRNAs associated with ENZ resistance acquisition and evaluating their dynamics during this process, using an in vitro 3D model that mimics disease progression. After inducing ENZ resistance in the LNCaP cell line, the researchers identified 12 overexpressed and 13 underexpressed miRNAs in the resistant cell line after screening 799 miRNAs, and defined a top 5 overexpressed miRNA profile using miRTarbase and Cytoscape (based on the the greatest impact on protein-protein interaction networks). The results demonstrated that hsa-miR-21-5p, hsa-miR-22-3p, hsa-miR-200a-3p, hsa-miR-221-3p, and hsa-miR-222-3p were packaged into EVs during the progression of resistance. A progressive increase in intracellular and EV-derived expression levels of hsa-miR-221-3p and hsa-miR-222-3p was observed in the 3D models resistant to increasing concentrations of ENZ, highlighting an association between the expression of these miRNAs and the acquisition of this phenotype. The results demonstrated that the developed 3D spheroid model effectively replicated ENZ resistance, highlighting the involvement of prostate cancer EV-derived miRNAs in this resistance process and their potential applicability as a source of circulating biomarkers for future liquid biopsies.
Authors and Affiliations:
Inês Tavares1,2*, Mariana Morais1,2*, Francisca Dias1, Mariana Ferreira1,2, Gabriela Martins3, Rui Fernandes4,5, Sílvia Joana Bidarra6,7, Rui Medeiros1,2,8,9,10, Ana Luísa Teixeira1
1Molecular 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 (Porto.CCC), Porto, Portugal;
2ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal
3Department of Immunology, Portuguese Oncology Institute of Porto (IPO-Porto), Porto, Portugal;
4HEMS-Histology and Electron Microscopy, i3S-Instituto de Investigação e Inovação em Saúde, University of Porto, 4200-135 Porto, Portugal.
5IBMC, Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal.
6Bioengineered 3D Microenvironment group, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
7 INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal
8Biomedical Reasearch Center (CEBIMED, Faculty of Health Sciences, Fernando Pessoa University (UFP), Porto, Portugal;
9Research Department, LPCC- Portuguese League Against Cancer (NRNorte);
10Faculty of Medicine, University of Porto (FMUP), University of Porto, Porto, Portugal
*These authors contributed equally to this work.
Abstract:
Enzalutamide (ENZ) has emerged as a major treatment advance in castration-resistant prostate cancer (CRPC) patients; however the development resistance remains a key challenge. The extracellular vesicles (VEs)-derived miRNAs play crucial roles tumor microenvironment 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 this model, we provide a comprehensive platform for elucidating the molecular alterations involved.
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, and analyzed in EVs derived from ENZ resistance 3D spheroid model.
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 adaptative process.
Journal: International Journal of Biological Macromolecules
Link: https://www.sciencedirect.com/science/article/abs/pii/S0141813024088044?via%3Dihub