Elucidating the Role of FASN in Lung Cancer Stem Cells in Sensitive and Resistant EGFR-Mutated Non-Small Cell Lung Cancer Cells

Emma Polonio-Alcalá,1,2 Sira Ausellé-Bosch,1 Gerard Riesco-Llach,3 Pablo Novales,1 Lidia Feliu,3 Marta Planas,3 Joaquim Ciurana,2 Teresa Puig1 1New Therapeutic Targets Laboratory (Targetslab) – Oncology Unit, Department of Medical Sciences, University of Girona, Girona, Spain; 2Product, Process, and Production Engineering Research Group (GREP), Department of Mechanical Engineering and Industrial Construction, University of Girona, Girona, Spain; 3Laboratori d’Innovació en Processos i Productes de Síntesi Orgànica (LIPPSO), Department of Chemistry, University of Girona, Girona, SpainCorrespondence: Teresa Puig, Email [email protected]: Cancer stem cells (CSCs) drive tumor initiation, relapse, and metastasis. Our research team developed polycaprolactone electrospun (PCL-ES) scaffolds for enriching lung CSCs (LCSCs) since monolayer culture do not allow the study of this malignant population. The upregulation of fatty acid synthase (FASN) correlates with resistance to tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR), and its inhibition induces cytotoxicity in EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC) cells. Therefore, this study aims to elucidate the role of FASN and related signaling pathways in LCSCs cultured in PCL-ES scaffolds and to evaluate the effectiveness of FASN inhibitor G28, a synthetic derivative of (−)‐epigallocatechin‐3‐gallate (EGCG), against this population.Methods: EGFR-TKI-sensitive and -resistant cell modes were used. FASN expression and function were studied by RT-qPCR, Western blotting, and free fatty acid quantification, while related signaling pathways (EGFR, MAPK, AKT, and STAT3) were examined by Western blotting. The effects of G28 on LCSCs —including its impact on FASN and related signaling—were evaluated using the MTT assay and Western blotting.Results: LCSCs cultured in PCL-ES scaffolds showed a significant FASN upregulation, supporting their proliferation and maintenance. Despite reduced EGFR activation in 3D-cultured cells, downstream signaling responses differed: PC9 cells exhibited higher levels of p-AKT, p-MAPK, and p-STAT3, while PC9-GR3 cells showed reduced p-MAPK and p-AKT, with no changes in p-STAT3. Regarding G28 treatment, it exhibited cytotoxic effects in both 2D- and 3D-cultured cells, suggesting potential efficacy in targeting both non-LCSCs and LCSCs. Furthermore, the treatment downregulated FASN and AKT, reducing or avoiding the proliferation of this malignant population.Conclusion: Our results highlight the potential of G28 as a therapeutic option for targeting LCSCs in both sensitive and resistant EGFRm NSCLC cells, though additional studies are required to validate these results and assess their clinical applicability. Keywords: lung cancer stem cells, FASN, EGCG synthetic derivative, NSCLC, 3D cell culture, AKT