Lysosomes have recently gained prominence as pivotal signaling hubs implicated in drug resistance within cancer cells. However, the precise role of Transient receptor potential mucolipin 3 (TRPML3), an endo-lysosomal Ca2+-permeable channel known to regulate lysosomal trafficking during endocytosis and autophagy, remains enigmatic in the context of cancer progression. This study aimed to elucidate the involvement of TRPML3 in modulating exosomal release triggered by lysosomal exocytosis during the development of gefitinib resistance in non-small cell lung cancer (NSCLC).

Exosomal counts and cell-cycle analysis were conducted by flow cytometry and western blot analysis. Ratiometric assays and enzymatic activity assessments were performed to investigate Ca2+ signaling, lysosomal pH, and lysosomal exocytosis

Our finding revealed that gefitinib-resistant NSCLC cells, HCC827/GR, exhibited significantly higher basal exosomal release and lysosomal exocytosis compared to gefitinib-sensitive NSCLC cells, HCC827. This difference was associated with an increased expression of TRPML3 in HCC827/GR cells. Furthermore, we observed a close correlation between the elevated exosomal release and lysosomal exocytosis and the upregulation of TRPML3 expression. Notably, the triggering of lysosomal Ca2+ release through TRPML3 was facilitated by gefitinib-induced elevation of lysosomal pH. Our investigation demonstrated that deficiency of TRPML3 resulted in gefitinib-induced cell death, as indicated by the accumulation of Sub-G0 population, hindered cell proliferation, and facilitated poly (ADP-ribose) polymerase cleavage.

In summary, our data demonstrate the emerging role of TRPML3 as a molecular factor in anti-cancer drug resistance. By effectively sensing lysosomal pH acidification, TRPML3 orchestrates lysosomal Ca2+ release, subsequently influencing lysosomal trafficking, exocytosis, and exosomal release. This study contributes to comprehending the defense mechanisms that developed by acquired drug resistance to tyrosine kinase inhibitor gefitinib in NSCLC cells.