Autophagy-lysosomal dependency defines a vulnerable physiological state in drug-tolerant persister cells of triple-negative breast cancer.

Triple-negative breast cancer (TNBC), a subtype of aggressive breast cancer, has limited treatment options. Recurrent disease caused by drug-tolerant persister cells (DTPs) that evade chemotherapeutic agents (e.g., doxorubicin hydrochloride (DOX) and paclitaxel (PTX)) is a significant challenge in treating TNBC. Recent studies highlight both autophagy and lysosomal function as key mechanisms supporting cancer cell survival; however, their precise roles in mediating drug tolerance in TNBC remain largely unexplored. This study aimed to elucidate the mechanisms by which autophagic and lysosomal activities support the survival of TNBC DTPs following exposure to DOX and PTX. Compared with parental cells in both 2D and 3D cellular topologies, our results showed that treatment with DOX and PTX produced a fraction of latent DTPs that exhibited increased autophagic induction and improved lysosomal protein expression. The results revealed that pharmacological inhibition of autophagy (hydroxychloroquine) or lysosomal activity (bafilomycin A1) compromised DTPs' survival. Similarly, disrupting lysosomal integrity with L-leucyl-L-leucine methyl ester (LLOME) decreased DTPs' viability. Additionally, knockdown of the lysosomal protein LAMP1 (lysosomal-associated membrane protein 1) significantly reduced persister cells' viability and enhanced the cytotoxic effects of DOX and PTX. In a xenograft model, depleting LAMP1 in TNBC cells slowed tumor proliferation and delayed tumor initiation. Our results demonstrated that increased autophagy-lysosomal process renders DTP cell survival in TNBC by maintaining mitochondrial reactive oxygen species (mROS) generation, which, in turn, contributes to chemotherapy resistance. A potential treatment strategy for eradicating DTP cells and preventing tumor recurrence in TNBC involves targeting these mechanisms.