Dynorphin B induces autophagy and cytotoxicity in thyroid cancer cells via mTORC1-TFE3 axis

Autophagy, a critical cellular self-degradation process, exhibits a complex, dual role in cancer, either sustaining tumor survival or triggering cell death. This makes it a promising, yet challenging, therapeutic target in oncology. Current thyroid cancer treatments often face limitations, necessitating the exploration of novel agents that can precisely modulate cellular pathways. Dynorphin B, an endogenous opioid peptide, is being investigated for its potential to selectively induce autophagy-mediated cell death, addressing the need for targeted therapies that can shift autophagy's balance towards tumor suppression.

Researchers investigated Dynorphin B's effects and mechanisms in TPC-1 thyroid cancer cells. Dynorphin B was applied to cells at various concentrations to assess dose-dependent effects on cellular processes. Key assays included LC3-II/I ratio analysis and p62 degradation to measure autophagic flux, monodansylcadaverine staining for autophagosome formation, and assessments of mitochondrial membrane potential and ATP production. Oxidative stress markers like 8-OHdG levels and SOD activity were evaluated. Mechanistic studies involved analyzing p-S6K phosphorylation (an mTORC1 readout), TFE3 phosphorylation at Ser321, nuclear translocation of TFE3, and qPCR for autophagy-related genes (ATG5, LC3) and lysosomal genes (LAMP1, CTSD). TFE3 knockdown experiments, chromatin immunoprecipitation (ChIP), and luciferase assays were used to confirm TFE3's role. Finally, immunohistochemical evaluation of human thyroid biopsy specimens assessed KOR-1 expression.

Dynorphin B significantly triggered autophagic flux in TPC-1 cells, evidenced by increased LC3-II/I ratios and enhanced p62 degradation. Autophagosome formation also rose, confirmed by monodansylcadaverine staining. Dynorphin B induced dose-dependent cytotoxicity, disrupting mitochondrial membrane potential and reducing ATP production. Oxidative stress increased, marked by elevated 8-OHdG levels and reduced SOD activity. Mechanistically, Dynorphin B inhibited mTORC1 activity, leading to reduced p-S6K phosphorylation levels. This inhibition resulted in TFE3 dephosphorylation at Ser321, promoting TFE3's translocation to the nucleus. Once in the nucleus, TFE3 activated the transcription of key autophagy genes, including ATG5 and LC3, as well as lysosomal genes LAMP1 and CTSD. TFE3 knockdown experiments blocked these effects, confirming its essential role. Chromatin immunoprecipitation further showed TFE3 binding to CLEAR motifs within the ATG5 and p62 promoters, and luciferase assays validated CLEAR gene activation. > Clinically, immunohistochemical evaluation of biopsy specimens demonstrated markedly lower KOR-1 expression in malignant thyroid tissues compared to adjacent normal tissues, suggesting a potential link between KOR-1 and thyroid cancer progression.