DACT1 inhibits cuproptosis and promotes cell malignancy via activation of PI3K/AKT signaling in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal cancer has one of the highest mortality rates of all head and neck cancers. DACT1 is a cuproptosis-related gene in laryngeal cancer and serves as a risk factor for patient prognosis. This study aimed to investigate the effects of DACT1 on the malignant behavior and cuproptosis of laryngeal squamous cell carcinoma (LSCC) cells. METHODS: DACT1 expression in LSCC cells was measured using RT-qPCR and western blotting. To establish cuproptosis cell model, TU212 and TU686 cells were incubated with elesclomol (20 nM) and CuCl(2) (20 nM) for 24 h. Transfection of shRNA or pcDNA3.1 vectors was performed to interfere DACT1 expression. LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) were used to silence and overexpress PI3K signaling in LSCC cells, respectively. A cuproptosis-specific inhibitor, tetrathiomolybdate, was used to suppress cuproptosis in LSCC cells. Cell viability, proliferation, migration, and invasion were assessed using CCK-8 assays, colony formation assays, Transwell migration, and Transwell invasion assays. Copper concentration and reactive oxygen species (ROS) level were also measured. Western blotting was performed to quantify protein levels of cuproptosis-related genes and factors involved in the PI3K/AKT pathway. RESULTS: DACT1 expression was upregulated in LSCC cells. DACT1 knockdown inhibited LSCC cell proliferation, migration, and invasion. DACT1 depletion enhanced cuproptosis, as evidenced by more pronounced decreases in cell viability, increased intracellular copper concentration and ROS levels, upregulation of HSP70, and downregulation of LIAS. Notably, treatment with the cuproptosis inhibitor tetrathiomolybdate reversed the pro-cuproptosis effects induced by DACT1 silencing. Furthermore, the silencing of DACT1 inactivated the PI3K/AKT signaling, as shown by reduced ratios of p-PI3K/PI3K and p-AKT/AKT. Conversely, DACT1 overexpression activated the PI3K/AKT pathway, an effect that was abolished by LY294002. Moreover, LY294002 reversed the promoting effects of DACT1 on LSCC cell malignancy and its inhibitory effects on cuproptosis. In contrast, activation of the PI3K signaling by 740Y-P reversed the enhancement of cuproptosis caused by DACT1 deficiency. CONCLUSION: DACT1 promotes the malignant behavior of LSCC cells and suppresses cuproptosis by activating the PI3K/AKT signaling.