Protective roles of cytoplasmic p21^Cip1/Waf1 in senolysis and ferroptosis of lung cancer cells

Objective Therapy-induced senescent cancer cells increase the expression of the cyclin-dependent kinase inhibitors p16(Ink4a) and p21(Cip1/Waf1). Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16-negative A549 human lung adenocarcinoma cell line. Methods Senescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT-263 and ABT-737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models. Results DXR-induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT-263 was augmented in p21-knockout A549 (A549-KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT-737. PEM alone induced a higher level of non-apoptotic cell death, ferroptosis, in A549-KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM-treated A549-KOp21 cells than in PEM-treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1A(high) lung adenocarcinoma patients had a poorer prognosis compared to CDKN1A(low) patients. Conclusion Cytoplasmic p21, which was increased in therapy-induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.