Cytoplasmic p21 promotes stemness of colon cancer cells via activation of the <scp>NFκB</scp> pathway

Cancer stem cells (CSCs) drive tumor initiation, metastasis, and therapy resistance. The role of cytoplasmic cyclin-dependent kinase inhibitor 1A (CDKN1A, p21) in CSC biology remains unclear. Since cytoplasmic p21 correlated with advanced stage and metastasis in colorectal cancer (CRC) patients, we investigated its causal role in CSC features in vitro and in vivo. Cytoplasmic p21 increased spheroid formation and CD133 expression in a mechanism partly dependent on AKT activation. Phosphomimetic p21 (p21^T145D) enhanced spheroid growth, CD133, and stemness factors (Oct3/4, Nanog, Sox2), whereas nuclear p21 (p21^T145A) reduced them. Immunoprecipitation, proximity ligation assays, and in silico modeling demonstrated that cytoplasmic p21 interacts with the NFκB-IκB complex, promoting NFκB release and activation. Consequently, NFκB targets BCL-xL and COX2 were upregulated in p21^T145D- and AKT^T308D,S473D CRC cells in vitro and in a chorioallantoic membrane (CAM) model, supporting their role as downstream effectors of cytoplasmic p21. Our findings uncover a new function of cytoplasmic p21 in regulating CSC properties through NFκB modulation. Screening p21 subcellular localization may stratify CRC patients with high metastatic risk providing a basis for CSC-targeted therapeutic strategies.