The homeobox transcription factor Nanog has a vital role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). Stabilization of Nanog proteins is essential for ESCs. The ubiquitin-proteasome pathway mediated by E3 ubiquitin ligases and deubiquitylases is one of the key ways to regulate protein levels and functions. Although ubiquitylation of Nanog catalyzed by the ligase FBXW8 has been demonstrated, the deubiquitylase that maintains the protein levels of Nanog in ESCs yet to be defined. In this study, we identify the ubiquitin-specific peptidase 21 (USP21) as a deubiquitylase for Nanog, but not for Oct4 or Sox2. USP21 interacts with Nanog protein in ESCs in vivo and in vitro. The C-terminal USP domain of USP21 and the C-domain of Nanog are responsible for this interaction. USP21 deubiquitylates the K48-type linkage of the ubiquitin chain of Nanog, stabilizing Nanog. USP21-mediated Nanog stabilization is enhanced in mouse ESCs and this stabilization is required to maintain the pluripotential state of the ESCs. Depletion of USP21 in mouse ESCs leads to Nanog degradation and ESC differentiation. Overall, our results demonstrate that USP21 maintains the stemness of mouse ESCs through deubiquitylating and stabilizing Nanog.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.