Cisplatin-based chemotherapy remains a standard treatment for advanced bladder cancer (BCa), however, the development of chemoresistance substantially compromises its clinical efficacy and patient prognosis. In this study, we investigated the functional role of NEK8 in cisplatin resistance in BCa and elucidated its underlying molecular mechanisms. We found that NEK8 was significantly upregulated in cisplatin-resistant BCa and was associated with unfavorable prognosis. Functional studies demonstrated that NEK8 promoted cisplatin resistance and tumor progression both in vitro and in vivo. Mechanistically, NEK8 directly interacted with ALYREF and promoted its phosphorylation and subsequent nuclear translocation. Nuclear ALYREF recognized m5C-modified CDK18 mRNA and enhanced its stability, resulting in increased CDK18 expression. Elevated CDK18 facilitated DNA damage repair, ultimately driving cisplatin resistance in BCa. Collectively, we identified a NEK8-mediated mechanism in which ALYREF phosphorylation and nuclear translocation enhance CDK18 mRNA stability and DNA damage repair, thereby driving cisplatin resistance in BCa.
Resistance to next-generation androgen receptor antagonists represents a major clinical challenge in prostate cancer (PCa), underscoring the need to define its underlying mechanisms. Here, through an integrated analysis of multiple cohorts, we identify PABPC1 as an RNA-binding protein upregulated in therapy-resistant tumors, whose expression correlates with adverse clinicopathological features. Elevated PABPC1 confers enzalutamide (Enz) resistance by suppressing tumor-cell ferroptosis and CD8+ T-cell cytotoxicity. Mechanistically, PABPC1 promotes the cytoplasmic colocalization of EIF4A1 with ATF4 mRNA and enhances EIF4A1-associated translation of ATF4 mRNA. Subsequently, ATF4 binds the BCAT1 promoter and transcriptionally upregulates BCAT1, thereby promoting branched-chain amino acid (BCAA) metabolic remodeling and ferroptosis resistance, with leucine emerging as a major functional contributor to both ferroptosis regulation and CD8+ T-cell effector responses. In preclinical models, ixazomib restores ferroptotic vulnerability, reinvigorates T-cell activity, and synergizes with Enz, without overt histopathological abnormalities in major organs. These results identify the PABPC1-ATF4-BCAT1 axis as a key regulatory pathway linking RNA regulation with metabolic and immune reprogramming to facilitate treatment resistance in PCa.
Premortem exposure of calreticulin (CALR) on stressed cancer cells is a cardinal damage-associated molecular pattern that licenses anticancer immunity, yet its immunological sufficiency has remained difficult to define because it normally coincides with cellular stress and cell death. Here, we engineered a chemical-genetic system enabling the conditional, biotin-driven translocation of a membrane-anchored CALR chimera from the endoplasmic reticulum to the plasma membrane, thereby uncoupling CALR exposure from cytotoxicity and other hallmarks of immunogenic cell death. This reductionist approach revealed that isolated CALR surface presentation is sufficient to promote CD91/LRP1-dependent engulfment of tumor cells by conventional dendritic cells, particularly cDC1 and cDC2, leading to enhanced dendritic cell activation, cytotoxic lymphocyte responses, and immunological memory. Across multiple syngeneic tumor models, enforced CALR exposure exerted modest standalone antitumor activity but robustly sensitized poorly immunogenic cancers to cisplatin and PD-1 blockade, culminating in T cell-dependent tumor eradication and durable protective immunity. Remarkably, concurrent inhibition of the CD47–SIRPα axis facilitated complete tumor eradication in all treated animals, highlighting the functional cooperation between eat-me and don’t-eat-me signals in orchestrating antigen presentation and adaptive immune priming. These findings further suggest that maximizing the therapeutic activity of CD47 blockade may require concomitant reinforcement of pro-phagocytic signals such as CALR exposure, providing a conceptual framework for the rational design of next-generation combination immunotherapies. Collectively, these findings establish surface-exposed CALR as a cell-autonomous immunogenic signal with instructive activity for dendritic cell-mediated cross-priming and provide a tractable platform to mechanistically deconvolute immunogenic stress responses, with immediate implications for the rational design of combinatorial chemo- and immunotherapeutic regimens in preclinical and potentially clinical oncology.
Nucleotide-binding oligomerization domain containing 2 (NOD2) recognizes conserved bacterial components in the cytoplasm to initiate innate immune responses, and its mutations are closely associated with Crohn’s disease (CD). Here, we discover that the NOD2 signaling is regulated by the deubiquitinase USP25. Macrophages lacking USP25 exhibit impaired NOD2 signaling and are defective in cytokine production upon stimulation with muramyl dipeptide (MDP). USP25 enhances NOD2-initiated signal transduction by increasing the expression and recruitment of X-linked inhibitor of apoptosis protein (XIAP). In mice, USP25 deficiency exacerbates intestinal inflammation induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS), whereas USP25 overexpression alleviates TNBS-induced colitis. Moreover, USP25 deficiency leads to spontaneous intestinal inflammation in aged mice. Importantly, a known single nucleotide polymorphism associated with human inflammatory bowel disease compromises USP25 expression, and lower USP25 expression is detected in patients with CD. Thus, USP25 plays a critical role in NOD2 signaling and may represent a potential therapeutic target for CD.
RIPK1 paradoxically orchestrates pro-survival and pro-death pathways through its scaffolding and kinase activities. Downstream of TNFR1, RIPK1 initiates the formation of two distinct multiprotein complexes: complex I for cell survival and complex IIa/IIb for apoptosis or necroptosis. However, the molecular architecture that initiates RIPK1-mediated assembly of these multiprotein signaling complexes remains unknown. Here, we elucidate the filamentous structure of the death domain of RIPK1 (RIPK1DD) at 2.5 Å resolution using cryogenic electron microscopy (cryo-EM). RIPK1DD filaments comprise three helical chains assembled via four distinct types of interactions, including a unique, hitherto undescribed Type IV interaction. Structure-guided mutagenesis of interface residues effectively disrupts RIPK1DD filament formation, impairs RIPK1 recruitment and ubiquitination within TNFR1 complex I, and abrogates TNF-induced transcriptional responses. Furthermore, death domain-mediated higher-order oligomerization drives RIPK1 autophosphorylation, which consequently licenses the assembly of complex IIa and complex IIb to execute apoptosis and necroptosis, respectively. Together, these findings reveal a critical structural basis of death domain-mediated RIPK1 oligomerization and provide fundamental mechanistic insights into RIPK1-mediated assembly of signaling complexes downstream of TNFR1.
