Resistance to nucleoside-like compounds in cancer treatment is a significant problem. However, the transcriptional pathways by which cancer cells adapt under increasing drug stress are not yet sufficiently elucidated for many clinically promising molecules. 5-Ethynyl-2′-deoxyuridine (EdU) is a thymidine analogue that exhibits a direct antitumor effect and can cross the blood-brain barrier. This compound leads to cell death by inhibiting the thymidylate synthase enzyme, arresting the replication fork, and initiating a futile cycle caused by nucleotide excision repair. To fully map the resistance process to EdU at a single concentration level, adaptive laboratory evolution (ALE) was applied to HeLa cells, and six different resistant cell line stages were obtained with gradually increasing doses of resistance from 0.1 µM to 80 µM EdU. Resistance was characterized by: cell viability tests, quantification of EdU incorporation into genomic DNA by immune-slot blot, morphological examination by bright-field microscopy, and RNA sequencing of all lines under both untreated and acute EdU application (10 µM, 24 h). In this study, five resistance phases differing in mechanism were identified: stress readiness, persistent cell-like AP-1/EGR arrest, AP-1 signaling re-stimulation and increased complexity, the phase where the integrated stress response led by ATF4 reaches its peak, and finally, transcriptional silence in EdU response. Remarkably, cells that have acquired almost complete resistance to EdU-induced cell death do not develop any canonical AP-1, inflammatory, or DNA damage-related transcriptional responses after acute drug administration; we define this as transcriptomic desensitization. This state differs entirely in its mechanism from conventional nucleoside analog resistance: there is no upregulation in either thymidylate synthase or genes involved in DNA repair. Instead, the resistance mechanism relies on two key elements: metabolic exclusion resulting from the suppression of the SLC22A17 transporter and the stepwise remodeling of cell identity towards a partial epithelial-mesenchymal transition (EMT) phenotype. These findings make EdU resistance a useful and investigable model for studying transcriptional adaptation processes to genotoxic nucleoside analogs, particularly those directly associated with glioblastoma combination therapies.
Hypoxia is a hallmark feature of the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC). Hypoxia-inducible factor-1α (HIF-1α) and hypoxia-inducible factor-2α (HIF-2α) are frequently overexpressed across multiple subtypes of head and neck tumors and are strongly associated with poor prognosis. While immune checkpoint inhibitors (ICIs) have revolutionized treatment for recurrent/metastatic HNSCC, their objective response rate (ORR) remains limited to 14
ATG5 (Autophagy-Related Gene 5) is a central regulator of cellular autophagy and a critical node at the intersection of autophagy, apoptosis and therapy resistance. Beyond its canonical role in autophagosome formation, ATG5 engages key apoptotic regulators and multiple signaling pathways to shape cancer cell fate. Although ATG5 is ubiquitously expressed in normal tissues, its impact in cancer is highly context specific. In numerous solid tumors, high ATG5 expression is associated with worse overall survival and therapy resistance, whereas in other cancer types elevated ATG5 predicts improved outcomes, reflecting its capacity to act as either a tumor suppressor or a tumor-protective factor. In this Review, we synthesize current knowledge on ATG5 as a context-dependent molecular switch that reshapes the balance between cellular adaptation and therapeutic vulnerability. We map the signaling networks that crosstalk with ATG5 to sustain autophagy-dependent survival and rewire stress responses to promote drug resistance across therapeutic modalities. We further delineate the regulatory and protein-interaction interfaces that maintain this dependency, and the treatment contexts in which autophagy inhibition is most likely to be effective. Together, these insights position ATG5 as both a mechanistic linchpin in therapy resistance and a translational opportunity. Defining tumors that are functionally dependent on ATG5-mediated autophagy, and the specific interactome nodes that sustain this state, will be essential for developing biomarker-driven strategies that convert ATG5 dependence into a therapeutic vulnerability.
Hypoopharyngeal squamous cell carcinoma (HPSCC) is one of the most aggressive cancers and is characterized by an extremely poor prognosis. We screened TROP2 to be highly expressed in HPSCC, but its effect on HPSCC remains unclear. This study is intended to determine the underlying mechanism by which TROP2 regulates HPSCC. Detecting TROP2 expression in HPSCC and normal tissues to clarify its distribution in tumor epithelial cells. Using TROP2 overexpression/knockdown to assess its effects on HPSCC cell progression phenotypes and pyroptosis. Applying molecular biology techniques to analyze TROP2’s regulation of the NLR signaling pathway, pyroptosis-related proteins and the PI3K/AKT pathway. And verifying via chromatin immunoprecipitation (ChIP) the binding of transcription factor JUND to the TROP2 promoter and its role in activating TROP2 expression. We showed that TROP2 is significantly upregulated in HPSCC, particularly in tumor epithelial cell. Our study revealed that TROP2 promotes HPSCC progression by suppressing pyroptosis. Mechanistically, TROP2 was found to regulate NOD-like receptor (NLR) signaling pathway and downregulate key pyrotosis-related protein, including GSDMD-N, Cleaved Caspase-1 (p20), IL18 and IL-1β. Additionally, we identified that PI3K/AKT signaling pathway is critically involved in TROP2-mediated inhibition of pyroptosis. Upstream analysis further revealed that the transcription factor JUND directly binds to and activates the TROP2 promoter, thereby contributing to its overexpression in HPSCC. Our findings highlight the JUND-TROP2-PI3K/AKT-pyrotosis axis as a potential therapeutic target for the treatment of HPSCC.
