The aggressive and immunotherapy-resistant characteristics of anaplastic thyroid carcinoma (ATC) are driven by an immunosuppressive tumor microenvironment and intercellular crosstalk; however, its regulatory mechanisms remain poorly understood. Here, we identified Glucose-6-Phosphate Isomerase (GPI) as a pivotal metabolic immune checkpoint that orchestrates myeloid cells-driven immunosuppression in ATC. We demonstrated that GPI enhances the hexosamine biosynthesis pathway to promote O-GlcNAcylation of thrombospondin-1 (THBS1) at serine-1068. This site-specific modification competes with ubiquitination to stabilize THBS1 and augment its secretion. Released THBS1 engages macrophages to trigger a CEBPB-dependent transcriptional program that drives the expression of the chemokine CCL2. Macrophage-derived CCL2 then acts on tumor-associated neutrophils, promoting their STAT3-dependent differentiation into polymorphonuclear myeloid-derived suppressor cells, which ultimately suppresses CD8+ T cell function. Genetic deletion of either GPI or THBS1 robustly inhibited tumor growth and reversed immunosuppression in vivo. To intervene this axis, we repurposed the multi-kinase inhibitor Regorafenib as a novel GPI inhibitor. We confirmed that Regorafenib disrupts this entire axis and, in combination with anti-PD-1 therapy in ATC, overcomes immunosuppression to elicit potent anti-tumor immunity. Our studies revealed the GPI/O-GlcNAcylation/THBS1 signal as a master regulator of myeloid cell crosstalk and established a novel therapeutic strategy for targeting this metabolic checkpoint to potentiate ATC immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a 5-year survival rate of merely 10%, underscoring the urgent need for new therapeutic targets. In this study, we investigated the driving roles of adhesion G-protein coupled receptor F1 (ADGRF1) in PDAC progression and its biological relevance. By bioinformatics analysis, we found that ADGRF1 was abnormally expressed in PDAC tissues, and high expression of ADGRF1 was significantly negatively correlated with patient prognosis. We further determined that ADGRF1 promotes malignant phenotypes such as proliferation, stemness, migration and invasion of PDAC cells. And in vivo experiments have shown that deletion of ADGRF1 inhibit the tumor progression of PDAC. We demonstrated the molecular mechanism that ADGRF1 regulates the expression of Yin-Yang 1 (YY1) to promote the transcriptional activation of Cathepsin D (CTSD), and thereby upregulates of the mTOR signaling pathway. Taken together, our results describe the promoting effect of ADGRF1 on PDAC progression and may serve as a potential therapeutic target for PDAC.

Mutational heterogeneity at drug targets both undermines small-molecule efficacy and contributes to disease. A practical response is to discover scaffolds that maintain potency in the presence of mutations; however, prevailing biological research strategies are not readily scalable, constraining high-throughput design and discovery. We present PSeMut, a structure-free Siamese model that contrasts wild-type and mutant protein-ligand fingerprints (PSICHIC-derived) to predict mutation-induced activity changes. On a variant-resolved benchmark, PSeMut attains a test RMSE of 0.400 ± 0.025 and consistently outperforms classical baselines trained on identical features and splits; removing the exchange-consistency constraint degrades performance. To evaluate the feasibility of the framework in a prospective setting, we applied a structure-free prioritization pipeline-combining scaffold-novelty filtering, PSICHIC activity scoring, PSeMut-based mutation-tolerance ranking, clustering-based selection, and biological validation. This workflow prioritized SNS-314 for follow-up testing. In the selected validation assays, SNS-314 showed mutation-selective cellular activity (IC50 = 0.45 μM in BRAFV600E; 7.5-fold vs. BRAFWT), suppressed the BRAF-MEK-ERK signaling axis, and produced about 50% tumor growth inhibition in vivo without overt toxicity. Together, these results validate the effectiveness of PSeMut in a structure-free, mutation-aware screening workflow that links sequence-driven modeling to experimental confirmation and enables rational prioritization of mutation-resilient scaffolds across heterogeneous disease settings.

BACKGROUND:Papillary thyroid cancer (PTC) exhibits highly variable clinical behavior, ranging from indolent growth to aggressive lymph node (LN) metastasis. However, the specific molecular mechanisms involved in PTC evolution, driven by metabolic reprogramming and tumor-microenvironmental interactions, remain poorly understood. This study aims to elucidate the spatial metabolic and transcriptional mechanisms underlying PTC tumorigenesis and LN metastasis at multiple molecular levels. METHODS:An integrated spatial multi-omics strategy combining spatial metabolomics and spatial transcriptomics was employed to map the distribution of metabolites and gene expression in heterogeneous PTC tissues. Metabolite profiles and transcriptomic data were analyzed to identify dysregulated pathways and to uncover the key molecular features contributing to PTC tumorigenesis and LN metastasis. Genes related to metastasis-driving metabolites were validated using the TCGA dataset, and further substantiated by zebrafish xenograft models. RESULTS:Spatial mapping revealed unique metabolic and transcriptional signatures across heterogeneous regions of PTC tissue. Key metabolic pathways, such as the arginine-polyamine axis, glycolysis and lipid metabolism, were prominently dysregulated in cancer regions. The surrounding stroma also exhibited metabolic adaptations, like polyamine recycling and glucose storage, which supported tumor growth via metabolic crosstalk. Most importantly, greater spatial heterogeneity, spatial evolutionary routes and 5 potential metastasis-driving metabolites, including FA (22:6), PC (36:4), PC (34:1), N-acetylaspartate and ascorbic acid, were discovered in the primary PTC tissue with LN metastasis. Knockdown of key genes NAT8L and SVCT-2 obviously decreased the metastatic ability of PTC cells. The TCGA database further confirmed that high expression of 10 pro-metastatic metabolite-related genes was significantly associated with poorer prognosis in PTC patients. CONCLUSIONS:This integrated spatial multi-omics approach provides novel insights into the molecular mechanisms underlying PTC evolution, potentially guiding the development of more effective diagnostic and therapeutic strategies for PTC patients.

Cancer drug resistance arises not only from selection of resistant clones, but also through rapid activation of adaptive transcriptional programs. One mechanism of transcriptional regulation involves N6-methyladenosine (m6A) RNA modification, which dynamically regulates mRNA processing and alternative splicing, ultimately impacting cell fate and differentiation. In prostate cancer (PC), resistance to systemic therapies such as the androgen receptor pathway inhibitor (ARPI) enzalutamide is associated with a host of well-documented androgen receptor (AR) alterations, including amplification, mutation, and alternative splicing. Given these functions, we hypothesized that m6A modifications play a role in the transition to enzalutamide resistance in PC. To test this, we used methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) in parallel with RNA-seq to identify gene transcripts that were both differentially methylated and differentially expressed between enzalutamide-sensitive and enzalutamide-resistant PC cells. We filtered and prioritized these genes using clinical and functional database tools, including Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA), and the Oncology Research Information Network (ORIEN) avatar. Using this approach, we identified 487 transcripts that were both differentially methylated and differentially expressed and validated six of the top 12 candidates via targeted qPCR and MeRIP-PCR. One of these, THBS1, was found to have increased m6A level associated with decreased transcript levels in enzalutamide-resistant cells, a finding recapitulated in publicly available preclinical and clinical data. Moreover, in enzalutamide-sensitive cells, depletion of THBS1 by siRNA-knockdown induced resistance to enzalutamide. While THBS1 has previously been implicated in aggressive PC phenotypes, we now show that THBS1 downregulation directly contributes to a rapid transition to enzalutamide resistance, suggesting a novel role for this gene in PC hormonal therapy resistance. These results constitute the first comprehensive epitranscriptomic profiling of ARPI resistance and identify THBS1 as a potential driver of acute resistance in prostate cancer.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by uncontrolled proliferation of immature myeloid blasts, leading to hematopoietic suppression and bone marrow failure. Advances in understanding the pathogenesis of AML have fueled the development of precision medicine approaches, with notable successes in targeting specific mutant proteins (e.g., FLT3, IDH1, IDH2), apoptotic regulators (e.g., BCL-2, MCL1), and cell-surface antigens (e.g., CD33, CD123, CD47). These targeted inhibitors exhibit moderate antileukemic activity as monotherapies and their clinical responses are often limited due to the emergence of drug resistance and disease relapse. Nevertheless, synergistic effects have been observed when these agents are combined with conventional chemotherapy or oncogenic pathway inhibitors. This review analyzes the current limitations of targeted therapies and explores multifaceted resistance drivers, encompassing on-target mutations, compensatory signaling pathway activation, drug-efflux mechanisms mediated by metabolic enzymes or transporters, intrinsic adaptive changes, and interactions with the tumor microenvironment. Corresponding therapeutic counterstrategies are also examined, such as mutation-specific molecular targeting, combinatorial suppression of alternative pathways, disruption of intrinsic adaptive responses, and immunotherapeutic approaches. These evolving interventions aim to overcome specific resistance mechanisms and reduce relapse rates. Future research integrating these strategies holds significant promise for addressing persistent challenges in AML management, ultimately advancing treatment paradigms and patient survival.

BACKGROUND:Clear cell renal cell carcinoma (ccRCC) is a prevalent and aggressive form of kidney cancer. Recently, the identification of suitable subtypes in ccRCC for the prediction of prognosis and immune infiltration remains limited. Kinesin superfamily proteins (KIFs), a group of molecular motor proteins, have been found to play crucial roles in tumor progression and patient prognosis in various cancers. However, the subtypes in ccRCC based on KIFs remain poorly understood. METHODS:In this study, transcriptional profiles of ccRCC were analyzed using data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Differential expression of KIFs was identified using R software. Subsequently, ccRCC patients were stratified into two distinct subgroups based on non-negative matrix factorization (NMF) analysis. Comparative analyses were performed to evaluate prognosis, mutations, and immune cell infiltration between these subtypes. Furthermore, signature genes associated with the identified subtypes were determined, followed by an investigation into their relationship with clinical characteristics and response to immune checkpoint inhibitors. Validation studies involving immunohistochemical staining, malignant phenotype assays, and immunofluorescence were conducted to assess the expression and function of these signature genes. RESULTS:Five KIFs genes, namely, KIF21B, KIF18B, KIF20A, KIF4A, and KIF13B, were identified as classifiers for categorizing ccRCC patients into two distinct subtypes known as KPCS1 and KPCS2. The aggressive subtype, KPCS2, was found to be associated with poorer survival outcomes. Furthermore, higher immune infiltration and copy number variations were observed in the KPCS2 subtype. Four signature genes (SLCA15, WDR72, PSAT1, and HJURP) displayed significant correlations with clinical characteristics and were determined to be linked to the ccRCC subtypes. The expression patterns and functional roles of these signature genes were subsequently validated in both ccRCC cells and tissues. CONCLUSION:KIFs-associated subtypes provide valuable insights into the molecular characteristics and prognostic implications of ccRCC, thereby suggesting potential therapeutic targets for intervention.

Poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) present major challenges in treatment owing to extreme aggressiveness and high heterogeneity. In this study, deep-scale analyses spanning genomic, proteomic, and phosphoproteomic data are performed on 348 thyroid-cancer and 119 tumor-adjacent samples. TP53 (48%), TERT promoter (36.5%), and BRAF (23%) are most frequently mutated in PDTC and ATC. Ribosome biogenesis is identified as a common hallmark of ATC, and RRP9 silencing dramatically inhibits tumor growth. Proteomic clustering identified three ATC/PDTC subtypes. Pro-I subtype is characterized with aberrant insulin signaling and low immune cell infiltration, and Pro-II is featured with DNA repair signaling, while Pro-III harbors high frequency of TP53 and BRAF mutation and intensive C5AR1+ myeloid infiltration. Targeting C5AR1 synergistically improves antitumor effect of PD-1 blockade against ATC cell-derived tumors. These findings provide systematic insights into tumor biology and opportunities for drug discovery, accelerating precision therapy for virulent thyroid cancers.

Anaplastic thyroid carcinoma (ATC), an exceptionally aggressive and rare subtype of thyroid cancer, accounts for 1–2% of all thyroid cancers yet carries a high mortality rate, with a median survival time of less than one year. Despite significant advancements in in the field of thyroid cancer research, effective therapeutic options for ATC remain notably limited. Recently, targeting deubiquitinating enzymes (DUBs) has emerged as a promising strategy in cancer therapy. In this study, we investigated the roles of two DUBs, USP14 and UCHL5, in the progression of ATC. Our findings revealed that both USP14 and UCHL5 were upregulated at both mRNA and protein levels in ATC. Individually silencing USP14 or UCHL5 significantly inhibited the malignant characteristics of ATC, while the simultaneous knockdown of both DUBs proved to be even more efficacious. Furthermore, b-AP15, a dual-targeting inhibitor acting on USP14 and UCHL5, effectively suppressed tumor growth in nude mice. Mechanistically, USP14 and UCHL5 cooperate to stabilize PKCα by concurrently removing K48-linked ubiquitination chains from PKCα, thereby facilitating the nuclear translocation of transcription factor NF-κB and activating the expression of pro-oncogenic and anti-apoptotic genes, such as C-MYC and BCL-XL. These findings suggest that targeting the USP14/UCHL5-PKCα-NF-κB axis may represent a novel therapeutic approach for ATC, offering promising prospects for the development of innovative treatment strategies against this highly lethal disease.

The efficacy of cancer therapy largely depends on the ability of drugs to penetrate the tumor tissues. However, therapeutic outcomes are often limited by formidable physical barriers in the tumor microenvironment (TME), including the blood-brain barrier (BBB), vascular barriers, extracellular matrix (ECM), elevated interstitial fluid pressure (IFP), and solid stress (SS). These barriers collectively restrict drug penetration, reducing the treatment effectiveness. Drug delivery systems (DDSs) have emerged as promising strategies to enhance drug penetration and distribution within tumors by overcoming these physical barriers. This review provides an in-depth examination of the characteristics of TME physical barriers and their impact on therapy as well as DDSs designed to overcome these barriers and improve drug delivery efficiency. Additionally, we discuss nanomaterials that have successfully reached the market or clinical trial phase, highlighting their challenges and significance. Overall, this review aims to inform and inspire the development of more effective DDSs, guiding future research and clinical applications to optimize tumor penetration and therapeutic outcomes.

Prostate cancer (PCa) is a common and aggressive malignancy in men, often diagnosed at advanced stages with a poor five-year survival rate. Despite therapeutic advances, effective treatments for castration-resistant PCa remain lacking. Timosaponin A3 (TA3), a natural steroidal saponin derived from Anemarrhena asphodeloides Bunge, has shown potential anti-tumor properties, but its role in PCa and the underlying mechanisms have not been fully elucidated. In this study, we demonstrate that TA3 significantly inhibits the proliferation, migration, and invasion of PCa cells in vitro, and suppresses tumor growth in xenograft models. Transcriptomic analysis revealed that TA3 exerts its anti-tumor effects by modulating cholesterol metabolism. Elevated cholesterol levels were observed in PCa patients, and exogenous cholesterol administration reduced tumor growth in vivo. Notably, TA3 treatment upregulated the lipid transporter StAR related lipid transfer domain containing 4 (STARD4), a key regulator of cholesterol transport, which was confirmed to mediate the inhibitory effects of TA3 on PCa progression. Overexpression of STARD4 attenuated PCa development both in vitro and in vivo, while STARD4 knockdown abolished these effects. Collectively, our findings suggest that TA3 suppresses PCa progression by enhancing cholesterol metabolism via STARD4 upregulation, supporting its potential as a novel therapeutic agent for prostate cancer.

V-set and immunoglobulin domain-containing 4 (VSIG4) positive tumor-associated macrophage (VSIG4+ TAM) is an immunosuppressive subpopulation newly identified in aggressive cancers. However, the mechanism how VSIG4+ TAMs mediate immune evasion in aggressive cancers have not been fully elucidated. In our study, we found targeting VSIG4+ TAMs by VSIG4 deficiency or blockade remarkably limited tumor growth and metastasis, especially those derived from anaplastic thyroid cancer (ATC) and pancreatic cancer, two extremely aggressive types. Moreover, the combination of VSIG4 blockade with a BRAF inhibitor synergistically enhanced anti-tumor activity in ATC-tumor bearing mice. VSIG4 deficiency recovered the antigen presentation (B2m, H2-k1, H2-d1) of TAMs and activated antigen-specific CD8+ T cells by promoting their in vivo proliferation and intratumoral infiltration. Notably, loss of VSIG4 in TAMs significantly reduced the production of lactate and histone H3 lysine 18 lactylation, resulting the decreased transcription of SPP1 mediated by STAT3, which collectively disrupted the cell-cell interactions between TAMs and neutrophils. Further combination of VSIG4 with SPP1 blockade synergistically boosted anti-tumor activity. Overall, our studies demonstrate the epigenetic regulation function of VSIG4 confers on TAMs an alternative pattern, beyond the checkpoint role of VSIG4, to shape the immunosuppressive tumor microenvironment and impair antigen-specific immunity against aggressive cancers.

Histone deacetylation represents a significant epigenetic mechanism that involves the removal of acetyl groups from histones, subsequently influencing gene transcription. Overexpression of histone deacetylases (HDACs) is prevalent across various cancer types, positioning HDAC inhibitors as broadly applicable therapeutic agents. These inhibitors are known to enhance tumor immune antigenicity, potentially slowing tumor progression. Furthermore, the tumor microenvironment, which is intricately linked to cancer development, acts as a mediator in the proliferation of numerous cancers and presents a viable target for oncological therapies. This paper primarily explores how HDAC inhibitors can regulate cancer progression via the tumor microenvironment and suppress tumor growth through multiple pathways, in addition to examining the synergistic effects of combined drug therapies involving HDAC inhibitors.

The poor prognosis of clear cell renal cell carcinoma (ccRCC) is primarily attributed to inherent resistance and malignant progression, yet the underlying mechanism and effective strategies remain poorly understood. Renal drug transporters play an indispensable role in regulating the intracellular concentration of tumor cells. Therefore, this study aims to investigate the role of proton-coupled oligopeptide transporter 2 (PEPT2) in ccRCC. PEPT2 was found to be downregulated in ccRCC tissues and cell lines, and its low expression was associated with an unfavorable prognosis in ccRCC patients. Overexpression of PEPT2 inhibited cell proliferation and metastasis both in vitro and in vivo. Furthermore, the transcriptional repression of PEPT2 was attributed to DNMT3A/B mediated methylation of its promoter, which could be reversed by the epigenetic inhibitor decitabine, leading to the restored expression and functional transport activity of PEPT2. Importantly, the combination treatment with decitabine (an epigenetic inhibitor) and a lenalidomide-dipeptide conjugate (a substrate for PEPT2) significantly enhanced cytotoxicity against ccRCC cells both in vitro and in vivo. Our investigation into the epigenetic repression of PEPT2 promoters has revealed a significant role in the progression of ccRCC. Furthermore, our findings demonstrate that the combination of PEPT2-targeted lenalidomide with epigenetic therapy effectively enhances cytotoxicity in ccRCC cells. This study provides compelling experimental evidence for the potential therapeutic benefit of targeting PEPT2 in the treatment of ccRCC.

Lymph node metastasis (LNM) holds substantial implications for the recurrence and survival of cancer patients, but the intricate regulatory mechanisms underlying LNM remain poorly understood. MTOGB was dominantly increased in LNM of pan-cancer, significantly activated in epithelial cells and enriched in LNM. Subsequently, we identified a specific epithelial cell subpopulation, EC4, located at the terminal of the LNM differentiation trajectory Lineage2. By intersecting differentially expressed genes in cluster 2, EC4 and Lineage2, we identified six crucial genes. Notably, the expression of Steroid 5α-reductase 3 (SRD5A3) increased with the progression of LNM stages. Knockdown of SRD5A3 effectively suppressed the MTOGB, blocking metastasis in both cell and animal models. Nilotinib was screened as a candidate inhibitor of SRD5A3 and was confirmed to remarkably decrease cancer cell metastasis. SOX4 was identified as a potential transcription factor of SRD5A3, modulated by a dramatic increase in cell communication of SPP1+ macrophages in the immune microenvironment. The supernatant from SPP1+ macrophage significantly enhanced the expression of SOX4/SRD5A3 and the metastatic ability of cancer cells, and this effect was reversed by the deletion of SPP1. Collectively, our findings illuminate the SPP1-SRD5A3 signaling as the crucial driver in LNM and suggest that its blockade could be a promising option for overcoming LNM.

Anaplastic thyroid cancer (ATC) is aggressive and has a high mortality rate. Doxorubicin (DOX), the first-line chemotherapy drug for ATC, has an insufficient effective concentration and severe cardiac toxicity, limiting its clinical application. Therefore, it is urgent to enhance the efficacy of DOX and reduce its toxicity. Designing stimulus-responsive drug delivery systems (DDSs) based on the characteristics of the tumor microenvironment may be a promising approach for the treatment of ATC. We found that heparanase (HPSE) is highly expressed in the tumor microenvironment of ATC, but not in normal thyroid tissue. In this study, we successfully constructed and characterized a HPSE-responsive self-assembled carrier, TET-HS-NI, which was modified with 3,3,5,5-tetraiodothyroacetic acid (TET) that targets αvβ3. DOX was loaded into TET-HS-NI to construct TET-HS-NI/DOX. Zebrafish cell-derived xenograft (CDX) and ATC orthotopic models showed that TET-HS-NI/DOX significantly improved the antitumor efficacy and safety of DOX. In a word, we have constructed a drug delivery system that sensitively releases DOX in the ATC environment where HPSE is overexpressed, increasing DOX's effective concentration at the tumor site and providing a new method for the treatment of ATC.

Thyroid cancer is the most frequently observed endocrine-related malignancy among which anaplastic thyroid cancer (ATC) is the most fatal subtype. The synthesis of protein is active to satisfy the rapid growth of ATC tumor, but the mechanisms regulating protein synthesis are still unknown. Our research revealed that kinetochore protein NUF2 played an essential role in protein synthesis and drove the progression of ATC. The prognosis of patients with thyroid carcinoma was positively correlated with high NUF2 expression. Depletion of NUF2 in ATC cells notably inhibited the proliferation and induced apoptosis, while overexpression of NUF2 facilitated ATC cell viability and colony formation. Deletion of NUF2 significantly suppressed the growth and metastasis of ATC in vivo. Notably, knockdown of NUF2 epigenetically inhibited the expression of magnesium transporters through reducing the abundance of H3K4me3 at promoters, thereby reduced intracellular Mg2+ concentration. Furthermore, we found the deletion of NUF2 or magnesium transporters significantly inhibited the protein synthesis mediated by the PI3K/Akt/mTOR pathway. In conclusion, NUF2 functions as an emerging regulator for protein synthesis by maintaining the homeostasis of intracellular Mg2+, which finally drives ATC progression.

Aims Ovarian cancer (OC) remains a significant challenge in oncology due to high rates of drug resistance and disease relapse following standard treatment with surgery and platinum-based chemotherapy. Despite the widespread use of these treatments, no effective biomarkers currently exist to identify which patients will respond favorably to therapy. This study introduces a zebrafish patient-derived xenograft (PDX) system, capable of replicating both the carboplatin response and metastatic behavior observed in OC patients, within a rapid 3-day assay period. Methods Two OC cell lines: carboplatin-sensitive (A2780) and resistant (OVCAR8) were used to assess differential responses to treatment in murine and zebrafish xenograft models. Tumor tissues from 16 OC patients were implanted into zebrafish embryos to test carboplatin responses and predict metastasis. Additionally, eight clinical OC samples were directly implanted into zebrafish embryos as part of a proof-of-concept demonstration. Results The zebrafish xenografts accurately reflected the carboplatin sensitivity and resistance patterns seen in in vitro and murine models. The zebrafish PDX model demonstrated a 67% success rate for implantation and a 100% success rate for engraftment. Notably, the model effectively distinguished between metastatic and non-metastatic disease, with an area under the ROC curve (AUC) of 0.818. Furthermore, the zebrafish PDX model showed a high concordance with patient-specific responses to carboplatin. Conclusions This zebrafish PDX model offers a fast, accurate, and clinically relevant platform for evaluating carboplatin response and predicting metastasis in OC patients. It holds significant potential for advancing personalized medicine, allowing for more precise therapeutic outcome predictions and individualized treatment strategies.

Background: Anaplastic thyroid carcinoma (ATC), a rare but highly aggressive endocrine malignancy, is characterized by a significant presence of cancer stem-like cells (CSCs). These CSCs, known for their self-renewal and differentiation capacities, contribute to various aggressive tumor properties, including recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. Despite their critical role, the regulatory mechanisms of CSCs in ATC remain poorly elucidated, posing challenges in effectively targeting these cells for treatment. Methods: To delve into this, we employed the single sample gene set enrichment analysis (ssGSEA) algorithm to evaluate the stemness of samples in combined datasets. Samples were then classified into high and low stemness subgroups based on their average stemness scores. Differential gene expression between these subgroups was analyzed. We further explored the association of candidate genes with patient prognosis. Additionally, we conducted gene set enrichment analysis (GSEA) and a series of cell biology experiments to validate the role of DEP domain-containing protein 1 (DEPDC1) in fostering CSC-like traits and regulating the malignant phenotypes of ATC. Results: Our investigation demonstrated that DEPDC1 was significantly upregulated in CSCs and is abundantly expressed in ATC tissues. In vitro assays revealed that knockdown of DEPDC1 markedly inhibited tumor sphere formation and attenuated the proliferation, invasion, and migration of ATC cells. This silencing also resulted in reduced expression of stemness markers associated with CSCs. Furthermore, our GSEA findings linked high DEPDC1 expression to cell cycle progression and the maintenance of tumor cell stemness, with DEPDC1 knockdown disrupting these signaling pathways. Collectively, our results position DEPDC1 as a pivotal regulator of CSC-like characteristics in ATC, where aberrant DEPDC1 expression amplifies stemness properties and fuels the cancer's aggressive behavior. Consequently, DEPDC1 emerges as a promising therapeutic target for ATC management. In summary, this study underscores the pivotal role of DEPDC1 in modulating CSC-like features in ATC, offering new avenues for targeted therapy in this challenging malignancy.