肿瘤干细胞的代谢重编程和放疗抵抗

Recurrence of hepatocellular carcinoma (HCC) is closely related to the infection of hepatitis B virus (HBV). The HBV x protein (HBx) plays a key role in promoting the malignant transformation of hepatocytes and cancer heterogeneity, but the role of HBx in metabolism influencing the generation of cancer stem cells (CSCs) is still unclear. This study explores HBx‐induced glucose metabolic reprogramming of HCC cells to promote the generation of CSCs. Immunohistochemical analysis of the expression of glucose metabolic reprogramming‐related enzymes and stemness markers in HCC tissues and corresponding paracancer tissues of 30 patients; Western blotting, laser confocal microscopy, and metabolism‐detection kits were applied to analyse the expression of glucose metabolism‐related enzymes and cancer stemness markers and glucose metabolic products; the generation of CSCs was observed by stem cell pellet and soft agar colony formation experiments. Results indicated that the expression of PKM2, HK2, LDHA, CSC‐related proteins, and CD133 and CD44 in HCC tissues was significantly higher than that in the corresponding paracancerous tissues. HBx stimulated the expression of the key enzyme of the Warburg effect and CSC‐related proteins, and these proteins were significantly reduced after interference with the expression of the PKM2 protein. PKM2 and OCT4 interact in HCC cells, and PKM2 has a regulatory effect on OCT4 function. This study found that HBx stimulated the Warburg effect and induced HCC stemness reprogramming by activating the PI3K/AKT signalling pathway; PKM2 played a key role in promoting the initiation of HCC stem cells. Targeting HBx and PKM2 is a new strategy for the treatment of HCC.

Human hepatocellular carcinomas (HCCs) with cancer stem cell (CSC) features are a subclass of therapeutically challenging cancers. We recently showed that retrodifferentiation of hepatic cancer cells into CSC-like cells leads to metabolic reprogramming and chemoresistance. The molecular mechanisms whereby differentiated cancer cells switch towards a CSC phenotype are poorly understood. By studying metabolic reprogramming associated with HCC cell plasticity, we identified an unsuspected role of peroxisome proliferator-activated receptor (PPAR)γ in hepatic CSC phenotype acquisition. Gene expression and metabolic analyses performed throughout cell differentiation/retrodifferentiation process of human HepaRG and HBG-BC2 HCC cells show that metabolic reprogramming in hepatic CSCs is associated with fragmented mitochondrial network, decreased respiration, de novo lipogenesis, fatty acid oxidation, but increased glycolysis and lipid storage. Mitochondrial genes downregulated in HepaRG-CSCs are also downregulated in the STEM HCC subclass. While PPARα is the main isoform in differentiated hepatic cells, we find high PPARγ expression in hepatic CSCs. Accordingly, nuclear localization of PPARγ is detected in human HCC tumors and PPARγhigh/PPARαlow expression is associated with the STEM HCC subclass and a poor outcome in human HCC cohorts. PPARγ silencing or/and inhibition of its target gene pyruvate dehydrogenase kinase 4 reactivates cell respiration, increases reactive oxygen species production and sensitizes hepatic CSCs to chemotherapy. Conversely, PPARα activation synergizes with chemotherapy to induce cell death. Targeting PPARγ, a key regulator of metabolic reprogramming and stemness in hepatic CSCs, or modulating the PPARγ/PPARα balance that finely tunes the differentiation/retrodifferentiation process in HCC deserves further investigation for antitumor therapy. Implications heading and statement PPARγ, a key regulator of metabolic reprogramming and stemness in hepatic CSC, reduces oxidative phosphorylation and reactive oxygen species production, therefore contributing to HCC chemoresistance.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that are responsible for disease recurrence and chemoresistance. The mechanisms by which CSCs arise in endometrial cancer are not fully understood and elucidating key molecular events may provide insight into therapeutic targets. CD73, a cell surface 5’nucleotidase that generates adenosine, is frequently downregulated in endometrial tumorigenesis, and CD73 loss is associated with aggressive disease and poor patient outcomes. In developing and characterizing CRISPR/Cas-9 models for CD73, we discovered CD73 deletion (CD73−/−) in HEC-1-A cells causes growth arrest and prolonged survival which are features suggestive of a stemness phenotype. Thus, we hypothesized that loss of CD73 is critical for promoting CSCs in endometrial cancer. To evaluate CSC characteristics, we performed MTT and spheroid assays and interrogated CSC marker protein expression (e.g., CD44 and NOTCH3). CD73−/− HEC-1-A cells formed larger spheroids and expressed high levels of CD44 and NOTCH3 compared to wild-type (CD73+/+) cells. To assess whether loss of CD73 enzymatic activity is responsible for the enrichment of CSCs, CD73+/+ HEC-1-B and HEC-50 cells were treated with AoPCP, a catalytic inhibitor of CD73. Similar to genetic deletion of CD73 in HEC-1-A cells, AoPCP treatment resulted in HEC-1-B and HEC-50 cells forming larger spheroids and expressing high CD44 and NOTCH3. These data indicate that loss of CD73 enzyme activity is important to the enrichment of CSCs. In contrast, HEYA8 ovarian cancer cells showed the opposite effects, wherein treatment with AoPCP decreased spheroid size. These differences support the emerging paradigm that CD73 has tissue- and cancer-specific roles. As CD73 is a major node in purine metabolic signaling, we further hypothesized that loss of CD73 activity may be promoting CSC enrichment via changes to metabolic pathways. Mass spectrometry metabolomics revealed significantly different profiles of CD73+/+ and CD73−/− HEC-1-A cells. Conditioned media experiments showed CD73−/−-conditioned media induces growth arrest in HEC-1-A wild-type cells, whereas the growth arrest and CSC marker expression enrichment of CD73−/− cells was abrogated by CD73+/+-conditioned media or daily media replenishment. Metabolomic profiling of CD73+/+ and CD73−/− HEC-1-A cells with/without daily media replenishment revealed metabolic pathways, such as NAD+ biosynthesis, lipid metabolism, and sugar alcohol synthesis, are up-regulated by CD73 deficiency and parallels the enrichment of CSCs. Current studies are dissecting these metabolic dependencies in supporting endometrial cancer cell stemness. Altogether, these studies provide novel insight into how CSCs arise in endometrial cancer and why CD73 loss is detrimental in this disease. Citation Format: Emily M. Rabjohns, Blake R. Rushing, Sayali Joseph, Cyrus Vaziri, Jessica L. Bowser. Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B035.

Although controversial, cancer stem cells (CSCs) are thought to be one tumor component, being characterized by their strong self-renewal and survival properties. Cancer cells, CSCs included, are thought to rely mostly on glycolysis, even in the presence of oxygen, which confers them adaptive advantages. Adenine nucleotide translocator 2 (ANT2), responsible for the exchange of ADP and ATP in the mitochondrial inner membrane, has been correlated with a higher glycolytic metabolism and is known to be overexpressed in cancer cells. Using P19 embryonal carcinoma stem cells, we inhibit ANT2 translation by using siRNA. ANT2 protein levels were shown to be overexpressed in P19 undifferentiated cells (P19SCs) when compared to their differentiated counterparts (P19dCs). Furthermore, we showed here that the OXPHOS machinery and mitochondrial membrane potential are compromised after ANT2 depletion, leading to a metabolic adaptation towards a less oxidative phenotype. Interestingly, hexokinase II levels were downregulated, which was also accompanied by decreased cell growth, and decreased ability to form spheroids. Our findings underscore ANT2 as a key regulator of metabolic remodeling and cell survival of cancer stem-like cells, suggesting its potential as a therapeutic target for controlling CSC-driven tumor progression.

Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we show that the interferon-inducible (IFN-inducible) protein viperin drove metabolic alteration in cancer cells. Viperin expression was observed in various types of cancer and was inversely correlated with the survival rates of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster–binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid β-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.

Simple Summary Bladder cancer (BC) ranks second in incidence and mortality among all genitourinary cancers. The high recurrence of BC is attributed to the presence of cancer stem cells (CSCs), which are the driving force behind tumor growth. Increasing evidence suggests that stem cells exhibit a distinct metabolic program compared to differentiated cells. Understanding their metabolic preference for maintaining stem cell properties is essential for developing novel therapeutics targeting CSCs. The current work shows for the first time that the scaffold protein β-arrestin1 (ARRB1) functions as a metabolic switch regulating the metabolic reprogramming of CSC-like cells towards glycolysis by regulating the mitochondrial pyruvate carrier MPC1 and glucose transporter GLUT1. The balance between glycolysis and oxidative phosphorylation plays a crucial role in regulating the fate of stem cells. Our findings will potentially open new therapeutic avenues for targeting bladder cancer cells and/or the CSC-like cells within aggressive bladder tumors. Abstract β-arrestin 1 (ARRB1) is a scaffold protein that regulates signaling downstream of G protein-coupled receptors (GPCRs). In the current work, we investigated the role of ARRB1 in regulating the metabolic preference of cancer stem cell (CSC)-like cells in bladder cancer (BC). We show that ARRB1 is crucial for spheroid formation and tumorigenic potential. Furthermore, we measured mitochondrial respiration, glucose uptake, glycolytic rate, mitochondrial/glycolytic ATP production and fuel oxidation in previously established ARRB1 knock out (KO) cells and corresponding controls. Our results demonstrate that depletion of ARRB1 decreased glycolytic rate and induced metabolic reprogramming towards oxidative phosphorylation. Mechanistically, the depletion of ARRB1 dramatically increased the mitochondrial pyruvate carrier MPC1 protein levels and reduced the glucose transporter GLUT1 protein levels along with glucose uptake. Overexpression of ARRB1 in ARRB1 KO cells reversed the phenotype and resulted in the upregulation of glycolysis. In conclusion, we show that ARRB1 regulates the metabolic preference of BC CSC-like cells and functions as a molecular switch that promotes reprogramming towards glycolysis by negatively regulating MPC1 and positively regulating GLUT1/ glucose uptake. These observations open new therapeutic avenues for targeting the metabolic preferences of cancer stem cell (CSC)-like BC cells.

Clear cell renal carcinoma (ccRCC) is one of the most common urological tumors worldwide and metabolic reprogramming is its distinguishing feature. A systematic study on the role of the metabolism-related genes in ccRCC cancer stem cells (CSCs) is still lacking. Moreover, an effective metabolism-related prediction signature is urgently needed to assess the prognosis of ccRCC patients. Clear cell renal carcinoma (ccRCC) is one of the most common urological tumors worldwide and metabolic reprogramming is its distinguishing feature. A systematic study on the role of the metabolism-related genes in ccRCC cancer stem cells (CSCs) is still lacking. Moreover, an effective metabolism-related prediction signature is urgently needed to assess the prognosis of ccRCC patients. Gene expression profiles of GSE48550 and GSE84546 were analyzed for the role of metabolism-related gene in ccRCC-CSCs. The GSE22541 dataset were used to construct and validate an effective metabolism-related prediction signature to assess the prognosis of ccRCC patients. For glycolytic metabolism, we found that HKDC1, PFKM and LDHB were significantly upregulated in ccRCC-CSCs in GSE84546. For TCA cycle, ACO1, SDHA and MDH1 were significantly downregulated in ccRCC-CSCs in both GSE48550 and GSE84546. For fatty acid metabolism, CPT1A and ACACB were significantly upregulated in ccRCC-CSCs in GSE84546. It is worth noting that SCD was significantly downregulated in both GSE48550 and GSE84546. For glutamine metabolism, SLC1A5, GLS and GOT1 were significantly upregulated in GSE84546. An eight-gene CSCs metabolism-related risk signature including HKDC1, PFKM, LDHB, IDH1, OGDH, SDHA, GLS and GLUL were constructed to predict the overall survival (OS) of ccRCC patients. Patients could be separated into two groups, and the patients with lower risk scores had longer survival time. Our study indicated that metabolic reprogramming, including glycolytic metabolism, TCA cycle, fatty acid metabolism and glutamine metabolism, is more obvious in CD105+ renal cells (GSE84546) than CD133+ renal cells (GSE48550). An eight-gene metabolismrelated risk signature including HKDC1, PFKM, LDHB, IDH1, OGDH, SDHA, GLS and GLUL can effectively predict OS in ccRCC. N/A

PRMT3 orchestrates metabolic reprogramming in non–small cell lung cancer through a TFAP2A-IDO1 pathway that stimulates kynurenine synthesis to promote radioresistance and immunosuppression, highlighting this axis as a putative therapeutic target.

Nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates the expression of critical antioxidant proteins, was recently demonstrated to play a key role in cancer progression. Resistance to radiotherapy is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about the association between Nrf2 and radioresistance in OSCC. Two OSCC cell lines (SAS and HSC-2) and their clinically relevant radioresistant (CRR) clones (SAS-R, HSC-2-R) were used. The effects of Nrf2 downregulation on radiosensitivity and the involvement of glycolysis in Nrf2-mediated radioresistance were evaluated. Immunohistochemistry of phosphorylated Nrf2 (p-Nrf2) was performed in 110 patients with OSCC who underwent preoperative chemoradiotherapy and surgery. Nrf2 was stably upregulated in CRR cells in vitro and in a mouse xenograft model. Moreover, elevated Nrf2 expression was associated with radioresistance. The enhancement of Nrf2-dependent glycolysis and glutathione synthesis was involved in the development of radioresistance. Additionally, p-Nrf2 expression was closely related to the pathological response to chemoradiotherapy, and its expression was predictive of prognosis in patients with advanced OSCC. Our results suggest that Nrf2 plays an important role in the radioresistance of OSCC accompanied by metabolic reprogramming. Targeting Nrf2 antioxidant pathway may represent a promising treatment strategy for highly malignant OSCC.

Metabolic reprogramming and abnormal glucose metabolism are hallmarks of head and neck squamous cell carcinoma (HNSCC). Certain oncogenes can promote cancer‐related metabolic changes, but understanding their crosstalk in HNSCC biology and treatment is essential for identifying predictive biomarkers and developing target therapies.

No abstract available

Cancer stem cells (CSCs) are a major cause for the insufficient tumor eradication in the clinic, which universally present enhanced mitochondrial oxidative phosphorylation (OXPHOS) to facilitate stemness maintenance and drive treatment resistance. Herein, we report a nanointegrative radiation-triggerable bioreactor (RTB) that selectively remodels CSC-intrinsic arginine metabolism to bioenergetically reprogram CSCs towards a therapeutically-vulnerable differentiated state, leading to durable radio-immunotherapeutic responses in vivo. The RTB nanosystem was developed through the supramolecular integration of radioresponsive iNOS-expressing genetic circuits (pDNAiNOS) and β-lapachone (LAP) into CSC-targeting cationic liposomes. Low-dose radiotherapy (LDR)-induced Nrf2 upregulation readily activates pDNAiNOS to express excessive iNOS, which then depletes CSC-intrinsic arginine while generating abundant nitric oxide (NO) for in-situ amplification of LDR-mediated cytotoxicity. Meanwhile, LDR also upregulates NQO1 expression to promote LAP-mediated ROS generation. These effects could act in a cooperative manner to potently damage CSC mitochondria, which not only blocks OXPHOS activity to drive the differentiation of CSCs for abolishing their self-renewal and resistance capability, but also enhances their propensity towards immunogenic necroptosis to elicit adaptive antitumor immunity, showing significant potential for treating therapy-persistent tumors.

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. Implications: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

Lung cancer is the most lethal cancer, and 85% of cases are classified as non-small cell lung cancer (NSCLC). Metabolic rewiring is a cancer hallmark that causes treatment resistance, and lacks insights into serine/glycine pathway adaptations upon radiotherapy. We analyzed radiotherapy responses using mass-spectrometry-based metabolomics in NSCLC patient’s plasma and cell lines. Efficacy of serine/glycine conversion inhibitor sertraline with radiotherapy was investigated by proliferation, clonogenic and spheroid assays, and in vivo using a serine/glycine dependent NSCLC mouse model by assessment of tumor growth, metabolite and cytokine levels, and immune signatures. Serine/glycine pathway metabolites were significantly consumed in response to radiotherapy in NSCLC patients and cell models. Combining sertraline with radiotherapy impaired NSCLC proliferation, clonogenicity and stem cell self-renewal capacity. In vivo, NSCLC tumor growth was reduced solely in the sertraline plus radiotherapy combination treatment group. Tumor weights linked to systemic serine/glycine pathway metabolite levels, and were inhibited in the combination therapy group. Interestingly, combination therapy reshaped the tumor microenvironment via cytokines associated with natural killer cells, supported by eradication of immune checkpoint galectin-1 and elevated granzyme B levels. Our findings highlight that targeting serine/glycine metabolism using sertraline restricts cancer cell recovery from radiotherapy and provides tumor control through immunomodulation in NSCLC.

Mitochondrial metabolism is enhanced in GSC compared to their differentiated progeny. IR-induced dedifferentiation in GBM cells occurs via an early metabolic shift to OXPHOS. IR-induced metabolic shift links CA9 induction, extracellular acidification, and stemness. Glioblastomas (GBM) are brain tumors with the worst prognosis despite treatment with surgery and radio/chemotherapy, emphasizing the need for new therapies and improved treatment efficacy. Previously, we showed that clinically relevant ionizing radiation (IR) doses enhance GBM cell dedifferentiation into a stem-like phenotype, increasing stemness markers, self-renewal, and tumorigenic abilities. This work focuses on identifying early mechanisms driving this plasticity, particularly metabolic adaptations, as tumor metabolism may support therapy resistance and recurrence. In this study, primary cell lines were established from GBM biopsies of several patients and cultured either in restrictive medium to form neurospheres (NS) enriched in GBM stem-like cells (GSC) or in normal medium to obtain their differentiated progenies. These differentiated GBM cells were then subjected to a short-term dedifferentiation protocol after IR (24 to 72 h) to characterize their metabolic and phenotypic properties. We found that early stem marker increases after IR exposure coincide with higher oxygen consumption rate (OCR), mitochondrial ATP production, and extracellular acidification rate (ECAR). However, lactate production remained unchanged, and glucose uptake showed only a transient, nonspecific increase. This shift to oxidative mitochondrial metabolism, coupled with extracellular acidosis, promotes a stem-like phenotype and is associated with Carbonic Anhydrase IX (CA9) overexpression, an extracellular membrane protein producing H + to buffer intracellular pH. CA9 downregulation via ShRNA reduces extracellular acidification and blocks early IR-induced dedifferentiation, such as neurosphere (NS) formation and stem marker overexpression. Similarly, increased OCR and ECAR, often linked to CA9 overexpression, are observed in patient-derived GSC compared to their differentiated progenies. These findings highlight CA9 as a potential target to block IR-induced early dedifferentiation process as well as the specific metabolic state of GSC. Our study strengthens the therapeutic potential of CA9 inhibition to limit GBM-associated acidic extracellular environment and enhance radiotherapy efficiency by limiting tumor cell plasticity.

Supratentorial ependymomas (ST-EPN) are pediatric brain tumors found in the cerebral hemisphere, driven by the oncogenic fusion of ZFTA with RELA. Other than surgery and radiotherapy, these tumors do not have effective therapeutic options. Recurrent ST-EPN tumors are highly aggressive and hard to treat, which makes these tumors lethal. To understand the disease mechanism and potentially identify or develop therapeutic targets, we established patient-derived models and interrogated the genetic and metabolic dependencies using cutting-edge, near-genome-wide genetic screening and metabolomic tools. Our integrative transcriptomics and metabolic analysis identify that the ZFTA-RELA fusion drives methionine metabolic reprogramming in the cancer stem cells to regulate permissive epigenetic status and pyrimidine nucleotide synthesis to fuel tumor growth. Limiting methionine or inhibiting MAT2A, a rate-limiting enzyme of methionine metabolism in ST-EPN cells, results in profound cell cycle arrest and make the cells vulnerable to pyrimidine inhibitors compared to other pediatric brain tumors. Thus, for treatment, along with radiotherapy, blocking the methionine cycle by using the methionine limited diet or in combination with pyrimidine metabolism inhibitors can be a novel therapeutic strategy against the aggressive paediatric ST-EPN tumors.

Glioblastoma (GBM) is the most invasive and lethal primary brain melanoma. The existing treatment modality is unable to achieve thorough elimination of GBM due to the aggressive nature, blood‐brain barrier (BBB), hypoxic environment, and heterogeneous cellular components. Herein, a radio‐immunotherapy regimen based on a versatile nanoplatform (G/APH‐M) is proposed to effectively kill quiescent cancer stem cells (CSCs) and proliferative cancer cells in GBM in a simultaneous manner. Among o ur prepared G/APH‐M, the coating of GL261 cell membrane guarantees the BBB‐penetrable delivery and homologous GBM‐targeting of the hollow Prussian blue loaded with oxidative phosphorylation inhibitor Gboxin and catalase‐mimetic nanozymes. After substantial tumor accumulation, the released Gboxin inhibits mitochondrial oxidative phosphorylation to kill CSCs, while the nanozymes catalyze the production of oxygen to enhance radiotherapy. Consequently, potent immunogenic cell death (ICD) of GBM is induced, which in combination with immune checkpoint inhibitors (αPD‐L1), achieving a potent therapeutic effect with an 80% survival rate in the orthotopic GBM model even at 60 days after the treatment. The synergistic modulation of hypoxia and metabolism based on G/APH‐M greatly intensifies the radio‐immunotherapy of GBM, which would inspire more comprehensive strategies targeting the multiple characteristics of GBM cells for clinical benefits.

ABSTRACT Radiotherapy is one of the curative mainstays of prostate cancer; however, its efficacy is often diminished by tumor radioresistance. Depending on the stage of disease, tumors can relapse in approximately 50% of patients with prostate cancer after radiotherapy. Nevertheless, the mechanisms that drive tumor cell survival are not fully characterized, and reliable molecular prognostic markers of prostate cancer radioresistance are missing. Similar to other tumor entities, prostate cancer cells are heterogeneous in their capability to maintain tumor growth. The populations of cancer stem cells (CSCs) with self-renewal and differentiation properties are responsible for tumor development and recurrence after treatment. Eradication of these CSC populations is of utmost importance for efficient tumor cure. In a recently published study, we showed that prostate cancer cells could be radiosensitized by glutamine deprivation, resulting in DNA damage, oxidative stress, epigenetic modifications, and depletion of CSCs. Conversely, prostate cancer cells with resistance to glutamine depletion show an activation of ATG-mediated macroautophagy/autophagy as a survival strategy to withstand radiation-induced damage. Thus, a combination of targeting glutamine metabolism and autophagy blockade lead to more efficient prostate cancer radiosensitization. Abbreviations: ATG5: autophagy related 5; CSCs: cancer stem cells; GLS: glutaminase; TCA cycle: tricarboxylic acid cycle

Abstract Background The most common type of esophageal cancer, esophageal squamous cell carcinoma (ESCC), has a 5-year survival rate of only 15%. This low survival rate is to some extent attributed to a high relapse rate, which is, in part, linked to the presence of cancer stem cells (CSC). CSC are a subpopulation of tumor cells that show reduced sensitivity to conventional anticancer therapies, increased potency and self-renewal capacities. With that in mind, ESCC cell lines were exposed in a prolonged manner to anticancer treatments (radiotherapy, 5-FU chemotherapy, and combined therapy) in our laboratory. As expected, an increase in the proportion of CSC (CD44highCD24high) was observed by flow cytometry following the treatments. In pursuit of innovative therapeutic approaches, we conducted an in-depth investigation of the proteomic profiles of these cell lines by mass spectrometry (MS), seeking to unveil novel biological mechanisms. Interestingly, many proteins associated with metabolism and autophagy were modulated. Several alterations were observed by a metabolomic approach as well, namely an increase in intracellular lactate concentration. Recently, a novel role for lactate has been described: lactylation, which is a post-translational modification. Lactylome of those cell lines revealed alteration in proteins related to autophagy and those results led to my research project. Aims Investigate the role of lactylation in autophagy of ESCC. Methods Human ESCC cell lines (including the cell lines exposed to anticancer treatments) will be used as cell culture models. Cell and organoid cultures, mass spectrometry, qPCR, WB, and proliferation assay were used. Results MS analysis of proteins related to autophagy (i.e. RAB1B, CK2, SNX6 et GABARAP) suggests differential regulation of autophagy in cell lines exposed to prolonged treatments. A strong contribution of the 5-FU treatment to the autophagic modification is observable in the double-treated cells. Thus, we confirmed an increase in the LC3BII/I ratio by WB in the cell lines exposed to anticancer treatments. LC3BII/I WB used in combination with Bafilomycin A1 treatment for inhibition of autophagosome and lysosome fusion is a commonly used read-out for autophagic flux. To further study the link between lactylation, autophagy and CSC, we screened 10 human ESCC cell lines for their LC3BII/I ratio. Four cell lines were selected: TE1, TE2, HCE4 and TE5. Using Bafilomycin A1, we confirmed that the autophagic flux in those cell lines was not blocked. Those cell lines will now be used to perform lactylome analysis of sorted CSC cells. Conclusions In brief, our results showed that the autophagic flux is increased in cells exposed to prolonged treatments. This suggests that autophagy could play a role in ESCC response to treatment. Funding Agencies CIHRTRIANGLE, Chaires de recherche du Canada, CRCHUS, Université de Sherbrooke

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a median survival of less than two years despite surgery, chemotherapy, and radiotherapy. A subpopulation of glioma stem-like cells (GSCs) survives radiation and repopulates the tumor. Recent studies have shown that GSCs rely heavily on mitochondrial fatty-acid oxidation (FAO) for ATP production, NAD+ regeneration, and redox balance. Carnitine-palmitoyl-transferase-1 (CPT-1) catalyzes the rate limiting step of FAO by shuttling long-chain fatty acids into the mitochondrial matrix. We therefore hypothesized that pharmacologic inhibition of CPT-1 will limit GSC metabolism and sensitize GSCs to radiation. Human GBM cell line U-118 and a patient-derived GSC line (GNS144) were treated for 72 h with perhexiline (5 µM), etomoxir (10 µM), or vehicle control.Cells then received a single fraction of 0, 2, 4, 6, or 8 Gy. Cell viability was measured 48 h later. Sphere formation assays were performed 7-14 days after radiation; sphere number and mean diameter were recorded. Immunoblotting was performed to evaluate for stem cell markers CD44, Nestin, and Vimentin. All experiments were performed in triplicate; statistical significance was assessed by two-way ANOVA with Tukey post-hoc test (p < 0.05). Perhexiline + radiation and etomoxir + radiation produced a dose dependent decline in cell viability compared with radiation alone (p < 0.001). Combination treatment reduced sphere number by 55-70 % across the 4-8 Gy range (p ≤ 0.005) and lowered mean sphere diameter by 30 % (p ≤ 0.005). Immunoblotting analysis showed a 2-fold reductions in CD44, Nestin, and Vimentin expression in the drug + radiation arms versus radiation alone (p < 0.01). These results suggest that inhibition of CPT-1-mediated FAO with perhexiline or etomoxir enhances radiation induced cell death, suppresses sphere forming capacity, and downregulates key GSC markers in both standard and patient-derived GBM models. These findings should be investigated further and suggest that CPT-1 blockade could be integrated with current standard of care regimens to overcome stem cell driven radiation resistance. Kenneth M. Austin, Tingting Huang, Todd Miller, Kelli B. Pointer. Targeting CPT-1-mediated fatty acid oxidation causes radiation sensitization in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6612.

Glioblastoma (GBM) is a malignant primary brain tumor in which the standard treatment, ionizing radiation (IR), achieves a median survival of less than 15 months. GBM harbors glioblastoma stem cells (GSCs), which are crucial in therapeutic resistance and recurrence. Here, we report that GSCs that survive standard treatment radiation upregulate Speedy/RINGO cell cycle regulator family member A (Spy1) and downregulate CAP-Gly domain-containing linker protein 3 (CLIP3). We discovered that Spy1 activation and CLIP3 inhibition coordinately shift GBM cell glucose metabolism to favor glycolysis via two cellular processes: transcriptional regulation of CLIP3 and facilitating Glucose transporter 3 (GLUT3) trafficking to cellular membranes in GBM cells. Importantly, in combination with IR, glimepiride, an FDA-approved medication used to treat type 2 diabetes mellitus, disrupts GSCs maintenance and suppresses glycolytic activity by restoring CLIP3 function. In addition, combining radiotherapy and glimepiride significantly reduced GBM growth and improved survival in a GBM orthotopic xenograft mouse model. Collectively, our data suggest that repurposing glimepiride for GBM therapy could be an attractive strategy for overcoming tumor recurrence. [This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2020M2D9A2094156) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00207904).] Citation Format: Hyunkoo Kang, Eunguk Shin, Haksoo Lee, BuHyun Youn. Pharmacological activation of CLIP3 reduces radioresistance by suppressing stemness and glycolysis in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A106.

Background: Head and Neck Squamous Cell Carcinomas (HNSCCs) are heterogeneous malignancies that comprise 90% of the head and neck cancers. HNSCCs originate from the mucosal lining epithelium of the upper aerodigestive tract. Cancer stem cells (CSCs) that generate HNSCCs with the CD44, CD133, and ALDH phenotype and are resistant to radiotherapy and chemotherapy. In the current, the quantitative alteration in CD44 and CD133 expression pre- and post-tumor resection and radiotherapy was evaluated in HNSCC patients. Moreover, the alterations in the expression of Bax, Bak, Bcl-2, ALDH, and PTEN genes were measured. Materials and Methods: Flow cytometry was performed to evaluate the alterations in CD44 and CD133 surface markers pre- and posttumor resection and radiotherapy. Quantitative real-time RT-PCR (qRT-PCR) was conducted to investigate the mRNA expression levels of Bax, Bak, Bcl-2, ALDH, and PTEN. Results: The results indicated that the cancer stem cell CD44 surface marker significantly decreased after tumor resection and radiotherapy in HNSCC cases, while the decrease was insignificant for CD133 marker expression. mRNA expression level of Bcl-2 and ALDH was increased, but Bax and Bak gene expressions were reduced significantly Conclusion: The results also indicated that the expression of CD44 significantly decreased after tumor resection and radiotherapy. The upregulation of mRNA level of Bcl-2 and ALDH, and the downregulation of Bax and Bak gene expression were noted in these cases when compared to the healthy control group.

Cuproptosis-based cancer nanomedicine has received widespread attention recently. However, cuproptosis nanomedicine against pancreatic ductal adenocarcinoma (PDAC) is severely limited by cancer stem cells (CSCs), which reside in the hypoxic stroma and adopt glycolysis metabolism accordingly to resist cuproptosis-induced mitochondria damage. Here, we leverage hyperbaric oxygen (HBO) to regulate CSC metabolism by overcoming tumor hypoxia and to augment CSC elimination efficacy of polydopamine and hydroxyethyl starch stabilized copper-diethyldithiocarbamate nanoparticles (CuET@PH NPs). Mechanistically, while HBO and CuET@PH NPs inhibit glycolysis and oxidative phosphorylation, respectively, the combination of HBO and CuET@PH NPs potently suppresses energy metabolism of CSCs, thereby achieving robust tumor inhibition of PDAC and elongating mice survival importantly. This study reveals novel insights into the effects of cuproptosis nanomedicine on PDAC CSC metabolism and suggests that the combination of HBO with cuproptosis nanomedicine holds significant clinical translation potential for PDAC patients.

No abstract available

Introduction The presence of cancer stem cells (CSCs) significantly limits the therapeutic efficacy of radiotherapy (RT). Efficient elimination of potential CSCs is crucial for enhancing the effectiveness of RT. Methods In this study, we developed a biomimetic hybrid nano-system (PMC) composed of MnCO3 as the inner core and platelet membrane (PM) as the outer shell. By exploiting the specific recognition properties of membrane surface proteins, PMC enables precise targeting of CSCs. Sonodynamic therapy (SDT) was employed using manganese carbonate nanoparticles (MnCO3 NPs), which generate abundant reactive oxygen species (ROS) upon ultrasound (US) irradiation, thereby impairing CSC self-renewal capacity and eradicating CSCs. Subsequent RT effectively eliminates common tumor cells. Results Both in vitro cell experiments and in vivo animal studies demonstrate that SDT mediated by PMC synergistically enhances RT to selectively combat CSCs while inhibiting tumor growth without noticeable side effects. Discussion Our findings offer novel insights for enhancing the efficacy and safety profiles of RT.

Highly plastic cancer stem‐like cells (CSCs) in hepatocellular carcinoma (HCC) drive tumor heterogeneity, contributing to radiotherapy failure. Although inducing CSC differentiation is proven effective in leukemia, this approach is shown limited success in solid tumors due to the complex signaling networks that sustain CSC stemness. In this study, the synergistic effect of Pin1 and Notch1 in HCC is identified, which plays a pivotal role in maintaining the aggressiveness of CSCs and promoting radioresistance. Building on this discovery, biomimetic nanovesicles (CALT‐GM‐NVs) are engineered by infusing tumor cell membranes into liposomes, which exhibit superior binding affinity to CSCs. RNA sequencing reveals that CALT‐GM‐NVs downregulate oncogenic signaling pathways while upregulating those linked to differentiation and apoptosis. In vivo, CALT‐GM‐NVs significantly reduced CSC‐driven radiotolerance and improved radiotherapy efficacy in both cell line‐derived and patient‐derived HCC xenograft models. These findings highlight the potential of simultaneously targeting Pin1 and Notch1 to induce CSC differentiation and provide a promising radiosensitizer for improving HCC radiotherapy outcomes.

In Brief Chen et al. show that the pluripotency transcription factor NANOG contributes to liver cancer progression by reprogramming mitochondrial metabolism to promote self-renewal ability, tumor-initiation property, and chemoresistance of tumor-initiating stem-like cells (TICs). Restoration of OXPHOS activity and inhibition of fatty acid oxidation restores TIC susceptibility to chemotherapy drugs.

Our study was done to elucidate the mechanism of sex-dependent differences in radiotherapy (RT) response in males versus females, and then utilize this mechanism to help prevent intestinal radiation toxicity. More than 50% of patients with gastrointestinal (GI) cancers undergo abdominal radiotherapy. However, intestinal epithelial radiosensitivity is a major limiting factor to delivering a tumoricidal dose. Personalized differences, including sex-specific differences in radiosensitivity, is one of the key determining factors in radiotherapy outcome. Using a mouse model of abdominal irradiation and a human intestinal organoid model, we previously demonstrated that healthy male intestinal stem cells are more radiosensitive than females due to higher rates of oxidative phosphorylation (OXPHOS) and production of reactive oxidative species (ROS). In the present study, we demonstrate that these higher rates of OXPHOS in males are due to increased expression of the Mitochondrial Pyruvate Carrier (MPC), which transports pyruvate into the mitochondria for flux through the TCA cycle, and, ultimately, the OXPHOS pathway. Genetic deletion of the MPC in Lgr5-EGFP-positive ISCs increases ISC survival following the reduction in radiation-induced mitochondrial pyruvate oxidation in both male and female organoids. In both human intestinal organoids and a mouse model of radiation-induced gastrointestinal syndrome, treatment with MPC inhibitor, UK5099, normalized these differences in radiation responses between males and females. Moreover, our study in a mouse model of pancreatic adenocarcinoma also establishes UK5099 as a radio-modulator for pancreatic cancer, as combination of RT+ UK5099 treatment significantly reduces tumor growth and alters the immunosuppressive tumor immune microenvironment compared to irradiated control. These findings clearly suggest that pyruvate metabolism and MPC can be a potential target to promote therapeutic ratio of abdominal radiotherapy. Stacey Krepel, Payel Bhanja, Rishi Man Chugh, Shujah Hamid Rehman, Subhrajit Saha. Reprogramming of pyruvate metabolism overcomes sex-specific differences in intestinal stem cell radiosensitivity and improves the therapeutic ratio for abdominal irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5267.

Radiotherapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. Here we report that radioresistant HNSCC cells exhibit increased tumor aggressiveness. Using unbiased proteome profiler antibody arrays, we identify that upregulation of c-Met phosphorylation is one of the critical mechanisms for radioresistance in HNSCC cells. We further uncover that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but is suppressed by its genetic knockdown and pharmacologic inactivation. Mechanistically, the resulting upregulation of c-Met promotes elevated expression of plexin domain containing 2 (PLXDC2) through activating ERK1/2-ELK1 signaling, which in turn modulates cancer cell plasticity by epithelial–mesenchymal transition (EMT) induction and enrichment of the cancer stem cell (CSC) subpopulation, leading to resistance of HNSCC cells to radiotherapy. Depletion of PLXDC2 overcomes c-Met–mediated radioresistance through reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, the addition of SU11274, a selective and potent c-Met inhibitor, to radiation induces tumor shrinkage and limits tumor metastasis to lymph nodes in an orthotopic mouse model. Collectively, these significant findings not only demonstrate a novel mechanism underpinning radioresistance-associated aggressiveness but also provide a possible therapeutic strategy to target radioresistance in patients with HNSCC. Significance: This work provides novel insights into c-Met-PLXDC2 signaling in radioresistance-associated aggressiveness and suggests a new mechanism-informed therapeutic strategy to overcome failure of radiotherapy in patients with HNSCC.

The development of radioresistance in head and neck squamous cell carcinoma (HNSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in the past decades. One of the clinical challenges is that radioresistance often promotes tumor aggressiveness. However, the underlying mechanisms and molecular determinants are largely unknown. We report here that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but can be suppressed by its genetic knockdown and pharmacological inactivation. Through unbiased RNAseq data, we further uncovered that the resulting upregulation of c-Met signaling increases the expression of PLXDC2. a critical gene associated with the tumor microenvironment. Mechanistically, PLXDC2 is upregulated in radioresistant HNSCC cells through c-Met mediated activation of ERK1/2-ELK1 signaling cascade. Most importantly, PLXDC2 modulates cancer cell plasticity by inducing epithelial-mesenchymal transition (EMT) and the emergence of cancer stem cell (CSC) subpopulation. Additionally, depletion of PLXDC2 overcomes c-Met-mediated radioresistance by reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, a combination of the c-Met selective inhibitor SU11274 with radiation remarkably induces tumor shrinkage and constrains tumor metastasis to lymph nodes in vivo. Our study is novel in being the first to explore the role and mechanism of the c-Met-PLXDC2 axis in radioresistance-associated HNSCC aggressiveness and the first to our knowledge to evaluate how to take advantage of blocking this signaling to overcome radioresistance in preclinical mouse models of HNSCC. Citation Format: Fanghui Chen, Liwei Lang, Chloe Shay, Georgia Chen, Nabil Saba, Yong Teng. Met confers radioresistance-associated aggressiveness through enhancing PLXDC2-mediated cancer stem cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5793.

Cancer stem/tumor-initiating cells display stress tolerance and metabolic flexibility to survive in a harsh environment with limited nutrient and oxygen availability. The molecular mechanisms underlying this phenomenon could provide targets to prevent metabolic adaptation and halt cancer progression. Here, we showed in cultured cells and live human surgical biopsies of non-small cell lung cancer that nutrient stress drives the expression of the epithelial cancer stem cell marker integrin αvβ3 via upregulation of the β3 subunit, resulting in a metabolic reprogramming cascade that allows tumor cells to thrive despite a nutrient-limiting environment. Although nutrient deprivation is known to promote acute, yet transient, activation of the stress sensor AMP-activated protein kinase (AMPK), stress-induced αvβ3 expression via Src activation unexpectedly led to secondary and sustained AMPK activation. This resulted in the nuclear localization of peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α) and upregulation of glutamine metabolism, the tricarboxylic acid cycle, and oxidative phosphorylation. Pharmacological or genetic targeting of this axis prevented lung cancer cells from evading the effects of nutrient stress, thereby blocking tumor initiation in mice following orthotopic implantation of lung cancer cells. These findings reveal a molecular pathway driven by nutrient stress that results in cancer stem cell reprogramming to promote metabolic flexibility and tumor initiation. SIGNIFICANCE Upregulation of integrin αvβ3, a cancer stem cell marker, in response to nutrient stress activates sustained AMPK/PGC1α signaling that induces metabolic reprogramming in lung cancer cells to support their survival. See related article by xxxx, p. xx.

No abstract available

Radioresistance imposes a great challenge in reducing tumor recurrence and improving the clinical prognosis of individuals having oral squamous cell carcinoma (OSCC). OSCC harbors a subpopulation of CD44(+) cells that exhibit cancer stem-like cell (CSC) characteristics are involved in malignant tumor phenotype and radioresistance. Nevertheless, the underlying molecular mechanisms in CD44( + )-OSCC remain unclear. The current investigation demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in CD44(+) cells and promotes CSCs phenotype. Using RNA-sequencing analysis, we further showed that Spalt-like transcription factor 4 (SALL4) is involved in the maintenance of CSCs properties. Furthermore, the overexpression of SALL4 in CD44( + )-OSCC cells caused radioresistance in vitro and in vivo. In contrast, silencing SALL4 sensitized OSCC cells to radiation therapy (RT). Mechanistically, we illustrated that SALL4 is a direct downstream transcriptional regulation target of METTL3, the transcription activation of SALL4 promotes the nuclear transport of β-catenin and the expression of downstream target genes after radiation therapy, there by activates the Wnt/β-catenin pathway, effectively enhancing the CSCs phenotype and causing radioresistance. Herein, this study indicates that the METTL3/SALL4 axis promotes the CSCs phenotype and resistance to radiation in OSCC via the Wnt/β-catenin signaling pathway, and provides a potential therapeutic target to eliminate radioresistant OSCC.

Colorectal cancer stem/progenitor cells expressing the PROX1 transcription factor display increased radiation resistance, which may be targeted by small-molecule inhibitors of the nonhomologous end-joining DNA damage repair pathway.

Background: Triple-negative breast cancer (TNBC) frequently develops radioresistance, yet the mechanisms remain incompletely elucidated. This study is the first to investigate how β-catenin, transported by extracellular vesicles (EVs) from radioresistant TNBC cells, promotes radioresistance and enhances cancer stem cell (CSC) activity in recipient TNBC cells, offering a novel mechanism distinct from prior EV-related findings in other cancers. Methods and Results: A radioresistant cell line (231-RR) was developed from MDA-MB-231 cells, and EVs were isolated for characterization. EVs from 231-RR cells decreased radiosensitivity in parental MDA-MB-231 and two other TNBC cell lines (MDA-MB-468 and Hs578T), as shown by clonogenic assay. These EVs also enhanced CSC activity in MDA-MB-231 and Hs578T cells, demonstrated through primary and secondary mammosphere formation. The effects were nullified when using EVs from 231-RR cells treated with the EV secretion inhibitor GW4869. 231-RR-derived EVs showed elevated β-catenin levels and increased active β-catenin and stemness proteins (c-Myc, OCT4, SOX2) in recipient TNBC cells. The β-catenin inhibitor CCT-031374 prevented EV-mediated enhancement of radioresistance and CSC activity. Public data analysis from breast cancer patients revealed post-radiotherapy upregulation of the β-catenin pathway, with elevated CTNNB1, MYC, and CD44 expression, alongside reduced CDKN2A and CDH1 levels, supporting clinical relevance. Conclusions: This study uniquely demonstrates that EVs from radioresistant TNBC cells transfer β-catenin to confer radioresistance and enhance CSC activity in recipient cells, a mechanism not previously reported in TNBC. These findings suggest the potential of EV-β-catenin derived as a novel biomarker for predicting radiotherapy outcomes and recurrence risk in TNBC patients, pending development of sensitive detection methods.

Abstract Background Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and treatment‐resistant tumor. The biological implications and molecular mechanism of cancer stem‐like cells (CSCs) in ESCC, which contribute to therapeutic resistance such as radioresistance, remain elusive. Methods Quantitative real‐time polymerase chain reaction, western blotting, immunohistochemistry, and in situ hybridization assays were used to detect methyltransferase‐like 14 miR‐99a‐5p tribble 2 (METTL14/miR‐99a‐5p/TRIB2) expression in ESCC. The biological functions of METTL14/miR‐99a‐5p/TRIB2 were demonstrated in vitro and in vivo. Mass spectrum analysis was used to identify the downstream proteins regulated by TRIB2. Chromatin immunoprecipitation (IP), IP, N6‐methyladenosine (m6A)‐RNA IP, luciferase reporter, and ubiquitination assays were employed to explore the molecular mechanisms underlying this feedback circuit and its downstream pathways. Results We found that miR‐99a‐5p was significantly decreased in ESCC. miR‐99a‐5p inhibited CSCs persistence and the radioresistance of ESCC cells, and miR‐99a‐5p downregulation predicted an unfavorable prognosis of ESCC patients. Mechanically, we unveiled a METTL14‐miR‐99a‐5p‐TRIB2 positive feedback loop that enhances CSC properties and radioresistance of ESCC cells. METTL14, an m6A RNA methyltransferase downregulated in ESCC, suppresses TRIB2 expression via miR‐99a‐5p‐mediated degradation of TRIB2 mRNA by targeting its 3′ untranslated region, whereas TRIB2 induces ubiquitin‐mediated proteasomal degradation of METTL14 in a COP1‐dependent manner. METTL14 upregulates miR‐99a‐5p by modulating m6A‐mediated, DiGeorge critical region 8‐dependent pri‐mir‐99a processing. Hyperactivation of TRIB2 resulting from this positive circuit was closely correlated with radioresistance and CSC characteristics. Furthermore, TRIB2 activates HDAC2 and subsequently induces p21 epigenetic repression through Akt/mTOR/S6K1 signaling pathway activation. Pharmacologic inhibition of HDAC2 effectively attenuates the TRIB2‐mediated effect both in vitro and in patient‐derived xenograft models. Conclusion Our data highlight the presence of the METTL14/miR‐99a‐5p/TRIB2 axis and show that it is positively associated with CSC characteristics and radioresistance of ESCC, suggesting potential therapeutic targets for ESCC treatment.

No abstract available

No abstract available

Glioma stem-like cells (GSCs) are key drivers of treatment resistance and recurrence in glioblastoma (GBM). Phosphoglycerate dehydrogenase (PHGDH), a crucial enzyme in the de novo serine synthesis pathway (SSP), is implicated in tumorigenesis and therapy resistance across various cancers. However, its specific role in GBM, particularly in radioresistance, remains poorly understood. In silico analysis of GBM patient data assessed SSP enrichment and PHGDH expression linked with tumor stemness. Comparative gene expression analysis focused on PHGDH in paired GBM specimens and GSCs. Genetic and pharmacological loss-of-function assays were performed in vitro and in vivo to evaluate PHGDH’s impact on GSC self-renewal and malignant progression. Comprehensive transcriptomic and metabolomic analyses, along with chromatin immunoprecipitation, mass spectrometry, and various other biochemical assays, were used to elucidate PHGDH-mediated mechanisms in GBM progression and radioresistance. PHGDH expression is significantly elevated in GSCs, associated with aggressive glioma progression and poor clinical outcomes. PHGDH activation enhances GSC self-renewal by regulating redox homeostasis, facilitating one-carbon metabolism, and promoting DNA damage response via SSP activation. Importantly, MYC was identified as a crucial transcriptional regulator of PHGDH expression. Furthermore, genetic ablation or pharmacological inhibition of PHGDH markedly reduced tumor growth and increased tumor sensitivity to radiotherapy, thereby improving survival outcomes in orthotopic GSC-derived and patient-derived GBM xenograft models. This study underscores the pivotal role of MYC-mediated PHGDH activation in driving GSC malignant progression and radioresistance in GBM. Targeting PHGDH presents a promising approach to enhance radiotherapy efficacy in GBM patients.

No abstract available

ABSTRACT Cancer stem-like cells (CSCs), featuring high tumorigenicity and invasiveness, are one of the critical factors leading to the failure of clinical cancer treatment such as metastasis and recurrence. However, current strategies suffer from the low stemness-inhibiting efficacy on CSCs by conventional molecular agents and the poor lethal effects against bulk tumor cells. Here we engineer a coordination nanomedicine by 2,5-dihydroxyterephthalic acid (DHT) complexing zinc ions (Zn2+) as a double-effect nanodisrupter of tumor iron (Fe) and redox homeostasis for catalysis-boosted tumor therapy with stemness inhibition. Taking advantage of the much higher binding force of DHT toward Fe3+, this nanomedicine can specifically chelate endogenous Fe3+ into its nanostructure and release Zn2+, and the in situ formed hexacoordinated Fe-DHT conformation is of much enhanced reducibility in order to promote reactive oxygen species (ROS) production in tumors. The nanomedicine-mediated Fe depletion and ROS generation collectively induce CSC differentiation via downregulating the Wnt signaling and inducing forkhead box O3 (FoxO3) activation, respectively. Notably, the combined tumor-selective ROS generation and Zn2+-induced antioxidation dysfunction potently trigger intratumoral oxidative damage leading to both cellular apoptosis and ferroptosis. This nanomedicine, capable of synchronously treating CSCs and bulk tumor cells, has been demonstrated to effectively inhibit the growth, postoperative recurrence and metastasis of orthotopic triple-negative breast tumors in vivo, offering an encouraging candidate of cancer therapeutic agents for treating CSCs-enriched malignancy.

BACKGROUND Triple-negative breast cancer (TNBC) recurrence and metastasis are the major causes of failure in TNBC therapy. The difficulties in treating TNBCs may be because of increased cancer cell plasticity that involves the fine-tuning of cellular redox homeostasis, mitochondrial bioenergetics, metabolic characteristics, and the development of cancer stem cells (CSCs). PURPOSE To investigate the effects and the underlying mechanisms of the phytosesquiterpene lactone deoxyelephantopin (DET) and its semi-synthesized derivative (DETD-35) in suppressing different phenotypic TNBC cell populations that contribute to tumor metastasis. METHODS A timelapse microfluidic-based system was established to analyze the effects of DETD-35 and DET on cell migration behavior in an oxygen gradient. Seahorse real-time cell metabolic analyzer and gas chromatography/quadrupole-time-of-flight mass spectrometry (GC/Q-TOF MS) were utilized to analyze the effects of the compounds on mitochondrial bioenergetics in TNBC cells. A miRNA knockout technique and miRNA sponges were employed to evaluate the miR-4284 involvement in the anti-TNBC cell effect of either compound. RESULTS DETD-35 and DET attenuated TNBC cell migration toward hypoxic regions under a 2-19 % oxygen gradient in a timelapse microfluidic-based system. DETD-35 and DET also suppressed CSC-like phenotypes, including the expression of Sox2, Oct4, and CD44 in TNBC cells under hypoxic conditions. DETD-35 and DET affected mitochondrial basal respiration, ATP production, proton leak, and primary metabolism, including glycolysis, the TCA cycle, and amino acid metabolism in the lung-metastatic TNBC cells. Furthermore, the expression of mitophagy markers PARKIN, BNIP3, PINK1, LC3-II, and apoptotic markers Bax, cleaved caspase 7, and cleaved PARP in hypoxic and lung-metastatic TNBC cells was also regulated by treatment with either compound. In miR-4284 knockout cells or miR-4284 inhibitor co-treated TNBC cells, DET- and DETD-35-induced over-expression of mitophagic and apoptotic markers was partially reversed, indicating miR-4284 involved with the compounds caused programmed cell death. CONCLUSION This study demonstrated the novel activities of DETD-35 and DET in suppressing CSC-like phenotypes and metastatic TNBC cells through the de-regulation of mitochondrial bioenergetics.

Cancer stem cells (CSCs) have been recognized as the culprit for tumor progression, treatment resistance, metastasis, and recurrence while redox homeostasis represents the Achilles' Heel of CSCs. However, few drugs or formulations that are capable of elevating oxidative stress have achieved clinical success for eliminating CSCs. Here, we report hydroxyethyl starch stabilized copper-diethyldithiocarbamate nanoparticles (CuET@HES NPs), which conspicuously suppress CSCs not only in vitro but also in numerous tumor models in vivo. Furthermore, CuET@HES NPs effectively inhibit CSCs in fresh tumor tissues surgically excised from hepatocellular carcinoma patients. Mechanistically, we uncover that hydroxyethyl starch stabilized copper-diethyldithiocarbamate nanocrystals via copper‑oxygen coordination interactions, thereby promoting copper-diethyldithiocarbamate colloidal stability, cellular uptake, intracellular reactive oxygen species production, and CSCs apoptosis. As all components are widely used in clinics, CuET@HES NPs represent promising treatments for CSCs-rich solid malignancies and hold great clinical translational potentials. This study has critical implications for design of CSCs targeting nanomedicines.

Abstract Cancer stem cell (CSC) radioresistance is a major cause of radiotherapy (RT) failure and tumor recurrence. The molecular target for eradicating CSCs has not been identified despite research efforts to overcome tumor radioresistance. The adenosine monophosphate-activated protein kinase (AMPK) is responsible for transmitting nuclear DNA damage signals to the mitochondria, which in turn generate adenosine triphosphate to execute a DNA damage response. Disruption of this mitochondria-mediated genomic defense mechanism may be an effective strategy to enhance the cytotoxic efficacy of RT. Here, we investigated the potential efficacy of the pan-AMPK inhibitor dorsomorphin (Dor) in preventing CSC radioresistance. Radioresistant cancer stem-like cells were derived from the human liver cancer cell line HepG2 (HepG2 82FR-31NR). The radiosensitizing effect of Dor was then examined in HepG2 82FR-31NR cell cultures by clonogenic assays. Low-dose Dor markedly suppressed the recovery of HepG2 cancer stem-like cells after radiation but had little effect on normal fibroblast proliferation and survival, whether applied alone or in combination with radiation. In conclusion, this study strongly suggests that Dor treatment can radiosensitize cancer stem-like cells at doses that have no significant cytotoxic effects on normal human fibroblasts.

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro, in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations.

Cancer stem cells (CSCs) play a pivotal role in driving colorectal cancer (CRC) progression and therapeutic resistance. However, the molecular mechanisms regulating CRC-CSC properties are not fully understood. Proline-rich Akt substrate 40 (PRAS40) is involved in various tumorigenic processes, yet little is known about its contribution to cancer stemness. In this study, we demonstrated that PRAS40 was overexpressed in CRC tissues and its elevated expression positively correlated with poor patient survival. Genetic ablation of PRAS40 suppressed tumorigenesis in CRC mouse models. Notably, PRAS40 enhanced the stemness of CRC cells, as evidenced by increased sphere formation, upregulation of stem cell markers, enrichment of the CD133+CD44+ cell population, and enhanced tumor initiation capacity in vivo. Mechanistically, PRAS40 induced a glycolytic phenotype by interacting with and activating the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Furthermore, PRAS40 enhanced the interaction between PGK1 and the acetyltransferase p300/CBP-associated factor (PCAF), thereby promoting PGK1 acetylation, which contributes to glycolysis activation and the maintenance of CRC stemness. Pharmacological inhibition of acetylation attenuated PRAS40-mediated CRC stemness and colorectal carcinogenesis. Collectively, our findings uncover a novel PRAS40/PGK1 regulatory axis that promotes CRC stemness and tumorigenesis through enhanced glycolysis, suggesting potential therapeutic strategies targeting this axis for CRC treatment.

Radiotherapy resistance remains a major barrier to effective treatment of triple-negative breast cancer (TNBC), highlighting the need to identify mechanisms driving resistance. Here, we identified SDC1 as a pivotal mediator of cancer-associated fibroblast (CAF)-induced radioresistance in breast cancer. SDC1 bound the TIM barrel domain of the glycolytic enzyme ENO1, preventing FBXW7-mediated degradation and driving aerobic glycolysis and lactate accumulation. The resulting lactate-rich microenvironment not only promoted tumor stemness but also significantly impaired the cytotoxic functions of both NK cells and CD8⁺ T cells. Pharmacologic inhibition of ENO1 or lactate export restored radiosensitivity. Targeting SDC1⁺ CAFs with the antibody-drug conjugate indatuximab ravtansine (BT062) synergized with radiotherapy in vivo, markedly reducing tumor burden, depleting stem-like tumor cells, and remodeling the immune microenvironment. These findings define a CAF metabolic program that fuels tumor stemness and rewires the immune microenvironment to confer radioresistance, supporting the therapeutic targeting of SDC1⁺ CAFs in TNBC.

Radiotherapy improves the survival and life quality of individuals diagnosed with locally advanced or terminal pancreatic ductal adenocarcinoma (PDAC). Nevertheless, the ubiquitous of radioresistance resulted in ineffective outcome, which commonly observed the recurrence and malignant progression within the radiation target area. Our clinical observation has shown that PDAC cells resident in rigid tumor niche, therefore to illuminate the underlying mechanism from the biomechanical perspective is of significant importance. To this end, PDAC models with tunable mechanical properties were constructed through 3D-bioprinted gelatin methacryloyl (GelMA) hydrogel, which enabled the precise manipulation of cellular behavior under physiological stiffness scopes. The effect of matrix stiffness on radiation tolerance of PDAC cells subjected to distinct X-ray radiation was assessed, and the results validated that stiff matrix promoted radioresistance. To elucidate the in-depth mechanism underlying this phenomenon, enrichment analysis was performed on DEGs between soft and stiff groups treated with 4 Gy X-ray radiation. The results showcased that glycolysis process was prominent enriched, further experiment demonstrated that matrix stiffness promoted the glycolysis level and lactate accumulation. The stiff group reinforced histone H3 lysine 18 lactylation (H3K18la) level, and the inhibition of histone lactylation efficiently suppressed PDAC radioresistance. shRAD51 assay corroborated that H3K18 lactylation-driven RAD51 transcriptional activation, which established a causal link to radioresistance. Overall, our research shed light on the matrix stiffness mediated histone lactylation, which exerted essential role in PDAC radioresistance and provided insight into future radiotherapy from the perspective of biomechanics.

Resistance to radiation therapy significantly affects the prognosis of solid tumors. Histone deacetylase 6 (HDAC6) is a stress-responsive lysine deacetylase that has emerged as a promising target for cancer therapy, with numerous clinical trials investigating interventions to modulate its activity. While HDAC6 appears to be linked to responses to DNA-damaging therapeutics, the reported responses and underlying mechanisms remain intriguing and varied. In this study, we elucidate a novel mechanism of radioresistance mediated by HDAC6 in non-small cell lung cancer (NSCLC). We classified nine NSCLC cell lines into three groups based on their radioresistance using survival assays and correlated this resistance with the induced expression of six deacetylases. We conducted gain-of-function experiments (GOF) using plasmid transfection and loss-of-function experiments (LOF) employing inhibitors or RNAi. Furthermore, we quantified the repair of damaged dsDNA through FACS-based GFP assays and assessed the mechanism of homologous recombination repair (HRR) end-resection via single-strand quantitative PCR. Co-immunoprecipitation assays, cancer stem cell enrichment, DNA damage-induced senescence assays, reverse-phase protein assays (RPPA), and RNA sequencing were also performed to investigate gene expression changes resulting from the loss of HDAC6. Our findings revealed that HDAC6 induction by radiation is strongly correlated with NSCLC resistance to radiation leading to CSC survival and escape from radiation led senescence LOF of HDAC6 through RNAi or inhibitors suppressed resistance, while GOF achieved through wild-type or deacetylase activity-deficient mutant transfection, induced resistance. Importantly, this resistance mechanism was independent of deacetylation activity. RNAi-mediated loss of HDAC6 reduced both HRR and non-homologous end-joining, whereas pharmacological inhibition of HDAC6 activity did not. Notably, DNA end-resection, a critical step in HRR, was decreased by RNA interference but not affected by the inhibitors. RPPA assays demonstrated that Histone demethylase 1A (LSD1/KDM1A) levels decreased significantly upon HDAC6 RNA interference but remained unaffected by pharmacological intervention. Loss of LSD1 alone resulted in decreased HRR and end-resection, likely mediated through HDAC6-induced ubiquitination. Moreover, overexpression of LSD1 rescued the HDAC6 loss-induced sensitization of NSCLCs. In summary, our study unveils a novel mechanism by which HDAC6 mediates HRR after radiation through the stabilization of LSD1. This mechanism operates independently of deacetylase activity. Therefore, our findings suggest that interventions targeting both deacetylation and non-deacetylation roles of HDAC6 should be considered to overcome HDAC6-derived radioresistance in NSCLCs and enhances the radiation therapy efficacy. Citation Format: Sojung Ha, Hyejin Kim, Hani Lee, Seokgyeong Choi, Ho-young Lee, Woo-Young Kim. HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2883.

Ionizing radiation is a popular and effective treatment option for glioblastoma (GBM). However, resistance to radiation therapy inevitably occurs during treatment. It is urgent to investigate the mechanisms of radioresistance in GBM and to find ways to improve radiosensitivity. Here, we found that heat shock protein 90 beta family member 1 (HSP90B1) was significantly upregulated in radioresistant GBM cell lines. More importantly, HSP90B1 promoted the localization of glucose transporter type 1, a key rate-limiting factor of glycolysis, on the plasma membrane, which in turn enhanced glycolytic activity and subsequently tumor growth and radioresistance of GBM cells. These findings imply that targeting HSP90B1 may effectively improve the efficacy of radiotherapy for GBM patients, a potential new approach to the treatment of glioblastoma.

The potential for tumor occurrence triggered by cancer stem cells (CSCs) has emerged as a significant challenge for human colorectal cancer therapy. However, the underlying mechanism of CSC development remains controversial. Our study provided evidence that the bulk of tumor cells could dedifferentiate to CSCs and reacquire CSC‐like phenotypes if cultured in the presence of extracellular matrix reagents, such as Matrigel and fibrin gels. In these 3D gels, CD133− colorectal cancer cells can regain tumorigenic potential and stem‐like phenotypes. Mechanistically, the 3D extracellular matrix could mediate cytoskeletal F‐actin bundling through biomechanical force associated receptors integrin β1 (ITGB1), contributing to the release of E3 ligase tripartite motif protein 11 (TRIM11) from cytoskeleton and degradation of the glycolytic rate‐limiting enzyme phosphofructokinase (PFK). Consequently, PFK inhibition resulted in enhanced glycolysis and upregulation of hypoxia‐inducible factor 1 (HIF1α), thereby promoting the reprogramming of stem cell transcription factors and facilitating tumor progression in patients. This study provided novel insights into the role of the extracellular matrix in the regulation of CSC dedifferentiation in a cytoskeleton/glycolysis‐dependent manner.

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (−) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

Radiotherapy remains one of the principal therapeutic modalities for non-small cell lung cancer (NSCLC), yet its therapeutic efficacy is frequently compromised by tumor recurrence, metastasis, and the development of radioresistance. Accumulating evidence identifies cancer stem cells (CSCs) as a critical driver of radioresistance. Therefore, elucidating the underlying molecular mechanisms and developing therapeutic targets to enhance radiosensitivity are of critical importance. To identify key mediators of adaptive radioresistance in NSCLC, we established radioresistant NSCLC cell lines through repeated cycles of irradiation and subsequently performed RNA-seq analysis comparing them with their parental counterparts. The effects of SLFN5 on radiosensitivity were evaluated by flow cytometry and colony formation assays, and further validated in vivo using a nude mouse xenograft tumor model. Cancer stem-like properties were assessed by RT-PCR, Western blot, flow cytometry, sphere formation assay, and in vivo limiting dilution tumorigenesis assays. Subsequently, we employed mass spectrometry, co-immunoprecipitation, and proximity ligation assay to identify the interaction between SLFN5 and NOTCH1. Furthermore, phase separation assay, fluorescence recovery after photobleaching, CUT&Tag-seq, and ATAC-seq were used to investigate the role of NOTCH1 phase separation in promoting stemness and radioresistance, and the regulatory effect of SLFN5 on this process. Finally, the methylation status of the SLFN5 promoter was analyzed using bisulfite sequencing PCR. Our results demonstrate that SLFN5 was significantly downregulated in radioresistant NSCLC cell lines. Overexpressing SLFN5 effectively suppressed cancer stemness and epithelial-mesenchymal transition, thereby enhancing radiosensitivity both in vitro and in vivo. Conversely, knockdown of SLFN5 had significantly opposite effects. Mechanistically, liquid-liquid phase separation property is critical for NOTCH1 mediated stemness and radioresistance. Notably, SLFN5 interacts with NOTCH1 and induces the liquid-to-solid phase transition of NOTCH1, thereby impairing NOTCH1 signaling and the subsequent stemness and radioresistance. Finally, we identified DNMT3A and DNMT3B as the epigenetic regulators responsible for promoter hypermethylation and consequent silencing of SLFN5. Our study reveals that DNA methylation downregulated SLFN5 resulting in radioresistance of NSCLC via liberating NOTCH1 from gel-like phase to liquid droplet to potentiating stemness of cancer cells. This research provides a potential therapeutic strategy to overcome radioresistance in NSCLC. Mi Tang, Lu Zhang, Jiaxin Zhao, Hongji Dai, Zhiyong Yuan, Zeyun Mi, Zhiqiang Wu. SLFN5 mediates liquid-to-solid phase transition of NOTCH1 to suppress stemness and radioresistance of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2192.

We proposed that cancer stem cells (CSCs) survived and presented resistance to radiotherapy (RT) in hepatocellular carcinoma (HCC) cells. Interleukin 6 (IL‐6) has been reported to be particularly involved in HCC tumorigenesis. Therefore, we intended to validate that IL‐6 downstream STAT3‐mediated CSCs formation and immune checkpoint PD‐L1 expression in HCC, thus contributing to radioresistance. HBV‐positive HCC tumorspheres were formed and exposed with X‐ray irradiation, cell viability of which was measured consequently. Specific inhibitors targeting EGFR (by gefitinib), STAT3 (by BBI608), and HCC‐targeted therapy sorafenib were investigated to suppress tumorsphere formation. Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) was used for detecting STAT3‐downstream PD‐L1 and anti‐apoptosis MCL1 and BCL2 gene expression in the PLC5‐derived tumorspheres and STAT3‐knockdown PLC5. We found that RT significantly inhibited HBV‐positive Hep3B and PLC5 cell viability but not for HCC‐derived stem‐like tumorspheres cultured by EGF, IL‐6, bFGF, and HGF. It revealed that tumorspheres presented radioresistance compared with the parental cells. Specifically, RT induces IFNs, EGF, and IL‐6 expression, resulting in STAT3 phosphorylation. Kaplan–Meier plotter indicated that highly EGF (p = .0024), IL‐6 (p = .12), and FGF2 (p = .0041) were associated with poor survival probability in patients with HBV‐positive HCC. We further demonstrated that BBI608 and sorafenib significantly suppressed PLC5 cell viability and PLC5‐derived tumorsphere formation. To investigate the mechanism of CSC‐presented radioresistance, STAT3 and STAT3‐downstream genes, including PD‐L1 and anti‐apoptosis MCL1 and BCL2, were detected using qPCR. We demonstrated higher STAT3, PD‐L1, MCL1, and BCL2 in Hep3B‐ and PLC5‐derived CSCs compared to PLC5. In addition, knockdown of STAT3 reduced cell proliferation in PLC5 cells, resulting in down‐regulation of IL‐6‐mediated PD‐L1 and BCL‐2. Meanwhile, we found that knockdown of STAT3 significantly improved RT‐mediated suppression of tumorsphere formation. In conclusion, we found that CSCs presented radioresistance and figured out which may be mediated by STAT3 in HBV‐positive HCC.

No abstract available

Temozolomide (TMZ) resistance in glioblastoma (GBM) remains a substantial clinical challenge. Targeting glioma stem cells (GSCs) represents a promising strategy to overcome chemoresistance and tumor recurrence. In this study, we found that GSCs maintain chemoresistance by increasing pentose phosphate pathway (PPP) flux compared with differentiated tumor cells. Following TMZ treatment, the activity of glucose-6-phosphate isomerase (GPI), a key glycolytic enzyme that catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate, was significantly suppressed in GSCs. Mechanistically, Ataxia Telangiectasia Mutated (ATM), activated by TMZ-induced DNA damage, phosphorylates polo-like kinase 1 (PLK1), promoting its nuclear export. PLK1 subsequently phosphorylates GPI at T215, leading to suppression of GPI activity. Targeting the ATM/PLK1/GPI axis through combinational treatment with rigosertib may therefore represent a therapeutic strategy. Moreover, PLK1 expression and GPI pT215 levels may serve as potential candidate markers for GBM. Collectively, activation of the ATM/PLK1/GPI axis plays a critical role in regulating PPP flux and TMZ resistance in GSCs.

No abstract available

No abstract available

Although the clinical observation of hematologic toxicity related to radiotherapy has been recognized for a long time, the underlying mechanisms remain to be fully explored. Here, we established a mouse model of reduced dietary intake (dietary restriction, DR, 30% reduction in food intake compared to age-matched and gender-matched mice) following X-ray radiation exposure to investigate the impact of reduced dietary intake on hematopoiesis after irradiation. We found that post-irradiation DR significantly and persistently suppressed hematopoiesis and notably impaired the regenerative capacity of hematopoietic cells. Compared to ad libitum (AL) fed mice, post-irradiation DR led to sustained upregulation of the DNA damage response (DDR) signaling pathway in hematopoietic cells, even 14 days to 1 month after irradiation, along with delayed DNA repair. Further investigation revealed that DR suppressed the post-irradiation activation of the pentose phosphate pathway (PPP). Inhibition of PPP by 6-Aminonicotinamide (6-AN) in AL mice mimicked the impairment of hematopoiesis observed in DR mice, while activation of PPP by AG1 in DR mice rescued the impairment of DNA repair and hematopoiesis in these mice. Additionally, we conducted a retrospective analysis of 101 cancer patients who received pelvic radiotherapy and found that patients with lower Body Mass Index (BMI) experienced more severe reductions in white blood cells (WBCs), neutrophils, and lymphocytes. This study suggests that DR following irradiation inhibits hematopoiesis by suppressing PPP, providing a new approach to addressing radiotherapy-related myelosuppression and potentially offering solutions for improving refractory hematopoietic disorders associated with radiotherapy.

Tumor angiogenesis plays a vital role in tumor growth and metastasis. It is proven that in tumor vasculature, endothelial cells (ECs) originate from a small population of cancer cells introduced as cancer stem cells (CSCs). Autophagy has a vital role in ECs differentiation from CSCs and tumor angiogenesis. High levels of reactive oxygen species (ROS) increased autophagy by inhibition of glucose-6-phosphate dehydrogenase (G6PD) and inactivation of the pentose phosphate pathway (PPP). Previously, we suggested that cancer cells initially increase the glycolysis rate when encountering ROS, then the metabolic balance is changed from glycolysis to PPP, following the continuation of oxidative stress. In this study, we investigate the possible role of persistent oxidative stress in the differentiation of CSCs into tumor ECs by relying on the relationship between the ROS, PPP and autophagy. Because tumor angiogenesis plays an important role in the growth and development of cancer, understanding the mechanisms involved in differentiating ECs from CSCs can help find promising treatments for cancer.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem/progenitor cells. AML prognosis remains poor, due to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The PI3K/AKT pathway is often dysregulated in AML. We found while that PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes NRF2 nuclear accumulation, which induces PGD and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may therefore eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

Prostate cancer (PCa) frequently develops resistance to radiation therapy (RT), driven in part by DNA repair mechanisms. We previously found that glutamine (L-Gln) enables protein O-GlcNAcylation post-translational modifications linked to DNA repair and therapy resistance. This is highly relevant to irradiation of PCa metastatic sites like the bone and liver microenvironments that have markedly high concentrations of L-Gln and accordingly poor response to irradiation. Here, we investigated whether pharmacologic glutamine depletion using sodium phenylbutyrate (SPB) disrupts this axis and enhances radiosensitivity. Radio-resistant 22Rv1 and ARCaPM lines were generated by iterative chronic irradiation. Subcutaneous and liver xenografts were treated with vehicle, SPB, RT, or SPB+RT. Tumors analyzed by bulk RNA-seq, proteomics, and immunoblotting revealed the importance of NDRG1 and PRDX1 o-glycosylation. CRISPR/Cas9 mutagenesis was used to generate O-GlcNAc-deficient NDRG1 and PRDX1 variants. DNA-damage repair, cell-cycle dynamics, and mitochondrial function were assessed by γH2AX staining, flow cytometry, and Seahorse assays. SPB+RT produced the greatest tumor reduction and significantly reduced circulating and intratumoral glutamine, compared to either RT or SBP alone. Transcriptomic and proteomic analyses showed downregulation of amino-acid transport, fatty-acid metabolism, and histone demethylase activity, indicating broad metabolic and epigenetic reprogramming. Mass spectrometry identified NDRG1 and PRDX1 as radiation-induced O-GlcNAc targets suppressed by SPB. CRISPR-engineered O-GlcNAc-deficient NDRG1 and PRDX1 variants exhibited greater RT sensitivity as a result of persistent γH2AX foci, prolonged G2/M arrest, reduced nuclear localization, and decreased protein stability. RNA-seq of these variants showed enrichment of p53 signaling, endoplasmic reticular (ER) stress with metabolic compensation through increased c-Myc activity, and oxidative phosphorylation. Nucleoside supplementation did not reverse SPB-mediated radio-sensitization, indicating that SPB’s effects extended beyond nucleotide depletion. Instead, the data suggested the role of L-Gln addiction in irradiated PCa tumors was due to NDRG1 and PRDX1 in ER stress-response proteins and activating the unfolded protein response. O-GlcNAcylation of NDRG1 and PRDX1 stabilizes stress-response proteins to support DNA-repair and metabolic fitness after irradiation. SPB disrupts this L-Gln-driven O-GlcNAcylation axis, to impair protein translation supporting significant radio-sensitization. As SPB is used for chronic management of urea cycle disorders, repurposing to overcome radiation resistance provides a near-term therapeutic translation opportunity for PCa patients. Manish Thiruvalluvan, Sandrine Billet, Saravana Kumar Kailasam Mani, Joshua Watson, Neil A. Bhowmick, . Limiting O-GlcNAcylation support prostate cancer radiation sensitivity through metabolic and epigenetic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6616.

Gliomas represent a wide spectrum of brain tumors characterized by their high invasiveness, resistance to chemoradiotherapy, and both intratumoral and intertumoral heterogeneity. Recent advances in transomics studies revealed that enormous abnormalities exist in different biological layers of glioma cells, which include genetic/epigenetic alterations, RNA expressions, protein expression/modifications, and metabolic pathways, which provide opportunities for development of novel targeted therapeutic agents for gliomas. Metabolic reprogramming is one of the hallmarks of cancer cells, as well as one of the oldest fields in cancer biology research. Altered cancer cell metabolism not only provides energy and metabolites to support tumor growth, but also mediates the resistance of tumor cells to antitumor therapies. The interactions between cancer metabolism and DNA repair pathways, and the enhancement of radiotherapy sensitivity and assessment of radiation response by modulation of glioma metabolism are discussed herein.

Simple Summary Cancer stem cells (CSCs) are a tumor cell population maintaining tumor growth and promoting tumor relapse if not wholly eradicated during treatment. CSCs are often equipped with molecular mechanisms making them resistant to conventional anti-cancer therapies whose curative potential depends on DNA damage-induced cell death. An elevated expression of some key DNA repair proteins is one of such defense mechanisms. However, new research reveals that the role of critical DNA repair proteins is extending far beyond the DNA repair mechanisms. This review discusses the diverse biological functions of DNA repair proteins in CSC maintenance and the adaptation to replication and oxidative stress, anti-cancer immune response, epigenetic reprogramming, and intracellular signaling mechanisms. It also provides an overview of their potential therapeutic targeting. Abstract Cancer stem cells (CSCs) are pluripotent and highly tumorigenic cells that can re-populate a tumor and cause relapses even after initially successful therapy. As with tissue stem cells, CSCs possess enhanced DNA repair mechanisms. An active DNA damage response alleviates the increased oxidative and replicative stress and leads to therapy resistance. On the other hand, mutations in DNA repair genes cause genomic instability, therefore driving tumor evolution and developing highly aggressive CSC phenotypes. However, the role of DNA repair proteins in CSCs extends beyond the level of DNA damage. In recent years, more and more studies have reported the unexpected role of DNA repair proteins in the regulation of transcription, CSC signaling pathways, intracellular levels of reactive oxygen species (ROS), and epithelial–mesenchymal transition (EMT). Moreover, DNA damage signaling plays an essential role in the immune response towards tumor cells. Due to its high importance for the CSC phenotype and treatment resistance, the DNA damage response is a promising target for individualized therapies. Furthermore, understanding the dependence of CSC on DNA repair pathways can be therapeutically exploited to induce synthetic lethality and sensitize CSCs to anti-cancer therapies. This review discusses the different roles of DNA repair proteins in CSC maintenance and their potential as therapeutic targets.

Abstract Ovarian cancer (OC) remains one of the most lethal gynecologic malignancies due to its asymptomatic progression, frequent late-stage diagnosis, and high rates of chemoresistance and recurrence. Beyond genetic alterations, recent studies highlight the central role of metabolic reprogramming in driving OC initiation, progression, and therapy resistance. OC cells exhibit dynamic metabolic reprogramming, enabling dynamic shifts between glycolysis and oxidative phosphorylation depending on environmental conditions and treatment pressures. In this review, we synthesize current understanding of key metabolic pathways altered in ovarian tumors, including enhanced aerobic glycolysis, glutamine addiction, dysregulated lipid metabolism, and mitochondrial adaptations. These metabolic shifts support rapid proliferation, redox homeostasis, immune evasion, and metastatic potential. We also explore how the metabolic landscape of OC is shaped by interactions with the tumor microenvironment, particularly through crosstalk with immune cells, cancer-associated fibroblasts, and adipocytes. Importantly, metabolic adaptations have been implicated in the emergence of cancer stem-like cells and in the development of resistance to platinum-based chemotherapy and PARP inhibitors. We also further discuss emerging therapeutic strategies targeting metabolic vulnerabilities, as well as combinatorial approaches integrating metabolic therapy with immunotherapy and DNA damage repair inhibition. Finally, we highlight how advances in metabolomics and spatial profiling are improving our ability to map metabolic heterogeneity and guide precision therapies in OC. This review underscores metabolic plasticity as a promising therapeutic vulnerability for overcoming drug resistance and improving outcomes in OC patients.

Simple Summary Cancer stem cell have certain metabolic properties that are distinct from their differentiated counterparts. Our aim is to characterize CSC metabolism in oral cancer. Our study have several impacts: 1. Previous metabolic studies of CSC mainly focused on few energy metabolism pathway. Here we used novel transcription/metabolic joint analysis to reveal comprehensive metabolic alteration of CSC in oral cancer. 2. Assessing CSC metabolic phenotype in vivo is challenging. Here We used single-cell sequencing to explore the metabolic characteristics of CSC in vivo. 3. Our data suggested oral CSCs are metabolically inactive compared with differentiated cancer cells. This state may allow CSCs to resist the metabolic therapeutic strategies currently used for highly proliferative tumors. This knowledge may allow us to better develop metabolic therapy against CSC in oral cancer. Abstract Understanding the distinct metabolic characteristics of cancer stem cells (CSC) may allow us to better cope with the clinical challenges associated with them. In this study, OSCC cell lines (CAL27 and HSC3) and multicellular tumor spheroid (MCTS) models were used to generate CSC-like cells. Quasi-targeted metabolomics and RNA sequencing were used to explore altered metabolites and metabolism-related genes. Pathview was used to display the metabolites and transcriptome data in a KEGG pathway. The single-cell RNA sequencing data of six patients with oral cancer were analyzed to characterize in vivo CSC metabolism. The results showed that 19 metabolites (phosphoethanolamine, carbamoylphosphate, etc.) were upregulated and 109 metabolites (2-aminooctanoic acid, 7-ketocholesterol, etc.) were downregulated in both MCTS cells. Integration pathway analysis revealed altered activity in energy production (glycolysis, citric cycle, fatty acid oxidation), macromolecular synthesis (purine/pyrimidine metabolism, glycerophospholipids metabolism) and redox control (glutathione metabolism). Single-cell RNA sequencing analysis confirmed altered glycolysis, glutathione and glycerophospholipid metabolism in in vivo CSC. We concluded that CSCs are metabolically inactive compared with differentiated cancer cells. Thus, oral CSCs may resist current metabolic-related drugs. Our result may be helpful in developing better therapeutic strategies against CSC.

Background Radiotherapy (RT) is a critical component of treatment for locally advanced rectal cancer (LARC), though patient response varies significantly. The variability in treatment outcomes is partly due to the resistance conferred by cancer stem cells (CSCs) and tumor immune microenvironment (TiME). This study investigates the role of EIF5A in radiotherapy response and its impact on the CSCs and TiME. Methods Predictive models for preoperative radiotherapy (preRT) response were developed using machine learning, identifying EIF5A as a key gene associated with radioresistance. EIF5A expression was analyzed via bulk RNA-seq and single-cell RNA-seq (scRNA-seq). Functional assays and in vivo experiments validated EIF5A’s role in radioresistance and TiME modulation. Results EIF5A was significantly upregulated in radioresistant colorectal cancer (CRC) tissues. EIF5A knockdown in CRC cell lines reduced cell viability, migration, and invasion after radiation, and increased radiation-induced apoptosis. Mechanistically, EIF5A promoted cancer stem cell (CSC) characteristics through the Hedgehog signaling pathway. Analysis of the TiME revealed that the radiation-resistant group had an immune-desert phenotype, characterized by low immune cell infiltration. In vivo experiments showed that EIF5A knockdown led to increased infiltration of CD8+ T cells and M1 macrophages, and decreased M2 macrophages and Tregs following radiation therapy, thereby enhancing the radiotherapy response. Conclusion EIF5A contributes to CRC radioresistance by promoting CSC traits via the Hedgehog pathway and modulating the TiME to an immune-suppressive state. Targeting EIF5A could enhance radiation sensitivity and improve immune responses, offering a potential therapeutic strategy to optimize radiotherapy outcomes in CRC patients.

Renal cell carcinoma (RCC) is a highly heterogeneous malignancy with limited sensitivity to conventional radiotherapy. Although radiotherapy is increasingly employed in the management of oligometastatic and unresectable RCC, resistance to ionizing radiation remains a major clinical obstacle. The molecular mechanisms underlying radioresistance in RCC are not fully elucidated. Tumor-associated fibroblasts (CAFs), as key components of the tumor microenvironment, have been implicated in therapeutic resistance across multiple cancer types. However, their specific contribution to radiotherapy resistance in RCC remains poorly defined. Primary CAFs and normal fibroblasts (NFs) were isolated from human RCC tissues. Single-cell RNA sequencing of treatment-naïve tumors was performed to analyze stromal composition and CXCL12 expression. RCC cells were stimulated with recombinant CXCL12 and subjected to transcriptomic, proteomic, and metabolomic profiling to evaluate alterations in taurine metabolism and transporter expression. In vitro co-culture systems and clonogenic survival assays were utilized to assess radiosensitivity, while ferroptosis was evaluated by measuring lipid peroxidation. In vivo, RCC cells were co-injected with CAFs into immunodeficient mice, followed by radiotherapy with or without the CXCR4 inhibitor AMD3100. Clinicopathological analysis revealed that RCC patients with high CAF infiltration exhibited reduced radiotherapy responsiveness. Single-cell RNA sequencing showed increased CAF abundance and elevated CXCL12 expression in resistant cases. In vitro, co-culture with CAFs induced radioresistance in RCC cells. Metabolomic profiling revealed that CXCL12 stimulation elevated intracellular taurine levels, while transcriptomic and proteomic data indicated upregulation of the taurine transporter SLC6A6. Functional assays demonstrated that SLC6A6-mediated taurine accumulation suppressed ferroptosis and conferred radioresistance. Inhibition of CXCR4 with AMD3100 abrogated taurine accumulation, restored ferroptosis sensitivity, and enhanced radiotherapy efficacy both in vitro and in vivo. This study uncovers a previously unrecognized mechanism by which CAFs confer radiotherapy resistance in RCC via CXCL12/CXCR4-driven metabolic reprogramming. The CXCL12/CXCR4/SLC6A6 axis represents a potential therapeutic target to overcome RCC radioresistance and highlights the pivotal role of CAF-derived metabolic cues in modulating tumor response to radiation. Qiwen Pan, liru He. Tumor-associated fibroblasts drive taurine accumulation and radiotherapy resistance in renal cell caecinoma via the CXCL12/CXCR4/SLC6A6 axis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C094.

Single‐atom nanozymes have revolutionized catalytic tumor therapy by maximizing atomic efficiency and enabling enzyme‐mimetic reactive oxygen species (ROS) generation. Yet, their single active‐site architecture imposes fundamental limitations on catalytic selectivity and intermediate adsorption–desorption kinetics. Here, a manganese/ruthenium dual‐metal single‐atom nanozyme (Mn/Ru BMSA) is reported that overcomes these constraints through synergistic metal interactions, exhibiting unprecedented multienzyme activities, including catalase, peroxidase, and superoxide dismutase. By leveraging tumor‐overexpressed hydrogen peroxide, Mn/Ru BMSA simultaneously alleviates hypoxia and produces cytotoxic hydroxyl radicals, creating a self‐sustaining catalytic cycle that amplifies ROS for radiotherapy synergy. Density functional theory calculations reveal that Mn coordination modulates the d‐band center of Ru, optimizing electronic structure to enhance substrate activation. In radioresistant breast cancer models, Mn/Ru BMSA exhibits a synergistic triple‐modal antitumor action: 1) sustained oxygen generation alleviates tumor hypoxia, 2) X‐ray‐enhanced ROS production amplifies oxidative stress, and 3) hypoxia‐inducible factor 1α pathway inhibition suppresses cancer stem cell (CSC) stemness (as validated by transcriptomics). Patient‐derived organoid assays demonstrated complete radioresistance reversal while maintaining exceptional biosafety. This work establishes Mn/Ru BMSA as a potential radiosensitizer that overcomes the limitations of conventional high‐Z materials by targeting the hypoxia‐CSC axis, providing a strategy for developing multi‐functional nanozymes against treatment‐resistant cancers.

Rationale: Breast cancer recurrences and treatment failures can be attributed to intra-tumoral heterogeneity (ITH), which is characterize by the coexistence of diverse cellular states, including cancer stem cells (CSCs), within a single tumor.Recent insights suggest that ITH arises from non-genetic dynamics, enabling tumors to adapt and evolve into a therapy-tolerant state under treatment pressure. The aim of this work is to decipher the origin of persistent radiation tolerant cells (RTP) in breast tumors and to understand their mechanisms in order to find new strategies to avoid radiation resistance. Methods: To this aim,we developed a lineage tracing system and engineeredvarious breast cancer cell lines and patient-derived xenografts totracked radiation-induced cell plasticity. We combined lineage tracing with a unique RNAi screen under irradiation to identify and functionally validate the regulators of radio-induced cell plasticity. Results: We discovered that RTP cells, which possess CSC properties, emerge from radiotherapy-induced reprogramming of non-CSCs. From the combinatorial approach of the lineage tracing and the RNAi screen under irradiation, we then identified and functionally validated the LRP4/YAP axis as a crucial regulator of radio-induced cell plasticity. We further demonstrate that overexpression of LRP4 is common in residual disease post-treatment and is associated with breast tumors of poor prognosis. Conclusions: This work has demonstrated that the LRP4/YAP axis drives radioresistance by promoting the emergence of RTP cells through radiation-induced plasticity, and that modulation of the LRP4/YAP axis is a promising strategy for sensitizing breast cancers to radiotherapy, opening up a new avenue for improving patient outcomes.

The de novo purine biosynthesis (DNPB) pathway plays a critical role in the malignant progression of tumors. However, its specific contribution to esophageal squamous cell carcinoma (ESCC) growth, radioresistance, and response to targeted therapies remains poorly understood. In this study, the underlying metabolic change of ESCC were analyzed by untargeted metabolomics and single-cell RNA sequencing data analyses. The expression and function of PPAT were further evaluated through immunohistochemistry (IHC) and lentivirus-mediated gene manipulation. Additionally, patient-derived xenograft (PDX) and cell-derived xenograft (CDX) models were utilized to assess the role of PPAT and its inhibitor in ESCC tumor growth and radioresistance. In this study, we identified phosphoribosyl pyrophosphate amidotransferase (PPAT), a key rate-limiting enzyme in the DNPB pathway, as a critical modulator of ESCC malignancy. We demonstrated that PPAT promotes the production of energy-related nucleotides, including AMP, GMP, ADP, GDP, ATP, and GTP, thereby fueling ESCC tumor growth in vitro and in vivo. Moreover, we found that Cucurbitacin B (CuB) specifically targets PPAT and induces its polyubiquitin-mediated degradation via the E3 ligase TRIM38, leading to suppression of the DNPB pathway and inhibition of tumor growth. Importantly, CuB also functions as a radiosensitizer, significantly enhancing the therapeutic efficacy of radiotherapy in ESCC. Our findings reveal that targeting PPAT represents a promising therapeutic strategy to suppress ESCC progression and enhance the efficacy of radiotherapy. Mengqiu Song, Jing Guo, Huajie Jia, Jie Tian, Pan Li, Zigang Dong. Inhibition of de novo purine biosynthesis via PPAT targeting by cucurbitacin B restrains esophageal squamous cell carcinoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3042.

Resistance to radiotherapy is a major obstacle for effective cancer treatment. Both cancer-associated fibroblasts (CAF) within the tumor microenvironment (TME) and Notch signaling are implicated in radioresistance, but their potential interrelationship is unclear. Here, we report that irradiated samples obtained from luminal breast cancer patient tumors express higher levels of the Notch ligand Dll1 and have a greater number of αSMA- and FAP-expressing activated CAFs. Single cell transcriptomic profiles further revealed enrichment of an αSMA+ myofibroblastic subpopulation of CAF in Dll1+ tumors. In murine and human PDX models, Dll1+ tumor cells were more radioresistant than Dll1- tumor cells, and genetic and pharmacological blocking of Dll1-mediated Notch signaling decreased the number of Dll1+ cancer stem cells (CSC) and CAFs and increased Dll1+ tumor cell radiosensitivity. Dll1+ cells recruited CAFs in an IL-6-dependent fashion and promoted Wnt ligand secretion by Notch2/3-expressing CAFs, thereby driving Wnt/β-catenin-dependent increases in Dll1+ CSC function to promote metastasis. Treatment with the porcupine inhibitor LGK-974 that inhibits Wnt ligand secretion or pharmacological blockade of IL-6 or Dll1 suppressed CAF-dependent enhancement of Dll1+ CSC function and metastasis in radioresistant tumors. Together, these findings reveal an essential crosstalk between Dll1+ cancer cells and CAFs that promotes metastasis and radioresistance, which could be therapeutically exploited to improve the outcome of breast cancer patients.

Glioblastoma (GBM) remains one of the most aggressive and fatal cancers, with a median survival of only 12-18 months despite standard treatments, including surgical resection, temozolomide (TMZ) chemotherapy, and radiotherapy. Tumor recurrence and therapy resistance are significant barriers to improving patient outcomes. To address this, our lab investigated the molecular mechanisms driving resistance during TMZ therapy using a single-cell RNA-sequencing screen on a patient-derived xenograft (PDX) model. This approach enabled us to study tumor evolution pre-, during, and post-therapy. Our analysis identified 149 unique genes expressed during TMZ therapy, with the ribonucleotide reductase (RNR) family, particularly Ribonucleotide Reductase Regulatory Subunit 2 (RRM2), emerging as critical players. During TMZ treatment, GBM cells preferentially utilize RRM1-RRM2 interaction to support deoxynucleoside triphosphate (dNTP) biosynthesis, whereas post-therapy recurrent GBM shifts to RRM1-RRM2B complexes. Functional studies demonstrated that RRM2 knockdown sensitized GBM cells to TMZ, reducing DNA repair capacity, as evidenced by increased yH2AX fluorescence, while RRM1 or RRM2B knockdown conferred resistance (p<0.001). Metabolomic profiling revealed that RRM2 mediates dCTP and dGTP production during TMZ treatment, critical for chemoresistance. Supplementation with these dNTPs rescued TMZ susceptibility in RRM2-deficient cells, highlighting its pivotal role in DNA repair and therapeutic resistance. Recognizing the limitations of first-generation RNR inhibitors, we explored the second-generation inhibitor Triapine (3-AP). Preclinical studies showed that 3-AP effectively inhibits RRM2 activity, enhancing TMZ sensitivity and extending survival in PDX models (p<0.0001). Mice treated with a combination of 3-AP and TMZ demonstrated significantly longer survival compared to TMZ alone. These findings underscore the potential of targeting RRM2-mediated dNTP biosynthesis to overcome chemoresistance in GBM. We are conducting a Phase 1/1b clinical trial to assess the safety, toxicity, and maximum tolerated dose (MTD) of combining 3-AP with TMZ in recurrent GBM patients. Atique U. Ahmed, Karan Dixit, Ella N Perrault, Jack M Shireman, Priya Kumthekar, Roger Stupp. David C James. RRM2-driven dNTP biosynthesis: a therapeutic vulnerability in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4431.

OBJECTIVE The fact that certain oral carcinoma patients experience radiotherapy failure implies that a more radioresistant and aggressive phenotype of surviving cancer cells potentially occurs during treatment. Our study aimed to establish radioresistant oral cancer cells through a fractionated irradiation protocol that mimics clinically relevant radiotherapy dosing strategies and to investigate all-round alterations in the malignant phenotype. METHODS Radioresistant oral carcinoma cells were generated by exposing Cal27 and Detroit 562 cells to 60 Gy radiation in 10 dose-escalating fractions and verified by cell immunofluorescence. Specific markers related to the epithelial-mesenchymal transition (EMT) process and the cancer stem cell (CSC) phenotype were assessed by Western blotting. Cell invasion and migration were evaluated using Matrigel-coated transwell and wound healing assays, respectively. Nontargeted metabolomics was used to mechanistically delineate the potential metabolic patterns linked to EMT and CSCs; the CSC phenotype was also examined by sphere formation assays and cell immunofluorescence. RESULTS Radioresistant oral carcinoma cell lines were successfully established and validated. These cells exhibited enhanced EMT and increase in both cell invasion and migration. These radioresistant cells further demonstrated a high metabolic profile, notably marked by lipid metabolism reprogramming and functional enrichment of ATP-binding cassette (ABC) transporters. Consistently, enhanced CSC phenotype in radioresistant cells was confirmed by elevated expression of stemness markers and increased sphere-forming capacity. CONCLUSION Radioresistant oral carcinoma cells subjected to fractionated radiation exhibit an augmented malignant phenotype. The metabolic characteristics linked to enhanced EMT and CSC phenotypes provide potential targets for improving radiotherapy in oral carcinoma.

The metabolic reprogramming in high-grade serous ovarian carcinoma (HGSOC) affects the tumor stemness, which mediates tumor recurrence and progression. Knowledge of the stemness and metabolic characteristics of HGSOC is insufficient. Squalene epoxidase (SQLE), a key enzyme in cholesterol metabolism, was significantly upregulated in HGSOC samples with a fold change of about 4 in the RNA sequencing analysis. SQLE was positively related to peritoneal metastasis and poor prognosis of HGSOC patients. Functionally, SQLE drove cancer cell proliferation and inhibited apoptosis to accelerate HGSOC growth. SQLE was highly expressed in ALDH+CD133+ FACS-sorted cells derived from HGSOC cells and ovarian cancer stem cells (OCSCs)-enriched tumorspheres. SQLE overexpression resulted in enhanced CSC-like properties, including increased tumorsphere formation and stemness markers expression. In vivo, SQLE not only promoted cell line-derived xenografts growth but extended the OCSCs subpopulation of single-cell suspension. Moreover, non-targeted metabolomics profiling from UPLC-MS/MS system identified 90 differential metabolites responding to SQLE overexpression in HGSOC cells. Among them, the dysfunctional metabolisms of cholesterol and glutathione were involved in the maintenance of HGSOC stemness. Previous studies showed the alteration of N6-Methyladenosine (m6A) modification in HGSOC development. Herein, the m6A modification in the 3’UTR and CDS regions of SQLE mRNA was increased due to upregulated methyltransferases WTAP and downregulated demethylases FTO, which was recognized by m6A-binding proteins IGF2BP3, rather than IGF2BP1 or IGF2BP2, thereby stabilizing the SQLE mRNA. These results suggested that SQLE was a novel potential clinical marker for predicting the HGSOC development and prognosis, as well as a potential therapeutic target of HGSOC.

Radiotherapy is a mainstay treatment for localized prostate cancer (PCa). Yet, radiation resistance remains a major clinical obstacle. Here, radiation induced a BMP/CD105-dependent metabolic shift in the tumor microenvironment that facilitates PCa cell survival. Using prostate tumor models and fibroblast cultures, we show that radiation enhances epithelial BMP ligand production which promotes fibroblastic CD105 signaling. Metabolomic analysis upon radiation revealed that fibroblastic CD105 signaling elevated key enzymes involved in mitochondrial biogenesis (PGC1α) and ketogenesis (HMGCS2). The increased production of β-hydroxybutyrate in the tumor microenvironment sustained PCa cell energy metabolism and enhanced DNA repair upon radiation stress. Blocking BMP signaling through carotuximab (ENV105), a CD105-targeting antibody, disrupted epithelial-fibroblast crosstalk, resulting in decreased β-hydroxybutyrate within the tumor microenvironment. This attenuation of fibroblast-mediated metabolic support increased DNA damage and apoptosis, sensitizing PCa cells to radiation. In subcutaneous mouse models, grafting PCa cells with CD105-KO or HMGCS2-KO fibroblasts yielded smaller tumors following radiation compared with wild-type fibroblast controls. Across subcutaneous and orthotopic models, combined treatment with carotuximab and irradiation reproducibly achieved superior tumor volume reduction relative to single-agent therapy. This study identified the BMP/CD105 axis as a key pathway in radiation resistance, highlighting the potential of targeting fibroblastic CD105 with carotuximab to enhance radiation sensitivity.

Triple-negative breast cancers are the most challenging to treat with targeted therapy because of their non-expression of cell surface receptors, unlike other types of breast cancer. However, conventional therapies such as chemotherapy, radiotherapy, and surgery are standard treatment modalities. Moreover, drug encapsulation strategies to deliver drugs at the targeted site have been sought-after alternatives. Hematopoietic Stem Cells (HSCs) have unique features to migrate towards inflammatory sites and other body organs during any injuries and release various cytokines, which lead to interaction with molecular pathways and results in reduced inflammation and tissue injuries. From this characteristic, we have hypothesized that hematopoietic stem cells can show tumor tropism and modulate the metabolic alteration in cancer and cancer stem cells, mainly focusing on Triple Negative Breast Cancer Stem Cells (TNBC CSCs). In this study, we have sorted both cancer stem cells and hematopoietic stem cells from TNBC and hematological cell lines, respectively. Sorted HSCs were cultured in specific conditions to prepare conditioned media. Then, both cell’ types were used for co-cultured assay and conditioned media cultured with TNBC-CSCs were performed. Metabolomics studies were performed to decipher the molecular interactions and TNBC-CSCs metabolic alterations. The result indicates that the HSCs are showing positive tropism towards TNBC stem cells. Furthermore, HSCs-derived conditioned media (CM) interact with the cell cycle pathway by downregulating the CDK genes and causing DNA breakage in the TNBC-CSCs. Moreover, the Metabolomics studies further confirmed that HSCs CM-derived cytokines interact with various pathways, dysregulating the TNBC-CSCs proliferation and altering the metabolic activities. Citation Format: Sumit Mallick, Sudheer Shenoy P, Bipasha Bose. Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2790.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization.

Metabolic and stress response adaptations in prostate cancer (PCa) mediate tumor resistance to radiation therapy (RT). Our study investigated the roles of glutamine (Gln) transporters SLC1A5, SLC7A5, and SLC38A1 in regulating NUPR1-mediated stress response, PCa cell survival, metabolic reprogramming, and response to RT. The radiosensitizing potential of GLS inhibition with CB-839 was analyzed in prostate cancer xenograft models. The level of gene expression was analyzed by RNA sequencing and RT-qPCR in the established cell lines or patient-derived tumor and adjacent non-cancerous tissues. Phosphoproteomic analysis was employed to identify the underlying signaling pathways. The publicly available PCa patient datasets, and a dataset for the patients treated with RT were analyzed by SUMO software. The key parameters of mitochondrial functions were measured by Seahorse analysis. Analysis of the general oxidative stress level and mitochondrial superoxide detection were conducted using flow cytometry. γH2A.X foci analysis was used to assess the DNA double strand break. Relative cell sensitivity to RT was evaluated by radiobiological clonogenic assays. Aldefluor assay and sphere-forming analysis were used to determine cancer stem cell (CSC) phenotype. A siRNA-mediated knockdown of Gln transporters SLC1A5, SLC7A5, and SLC38A1 resulted in significant radiosensitization of PCa cells. Consistently, the first-in-clinic glutaminase (GLS) inhibitor CB-839, combined with RT, demonstrated a synergistic effect with radiotherapy in vivo, significantly delaying tumor growth. Inhibition of Gln metabolism or knockdown of Gln transporters SLC1A5, SLC7A5, or SLC38A1 induces expression of NUPR1, a stress response transcriptional regulator, but simultaneously uncouples the NUPR1-driven metabolic stress-adaptation program. Similarly to the effect from NUPR1 knockdown, depletion of these Gln transporters led to reduced cell viability, accumulation of mitochondrial ROS, and increased PCa radiosensitivity. This effect is more pronounced in PCa cells with high dependency on OXPHOS for energy production. Our work underscores the role of Gln transporters and the NUPR1-mediated stress response in PCa cell survival, oxidative stress, mitochondrial functions, and radioresistance. Our findings provide a potential therapeutic in vivo strategy to enhance the efficacy of RT and suggest a potential synergism between the depletion of Gln transporters or NUPR1 and OXPHOS inhibition.

Cancer stem cells (CSCs) are able to self-renew and are refractory to cancer treatment. To investigate the effects of resveratrol on CSCs of nasopharyngeal carcinoma (NPC), we employed a behavior selection strategy to isolate CSCs based on radioresistance, chemoresistance, and tumor sphere formation ability. These NPC CSCs displayed stem cell properties and underwent metabolic shift to predominately rely on glycolysis for energy supply. Intriguingly, we found that resveratrol turned off the metabolic switch, increased the reactive oxygen species (ROS) level, and depolarized mitochondrial membranes. These alterations in metabolism occurred concomitantly with the suppression of CSC properties including resistance to therapy, self-renewal capacity, tumor initiation capacity, and metastatic potential in NPC CSCs. We found that resveratrol impeded CSC properties through the activation of p53 and this effect could be reversed by knockdown of p53. Furthermore, resveratrol suppressed the stemness and EMT through reactivating p53 and inducing miR-145 and miR-200c, which were downregulated in NPC CSCs. In conclusion, we demonstrated that resveratrol employed the p53 pathway in regulating stemness, EMT, and metabolic reprogramming. Further investigation of the molecular mechanism of p53 activation by resveratrol may provide useful information for the development of novel therapies for cancer treatment through targeting to CSCs.

Whether stem-cell-like cancer cells avert ferroptosis to mediate therapy resistance remains unclear. In this study, using a soft fibrin gel culture system, we found that tumor-repopulating cells (TRCs) with stem-cell-like cancer cell characteristics resist chemotherapy and radiotherapy by decreasing ferroptosis sensitivity. Mechanistically, through quantitative mass spectrometry and lipidomic analysis, we determined that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4 to drive ferroptosis-associated phospholipid remodeling. TRCs downregulate the PCK2 expression to confer themselves on a structural ferroptosis-resistant state. Notably, in addition to confirming the role of PCK2-pACSL4(T679) in multiple preclinical models, we discovered that higher PCK2 and pACSL4(T679) levels are correlated with better response to chemotherapy and radiotherapy as well as lower distant metastasis in nasopharyngeal carcinoma cohorts.

Cancer stem cells (CSCs) play an essential role in tumor initiation and therapy resistance. Histone lactylation as a novel epigenetic modification could regulate the gene transcription process during tumor progression. Nevertheless, researches have not well examined its role in maintaining CSC properties. Our study identified Minichromosome maintenance complex component 7 (MCM7) as a candidate gene in Hepatocellular carcinoma (HCC) with diagnostic and prognostic values, and Real-time quantitative PCR (qRT-PCR), Western blot (WB), and Immunohistochemistry (IHC) assays ascertained its obviously higher expressions in HCC cells and tissues. Ectopic of MCM7 could increase the expression of CSC-related genes and enhance spheroid both in size and in number. Suppression of MCM7 could strengthen the efficacy of radiotherapy verified by Cell counting kit-8 (CCK-8) and colony formation assays. The subcutaneous xenograft model indicated that suppression of MCM7 could inhibit CSC properties and the efficacy of radiotherapy in vivo. Mechanistically, histone lactylation could facilitate MCM7 expression, and both messenger RNA (mRNA) and protein level of MCM7 expression presented an obvious decrease due to 2-DG (glycolysis inhibitor) treatment and an obvious increase due to Rotenone (glycolysis activator) treatment. Rescue experiments verified that histone lactylation was necessary for MCM7 to promote CSC properties and radio-resistance in HCC. Arsenic trioxide (ATO) targeting MCM7 could inhibit the CSC phenotypes and enhance the efficacy of radiotherapy in vivo and in vitro. Collectively, histone lactylation could transcriptionally activate MCM7 to accelerate proliferation and radio-resistance through enhancing CSC properties. ATO targeting MCM7 could inhibit CSCs phenotypes and synergistically increase the efficacy of radiation therapy.

Pancreatic ductal adenocarcinoma (PDAC) cells utilize a novel non-canonical pathway of glutamine metabolism that is essential for tumor growth and redox balance. Inhibition of this metabolic pathway in PDAC can potentially synergize with therapies that increase intracellular reactive oxygen species (ROS) such as radiation. Here, we evaluated the dependence of pancreatic cancer stem cells (PCSCs) on this non-canonical glutamine metabolism pathway and researched whether inhibiting this pathway can enhance radiosensitivity of PCSCs. We showed that glutamine deprivation significantly inhibited self-renewal, decreased expression of stemness-related genes, increased intracellular ROS, and induced apoptosis in PCSCs. These effects were countered by oxaloacetate, but not α-ketoglutarate. Knockdown of glutamic-oxaloacetic transaminase dramatically impaired PCSCs properties, while glutamate dehydrogenase knockdown had a limited effect, suggesting a dependence of PCSCs on non-canonical glutamine metabolism. Additionally, glutamine deprivation significantly increased radiation-induced ROS and sensitized PCSCs to fractionated radiation. Moreover, transaminase inhibitors effectively enhanced ROS generation, promoted radiation sensitivity, and attenuated tumor growth in nude mice following radiation exposure. Our findings reveal that inhibiting the non-canonical pathway of glutamine metabolism enhances the PCSC radiosensitivity and may be an effective adjunct in cancer radiotherapy.

To assess whether anaerobic metabolism, proliferation activity and stem cell content are linked with radioresistance in bladder cancer. Tissue sections from 66 patients with invasive transitional cell bladder cancer treated with hypofractionated accelerated radiotherapy, was immunohistochemically analyzed for the Hypoxia-Inducible Factor 1α (HIF1α) and the anaerobic glycolysis enzyme lactate dehydrogenase 5 (LDH5). Proliferation index (Ki-67) and stem-cell marker (cluster of differentiation CD44, aldehyde dehydrogenase ALDH1) expression was also examined. Both HIF1α and LDH5 expression were linked with high CD44 stem cell population (p = 0.001 and 0.05, respectively), while high Ki-67 proliferation index was linked with nuclear LDH5 expression (p = 0.03) and high histological grade (p = 0.02). A strong significant association of HIF1α (p = 0.0009) and of LDH5 (p < 0.0001) with poor local relapse free survival (LRFS) was noted, which was also confirmed in multivariate analysis. A significant association with overall survival was also noted. Silencing of lactate dehydrogenase LDHA gene in the human RT112 bladder cancer cell line, or exposure to oxamate (LDH activity inhibitor), resulted in strong radio-sensitization. HIF1α and LDH5 are markers of poor outcome in patients with bladder cancer treated with radiotherapy. Blockage of anaerobic metabolism may prove of importance in clinical radiotherapy.

The high fatality rate of glioblastoma (GBM) is attributed to glioblastoma stem cells (GSCs), which exhibit heterogeneity and therapeutic resistance. Metabolic plasticity of mitochondria is the hallmark of GSCs. Targeting mitochondrial biogenesis of GSCs is crucial for improving clinical prognosis in GBM patients. SMYD2-induced PGC1α methylation and followed nuclear export are confirmed by co-immunoprecipitation, cellular fractionation, and immunofluorescence. The effects of SMYD2/PGC1α/CRM1 axis on GSCs mitochondrial biogenesis are validated by oxygen consumption rate, ECAR, and intracranial glioma model. PGC1α methylation causes the disabled mitochondrial function to maintain the stemness, thereby enhancing the radio-resistance of GSCs. SMYD2 drives PGC1α K224 methylation (K224me), which is essential for promoting the stem-like characteristics of GSCs. PGC1α K224me is preferred binding with CRM1, accelerating PGC1α nuclear export and subsequent dysfunction. Targeting PGC1α methylation exhibits significant radiotherapeutic efficacy and prolongs patient survival. These findings unveil a novel regulatory pathway involving mitochondria that govern stemness in GSCs, thereby emphasizing promising therapeutic strategies targeting PGC1α and mitochondria for the treatment of GBM.

Tumor recurrence after radiotherapy due to the presence of breast cancer stem cells (BCSCs) is a clinical challenge, and the mechanism remains unclear. Low levels of ROS and enhanced antioxidant defenses are shown to contribute to increasing radioresistance. However, the role of Nrf2-Keap1-Bach1 signaling in the radioresistance of BCSCs remains elusive. Fractionated radiation increased the percentage of the ALDH-expressing subpopulation and their sphere formation ability, promoted mesenchymal-to-epithelial transition and enhanced radioresistance in BCSCs. Radiation activated Nrf2 via Keap1 silencing and enhanced the tumor-initiating capability of BCSCs. Furthermore, knockdown of Nrf2 suppressed ALDH

Despite available treatment approaches, including surgical resection along with chemotherapy and radiotherapy, glioblastoma (GBM), the most prevalent primary brain tumor, remains associated with a grim prognosis. Although radiotherapy is central to GBM treatment, its combination with bioenergetics regulators has not been validated in clinical practice. Here, we hypothesized that bioenergetics regulators can enhance the radio-sensitivity of GBM tumorspheres (TSs). Gene expression profiles of GBM patient-derived TSs were obtained through microarray and RNA-seq. In vitro treatment efficacy was assessed using clonogenic assay, 3D invasion assay, neurosphere formation assay, and flow cytometry. Protein expression was measured via western blot, and γH2AX foci were detected via immunofluorescence. In vivo efficacy was confirmed in an orthotopic xenograft model. Based on radiation response-associated gene expression, GBM TSs were classified into high- or low-radioresistant groups. Among the five bioenergetics regulators, the pentose phosphate pathway inhibitor DHEA and the glycolysis inhibitor 2-DG notably enhanced the efficacy of ionizing radiation (IR) efficacy in vitro, reducing the survival fraction, stemness, and invasiveness in high- and low-radioresistant TSs. Combination with 2-DG further stimulated IR-induced DNA damage response and apoptosis in low-radioresistant GBM TSs. RNA-seq analysis revealed a downregulation of bioenergetics- and cell cycle-associated genes, whereas extracellular matrix- and cell adhesion-associated genes were enhanced by combined IR and 2-DG treatment. This therapeutic regimen extended survival and diminished tumor size in mouse xenograft models. Our data suggest that combination with bioenergetics regulator 2-DG enhances the radio-sensitivity of GBM TSs, highlighting the clinical potential of this combined regimen.