RESUMEN
Targeting CDC20 can enhance the radiosensitivity of tumor cells, but the function and mechanism of CDC20 on DNA damage repair response remains vague. To examine that issue, tumor cell lines, including KYSE200, KYSE450, and HCT116, were utilized to detect the expression, function, and underlying mechanism of CDC20 in radio-chemoresistance. Western blot and immunofluorescence staining were employed to confirm CDC20 expression and location, and radiation could upregulate the expression of CDC20 in the cell nucleus. The homologous recombination (HR) and non-homologous end joining (NHEJ) reporter gene systems were utilized to explore the impact of CDC20 on DNA damage repair, indicating that CDC20 could promote HR repair and radio/chemo-resistance. In the early stages of DNA damage, CDC20 stabilizes the RPA1 protein through protein-protein interactions, activating the ATR-mediated signaling cascade, thereby aiding in genomic repair. In the later stages, CDC20 assists in the subsequent steps of damage repair by the ubiquitin-mediated degradation of RPA1. CCK-8 and colony formation assay were used to detect the function of CDC20 in cell vitality and proliferation, and targeting CDC20 can exacerbate the increase in DNA damage levels caused by cisplatin or etoposide. A tumor xenograft model was conducted in BALB/c-nu/nu mice to confirm the function of CDC20 in vivo, confirming the in vitro results. In conclusion, this study provides further validation of the potential clinical significance of CDC20 as a strategy to overcome radio-chemoresistance via uncovering a novel role of CDC20 in regulating RPA1 during DNA damage repair.
Asunto(s)
Proteínas Cdc20 , Daño del ADN , Resistencia a Antineoplásicos , Tolerancia a Radiación , Proteína de Replicación A , Humanos , Animales , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Ratones , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Resistencia a Antineoplásicos/genética , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Línea Celular Tumoral , Ratones Endogámicos BALB C , Ratones Desnudos , Reparación del ADN/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Células HCT116 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
BACKGROUND: Radioresistance is the leading cause of death in advanced cervical cancer (CC). Dysregulation of RNA modification has recently emerged as a regulatory mechanism in radiation and drug resistance. We aimed to explore the biological function and clinical significance of 5-methylcytosine (m5C) in cervical cancer radiosensitivity. METHODS: The abundance of RNA modification in radiotherapy-resistant and sensitive CC specimens was quantified by liquid chromatography-tandem mass spectrometry. The essential RNA modification-related genes involved in CC radiosensitivity were screened via RNA sequencing. The effect of NSUN6 on radiosensitivity was verified in CC cell lines, cell-derived xenograft (CDX), and 3D bioprinted patient-derived organoid (PDO). The mechanisms of NSUN6 in regulating CC radiosensitivity were investigated by integrative m5C sequencing, mRNA sequencing, and RNA immunoprecipitation. RESULTS: We found a higher abundance of m5C modification in resistant CC samples, and NSUN6 was the essential m5C-regulating gene concerning radiosensitivity. NSUN6 overexpression was clinically correlated with radioresistance and poor prognosis in cervical cancer. Functionally, higher NSUN6 expression was associated with radioresistance in the 3D PDO model of cervical cancer. Moreover, silencing NSUN6 increased CC radiosensitivity in vivo and in vitro. Mechanistically, NDRG1 was one of the downstream target genes of NSUN6 identified by integrated m5C-seq, mRNA-seq, and functional validation. NSUN6 promoted the m5C modification of NDRG1 mRNA, and the m5C reader ALYREF bound explicitly to the m5C-labeled NDRG1 mRNA and enhanced NDRG1 mRNA stability. NDRG1 overexpression promoted homologous recombination-mediated DNA repair, which in turn led to radioresistance in cervical cancer. CONCLUSIONS: Aberrant m5C hypermethylation and NSUN6 overexpression drive resistance to radiotherapy in cervical cancer. Elevated NSUN6 expression promotes radioresistance in cervical cancer by activating the NSUN6/ALYREF-m5C-NDRG1 pathway. The low expression of NSUN6 in cervical cancer indicates sensitivity to radiotherapy and a better prognosis.
Asunto(s)
5-Metilcitosina , Proteínas de Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , ARN Mensajero , Tolerancia a Radiación , Neoplasias del Cuello Uterino , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/radioterapia , Neoplasias del Cuello Uterino/patología , Humanos , Femenino , Tolerancia a Radiación/genética , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Línea Celular Tumoral , Pronóstico , Ensayos Antitumor por Modelo de Xenoinjerto , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.
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Reparación del ADN , Proteínas de Unión al ADN , Radiación Ionizante , Tardigrada , Transcriptoma , Tardigrada/genética , Tardigrada/metabolismo , Animales , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Daño del ADN , Tolerancia a Radiación/genéticaRESUMEN
Long non-coding RNAs (lncRNAs) have been implicated in cancer progression and drug resistance development. Moreover, there is evidence that lncRNA HOX transcript antisense intergenic RNA (HOTAIR) is involved in colorectal cancer (CRC) progression. The present study aimed to examine the functional role of lncRNA HOTAIR in conferring radiotherapy resistance in CRC cells, as well as the underlying mechanism. The relative expression levels of HOTAIR were examined in 70 pairs of CRC tumor and para-cancerous tissues, as well as in radiosensitive and radioresistant samples. The correlations between HOTAIR expression levels and clinical features of patients with CRC were assessed using the Chi-square test. Functional assays such as cell proliferation, colony formation and apoptosis assays were conducted to determine the radiosensitivity in CRC cells with HOTAIR silencing after treatment with different doses of radiation. RNA pull-down assay and fluorescence in situ hybridization (FISH) were used to determine the interaction between HOTAIR and DNA damage response mediator ataxia-telangiectasia mutated- and Rad3-related (ATR). HOTAIR was significantly upregulated in CRC tumor tissues, especially in radioresistant tumor samples. The elevated expression of HOTAIR was correlated with more advanced histological grades, distance metastasis and the poor prognosis in patients with CRC. Silencing HOTAIR suppressed the proliferation and promoted apoptosis and radiosensitivity in CRC cells. HOTAIR knockdown also inhibited the tumorigenesis of CRC cells and enhanced the sensitivity to radiotherapy in a mouse xenograft model. Moreover, the data showed that HOTAIR could interact with ATR to regulate the DNA damage repair signaling pathway. Silencing HOTAIR impaired the ATR-ATR interacting protein (ATRIP) complex and signaling in cell cycle progression. Collectively, the present results indicate that lncRNA HOTAIR facilitates the DNA damage response pathway and promotes radioresistance in CRC cells by targeting ATR.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Proliferación Celular , Neoplasias Colorrectales , Daño del ADN , Reparación del ADN , ARN Largo no Codificante , Tolerancia a Radiación , ARN Largo no Codificante/genética , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/radioterapia , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Tolerancia a Radiación/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ratones , Proliferación Celular/genética , Apoptosis/genética , Línea Celular Tumoral , Femenino , Masculino , Regulación Neoplásica de la Expresión Génica , Pronóstico , Persona de Mediana Edad , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones DesnudosRESUMEN
Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.
Asunto(s)
Resistencia a Antineoplásicos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Factor Neurotrófico Derivado de la Línea Celular Glial , Glioblastoma , Tolerancia a Radiación , Transducción de Señal , Temozolomida , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Resistencia a Antineoplásicos/genética , Temozolomida/farmacología , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Lomustina/farmacología , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T (UBE2T) on radiosensitivity of hepatocellular carcinoma (HCC). METHODS: Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector, and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay. The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice, and tumor growth following irradiation were recorded. The xenografts were collected for analyzing infiltration of CD4+ T cells and regulatory T cells (Tregs) using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting. The correlations of UBE2T expression with immune cell infiltration, glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database, and the results were verified in a co-culture system of Hepa1-6 cells and Tregs. RESULTS: UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models (especially in C57BL/6 mice). CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells, T follicular helper cells, M2 macrophages, monocytes, lymphocytes and Tregs in HCC. The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA, and irradiation increased infiltration of CD4+ T cells and Tregs in the tumor microenvironment. Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration. GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC. In the cell co-culture system, UBE2T overexpression significantly enhanced lactate production, proliferation and immunosuppressive functions of Tregs. CONCLUSION: A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratones Endogámicos C57BL , Ratones Desnudos , Tolerancia a Radiación , Linfocitos T Reguladores , Microambiente Tumoral , Enzimas Ubiquitina-Conjugadoras , Animales , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/inmunología , Ratones , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Línea Celular Tumoral , Tolerancia a Radiación/genética , Humanos , GlucólisisRESUMEN
PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.
Asunto(s)
Proteína-Arginina N-Metiltransferasas , Tolerancia a Radiación , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Animales , Humanos , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Línea Celular Tumoral , Ratones , Reparación del ADN , Proliferación Celular/efectos de la radiación , Ensayos Antitumor por Modelo de Xenoinjerto , Roturas del ADN de Doble Cadena/efectos de la radiación , Ratones DesnudosRESUMEN
Radiotherapy is a key treatment option for a wide variety of human tumors, employed either alone or alongside with other therapeutic interventions. Radiotherapy uses high-energy particles to destroy tumor cells, blocking their ability to divide and proliferate. The effectiveness of radiotherapy is due to genetic and epigenetic factors that determine how tumor cells respond to ionizing radiation. These factors contribute to the establishment of resistance to radiotherapy, which increases the risk of poor clinical prognosis of patients. Although the mechanisms by which tumor cells induce radioresistance are unclear, evidence points out several contributing factors including the overexpression of DNA repair systems, increased levels of reactive oxygen species, alterations in the tumor microenvironment, and enrichment of cancer stem cell populations. In this context, dysregulation of microRNAs or miRNAs, critical regulators of gene expression, may influence how tumors respond to radiation. There is increasing evidence that miRNAs may act as sensitizers or enhancers of radioresistance, regulating key processes such as the DNA damage response and the cell death signaling pathway. Furthermore, expression and activity of miRNAs have shown informative value in overcoming radiotherapy and long-term radiotoxicity, revealing their potential as biomarkers. In this review, we will discuss the molecular mechanisms associated with the response to radiotherapy and highlight the central role of miRNAs in regulating the molecular mechanisms responsible for cellular radioresistance. We will also review radio-miRs, radiotherapy-related miRNAs, either as sensitizers or enhancers of radioresistance that hold promise as biomarkers or pharmacological targets to sensitize radioresistant cells.
Asunto(s)
MicroARNs , Neoplasias , Tolerancia a Radiación , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Tolerancia a Radiación/genética , Neoplasias/radioterapia , Neoplasias/genética , Neoplasias/metabolismo , Reparación del ADN , Animales , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Daño del ADNRESUMEN
Background: Cellular senescence in response to ionizing radiation (IR) limits the replication of damaged cells by causing permanent cell cycle arrest. However, IR can induce pro-survival signaling pathways that reduce the extent of radiation-induced cytotoxicity and promote the development of radioresistance. The differential incorporation of histone variant H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of radiation-induced senescence. However, the precise epigenetic mechanism and function of H2A.J overexpression in response to IR exposure still needs to be elucidated. Methods: Primary (no target, NT) and genetically modified fibroblasts overexpressing H2A.J (H2A.J-OE) were exposed to 20 Gy and analyzed 2 weeks post-IR for radiation-induced senescence by immunohistochemistry and immunofluorescence microscopy. Transcriptome signatures were analyzed in (non-)irradiated NT and H2A.J-OE fibroblasts by RNA sequencing. Since H2A.J plays an important role in the epidermal homeostasis of human skin, the oncogenic potential of H2A.J was investigated in cutaneous squamous cell carcinoma (cSCC). The tissue microarrays of cSCC were analyzed for H2A.J protein expression pattern by automated image analysis. Results: In response to radiation-induced DNA damage, the overexpression of H2A.J impairs the formation of senescence-associated heterochromatin foci (SAHF), thereby inhibiting the SAHF-mediated silencing of proliferation-promoting genes. The dysregulated activation of cyclins and cyclin-dependent kinases disturbs cell cycle arrest in irradiated H2A.J-OE fibroblasts, thereby overcoming radiation-induced senescence. Comparative transcriptome analysis revealed significantly increased WNT16 signaling in H2A.J OE fibroblasts after IR exposure, promoting the fundamental mechanisms of tumor development and progression, including the activation of the epithelial-mesenchymal transition. The quantitative analysis of cSCCs revealed that undifferentiated tumors are associated with high nuclear H2A.J expression, related with greater oncogenic potential. Conclusion: H2A.J overexpression induces radioresistance and promotes oncogenic transformation through the activation of WNT16 signaling pathway functions. H2A.J-associated signatures may improve risk stratification by identifying patients with more aggressive cSCC who may require radiotherapy with increased doses.
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Carcinoma de Células Escamosas , Transformación Celular Neoplásica , Senescencia Celular , Fibroblastos , Histonas , Neoplasias Cutáneas , Humanos , Histonas/metabolismo , Histonas/genética , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Senescencia Celular/genética , Senescencia Celular/efectos de la radiación , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/efectos de la radiación , Tolerancia a Radiación/genética , Radiación Ionizante , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células Cultivadas , Epigénesis GenéticaRESUMEN
BACKGROUND: Artemis deficiency is an autosomal recessive disorder characterized by a combined immunodeficiency with increased cellular radiosensitivity. In this review, the clinical and genetic characteristics of 15 patients with DCLRE1C variants are presented. METHODS: The demographic, clinical, immunologic, and genetic characteristics of patients with confirmed DCLRE1C variants diagnosed between 2013 and 2023 were collected retrospectively. Three patients were evaluated for radiosensitivity by the Comet assay, compared with age- and sex-matched healthy control. RESULTS: Seven patients who had severe infections in the first 6 months of life were diagnosed with T-B-NK+ SCID (severe combined immunodeficiency). Among them, four individuals underwent transplantation, and one of those died due to post-transplant complications in early life. Eight patients had hypomorphic variants. Half of them were awaiting a suitable donor, while the other half had already undergone transplantation. The majority of patients were born into a consanguineous family (93.3%). Most patients had recurrent sinopulmonary infections (73.3%), and one patient had no other infection than an acute respiratory infection before diagnosis. Two patients (13.3%) had autoimmunity in the form of autoimmune hemolytic anemia. Growth retardation was observed in only one patient (6.6%), and no malignancy was detected in the surviving 11 patients during the median (IQR) of 21.5 (12-45) months of follow-up. Three patients who had novel variants exhibited increased radiosensitivity and compromised DNA repair, providing a potential vulnerability to malignant transformation. CONCLUSION: Early diagnosis, radiation avoidance, and careful preparation for transplantation contribute to minimizing complications, enhancing life expectancy, and improving the patient's quality of life.
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Proteínas de Unión al ADN , Tolerancia a Radiación , Inmunodeficiencia Combinada Grave , Humanos , Tolerancia a Radiación/genética , Masculino , Femenino , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Lactante , Proteínas de Unión al ADN/genética , Preescolar , Estudios Retrospectivos , Endonucleasas/genética , Proteínas Nucleares/genética , Niño , Estudios de CohortesRESUMEN
Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.
Asunto(s)
Autofagia , Hipoxia de la Célula , Tolerancia a Radiación , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Autofagia/efectos de la radiación , Autofagia/genética , Tolerancia a Radiación/genética , Línea Celular Tumoral , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Hipoxia de la Célula/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Beclina-1/metabolismo , Beclina-1/genética , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Reparación del ADN/efectos de la radiación , Reparación del ADN/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Rayos X , Roturas del ADN de Doble Cadena/efectos de la radiación , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive. METHOD: Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS. RESULT: We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model. CONCLUSION: These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.
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Células Madre Neoplásicas , Tolerancia a Radiación , Ubiquitina Tiolesterasa , Humanos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Tolerancia a Radiación/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Animales , Ratones , Línea Celular Tumoral , Glioma/patología , Glioma/genética , Glioma/radioterapia , Glioma/metabolismo , Apoptosis/genética , Apoptosis/efectos de la radiación , Ubiquitinación , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Ratones Desnudos , Fenotipo , Regulación Neoplásica de la Expresión Génica , PronósticoRESUMEN
OBJECTIVE: Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer. However, the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer (CRC) cells is unclear. This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells. METHODS: The expression of TOPK was detected in CRC tissues by immunohistochemistry, and the effect of TOPK knockdown was detected in CRC cells by Western blotting. CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells. Furthermore, proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy. DNA damage was detected by the comet assay. Changes in the DNA damage response signaling pathway were analyzed by Western blotting, and apoptosis was detected by flow cytometry. RESULTS: The expression of TOPK was significantly greater in CRC tissues at grades 2-4 than in those at grade 1. After irradiation, CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins, including phospho-cyclin-dependent kinase 1 (p-CDK1), phospho-ataxia telangiectasia-mutated (p-ATM), poly ADP-ribose polymerase (PARP), and meiotic recombination 11 homolog 1 (MRE11). CONCLUSIONS: TOPK was overexpressed in patients with moderately to poorly differentiated CRC. Moreover, TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.
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Apoptosis , Neoplasias Colorrectales , Daño del ADN , Tolerancia a Radiación , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/radioterapia , Neoplasias Colorrectales/patología , Daño del ADN/efectos de la radiación , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de los fármacos , Línea Celular Tumoral , Masculino , Técnicas de Silenciamiento del Gen , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transducción de Señal , Femenino , Fosforilación , Quinasas de Proteína Quinasa Activadas por MitógenosRESUMEN
We aimed to determine whether monitoring tumor-derived exosomal microRNAs (miRNAs) could be used to assess radiotherapeutic sensitivity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). RNA sequencing was employed to conduct a comparative analysis of miRNA expression levels during radiotherapy, focusing on identifying miRNAs associated with progression. Electron microscopy confirmed the existence of exosomes, and co-cultivation assays and immunofluorescence validated their capacity to infiltrate macrophages. To determine the mechanism by which exosomal miR-143-3p regulates the interplay between ESCC cells and M2 macrophages, ESCC cell-derived exosomes were co-cultured with macrophages. Serum miR-143-3p and miR-223-3p were elevated during radiotherapy, suggesting resistance to radiation and an unfavorable prognosis for ESCC. Increased levels of both miRNAs independently predicted shorter progression-free survival (p = 0.015). We developed a diagnostic model for ESCC using serum microRNAs, resulting in an area under the curve of 0.751. Radiotherapy enhanced the release of miR-143-3p from ESCC cell-derived exosomes. Immune cell infiltration analysis at the Cancer Genome Atlas (TCGA) database revealed that ESCC cell-derived miR-143-3p triggered M2 macrophage polarization. Mechanistically, miR-143-3p upregulation affected chemokine activity and cytokine signaling pathways. Furthermore, ESCC cell exosomal miR-143-3p could be transferred to macrophages, thereby promoting their polarization. Serum miR-143-3p and miR-223-3p could represent diagnostic and prognostic markers for patients with ESCC undergoing radiotherapy. Unfavorable prognosis could be linked to the increased levels of ESCC cell-derived exosomal miR-143-3p, which might promote tumor progression by interacting with macrophages.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Exosomas , Regulación Neoplásica de la Expresión Génica , Macrófagos , MicroARNs , Tolerancia a Radiación , MicroARNs/genética , Humanos , Exosomas/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/radioterapia , Carcinoma de Células Escamosas de Esófago/metabolismo , Macrófagos/metabolismo , Tolerancia a Radiación/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/metabolismo , Línea Celular Tumoral , Masculino , Femenino , Persona de Mediana Edad , Pronóstico , Anciano , Activación de Macrófagos/genéticaRESUMEN
This study explored the role of 14-3-3σ in carbon ion-irradiated pancreatic adenocarcinoma (PAAD) cells and xenografts and clarified the underlying mechanism. The clinical significance of 14-3-3σ in patients with PAAD was explored using publicly available databases. 14-3-3σ was silenced or overexpressed and combined with carbon ions to measure cell proliferation, cell cycle, and DNA damage repair. Immunoblotting and immunofluorescence (IF) assays were used to determine the underlying mechanisms of 14-3-3σ toward carbon ion radioresistance. We used the BALB/c mice to evaluate the biological behavior of 14-3-3σ in combination with carbon ions. Bioinformatic analysis revealed that PAAD expressed higher 14-3-3σ than normal pancreatic tissues; its overexpression was related to invasive clinicopathological features and a worse prognosis. Knockdown or overexpression of 14-3-3σ demonstrated that 14-3-3σ promoted the survival of PAAD cells after carbon ion irradiation. And 14-3-3σ was upregulated in PAAD cells during DNA damage (carbon ion irradiation, DNA damaging agent) and promotes cell recovery. We found that 14-3-3σ resulted in carbon ion radioresistance by promoting RPA2 and RAD51 accumulation in the nucleus in PAAD cells, thereby increasing homologous recombination repair (HRR) efficiency. Blocking the HR pathway consistently reduced 14-3-3σ overexpression-induced carbon ion radioresistance in PAAD cells. The enhanced radiosensitivity of 14-3-3σ depletion on carbon ion irradiation was also demonstrated in vivo. Altogether, 14-3-3σ functions in tumor progression and can be a potential target for developing biomarkers and treatment strategies for PAAD along with incorporating carbon ion irradiation.
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Proteínas 14-3-3 , Ratones Endogámicos BALB C , Neoplasias Pancreáticas , Reparación del ADN por Recombinación , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Animales , Humanos , Ratones , Línea Celular Tumoral , Regulación hacia Abajo , Tolerancia a Radiación/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Radioterapia de Iones Pesados , Carbono , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Masculino , Daño del ADN , FemeninoRESUMEN
This paper reports the results of gamma irradiation experiments and whole genome sequencing (WGS) performed on vegetative cells of two radiation resistant bacterial strains, Metabacillus halosaccharovorans (VITHBRA001) and Bacillus paralicheniformis (VITHBRA024) (D10 values 2.32 kGy and 1.42 kGy, respectively), inhabiting the top-ranking high background radiation area (HBRA) of Chavara-Neendakara placer deposit (Kerala, India). The present investigation has been carried out in the context that information on strategies of bacteria having mid-range resistance for gamma radiation is inadequate. WGS, annotation, COG and KEGG analyses and manual curation of genes helped us address the possible pathways involved in the major domains of radiation resistance, involving recombination repair, base excision repair, nucleotide excision repair and mismatch repair, and the antioxidant genes, which the candidate could activate to survive under ionizing radiation. Additionally, with the help of these data, we could compare the candidate strains with that of the extremely radiation resistant model bacterium Deinococccus radiodurans, so as to find the commonalities existing in their strategies of resistance on the one hand, and also the rationale behind the difference in D10, on the other. Genomic analysis of VITHBRA001 and VITHBRA024 has further helped us ascertain the difference in capability of radiation resistance between the two strains. Significantly, the genes such as uvsE (NER), frnE (protein protection), ppk1 and ppx (non-enzymatic metabolite production) and those for carotenoid biosynthesis, are endogenous to VITHBRA001, but absent in VITHBRA024, which could explain the former's better radiation resistance. Further, this is the first-time study performed on any bacterial population inhabiting an HBRA. This study also brings forward the two species whose radiation resistance has not been reported thus far, and add to the knowledge on radiation resistant capabilities of the phylum Firmicutes which are abundantly observed in extreme environment.
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Rayos gamma , Genoma Bacteriano , Tolerancia a Radiación , Tolerancia a Radiación/genética , Radiación de Fondo , Secuenciación Completa del Genoma , India , Bacillus/genética , Bacillus/efectos de la radiación , Bacillus/metabolismo , Reparación del ADNRESUMEN
Lung cancer is one of the most significant malignancies, with both high morbidity and mortality. CDK10 is closely related to cancer progression and metastasis. However, its role in lung cancer radioresistance demands further clarification. In this study, we demonstrated that CDK10 was downregulated in lung cancer tissues, and CDK10 expression level was associated with the clinical prognosis in lung cancer patients. We also found that silencing CDK10 promoted lung cancer cell proliferation, migration, and radioresistance. We further verified that silencing CDK10 facilitated the activation of JNK/c-Jun signaling, and c-Jun depletion could reverse the effects of CDK10 knockdown in lung cancer cells. Our findings revealed that CDK10 plays an important role in cell growth and radioresistance by inhibiting JNK/c-Jun signaling pathway in lung cancer. Therefore, CDK10 might be a promising therapeutic target in lung cancer. [BMB Reports 2024; 57(7): 336-341].
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Proliferación Celular , Quinasas Ciclina-Dependientes , Progresión de la Enfermedad , Regulación hacia Abajo , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/genética , Regulación hacia Abajo/genética , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Tolerancia a Radiación/genética , Movimiento Celular/genética , Sistema de Señalización de MAP Quinasas/genética , Transducción de Señal , Regulación Neoplásica de la Expresión Génica , PronósticoRESUMEN
OBJECTIVE: This study aimed to investigate the role of JMJD2A in radiotherapy tolerance of esophageal squamous cell carcinoma (ESCC). METHODS: The levels of H3K9me3 modification were analyzed in anti-PD-1 therapy non-responder or responder patients, and the expression differences of H3K9me3-related modifying enzymes were assessed in TCGA-ESCC and ICGC cohorts. Subsequently, JMJD2A was knocked down in ESCC cells using CRISPR-Cas9 or lentivirus-mediated shRNA, and changes in malignant behavior of ESCC cells were observed. RNA-seq, ATAC-seq, and ChIP-seq analyses were then conducted to investigate the genes and downstream signaling pathways regulated by JMJD2A, and functional validation experiments were performed to analyze the role of downstream regulated genes and pathways in ESCC malignant behavior and immune evasion. RESULTS: JMJD2A was significantly overexpressed in ESCC and anti-PD-1 therapy non-responders. Knockdown or deletion of JMJD2A significantly promoted the malignant behavior and immune evasion of ESCC. JMJD2A facilitated the structural changes in chromatin and promoted the binding of SMARCA4 to super-enhancers, thereby inducing the expression of GPX4. This resulted in the inhibition of radiation-induced DNA damage and cell ferroptosis, ultimately promoting the malignant behavior and immune evasion of ESCC cells. CONCLUSION: JMJD2A plays an indispensable role in the malignant behavior and immune evasion of ESCC. It regulates the binding of SMARCA4 to super-enhancers and affects the chromatin's epigenetic landscape, thereby promoting the expression of GPX4 and attenuating iron-mediated cell death caused by radiotherapy. Consequently, it triggers the malignant behavior and immune evasion of ESCC cells.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji , Humanos , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/inmunología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/inmunología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Línea Celular Tumoral , Tolerancia a Radiación/genética , Evasión Inmune , Escape del Tumor , Ferroptosis/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Histonas/metabolismo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismoRESUMEN
The efficacy of radiotherapy, a cornerstone in the treatment of lung adenocarcinoma (LUAD), is profoundly undermined by radiotolerance. This resistance not only poses a significant clinical challenge but also compromises patient survival rates. Therefore, it is important to explore this mechanism for the treatment of LUAD. Multiple public databases were used for single-cell RNA sequencing (scRNA-seq) data. We filtered, normalized and downscaled scRNA-seq data based on the Seurat package to obtain different cell subpopulations. Subsequently, the ssGSEA algorithm was used to assess the enrichment scores of the different cell subpopulations, and thus screen the cell subpopulations that are most relevant to radiotherapy tolerance based on the Pearson method. Finally, pseudotime analysis was performed, and a preliminary exploration of gene mutations in different cell subpopulations was performed. We identified HIST1H1D+ A549 and PIF1+ A549 as the cell subpopulations related to radiotolerance. The expression levels of cell cycle-related genes and pathway enrichment scores of these two cell subpopulations increased gradually with the extension of radiation treatment time. Finally, we found that the proportion of TP53 mutations in patients who had received radiotherapy was significantly higher than that in patients who had not received radiotherapy. We identified two cellular subpopulations associated with radiotherapy tolerance, which may shed light on the molecular mechanisms of radiotherapy tolerance in LUAD and provide new clinical perspectives.