The AEG-1-USP10-PARP1 axis confers radioresistance in esophageal squamous cell carcinoma via facilitating homologous recombination-dependent DNA damage repair.

Radiotherapy is the standard adjuvant treatment for esophageal squamous cell carcinoma (ESCC), yet radioresistance remains a major obstacle leading to treatment failure and unfavorable prognosis. Previous reports have demonstrated the involvement of astrocyte elevated gene-1 (AEG-1) in tumorigenesis and progression of multiple malignancies. Nevertheless, the precise role of AEG-1 in the radioresistance of ESCC remains elusive. Here, we unveiled a strong correlation between aberrant AEG-1 gene overexpression and malignant progression as well as adverse prognosis in ESCC patients. Moreover, both in vitro and in vivo investigations revealed that AEG-1 significantly alleviated irradiation-induced DNA damage and enhanced radiation resistance in ESCC cells. Mechanistically, AEG-1 recruited the deubiquitinase USP10 to remove the K48-linked polyubiquitin chains at the Lys425 of PARP1, thus preventing its proteasomal degradation. This orchestrated process facilitated homologous recombination-mediated DNA double-strand breaks (DSBs) repair, culminating in mitigated DNA damage and acquired radioresistance in ESCC cells. Notably, PARP1 overexpression reversed the radiosensitizing effect caused by AEG-1 deficiency. Collectively, these findings shed new light on the mechanism of ESCC radioresistance, providing potential therapeutic targets to enhance the efficacy of radiotherapy in ESCC.

Radiation induces IRAK1 expression to promote radioresistance by suppressing autophagic cell death via decreasing the ubiquitination of PRDX1 in glioma cells.

Radiotherapy is the standard adjuvant treatment for glioma patients; however, the efficacy is limited by radioresistance. The function of Interleukin-1 receptor associated kinase 1 (IRAK1) in tumorigenesis and radioresistance remains to be elucidated. IRAK1 expression and its correlation with prognosis were analyzed in glioma tissues. We found that glioma patients with overexpressed IRAK1 show a poor prognosis. Notably, ionizing radiation (IR) remarkably induces IRAK1 expression, which was decreased by STING antagonist H-151 treatment. JASPAR prediction, ChIP assays, and dual luciferase reporter assays indicated that transcription factor FOXA2, suppressed by STING inhibition, directly binds to the IRAK1 promoter region and activates its transcription. IRAK1 knockdown inhibits malignancy and enhances the radiosensitivity of glioma in vitro and in vivo. To explore the potential IRAK1 interacting targets mediating the radioresistance of glioma cells, IP/Co-IP, LC-MS/MS, GST pull-down, and ubiquitination analyses were conducted. Mechanistically, IRAK1 bound to PRDX1, a major member of antioxidant enzymes, and further prevents ubiquitination and degradation of PRDX1 mediated by E3 ubiquitin ligase HECTD3; Both the DOC and HECT domains of HECTD3 directly interacted with PRDX1 protein. Overexpression of PRDX1 reverses the radiotherapy sensitization effect of IRAK1 depletion by diminishing autophagic cell death. These results suggest the IRAK1-PRDX1 axis provides a potential therapeutic target for glioma patients.

Comprehensive Pan-Cancer Analysis of IRAK Family Genes Identifies IRAK1 as a Novel Oncogene in Low-Grade Glioma.

BACKGROUND: The interleukin-1 receptor-associated kinases (IRAK) family genes, indispensable mediators of interleukin-1 receptor (IL1R) and Toll-like receptor (TLR)-inflammatory signaling, may be involved in the biological function of human cancers due to the crucial roles of inflammation in tumor development. Though a little research has demonstrated the function of individual IRAK family members in specific tumors, comprehensive analysis is still lacking in pan-cancer. METHODS: We analyzed the mRNA expression landscape, mutation, and prognosis value of IRAK genes based on The Cancer Genome Atlas (TCGA), cBioPortal, GlioVis, and Rembrandt databases. The correlation between the expression of IRAK genes and tumor microenvironment (TME), Stemness score, and immune subtypes was explored. Western blot, cell proliferation, apoptosis, migration assays, and xenograft models were utilized in this study. RESULTS: We found that the expression of IRAK genes extensively changed and was related to patient survival in pan-cancer. Besides, IRAK family genes were correlated with TME, Stemness score, and immune subtypes in most cases. Given that high expression of all IRAK family members predicted poor prognosis in low-grade glioma (LGG), the oncogenic function of the highest expressed IRAK1 in LGG has been confirmed in vitro and in vivo. IRAK1 was uncovered to inhibit cell apoptosis and augment malignancy of LGG in vitro and in vivo. CONCLUSION: These findings revealed the potential targets of IRAK family genes in pan-cancer and provided insights for further investigation of IRAK1 as a novel oncogenic gene in LGG.

Radiation induces NORAD expression to promote ESCC radiotherapy resistance via EEPD1/ATR/Chk1 signalling and by inhibiting pri-miR-199a1 processing and the exosomal transfer of miR-199a-5p.

BACKGROUND: Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and subsequent local recurrence, threatening a large proportion of patients with ESCC. To date, lncRNAs have been reported to be involved in diverse biological processes, including radioresistance. METHODS: FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a1 and miR-199a-5p. Electron microscopy and nanoparticle tracking analysis (NTA) were conducted to observe and identify exosomes. High-throughput microRNAs sequencing and TMT mass spectrometry were performed to identify the functional miRNA and proteins. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. ChIP, RIP-qPCR, co-IP and dual-luciferase reporter assays were conducted to explore the interaction of related RNAs and proteins. RESULTS: We show here that DNA damage activates the noncoding RNA NORAD, which is critical for ESCC radioresistance. NORAD was expressed at high levels in radioresistant ESCC cells. Radiation treatment promotes NORAD expression by enhancing H3K4me2 enrichment in its sequence. NORAD knockdown cells exhibit significant hypersensitivity to radiation in vivo and in vitro. NORAD is required to initiate the repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon radiation treatment. Mechanistically, NORAD inhibits miR-199a-5p expression by competitively binding PUM1 from pri-miR-199a1, inhibiting the processing of pri-miR-199a1. Mature miR-199a-5p in NORAD knockdown cells is packaged into exosomes; miR-199a-5p restores the radiosensitivity of radioresistant cells by targeting EEPD1 and then inhibiting the ATR/Chk1 signalling pathway. Simultaneously, NORAD knockdown inhibits the ubiquitination of PD-L1, leading to a better response to radiation and anti-PD-1 treatment in a mouse model. CONCLUSIONS: Based on the findings of this study, lncRNA-NORAD represents a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.

AEG-1 silencing attenuates M2-polarization of glioma-associated microglia/macrophages and sensitizes glioma cells to temozolomide.

Glioma is the most frequent primary malignancy in the brain; temozolomide (TMZ) is the first-line chemotherapeutic agent used to combat this tumor. We showed here that astrocyte elevated gene-1 (AEG-1) was overexpressed in glioma tissues and associated with a worse subtype and a poor prognosis. CCK-8 proliferation assays and clone formation experiments presented that AEG-1 knockdown sensitizes glioma cells to TMZ. The γH2AX foci formation assays indicated that AEG-1 silencing promotes TMZ-induced DNA damage in glioma cells. Glioma-associated microglia/macrophages (GAMs), the largest subpopulation infiltrating glioma, play important roles in the tumor microenvironment. Bioinformatics analyses and functional studies demonstrated that AEG-1 silencing decreased M2-polarization of HMC3 microglia and the secretion of tumor supportive cytokines IL-6 and TGF-ß1. The expression of AEG-1 was positively associated with M2 markers in glioma tissues varified by IHC staining. Based on the results of Affymetrix microarray and GSEA analyses, Western blot and Co-Immunoprecipitation assays were conducted to show that AEG-1 activates Wnt/ß-catenin signaling by directly interacting with GSK-3ß. The co-localization of AEG-1 and GSK-3ß in the cytoplasm of glioma cells was detected through immunofluorescence staining. This study raises the possibility that targeting AEG-1 might improve the efficiency of chemotherapy and reduce immunosuppressive M2 GAMs in glioma.

AEG-1 Knockdown Sensitizes Glioma Cells to Radiation Through Impairing Homologous Recombination Via Targeting RFC5.

Radiotherapy is the most important adjuvant treatment for glioma; however, radioresistance is the major cause for inevitable recurrence and poor survival of glioma patients. Thus, this study aims to investigate the effect of astrocyte elevated gene-1 (AEG-1) on the radiosensitivity of glioma cells. Immunohistochemistry assay found that AEG-1 was generally overexpressed in glioma tissues and was correlated with poor clinicopathological features of glioma patients. AEG-1 knockdown inhibited proliferation of glioma cells. And γ-H2AX foci assay, colony formation assay, and flow cytometry analysis demonstrated that AEG-1 depletion enhanced radiosensitivity and promoted apoptosis as well as cell cycle arrest in G2 phase of glioma cells treated by ionizing radiation. Moreover, replication factor C5 (RFC5) was screened as the target of AEG-1 by using Affymetrix human gene expression array, and RFC5 expression was downregulated in AEG-1 knockdown glioma cells. Mechanistically, AEG-1 knockdown impaired homologous recombination repair activity induced by radiation through inhibiting RFC5 expression. Furthermore, the Kaplan-Meier analysis and multivariate Cox regression analysis indicated that high levels of AEG-1 and RFC5 were related to poor prognosis of glioma patients treated with radiotherapy. Taken together, our findings indicate that AEG-1 may serve as a reliable radiosensitizing target for glioma radiotherapy.

SOCS6 promotes radiosensitivity and decreases cancer cell stemness in esophageal squamous cell carcinoma by regulating c-Kit ubiquitylation.

BACKGROUND: Radiotherapy is a major treatment for esophageal squamous cell carcinoma (ESCC). However, HPV infection related radioresistance caused poor prognosis of ESCC. The function of SOCS6, which has been shown to be a tumor suppressor in several cancers, has not been fully investigated up till now. In this manuscript, we aim to further investigate the role of SOCS6 in regulating ESCC radioresistance. METHODS: Fifty-seven ESCC patients were enrolled for survival analysis. SOCS6 was stably overexpressed in HPV+ ESCC and ESCC cells, and cells were treated with radiation and then subjected to colony formation assays. Expression of DNA damage repair regulating proteins were examined by Western blotting. Cell growth, cell migration and cisplatin sensitivity were then analyzed. Sphere formation assays and flow cytometry were used to investigate changes in cancer stem cell (CSC) properties. Immunofluorescent staining and confocal microscopy were used to locate SOCS6 and c-Kit. Ubiquitylation level of c-Kit were analyzed after immunoprecipitation. Then, coimmunoprecipitation (CoIP) of SOCS6 and c-Kit were performed. In vivo, xenograft animal models were treated with radiation to examine the radiosensitivity. RESULTS: SOCS6 is correlated with better prognosis in ESCC patients. Radioresistance is impaired by SOCS6 upregulation, which inhibited cell growth, migration and increased sensitivity to cisplatin. SOCS6 significantly decreased the population of CSCs expressing the surface biomarker CD271 or CD24low/CD44high and their ability of sphere formation. SOCS6 and c-Kit were collocated in the cytoplasm. Blotting of ubiquitin and CoIP experiments indicated that the mechanism was related to ubiquitylation and degradation of the receptor c-Kit. Xenograft tumor mouse model showed that SOCS6 inhibited tumor growth and promoted radiosensitivity in vivo. CONCLUSIONS: Our findings suggest that SOCS6 can promote the radiosensitivity of HPV+ ESCC and ESCC cells and reduce their stemness via ubiquitylation and degradation of c-Kit. Thus, SOCS6 is a potential target for overcoming radioresistance of ESCC.

pH-Sensitive and Long-Circulation Nanoparticles for Near-Infrared Fluorescence Imaging-Monitored and Chemo-Photothermal Synergistic Treatment Against Gastric Cancer.

Gastrectomy is the primary therapeutic option for gastric cancer. Postoperative treatment also plays a crucial role. The strategy to improve the postoperative prognosis of gastric cancer requires a combined system that includes a more efficient synergistic treatment and real-time monitoring after surgery. In this study, photothermal-chemotherapy combined nanoparticles (PCC NPs) were prepared via π-π stacking to perform chemo-photothermal synergistic therapy and continuous imaging of gastric cancer. PCC NPs had a spherical morphology and good monodispersity under aqueous conditions. The hydrodynamic diameter of PCC NPs was 59.4 ± 3.6 nm. PCC NPs possessed strong encapsulation ability, and the maximum drug loading rate was approximately 37%. The NPs exhibited extraordinary stability and pH-response release profiles. The NPs were rapidly heated under irradiation. The maximum temperature was close to 58°C. PCC NPs showed good biocompatibility both in vitro and in vivo. Moreover, the NPs could effectively be used for in vivo continuous monitoring of gastric cancer. After one injection, the fluorescent signal remained in tumor tissues for nearly a week. The inhibitory effect of PCC NPs was evaluated in a gastric cancer cell line and xenograft mouse model. Both in vitro and in vivo evaluations demonstrated that PCC NPs could be used for chemo-photothermal synergistic therapy. The suppression effect of PCC NPs was significantly better than that of single chemotherapy or photothermal treatment. This study lays the foundation for the development of novel postoperative treatments for gastric cancer.

Genome-wide analysis of lncRNAs, miRNAs, and mRNAs forming a prognostic scoring system in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal carcinoma. Protein coding genes and non-coding RNAs can be powerful prognostic factors in multiple cancers, including ESCC. However, there is currently no model that integrates multiple types of RNA expression signatures to predict clinical outcomes. METHODS: The sequencing data (RNA-sequencing and miRNA-sequencing) and clinical data of ESCC patients were obtained from The Cancer Genome Atlas (TCGA) database, and Differential gene expression analysis, Cox regression analysis and Spearman correlation analysis were used to construct prognosis-related lncRNA-mRNA co-expression network and scoring system with multiple types of RNA. The potential molecular mechanisms of prognostic mRNAs were explored by functional enrichment analysis. RESULTS: A total of 62 prognostic lncRNAs, eight prognostic miRNAs and 66 prognostic mRNAs were identified in ESCC (P-value < 0.05) and a prognosis-related lncRNA-mRNA co-expression network was created. Five prognosis-related hub RNAs (CDCA2, MTBP, CENPE, PBK, AL033384.1) were identified. Biological process analysis revealed that mRNAs in prognosis-related co-expression RNA network were mainly enriched in cell cycle, mitotic cell cycle and nuclear division. Additionally, we constructed a prognostic scoring system for ESCC using ten signature RNAs (MLIP, TNFSF10, SIK2, LINC01068, LINC00601, TTTY14, AC084262.1, LINC01415, miR-5699-3p, miR-552-5p). Using this system, patients in the low-risk group had better long-term survival than those in the high-risk group (log-rank, P-value < 0.0001). The area under the ROC curve (AUCs) revealed that the accuracy of the prediction model was higher than the accuracy of single type of RNA prediction model. CONCLUSION: In brief, we constructed a prognostic scoring system based on multiple types of RNA for ESCC that showed high predicting prognosis performance, and deeply understood the regulatory mechanism of prognosis-related lncRNA-mRNA co-expression network.

Impact of the Time Proportion of Respiratory Phases on Dosimetry in SBRT of Lung Tumor Near the Chest Wall or Diaphragm.

PURPOSE: Large tumor motion often leads to larger dosimetric variation, especially in lung tumors located in lower lobe and adhered to chest wall or diaphragm. The purpose of this work is to discuss the impact of the time proportion of each respiratory phase on dosimetry in stereotactic body radiation therapy with lung cancer tumor close to chest wall or diaphragm. METHODS: Participants include 14 patients with lung cancer located in the lower lobe. Each patient received treatment planning 3-dimensional computed tomography and an additional 4-dimensional computed tomography simulation under free-breathing condition. The percentage of time intervals for each respiratory phase in a whole respiratory cycle was calculated from respiratory motion curves recorded during 4-dimensional computed tomography scanning. Treatment plan was made upon treatment planning 3-dimensional computed tomography and then transformed onto each image of 4-dimensional computed tomography. The transformed doses on each image of 4-dimensional computed tomography were accumulated with equal weight or with weight of time proportion for each respiratory phase. RESULTS: Compared to treatment planning 3-dimensional computed tomography dose, the mean dose of tumor, affected lung, contralateral lung, bilateral lungs, and V20 of affected lung decreased by 2.7%, 4.5%, 1.5%, 1.2%, and 4.1%, respectively, after equal-weighted accumulation, while mean dose of heart increased by 3.6% (P < .05). Accumulated dose with weight of actual time proportion decreased in the mean dose of tumor, affected lung, contralateral lung, bilateral lungs, and V20 of affected lung by 2.37%, 5.19%, 3.61%, 3.46%, and 5.08%, respectively compared to treatment planning 3-dimensional computed tomography dose, but mean dose of heart increased by 5.12% (P < .05). CONCLUSIONS: Doses received by tumor, lungs, and heart changed more significantly after time-weighted 4-dimensional accumulation than equal-weight 4-dimensional accumulation. Utilizing 4-dimensional computed tomography and deformable image registration to introduce time proportions of each respiratory phase to dose distribution evaluation is of significance for accuracy in lung cancer during stereotactic body radiation therapy treatment.

Nomograms to predict survival rates for esophageal cancer patients with malignant behaviors based on ICD-0-3.

AIM: We aimed to investigate the effect of current treatment based on stage and histology type, which were important factors for treating esophageal cancer. METHODS: Log-rank test, COX and nomograms were used for survival analysis. DCA, C-index and calibration curves were used for validation. RESULTS: A total of 3224 patients were recruited. As for cT2-T4aM0 patients, chemotherapy and radiation prolonged overall survival (OS) for esophageal squamous cell carcinoma (ESCC) and chemotherapy improved OS for esophageal adenocarcinoma (EAC). Meanwhile, neoadjuvant radiotherapy had longer OS than adjuvant radiotherapy for ESCC. As for T4b patients, radiation and chemotherapy correlated with better OS for ESCC and chemotherapy prolonged OS for EAC. CONCLUSION: Neoadjuvant radiotherapy might be optimal for cT2-T4aM0 ESCC. Radiation was recommended for T4b ESCC while chemotherapy was recommended for T4b EAC.

LINC00657 played oncogenic roles in esophageal squamous cell carcinoma by targeting miR-615-3p and JunB.

BACKGROUND: The prognosis of esophageal squamous cell carcinoma (ESCC) is relatively poor due to the absence of efficient treatment. In this manuscript, we have investigated the specific roles and molecular mechanisms of LINC00657 to order to identify novel therapeutic targets for ESCC. METHOD: The LINC00657 expression in ESCC tissues and cell lines were evaluated by quantitative real-time PCR. The expression of LINC00657 in ESCC cells was regulated by lentivirus transfection. Online bioinformatics analysis tools were used to predict the potential targets of LINC00657 and miR-615-3p. TCGA database was used to analyze the prognosis of ESCC patients. Transwell, wound healing assay and MTT were performed to investigate the ESCC cells' biological functions. JunB expression was evaluated by Western blot. RESULT: LINC00657 was moderately increased in ESCC both in vivo and in vitro and up regulated by irradiation. LINC00657 knockdown could inhibit the migration and proliferation of ESCC cells. And downregulation of LINC00657 significantly enhanced the radio-sensitivity. Moreover, LINC00657 could act as a ceRNA to increase the expression of JunB by binding to miR-615-3p. Meanwhile, overexpression of miR-615-3p resulted in anti-tumor effects and led to the down-regulation of JunB. Survival analysis from TCGA indicated that ESCC patients with higher JunB expression had significant poorer prognosis. CONCLUSION: LINC00657 might be involved in regulating ESCC's response to radiation; and it functioned as an oncogene in ESCC by targeting miR-615-3p and JunB, providing novel potential therapeutic targets.