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BACKGROUND: Aberrant glycosylation has been recognized as a hallmark of cancer and N-glycosylation is one of the main types of glycosylation in eukaryotes. Although N-glycoproteomics has made contributions to the discovery of biomarkers in a variety of cancers, less is known about the abnormal glycosylation signatures in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, we reported the proteomics and N-glycoproteomic site-mapping analysis of eight pairs of ESCC tissues and adjacent normal tissues. With zic-HILIC enrichment, TMT-based isobaric labeling, LC-MS/MS analysis, differentially expressed N-glycosylation was quantitatively characterized. Lectin affinity enrichment combined with western blot was used to validate the potential biomarkers in ESCC. RESULTS: A series of differentially expressed glycoproteins (e.g., LAMP2, PLOD2) and enriched signaling pathways (e.g., metabolism-related pathway, ECM-receptor interaction, focal adhesion) were identified. Besides that, seven significantly enriched motifs were found from the identified N-glycosylation sites. Three clusters were identified after conducting the dynamic profiling analysis of glycoprotein change during lymph node metastasis progression. Further validation found that the elevated fucosylation level of ITGB1, CD276 contributed to the occurrence and development of ESCC, which might be the potential biomarkers in ESCC. CONCLUSION: In summary, we characterized the N-glycosylation and N-glycoprotein alterations associated with ESCC. The typical changes in glycoprotein expression and glycosylation occupancy identified in our study will not only be used as ESCC biomarkers but also improve the understanding of ESCC biology.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Antígenos B7 , Biomarcadores , Biomarcadores Tumorais/metabolismo , Cromatografia Líquida , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Glicoproteínas/metabolismo , Humanos , Espectrometria de Massas em TandemRESUMO
Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs.
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Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Estudos de Associação Genética/métodos , Variação Genética , Linhagem Celular , Ciclina D1/genética , Variações do Número de Cópias de DNA , Receptores ErbB/genética , Carcinoma de Células Escamosas do Esôfago , Deleção de Genes , Rearranjo Gênico , Genes p16 , Genoma Humano , Genômica , Humanos , Hibridização in Situ Fluorescente , Receptor ErbB-2/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Notch1/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Translocação GenéticaRESUMO
Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.
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Carcinoma de Células Escamosas/genética , Citidina Desaminase/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença/genética , Genoma Humano/genética , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética , Desaminase APOBEC-1 , Análise de Variância , Sequência de Bases , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , China , Classe I de Fosfatidilinositol 3-Quinases , Variações do Número de Cópias de DNA/genética , Carcinoma de Células Escamosas do Esôfago , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas com Domínio LIM/genética , Ligases , Dados de Sequência Molecular , Receptores Patched , Receptor Patched-1 , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Sais de Tetrazólio , Tiazóis , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVE: To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. METHODS: Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. RESULTS: In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. CONCLUSIONS: Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.
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Proteínas de Ciclo Celular/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Mutação , Fenótipo , Transdução de Sinais , TransfecçãoRESUMO
Tumor-infiltrating B-lineage cells have become predictors of prognosis and immunotherapy responses in various cancers. However, limited knowledge about their infiltration and migration patterns has hindered the understanding of their anti-tumor functions. Here, we examined the immunoglobulin heavy chain (IGH) repertoires in 496 multi-regional tumor, 107 normal tissue, and 48 metastatic lymph node samples obtained from 107 patients with esophageal squamous cell carcinoma (ESCC). Our study revealed higher IgG-type B-lineage cells infiltration in tumors than in healthy tissue, which was associated with improved patient outcomes. Genes such as ACTN1, COL6A5, and pathways like focal adhesion, which shapes the physical structure of tumors, could affect B-lineage cell infiltration. Notably, the IGH sequence was used as an identity-tag to monitor B cell migration, and their infiltration schema within the tumor were depicted based on our multi-regional tumor specimens. This analysis revealed an escalation in B cell clones overlapped between metastatic lymph nodes and tumors. Therefore, the Lymph Node Activation Index was defined, which could predict the outcomes of patients with lymph node metastasis. This research introduces a novel framework for probing B cell infiltration and migration within the tumor microenvironment using large-scale transcriptome data, while simultaneously providing fresh perspectives on B cell immunology within ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Neoplasias Esofágicas/patologia , Prognóstico , Metástase Linfática/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Mitochondria play essential roles in tumorigenesis; however, little is known about the contribution of mitochondrial DNA (mtDNA) to esophageal squamous cell carcinoma (ESCC). Whole-genome sequencing (WGS) is by far the most efficient technology to fully characterize the molecular features of mtDNA; however, due to the high redundancy and heterogeneity of mtDNA in regular WGS data, methods for mtDNA analysis are far from satisfactory. METHODS: Here, we developed a likelihood-based method dMTLV to identify low-heteroplasmic mtDNA variants. In addition, we described fNUMT, which can simultaneously detect non-reference nuclear sequences of mitochondrial origin (non-ref NUMTs) and their derived artifacts. Using these new methods, we explored the contribution of mtDNA to ESCC utilizing the multi-omics data of 663 paired tumor-normal samples. RESULTS: dMTLV outperformed the existing methods in sensitivity without sacrificing specificity. The verification using Nanopore long-read sequencing data showed that fNUMT has superior specificity and more accurate breakpoint identification than the current methods. Leveraging the new method, we identified a significant association between the ESCC overall survival and the ratio of mtDNA copy number of paired tumor-normal samples, which could be potentially explained by the differential expression of genes enriched in pathways related to metabolism, DNA damage repair, and cell cycle checkpoint. Additionally, we observed that the expression of CBWD1 was downregulated by the non-ref NUMTs inserted into its intron region, which might provide precursor conditions for the tumor cells to adapt to a hypoxic environment. Moreover, we identified a strong positive relationship between the number of mtDNA truncating mutations and the contribution of signatures linked to tumorigenesis and treatment response. CONCLUSIONS: Our new frameworks promote the characterization of mtDNA features, which enables the elucidation of the landscapes and roles of mtDNA in ESCC essential for extending the current understanding of ESCC etiology. dMTLV and fNUMT are freely available from https://github.com/sunnyzxh/dMTLV and https://github.com/sunnyzxh/fNUMT , respectively.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , DNA Mitocondrial/genética , DNA Mitocondrial/análise , DNA Mitocondrial/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Funções Verossimilhança , Mitocôndrias/genética , CarcinogêneseRESUMO
Esophageal squamous cell carcinoma (ESCC) is a poor-prognostic cancer type with extensive intra- and inter-patient heterogeneity in both genomic variations and tumor microenvironment (TME). However, the patterns and drivers of spatial genomic and microenvironmental heterogeneity of ESCC remain largely unknown. Here, we generated a spatial multi-omic atlas by whole-exome, transcriptome, and methylome sequencing of 507 tumor samples from 103 patients. We identified a novel tumor suppressor PREX2, accounting for 22% of ESCCs with frequent somatic mutations or hyper-methylation, which promoted migration and invasion of ESCC cells in vitro. Analysis of the TME and quantification of subclonal expansion indicated that ESCCs undergo spatially directed evolution, where subclones mostly originated from the tumor center but had a biased clonal expansion to the upper direction of the esophagus. Interestingly, we found upper regions of ESCCs often underwent stronger immunoediting with increased selective fitness, suggesting more stringent immune selection. In addition, distinct TMEs were associated with variable genomic and clinical outcomes. Among them, hot TME was associated with high immune evasion and subclonal heterogeneity. We also found that immunoediting, instead of CD8+ T cell abundance, acts as an independent prognostic factor of ESCCs. Importantly, we found significant heterogeneity in previously considered potential therapeutic targets, as well as BRCAness characteristics in a subset of patients, emphasizing the importance of focusing on heterogeneity in ESCC targeted therapy. Collectively, these findings provide novel insights into the mechanisms of the spatial evolution of ESCC and inform precision therapeutic strategies.
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Esophageal squamous cell carcinoma (ESCC) is a malignant upper digestive tract cancer, and its pathogenesis and etiology are poorly understood. Because gut microbes commonly impact progression, metastasis, and immunotherapy responses in colorectal cancer (CRC), the roles of the esophageal microbiota in ESCC have gradually drawn attention. As reported previously, Fusobacterium nucleatum (Fn), the notable "culprit" of CRC, can also influence the prognosis of ESCC in clinical studies. However, thus far, the underlying mechanism is unclear. In this study, 73 Chinese ESCC samples were collected. In those clinical samples, the abundance of Fn was found to be higher in tumors than in adjacent normal tissues, and a high abundance of Fn was correlated with shorter survival. Furthermore, using in vitro experiments, we demonstrated that Fn can invade ESCC cells, enhancing their proliferation capacity. The mechanism study revealed that Fn can produce high levels of putrescine after invasion, which disturbs polyamine metabolism and promotes the malignant proliferation of ESCC cells. In conclusion, Fn infection was found in Chinese ESCC tumor tissue samples and may promote ESCC progression by disturbing the polyamine metabolism pathway. IMPORTANCE Nowadays, the complex and varied interactions between microbes and human body are known to be crucial for maintaining the health of the human body. However, knowledge concerning the influence of esophageal microbes on the progression of esophageal squamous cell carcinoma is limited. Here, in our study, we confirmed that F. nucleatum can invade ESCC cells and consequently promote their proliferation, suggesting that esophageal microbes likely influence the progression of ESCC in clinical settings. Because the esophagus connects the oral cavity and stomach, acting as a canal for transporting foods, its special physical location makes it easily exposed to microorganisms. Thus, it is necessary to explore the interaction between esophageal microbes and ESCC.
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Increased rates of ribosome biogenesis have been recognized as hallmarks of many cancers and are associated with poor prognosis. Using a CRISPR synergistic activation mediator (SAM) system library targeting 89 ribosomal proteins (RPs) to screen for the most oncogenic functional RPs in human esophageal squamous cell carcinoma (ESCC), we found that high expression of RPS15 correlates with malignant phenotype and poor prognosis of ESCC. Gain and loss of function models revealed that RPS15 promotes ESCC cell metastasis and proliferation, both in vitro and in vivo. Mechanistic investigations demonstrated that RPS15 interacts with the K homology domain of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which recognizes and directly binds the 3'-UTR of MKK6 and MAPK14 mRNA in an m6A-dependent manner, and promotes translation of core p38 MAPK pathway proteins. By combining targeted drug virtual screening and functional assays, we found that folic acid showed a therapeutic effect on ESCC by targeting RPS15, which was augmented by the combination with cisplatin. Inhibition of RPS15 by folic acid, IGF2BP1 ablation, or SB203580 treatment were able to suppress ESCC metastasis and proliferation via the p38 MAPK signaling pathway. Thus, RPS15 promotes ESCC progression via the p38 MAPK pathway and RPS15 inhibitors may serve as potential anti-ESCC drugs.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Integrated molecular analysis of human cancer has yielded molecular classification for precise management of cancer patients. Here, we analyzed the whole genomic, epigenomic, transcriptomic, and proteomic data of 155 esophageal squamous cell carcinomas (ESCCs). Multi-omics analysis led to the classification of ESCCs into four subtypes: cell cycle pathway activation, NRF2 oncogenic activation, immune suppression (IS), and immune modulation (IM). IS and IM cases were highly immune infiltrated but differed in the type and distribution of immune cells. IM cases showed better response to immune checkpoint blockade therapy than other subtypes in a clinical trial. We further developed a classifier with 28 features to identify the IM subtype, which predicted anti-PD-1 therapy response with 85.7% sensitivity and 90% specificity. These results emphasize the clinical value of unbiased molecular classification based on multi-omics data and have the potential to further improve the understanding and treatment of ESCC.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/genética , Multiômica , ProteômicaRESUMO
ESCC is a highly malignant tumor, and its morbidity and mortality in China account for more than 50% of the world's total rates. As effective treatments are lacking, the 5-year survival rate of patients does not exceed 30%. CAR-T-cell-based immunotherapy has emerged as one of the most promising cancer treatments; however, there are relatively fewer reports regarding its application for ESCC. In this study, we conducted large-sample whole-genome sequencing (WGS) and RNA-seq analysis of patients with ESCC from China to examine the feasibility of EGFR-targeting CAR-T cells in the treatment of ESCC. We found much higher levels of EGFR gene amplification and overexpression in tumors than in the normal tissues, indicating that EGFR could be a promising target of CAR-T-cell-based immunotherapy in ESCC. Therefore, we tested EGFR-targeting CAR-T cells for lytic activity against ESCC cells as a model to establish cellular immunotherapy for ESCC. Five types of CAR-T cells targeting EGFR were constructed, two of which, CAR1-T and CAR2-T, showed a strong cytotoxicity against ESCC in in vitro and in vivo experiments. The results of this study suggest that CAR1-T and CAR2-T have the potential to be used for anti-ESCC immunotherapy in clinics.
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Esophageal squamous cell carcinoma (ESCC) demonstrates high genome instability. Here, we analyze 528 whole genomes to investigate structural variations' mechanisms and biological functions. SVs show multi-mode distributions in size, indicating distinct mutational processes. We develop a tool and define five types of complex rearrangements with templated insertions. We highlight a type of fold-back inversion, which is associated with poor outcomes. Distinct rearrangement signatures demonstrate variable genomic metrics such as replicating time, spatial proximity, and chromatin accessibility. Specifically, fold-back inversion tends to occur near the centrosome; TD-c2 (Tandem duplication-cluster2) is significantly enriched in chromatin-accessibility and early-replication region compared to other signatures. Analyses of TD-c2 signature reveal 9 TD hotspots, of which we identify a hotspot consisting of a super-enhancer of PTHLH. We confirm the oncogenic effect of the PTHLH gene and its interaction with enhancers through functional experiments. Finally, extrachromosomal circular DNAs (ecDNAs) are present in 14% of ESCCs and have strong selective advantages to driver genes.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cromatina/genética , China , DNA CircularRESUMO
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors in China, and its prognosis remains poor. Autophagy is an evolutionarily conserved catabolic process involved in the occurrence and development of ESCC. In this study, we described the expression profile of autophagy-related genes (ARGs) in ESCC and developed a prognostic prediction model for ESCC patients based on the expression pattern of ARGs. We used four ESCC cohorts, GSE53624 (119 samples) set as the discovery cohort, The Cancer Genome Atlas (TCGA) ESCC set (95 samples) as the validation cohort, 155 ESCC cohort, and Oncomine cohort were used to screen and verify differentially expressed ARGs. We identified 34 differentially expressed genes out of 222 ARGs. In the discovery cohort, we divided ESCC patients into three groups that showed significant differences in prognosis. Then, we analyzed the prognosis of 34 differentially expressed ARGs. Three genes [poly (ADP-ribose) polymerase 1 (PARP1), integrin alpha-6 (ITGA6), and Fas-associated death domain (FADD)] were ultimately obtained through random forest feature selection and were constructed as an ARG-related prognostic model. This model was further validated in TCGA ESCC set. Cox regression analysis confirmed that the three-gene signature was an independent prognostic factor for ESCC patients. This signature effectively stratified patients in both discovery and validation cohorts by overall survival (P = 5.162E-8 and P = 0.052, respectively). We also constructed a clinical nomogram with a concordance index of 0.713 to predict the survival possibility of ESCC patients by integrating clinical characteristics and the ARG signature. The calibration curves substantiated fine concordance between nomogram prediction and actual observation. In conclusion, we constructed a new ARG-related prognostic model, which shows the potential to improve the ability of individualized prognosis prediction in ESCC.
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BACKGROUND: CDCA7 is a copy number amplified gene identified not only in esophageal squamous cell carcinoma (ESCC) but also in various cancer types. Its clinical relevance and underlying mechanisms in ESCC have remained unknown. METHODS: Tissue microarray data was used to analyze its expression in 179 ESCC samples. The effects of CDCA7 on proliferation, colony formation, and cell cycle were tested in ESCC cells. Real-time PCR and Western blot were used to detect the expression of its target genes. Correlation of CDCA7 with its target genes in ESCC and various SCC types was analyzed using GSE53625 and TCGA data. The mechanism of CDCA7 was studied by chromatin immunoprecipitation (ChIP), luciferase reporter assays, and rescue assay. RESULTS: The overexpression of CDCA7 promoted proliferation, colony formation, and cell cycle in ESCC cells. CDCA7 affected the expression of cyclins in different cell phases. GSE53625 and TCGA data showed CCNA2 expression was positively correlated with CDCA7. The knockdown of CCNA2 reversed the malignant phenotype induced by CDCA7 overexpression. Furthermore, CDCA7 was found to directly bind to CCNA2, thus promoting its expression. CONCLUSIONS: Our results reveal a novel mechanism of CDCA7 that it may act as an oncogene by directly upregulating CCNA2 to facilitate tumor progression in ESCC.
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Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancer types in China. In recent years, progress has been made in various types of cancer genomics including ESCC. However, the clinical significance of genomic variation of ESCC remains poorly defined. In the present study, genomic sequencing data from 469 ESCC cases were analyzed and potential therapeutic targets in the Druggable Genome Interaction Database (DGIdb) were screened. A series of potential therapeutic target genes and pathways were identified, of which treatment of ESCC with bortezomib (a specific inhibitor targeting proteasome) potently inhibited the proliferation of 5 ESCC cell lines and administration of bortezomib led to significant tumor xenograft regression in SCID mice. It was also identified that kinase insert domain receptor (KDR), which had drug recommendations from all 6 sources integrated by the DGldb and harbored significant amplification in ESCC, might be a downstream target of zinc finger protein 750 (ZNF750). ZNF750 acts as a transcription factor and has been demonstrated to harbor frequently inactivating mutations in ESCC by previous independent studies. In the present study, KDR was upregulated upon ZNF750 knockdown and the rescue of ZNF750 also led to marked restoration of KDR. KDR knockdown in stable ZNF750-knockdown KYSE150 and KYSE140 ESCC cells significantly attenuated the promotion of cell growth, colony formation, invasion and migration induced by ZNF750 knockdown. Further experiments found that apatinib treatment, a potent inhibitor of KDR, resulted in profound inhibition of cell proliferation and invasion. Collectively, the present study provided insight for genomic alterations as potential therapeutic targets in ESCC and supported the possibility of a therapeutic strategy targeting the proteasome in ESCC. The present results also suggested that targeting KDR may be an effective way to treat ESCC, not only in KDR variant cases, but also in individuals with ZNF750 mutations and deletions.
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Biomarcadores Tumorais , Carcinoma de Células Escamosas do Esôfago/metabolismo , Inibidores de Proteassoma/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Bases de Dados Genéticas , Modelos Animais de Doenças , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Terapia de Alvo Molecular , Mutação , Polimorfismo de Nucleotídeo Único , Inibidores de Proteassoma/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As a key enzyme in de novo pyrimidine biosynthesis, the expression level of dihydroorotate dehydrogenase (DHODH) has been reported to be elevated in various types of malignant tumors and its tumor-promoting effect was considered to relate to its pyrimidine synthesis function. Here, we revealed one intriguing potential mechanism that DHODH modulated ß-catenin signaling in esophageal squamous cell carcinoma (ESCC). We demonstrated that DHODH directly bound to the NH2 terminal of ß-catenin, thereby, interrupting the interaction of GSK3ß with ß-catenin and leading to the abrogation of ß-catenin degradation and accumulation of ß-catenin in the nucleus, which in turn, resulted in the activation of ß-catenin downstream genes, including CCND1, E2F3, Nanog, and OCT4. We further demonstrated that the regulation of ß-catenin by DHODH was independent of DHODH catalyzing activity. Univariate and multivariate analyses suggested that DHODH expression might be an independent prognostic factor for ESCC patients. Collectively, our study highlights the pivotal role of DHODH mediated ß-catenin signaling and indicates that DHODH may act as a multi-functional switcher from catalyzing pyrimidine metabolism to regulating tumor-related signaling pathways in ESCC.
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Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , beta Catenina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células/fisiologia , Di-Hidro-Orotato Desidrogenase , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/enzimologia , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Transdução de SinaisRESUMO
Background: TSTA3 gene encodes an enzyme responsible for synthesis of GDP-L-fucose as the only donor in fucosylation. This study was designed to explore clinical value, function and underlying mechanism of TSTA3 in the development of esophageal squamous cell carcinoma (ESCC). Methods: Whole genomic sequencing data from 663 ESCC patients and RNA sequencing data from 155 ESCC patients were used to analyze the copy number variation and mRNA expression of TSTA3 respectively. Immunohistochemistry based or not based on the tissue microarrays was used to detect its protein expression. Transwell assay and in vivo metastasis assay were used to study the effect of TSTA3 on invasion and metastasis of ESCC. Immunofluorescence was used to analyze fucosylation level. N-glycoproteomics and proteomics analysis, Lens Culinaris Agglutinin (LCA) and Ulex Europaeus Agglutinin I (UEA-I) affinity chromatography, immunoprecipitation, glycosyltransferase activity kit and rescue assay were used to explore the mechanism of TSTA3. Results: TSTA3 was frequently amplified and overexpressed in ESCC. TSTA3 amplification and protein overexpression were significantly associated with malignant progression and poor prognosis of ESCC patients. TSTA3 knockdown significantly suppressed ESCC cells invasion and tumor dissemination by decreasing fucosylation level. Conversely, exogenous overexpression of TSTA3 led to increased invasion and tumor metastasis in vitro and in vivo by increasing fucosylation level. Moreover, core fucosylated LAMP2 and terminal fucosylated ERBB2 might be mediators of TSTA3-induced pro-invasion in ESCC and had a synergistic effect on the process. Peracetylated 2-F-Fuc, a fucosyltransferase activity inhibitor, reduced TSTA3 expression and fucosylation modification of LAMP2 and ERBB2, thereby inhibiting ESCC cell invasion. Conclusion: Our results indicate that TSTA3 may be a driver of ESCC metastasis through regulating fucosylation of LAMP2 and ERBB2. Fucosylation inhibitor may have prospect to suppress ESCC metastasis by blocking aberrant fucosylation.
Assuntos
Carboidratos Epimerases/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Cetona Oxirredutases/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Receptor ErbB-2/metabolismo , Idoso , Animais , Carboidratos Epimerases/genética , Linhagem Celular Tumoral , Proliferação de Células , Variações do Número de Cópias de DNA , Progressão da Doença , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/mortalidade , Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Humanos , Cetona Oxirredutases/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Cancer genomic studies have identified Zinc Finger Protein 750 (ZNF750) was a novel significantly mutated gene in esophageal squamous cell carcinoma (ESCC). This study was designed to determine the clinical value and molecular mechanisms of ZNF750 in the development of ESCC. Methods: Genomic data from 4 reported ESCC cohorts were used to analyze the mutation profile of ZNF750. Tissue microarrays were used to detect its expression in 308 ESCC samples. Furtherly, the effects of ZNF750 on proliferation, colony formation, migration and invasion were tested in ESCC cells. PCR-array, chromatin immunoprecipitation (ChIP), luciferase reporter assays, and rescue assay were used to explore the mechanism of ZNF750. Correlation of ZNF750 with its target genes was analyzed in TCGA data from various SCC types. Results: ZNF750 was frequently mutated in ESCC and the most common type was nonsense mutation. Its nucleus/cytoplasm ratio in ESCC was significantly lower than that in paired non-tumor tissues; it was an independent and potential predictor for survival in ESCC patients. Furtherly, ZNF750 knockdown significantly promoted proliferation, colony formation, migration and invasion in ESCC cells. PCR-array showed epithelial-to-mesenchymal transition (EMT) was the main biologic process affected by ZNF750. Moreover, ZNF750 directly bound to the promoter region of SNAI1 and depressed its activity. Decreased ZNF750 up-regulated SNAI1 expression and promoted EMT phenotype. SNAI1 knockdown partially reversed the malignant phenotype induced by ZNF750 knockdown. Further TCGA data analyses showed ZNF750 expression was positively correlated with E-cadherin and negatively correlated with SNAI1, N-cadherin and Vimentin in ESCC and other SCC samples. Conclusion: Our results suggest that ZNF750 may act as a tumor suppressor by directly repressing SNAI1 and inhibiting EMT process in ESCC and other types of SCC.