Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.

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.

IGH repertoire analysis at scale: deciphering the complexity of B cell infiltration and migration in esophageal squamous cell carcinoma.

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.

RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m6A modification.

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.

Fusobacterium nucleatum Infection Induces Malignant Proliferation of Esophageal Squamous Cell Carcinoma Cell by Putrescine Production.

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.

Integrated multi-omics profiling yields a clinically relevant molecular classification for esophageal squamous cell carcinoma.

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.

Role of Epidermal Growth Factor Receptor-Specific CAR-T Cells in the Suppression of Esophageal Squamous Cell Carcinoma.

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.

Characterization of somatic structural variations in 528 Chinese individuals with Esophageal squamous cell carcinoma.

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.

An N-glycoproteomic site-mapping analysis reveals glycoprotein alterations in esophageal squamous cell carcinoma.

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.

CDCA7 Facilitates Tumor Progression by Directly Regulating CCNA2 Expression in Esophageal Squamous Cell Carcinoma.

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.

Autophagy-Related Three-Gene Prognostic Signature for Predicting Survival in Esophageal Squamous Cell Carcinoma.

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.

Author Correction: Multi-region sequencing unveils novel actionable targets and spatial heterogeneity in esophageal squamous cell carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Elevated DHODH expression promotes cell proliferation via stabilizing ß-catenin in esophageal squamous cell carcinoma.

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.

TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2.

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.

Potential Role of Targeting KDR and Proteasome Inhibitors in the Therapy of Esophageal Squamous Cell Carcinoma.

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.

Whole-genome sequencing of 508 patients identifies key molecular features associated with poor prognosis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a poor-prognosis cancer type with limited understanding of its molecular etiology. Using 508 ESCC genomes, we identified five novel significantly mutated genes and uncovered mutational signature clusters associated with metastasis and patients' outcomes. Several functional assays implicated that NFE2L2 may act as a tumor suppressor in ESCC and that mutations in NFE2L2 probably impaired its tumor-suppressive function, or even conferred oncogenic activities. Additionally, we found that the NFE2L2 mutations were significantly associated with worse prognosis of ESCC. We also identified potential noncoding driver mutations including hotspot mutations in the promoter region of SLC35E2 that were correlated with worse survival. Approximately 5.9% and 15.2% of patients had high tumor mutation burden or actionable mutations, respectively, and may benefit from immunotherapy or targeted therapies. We found clinically relevant coding and noncoding genomic alterations and revealed three major subtypes that robustly predicted patients' outcomes. Collectively, we report the largest dataset of genomic profiling of ESCC useful for developing ESCC-specific biomarkers for diagnosis and treatment.

Decreased ZNF750 promotes angiogenesis in a paracrine manner via activating DANCR/miR-4707-3p/FOXC2 axis in esophageal squamous cell carcinoma.

ZNF750 is one novel significantly mutated gene identified in esophageal squamous cell carcinoma (ESCC) using next-generation sequencing. However, its clinically relevant and potential mechanisms have remained elusive. Using genomic sequencing of 612 ESCC patients, we analyzed the associations of ZNF750 mutations with clinicopathologic features and its prognostic value. We further investigated the function and underlying mechanism of ZNF750 in angiogenesis. The results showed ZNF750 mutations/deletions are significantly associated with malignant progression and poor prognosis of ESCC patients. Decreased ZNF750 in ESCC cells induces enhanced angiogenesis of human umbilical vein endothelial cells (HUVECs) and human arterial endothelial cells (HAECs), and the effect may be indirectly mediated by FOXC2. RNA-seq and ChIP shows lncRNA DANCR is a direct downstream target of ZNF750. Furtherly, knockdown ZNF750 evokes DANCR expression, which prevents miR-4707-3p to interact with FOXC2 as a microRNA sponge in a ceRNA manner, leading to enhanced FOXC2 signaling and angiogenesis. In contrast, ZNF750 expression reverses the effect. Our study reveals a novel mechanism of ZNF750, highlights a significance of ZNF750 as a metastatic and prognostic biomarker, and offers potential therapeutic targets for ESCC patients harboring ZNF750 mutations.