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Background: Emerging evidence indicates that myristoylated alanine-rich C kinase substrate like 1 (MARCKSL1) is involved in the progression of esophageal squamous cell carcinoma (ESCC). However, the underpinning mechanism is unclear. Here, we investigated the mechanisms involving MARCKSL1 in ESCC progression., Methods: CCK8, Transwell and wound-healing assays were employed to test the effect of MARCKSL1 on proliferation, invasion and migration in vitro. Next, transcriptome profiling was conducted through RNA sequencing to reveal the underlying mechanism of MARCKSL1 in ESCC progression, which was subsequently verified by western blot and qPCR analysis. Moreover, immunofluorescence and gelatin degradation assays were performed to reveal the ability of MARCKSL1 to mediate invadopodia formation and extracellular matrix (ECM) degradation. Finally, the correlation between MARCKSL1 and the clinicopathological features of ESCC patients was assessed based on TCGA database analysis and immunohistochemistry staining of tissue microarrays., Results: Knockdown of MARCKSL1 markedly attenuated the cell motility capacity of ESCC cells in vitro, while MARCKSL1 overexpression had the opposite effect. Transcriptomic analysis showed that MARCKSL1 mediated the mobility and migration of ESCC cells. In addition, overexpression of MARCKSL1 increased the colocalization of F-actin and cortactin at the frontier edge of migrating cells and ECM degradation. Furthermore, in ESCC patients, the mRNA level of MARCKSL1 in esophageal carcinomas (n = 182) was found to be notably higher than that in adjacent esophageal epithelia (n = 286) and the expression levels of MARCKSL1 in the tumor tissues (n = 811) were significantly increased compared to those in noncancerous esophageal tissues (n = 442) with a large sample size. Higher expression of MARCKSL1 was positively correlated with lymph node metastasis and associated with worse survival rates of patients with ESCC., Conclusion: MARCKSL1 promotes cell migration and invasion by interacting with F-actin and cortactin to regulate invadopodia formation and ECM degeneration. High MARCKSL1 expression is positively correlated with poor prognosis in ESCC patients with lymph node metastasis., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Esophageal squamous cell carcinoma (ESCC) is one of the fatal malignancies worldwide. It has an increased propensity to metastasize via lymphogenous routes in an early stage. The prognosis of patients with lymph node metastases (LNM) is often worse than that of patients without metastases. Although several factors have been found to influence metastasis, the mechanisms of preference for specific metastatic routes remain poorly understood. Herein, we provide evidence that the intrinsic hypersensitivity of tumor cells to ferroptosis may proactively drive lymphatic metastasis. Serum autoantibodies associated with LNM of early ESCC were screened using a whole-proteome protein array containing 19 394 human recombinant proteins, and an anti-BACH1 autoantibody was first identified. Pan-cancer analysis of ferroptosis-related genes with preferential lymphatic metastasis and preferential hematogenous metastasis based on The Cancer Genome Atlas data was performed. Only BACH1 showed significant overexpression in tumors with preferential lymphatic metastasis, whereas it was downregulated in most tumors with preferential nonlymphatic metastasis. In addition, it was found that the serum levels of autoantibodies against BACH1 were elevated in early-stage patients with LNM. Interestingly, BACH1 overexpression and ferroptosis induction promoted LNM but inhibited hematogenous metastasis in mouse models. Transcriptomic and lipidomic analyses found that BACH1 repressed SCD1-mediated biosynthesis of monounsaturated fatty acids, especially oleic acid (OA). OA significantly attenuated the ferroptotic phenotypes and reversed the metastatic properties of BACH1-overexpressing cells. OA addition significantly rescued the ferroptotic phenotypes and reversed the metastatic properties of BACH1-overexpressing cells. Importantly, the concentration gradient of OA between primary lesions and the lymph resulted in the chemoattraction of tumor cells to promote invasion, thus facilitating lymphatic metastasis. BACH1-induced ferroptosis drives lymphatic metastasis via the BACH1-SCD1-OA axis. More importantly, this study confirms that ferroptosis is a double-edged sword in tumorigenesis and tumor progression. The clinical application of ferroptosis-associated agents requires a great caution., (© 2023. The Author(s).)

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment., (© 2022. The Author(s).)

Metastasis to regional lymph nodes or distal organs predicts the progression of the disease and poor prognosis in esophageal squamous cell carcinoma (ESCC). Previous studies demonstrated that BTB and CNC homology 1 (BACH1) participates in various types of tumor metastasis. However, the function of BACH1 in ESCC was rarely reported. The present study demonstrated that BACH1 protein was overexpressed in ESCC tissues compared with paired esophageal epithelial tissues according to immunohistochemical staining (IHC). Higher levels of BACH1 mRNA were associated with decreased overall survival (OS) and shorter disease-free survival (DFS) of ESCC patients based on an analysis of The Cancer Genome Atlas (TCGA) datasets. BACH1 significantly enhanced the migration and invasion of ESCC in vitro. Mechanistically, BACH1 promoted the epithelial-mesenchymal transition (EMT) by directly activating the transcription of CDH2, SNAI2, and VIM, as determined by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR). BACH1 overexpression significantly enhanced CDH2 promoter activity according to the results of a luciferase assay. The results of subsequent experiments indicated that BACH1 enhanced the growth of tumor xenografts. The density of CD31 + blood vessels and the expression of vascular endothelial growth factor C (VEGFC) in tumor xenografts were significantly associated with BACH1 levels according to the results of IHC and immunofluorescence (IF) analyses performed in vivo. Moreover, ChIP-qPCR analysis demonstrated that the transcriptional activity of VEGFC was also upregulated by BACH1. Thus, BACH1 contributes to ESCC metastasis and tumorigenesis by partially facilitating the EMT and angiogenesis, and BACH1 may be a promising therapeutic target or molecular marker in ESCC., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal cancers in the world. Dysregulation of purine-rich element binding protein alpha (PURα), which contributes to the initiation of PURΑ syndrome, is reportedly involved in the progression of multiple cancers, but its function and underlying mechanisms in ESCC progression remain unclear. Here, we first demonstrated that PURα promoted cell growth, migration and invasion in ESCC both in vitro and in vivo. An immunohistochemistry assay was then performed on 225 ESCC tissues, showing that high PURα expression was positively associated with lymph node metastasis and the AJCC stage, and the ESCC patients with positive PURα expression had worse survival. In addition, RNA sequencing implied that PURα induced epithelial-mesenchymal transition (EMT) in ESCC, which was further confirmed by qPCR, Western blotting and immunofluorescence analyses. Mechanistically, PURα enhanced the transcription of Snail2 by binding to its promoter region. Knockdown of Snail2 reversed PURα-induced EMT and inhibited the migration and invasion of ESCC cells. In conclusion, this study indicated that PURα promotes Snail2 transcriptional activity to induce EMT during ESCC progression., (Copyright © 2020. Published by Elsevier B.V.)

Stathmin-1 is a microtubule depolymerization protein that regulates cell division, growth, migration, and invasion. Overexpression of stathmin-1 has been observed to be associated with metastasis, poor prognosis, and chemoresistance in various human cancers. Our previous studies found that serum stathmin-1 was significantly elevated in patients with esophageal squamous cell carcinoma (ESCC) by ELISAs. Here, we constructed high-affinity monoclonal antibodies and then developed a competitive AlphaLISA for rapid, accurate quantitation of stathmin-1 in serum. Compared to ELISA, our homogeneous AlphaLISA showed better sensitivity and accuracy, a lower limit of detection, and a wider linear range. The measurements of nearly 1000 clinical samples showed that serum stathmin-1 level increased dramatically in patients with squamous cell carcinoma (SCC), especially in ESCC, with a sensitivity and a specificity of 81% and 94%, respectively. Even for early stage ESCC, stathmin-1 achieved an area under the receiver operating characteristic curve (AUC) of 0.88. Meanwhile, raised concentrations of stathmin-1 were associated with lymph node metastasis and advanced cancer stage. Notably, various types of SCC showed significantly higher AUCs in serum stathmin-1 detection compared to adenocarcinoma. Furthermore, we confirmed that stathmin-1 was enriched in the oncogenic exosomes, which can explain the reason why it enters into the blood to serve as a tumor surrogate. In conclusion, this large-scale and systematic study of serum stathmin-1 measured by our newly established AlphaLISA showed that stathmin-1 is a very promising diagnostic and predictive marker for SCC in the clinic, especially for ESCC., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Receptor interacting protein kinase 3 (RIP3) is a critical regulator of programmed necrotic cell death. Here, we observed that RIP3 was significantly down-regulated in esophageal cancer. And its remaining expression was associated with better response to chemotherapy and prolonged survival. Notably, re-expression of kinase-dead RIP3 also restored cisplatin sensitivity, suggesting that some roles of RIP3 beyond necroptosis may be involved in cisplatin-based chemosensitivity. To investigate the mechanisms, a large-scale quantitative proteomics study was performed after cisplatin treatment in RIP3-knockdown cells. In total, approximately 7000 protein groups were confidently identified, with a false discovery rate of 0.21% at the protein level. Of these proteins, 685 displayed RIP3-dependent changes in abundance. Bioinformatics analyses indicated that DNA repair pathway was stimulated after RIP3 depletion. Functional studies showed that deficient RIP3 upregulated FOSL1 and POLD1 through activation of the HSP90/CDC37 complex and ERK phosphorylation in multiple cell lines. Furthermore, via inhibition of the HSP90/CDC37 complex, ERK and FOSL1 reversed the cisplatin resistance phenotype. These results suggest that RIP3 regulates cisplatin sensitivity through both pronecrotic and non-necrotic functions. RIP3 may be a potential marker for predicting chemosensitivity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however β-catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and β-catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and  β-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application., Competing Interests: The authors have declared that no competing interests exist.

We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate is an antioxidant found in the testa of peanut seeds. Previous studies have shown that ethyl-3,4-dihydroxybenzoate can effectively reduce breast cancer cell metastasis by inhibiting prolyl-hydroxylase. In this study, we investigated the cytotoxic effect of ethyl-3,4-dihydroxybenzoate on esophageal squamous cell carcinoma cells in vitro and identified key regulators of ethyl-3,4-dihydroxybenzoate-induced esophageal cancer cell death through transcription expression profiling. Using flow cytometry analysis, we found that ethyl-3,4-dihydroxybenzoate induced S phase accumulation, a loss in mitochondrial membrane permeabilization, and caspase-dependent apoptosis. Moreover, an expression profile analysis identified 46 up- and 9 down-regulated genes in esophageal cancer KYSE 170 cells treated with ethyl-3,4-dihydroxybenzoate. These differentially expressed genes are involved in several signaling pathways associated with cell cycle regulation and cellular metabolism. Consistent with the expression profile results, the transcriptional and protein expression levels of candidate genes NDRG1, BNIP3, AKR1C1, CCNG2 and VEGFA were found to be significantly increased in treated KYSE 170 cells by reverse-transcription PCR and western blot analysis. We also found that protein levels of hypoxia-inducible factor-1α, BNIP3, Beclin and NDRG1 were increased and that enriched expression of BNIP3 and Beclin caused autophagy mediated by microtubule-associated protein 1 light chain 3 in the treated cells. Autophagy and apoptosis were activated together in esophageal cancer cells after exposed to ethyl-3,4-dihydroxybenzoate. Furthermore, knock-down of NDRG1 expression by siRNA significantly attenuated apoptosis in the cancer cells, implying that NDRG1 may be required for ethyl-3,4-dihydroxybenzoate-induced apoptosis. Together, these results suggest that the cytotoxic effects of ethyl-3,4-dihydroxybenzoate were mediated by the up-regulation of NDRG1, BNIP3, Beclin and hypoxia-inducible factor-1α, initiating BNIP3 and Beclin mediated autophagy at an early stage and ultimately resulting in esophageal cancer cell apoptosis.

Esophageal squamous cell carcinoma (ESCC) is the eighth most common cause of cancer-related death worldwide. However, previous genome-wide single nucleotide polymorphism association analyses have not explained the high heritability associated with ESCC. In this study, we performed genome-wide copy number variation (CNV) analysis on 128 discordant sibling pairs to identify novel genes that contribute to ESCC susceptibility. A total of 57 774 individual CNVs were identified, and an interactive network of common CNV-associated genes was constructed, which showed that several ABC transporter genes contain CNVs in ESCC patients. Independent validation of a CNV at 13q32.1 in 1048 northern Chinese Han subjects demonstrated that the amplification of ABCC4 significantly correlated with ESCC risk [odds ratio: 3.36 (1.65-7.93), P = 0.0013]. Immunohistochemistry staining suggested that high copy numbers correlated with increased protein levels. High expression of ABCC4 was an independent poor prognostic factor for ESCC [relative risk: 1.73 (1.10-2.73), P = 0.0181]. The CNV region showed strong enhancer activity. Furthermore, inhibition of ABCC4 protein in ESCC cells decreased cell proliferation and motility via the inhibition of COX-2, PGE2 receptors and c-Myc expression; AKT, extracellular signal-regulated kinase and cAMP response element-binding protein phosphorylation; and β-catenin nuclear translocation in ESCC cells. In conclusion, the CNV at 13q32.1 is associated with ESCC susceptibility, and a gene within this locus, ABCC4, activates the oncogenic pathways in ESCC and thus facilitates cancer cell development and progression. A direct genetic contribution of ESCC risk through CNV common variants was determined in this study, and ABCC4 might therefore have predictive and therapeutic potential for ESCC., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)