Esophageal Squamous Cell Carcinoma Serum Research Articles

SPI1-Mediated Upregulation of the CST1 Gene as an Independent Poor Prognostic Factor Accelerates Metastasis in Esophageal Squamous Cell Carcinoma (ESCC) by Interacting with MMP2.

Esophageal squamous cell carcinoma (ESCC) is a highly lethal tumor type, but studies on the ESCC tumor microenvironment are limited. We found that cystatin SN (CST1) plays an important role in the ESCC tumor microenvironment. CST1 has been reported to act as an oncogene in multiple human cancers, but its clinical significance and underlying mechanism in ESCC remain elusive. We performed ESCC gene expression profiling with data from RNA-sequencing and public databases and found CST1 upregulation in ESCC. Then, we assessed CST1 expression in ESCC by RT‒qPCR and Western blot analysis. In addition, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were used to estimate the expression of CST1 in ESCC tissue and serum. Moreover, further functional experiments were conducted to verify that the gain and loss of CST1 in ESCC cell lines significantly influenced the proliferation and metastasis of ESCC. Mass spectrometry, coimmunoprecipitation, and gelatin zymography experiments were used to validate the interaction between CST1 and matrix metalloproteinase 2 (MMP2) and the mechanism of CST1 influence on metastasis in ESCC. Here, we found that CST1 expression was significantly elevated in ESCC tissues and serum. Moreover, compared with patients with low CST1 expression, patients with high CST1 expression had a worse prognosis. Overall survival (OS) and disease-free survival (DFS) were significantly unfavorable in the high CST1 expression subgroup. Likewise, the CST1 level was significantly increased in ESCC serum compared with healthy control serum, indicating that CST1 may be a potential serum biomarker for diagnosis, with an area under the curve (AUC) = 0.9702 and p < 0.0001 by receiver operating curve (ROC) analysis. Furthermore, upregulated CST1 can promote the motility and metastatic capacity of ESCC in vitro and in vivo by influencing epithelial mesenchymal transition (EMT) and interacting with MMP2 in the tumor microenvironment (TME). Collectively, the results of this study indicated that high CST1 expression mediated by SPI1 in ESCC may serve as a potentially prognostic and diagnostic predictor and as an oncogene to promote motility and metastatic capacity of ESCC by influencing EMT and interacting with MMP2 in the TME.

MicroRNA-29b regulates the radiosensitivity of esophageal squamous cell carcinoma by regulating the BTG2-mediated cell cycle.

Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable due to old age or advanced stage; thus, radio- and chemotherapy are considered the standard treatments for these patients. However, due to the radiation resistance of tumor cells that may arise during radiotherapy, results are still not satisfactory. The authors' previous studies found that microRNA can affect radiosensitivity, and further microRNA research was conducted to improve the radiosensitivity of ESCC. Cells were treated with silent miR-29b (si-miR-29b). Thereafter,proliferation, colony formation, cell cycle, and apoptosis were determined. The luciferase reporting assay was used to confirm the direct interaction between miR-29b and BTG2. Serum samples and clinical follow-up data of 75elderly or advanced ESCC patients who could not tolerate surgery were collected. The expression level of miR-29 in ESCC serum was closely correlated to radiosensitivity (χ2 =8.36, p < 0.05) and correlated with overall survival (OS; hazard ratio [HR] 0.47, 95% confidence interval [CI] 0.24-0.90). Function assays demonstrated that the number of cell clones increased after radiometry radiation, and the cell cycle was blocked in the G0/G1 phase (from 37.2 to 56.9%) in the si-miR-29b transfection group. Expression of BTG2 was upregulated and expression of cyclin D1 was downregulated (p < 0.05). Transfection of si-BTG2 can reverse this result and restore the expression level of cyclin D1 (p < 0.05). The target gene BTG2 of miR-29b was predicted using abioinformatics tool and confirmed by dual-luciferase reporter assay. Silencing of miR-29b in ESCC cells can increase expression of BTG2 and decrease the level of intracellular cyclin D1, resulting in cell cycle arrest and accumulation in the G0/G1 phase. Because G0/G1-phase cells are insensitive to radiotherapy, the sensitivity of radiotherapy is reduced.

Upregulated expression of serum exosomal hsa_circ_0026611 is associated with lymph node metastasis and poor prognosis of esophageal squamous cell carcinoma.

Background: To identify the diagnostic and prognostic values of serum exosomal hsa_circ_0026611 in esophageal squamous cell carcinoma (ESCC).Methods: ESCC serum exosome global circRNA expression was detected using a circRNA microarray. The expression levels of candidate serum exosome circRNAs were detected by quantitative polymerase chain reaction (qRT-PCR). A receiver operating characteristic curve (ROC) was generated to confirm the diagnostic value. Survival data and their differences were observed by the Kaplan-Meier method and log-rank tests. The Cox regression analysis was employed to identify prognostic factors.Results: The expression levels of serum exosomal has_circ_0026611 in ESCC with lymph node metastasis were significantly higher than those in ESCC without lymph node metastasis (P =0.001). In addition, serum exosomal hsa_circ_0026611 expression could be used as a significant parameter to discriminate between non-lymph node-metastatic and lymph node-metastatic ESCC with an area under curve (AUC) of 0.724 (95% CI: 0.604~0.865). The multivariate Cox regression analysis indicated that survival was poor in patients with high serum exosomal hsa_circ_0026611 expression levels compared to those with low serum exosomal hsa_circ_0026611 levels (for OS, HR [95% CI]: 3.79 [1.27, 11.29], for DFS, HR [95% CI]: 2.77 [1.06, 7.22]).Conclusion: Serum exosomal hsa_circ_0026611 expression is significantly upregulated in ESCC with lymph node metastasis and is a predictor of ESCC prognosis.

Development of a Novel Serum Exosomal MicroRNA Nomogram for the Preoperative Prediction of Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma.

Preoperative prediction of lymph node (LN) metastasis is accepted as a crucial independent risk factor for treatment decision-making for esophageal squamous cell carcinoma (ESCC) patients. Our study aimed to establish a non-invasive nomogram to identify LN metastasis preoperatively in ESCC patients. Construction of the nomogram involved three sequential phases with independent patient cohorts. In the discovery phase (N = 20), LN metastasis-associated microRNAs (miRNAs) were selected from next-generation sequencing (NGS) assay of human ESCC serum exosome samples. In the training phase (N = 178), a nomogram that incorporated exosomal miRNA model and clinicopathologic was developed by multivariate logistic regression analysis to preoperatively predict LN status. In the validation phase (n = 188), we validated the predicted nomogram's calibration, discrimination, and clinical usefulness. Four differently expressed miRNAs (chr 8-23234-3p, chr 1-17695-5p, chr 8-2743-5p, and miR-432-5p) were tested and selected in the serum exosome samples from ESCC patients who have or do not have LN metastasis. Subsequently, an optimized four-exosomal miRNA model was constructed and validated in the clinical samples, which could effectively identify ESCC patients with LN metastasis, and was significantly superior to preoperative computed tomography (CT) report. In addition, a clinical nomogram consisting of the four-exosomal miRNA model and CT report was established in training cohort, which showed high predictive value in both training and validation cohorts [area under the receiver operating characteristic curve (AUC): 0.880 and 0.869, respectively]. The Hosmer–Lemeshow test and decision curve analysis implied the nomogram's clinical applicability. Our novel non-invasive nomogram is a robust prediction tool with promising clinical potential for preoperative LN metastasis prediction of ESCC patients, especially in T1 stage.

Downregulation of Ubiquitin Inhibits the Aggressive Phenotypes of Esophageal Squamous Cell Carcinoma.

Purpose:Esophageal cancer is one of the most common malignancies worldwide. Ubiquitin-dependent degradation of regulatory proteins reportedly plays a central role in diverse cellular processes. This study investigated the expression levels of ubiquitin in esophageal squamous cell carcinoma tissues and the functions of ubiquitin in the context of esophageal squamous cell carcinoma progression.Methods:The expression of ubiquitin in esophageal squamous cell carcinoma and normal esophageal samples was determined via immunohistochemistry. Serum ubiquitin levels were determined by enzyme-linked immunosorbent assay. The association between serum ubiquitin level and clinicopathological factors was analyzed. Real-time PCR analysis was employed to measure the mRNA levels of the ubiquitin coding genes ubiquitin B and ubiquitin C. Proliferation assays, colony formation assays, and Transwell-based assays were used to determine the influence of ubiquitin on cell growth and cell invasion. Proteomic analysis was performed to identify the proteins associated with ubiquitin.Results:Ubiquitin expression in esophageal squamous cell carcinoma tissues was markedly higher than that in normal and tumor adjacent tissues. The levels of ubiquitin in esophageal squamous cell carcinoma serum samples were significantly higher than those in healthy controls. Serum ubiquitin levels were correlated with tumor stage and lymph node metastasis. To silence the expression of ubiquitin, we knocked down the ubiquitin coding genes ubiquitin B and ubiquitin C in TE-1 and Eca-109 cells. Silencing ubiquitin resulted in the suppression of cell growth, chemoresistance, colony formation and cell migration in esophageal squamous cell carcinoma cells. Proteomic analysis in esophageal squamous cell carcinoma cells showed that knockdown of ubiquitin coding genes deregulated the expression of 159 proteins (92 were upregulated and 67 were downregulated) involved in multiple pathways. These proteins included ferritin light chain, ferritin heavy chain, cellular retinoic acid-binding protein 2, and DNA replication factor 1.Conclusion:Ubiquitin expression is upregulated in esophageal squamous cell carcinoma tissues and serum samples. Serum ubiquitin levels were correlated with tumor stage and lymph node metastasis. Downregulation of ubiquitin suppresses the aggressive phenotypes of esophageal squamous cell carcinoma cells by complex mechanisms; ubiquitin may represent a novel target for the treatment of esophageal squamous cell carcinoma.

Activation of choline kinase drives aberrant choline metabolism in esophageal squamous cell carcinomas

Esophageal squamous cell carcinoma (ESCC) is a major health threat worldwide. Research focused on molecular events associated with ESCC carcinogenesis for diagnosis, treatment and prevention is needed. Our goal is to discover novel biomarkers and investigate the underlying molecular mechanisms of ESCC progression by employing a global metabolomic approach. Sera from 34 ESCC patients and 32 age and sex matched healthy controls were profiled using two-dimensional liquid chromatography-mass spectrometry (2D LC–MS). We identified 120 differential metabolites in ESCC patient serums compared to healthy controls. Several amino acids, serine, arginine, lysine and histidine were significantly changed in ESCC patients. Most importantly, we found dysregulated lipid metabolism as an important characteristic in ESCC patients. Several free fat acids (FFA) and carnitines were found down-regulated in ESCC patients. Choline was significantly increased and phosphatidylcholines (PC) were significantly decreased in ESCC serum. The high expression of choline and low expression of total PC in patient serum were associated with the high expression of choline kinase (Chok) and activated Kennedy pathway in ESCC cells. Chok expression can serve as a significant biomarker for ESCC prognosis. In conclusion, metabolite profiles in the ESCC patient serum were significantly different from those in the healthy controls. Phosphatidylcholines and Chok, the key enzyme in the PC metabolism pathway, may serve as novel biomarkers for ESCC.

A novel serum microRNA signature to screen esophageal squamous cell carcinoma.

Circulating microRNAs (miRNAs) have been used as promising diagnostic biomarkers for esophageal squamous cell carcinoma (ESCC). We performed miRNA expression profiling using quantitative reverse transcription polymerase chain reaction (qRT‐PCR) based Exiqon panels from three ESCC pools and one normal control (NC) pool samples. Using qRT‐PCR, identified serum miRNAs were further confirmed in training (32 ESCC vs. 32 NCs) and testing stages (108 ESCC vs. 96 NCs). Consequently, five serum miRNAs (miR‐20b‐5p, miR‐28‐3p, miR‐192‐5p, miR‐223‐3p, and miR‐296‐5p) were significantly overexpressed in ESCC compared with NCs. The diagnostic value of the 5‐miRNA signature was validated by an external cohort (60 ESCC vs. 60 NCs). The areas under the receiver operating characteristic curve (ROC) of the 5‐miRNA signature were 0.753, 0.763, and 0.966 for the training, testing, and the external validation stages, respectively. The expression levels of the miRNAs were also determined in tissues, arterial serum, and exosomes. MiR‐20b‐5p, miR‐28‐3p, and miR‐192‐5p were significantly upregulated in ESCC tissues, while miR‐296‐5p was overexpressed in ESCC serum exosomes. In conclusion, we identified a 5‐miRNA signature in serum for the detection of ESCC.

LC–MS-based serum metabolomic analysis reveals dysregulation of phosphatidylcholines in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with poor prognosis. Here, we carried out liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–Q–TOF-MS)-based untargeted metabolomic analysis of ESCC serum samples. Statistical analysis resulted in the identification of 652 significantly dysregulated molecular features in serum from ESCC patients as compared to the healthy subjects. Phosphatidylcholines were identified as a major class of dysregulated metabolites in this study suggesting potential perturbation of phosphocholine metabolism in ESCC. By using a targeted MS/MS approach both in positive and negative mode, we were able to characterize and confirm the structure of seven metabolites. Our study describes a quantitative LC–MS approach for characterizing dysregulated lipid metabolism in ESCC. Biological significanceAltered metabolism is a hallmark of cancer. We carried out (LC–MS)-based untargeted metabolomic profiling of serum from esophageal squamous cell carcinoma (ESCC) patients to characterize dysregulated metabolites. Phosphatidylcholine metabolism was found to be significantly altered in ESCC. Our study illustrates the use of mass spectrometry-based metabolomic analysis to characterize molecular alterations associated with ESCC. This article is part of a Special Issue entitled: Proteomics in India.

Identification of tumor antigens that elicit a humoral immune response in the sera of Chinese esophageal squamous cell carcinoma patients by modified serological proteome analysis

Our aim was to identify novel tumor-associated antigens from the esophageal squamous cell carcinoma (ESCC) cell line EC0156, and related autoantibodies in sera from patients with ESCC. We used modified serological proteome analysis, involving one- and two-dimensional electrophoresis, Western blot, and MALDI-TOF/TOF-MS to identify 6 ESCC-associated antigens. From these, 105kDa heat shock protein (HSP105) and triosephosphate isomerase (TIM) were further evaluated and we determined they could induce autoantibody responses in ESCC sera and are highly expressed in ESCC tissues. Anti-HSP105 and anti-TIM autoantibodies were found in 39.1% (18/46) and 34.8% (16/46) of patients with ESCC, respectively, but only in two controls. A receiver operating characteristic curve constructed with HSP105 and TIM gave a sensitivity of 54.3% and 95% (38/40) specificity in discriminating ESCC from matched controls. Interestingly, we found that autoantibodies against TIM in ESCC serum mainly reacted with glycosylated but not deglycosylated TIM. The preliminary results suggest the potential utility of screening autoantibodies in sera for use as biomarkers for cancer diagnosis.

Using Proteomic Approach to Identify Tumor-Associated Proteins as Biomarkers in Human Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. The lower survival rate of ESCC is attributed to late diagnosis and poor therapeutic efficacy; therefore, the identification of tumor-associated proteins as biomarkers for early diagnosis, and the discovery of novel targets for therapeutic intervention, seems very important for increasing the survival rate of ESCC. To identify tumor-associated proteins as biomarkers in ESCC, we have analyzed ESCC tissues and adjacent normal tissues by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The results showed that a total of 104 protein spots with different expression levels were found on 2DE, and 47 proteins were eventually identified by MALDI-TOF MS. Among these identified proteins, 33 proteins including keratin 17 (KRT17), biliverdin reductase B (BLVRB), proteasome activator subunit 1 (PSME1), manganese superoxide dismutase (MnSOD), high-mobility group box-1(HMGB1), heat shock protein 70 (HSP70), peroxiredoxin (PRDX1), keratin 13 (KRT13), and so on were overexpressed, and 14 proteins including cystatin B (CSTB), tropomyosin 2 (TPM2), annexin 1 (ANX1), transgelin (TAGLN), keratin 19 (KRT19), stratifin (SFN), and so on were down-expressed in ESCC. Biological functions of these proteins are associated with cell proliferation, cell motility, protein folding, oxidative stress, and signal transduction. In the subsequent study using immunoassay on ESCC serum samples and tissue-array slides, two representative proteins, HSP70 and HMGB1, were selected as examples for the purpose of validation. The results showed that both HSP70 and HMGB1 can induce autoantibody response in ESCC sera and have higher expression in ESCC tissues. Especially, the frequency of antibodies to HSP70 in ESCC sera was significantly higher than that in normal human sera. The preliminary results suggest that some of these identified proteins might contribute to esophageal cell differentiation and carcinogenesis, certain proteins could be used as tumor-associated antigen (TAA) biomarkers in cancer diagnosis, and further studies on these identified proteins should provide more evidence of how these proteins are involved in carcinogenesis of ESCC.