Lymph Node Metastasis In Esophageal Squamous Cell Carcinoma Research Articles

Intravoxel incoherent motion diffusion-weighted imaging in evaluating preoperative staging of esophageal squamous cell carcinoma

BackgroundThe aim of this research is to prospectively investigate the diagnostic performance of intravoxel incoherent motion (IVIM) using the integrated slice-specific dynamic shimming (iShim) technique in staging primary esophageal squamous cell carcinoma (ESCC) and predicting presence of lymph node metastases from ESCC.MethodsSixty-three patients with ESCC were prospectively enrolled from April 2016 to April 2019. MR and IVIM using iShim technique (b = 0, 25, 50, 75, 100, 200, 400, 600, 800 s/mm2) were performed on 3.0T MRI system before operation. Primary tumour apparent diffusion coefficient (ADC) and IVIM parameters, including true diffusion coefficient (D), pseudodiffusion coefficient (D*), pseudodiffusion fraction (f) were measured by two independent radiologists. The differences in D, D*, f and ADC values of different T and N stages were assessed. Intraclass correlation coefficients (ICCs) were calculated to evaluate the interobserver agreement between two readers. The diagnostic performances of D, D*, f and ADC values in primary tumour staging and prediction of lymph node metastasis of ESCC were determined using receiver operating characteristic (ROC) curve analysis.ResultsThe inter-observer consensus was excellent for IVIM parameters and ADC (D: ICC = 0.922; D*: ICC = 0.892; f: ICC = 0.948; ADC: ICC = 0.958). The ADC, D, D* and f values of group T1 + T2 were significantly higher than those of group T3 + T4a [ADC: (2.55 ± 0.43) ×10− 3 mm2/s vs. (2.27 ± 0.40) ×10− 3 mm2/s, t = 2.670, P = 0.010; D: (1.82 ± 0.39) ×10− 3 mm2/s vs. (1.53 ± 0.33) ×10− 3 mm2/s, t = 3.189, P = 0.002; D*: 46.45 (30.30,55.53) ×10− 3 mm2/s vs. 32.30 (18.60,40.95) ×10− 3 mm2/s, z=-2.408, P = 0.016; f: 0.45 ± 0.12 vs. 0.37 ± 0.12, t = 2.538, P = 0.014]. The ADC, D and f values of the lymph nodes-positive (N+) group were significantly lower than those of lymph nodes-negative (N0) group [ADC: (2.10 ± 0.33) ×10− 3 mm2/s vs. (2.55 ± 0.40) ×10− 3 mm2/s, t=-4.564, P < 0.001; D: (1.44 ± 0.30) ×10− 3 mm2/s vs. (1.78 ± 0.37) ×10− 3 mm2/s, t=-3.726, P < 0.001; f: 0.32 ± 0.10 vs. 0.45 ± 0.11, t=-4.524, P < 0.001]. The combination of D, D* and f yielded the highest area under the curve (AUC) (0.814) in distinguishing group T1 + T2 from group T3 + T4a. D combined with f provided the highest diagnostic performance (AUC = 0.849) in identifying group N + and group N0 of ESCC.ConclusionsIVIM may be used as an effective functional imaging technique to evaluate preoperative stage of primary tumour and predict presence of lymph node metastases from ESCC.

High expression of the stemness-associated molecule Nanog in esophageal squamous cell carcinoma tissues promotes tumor invasion and metastasis by activating the TGF-β signaling pathway

To investigate the expression of Nanog and its regulatory relationship with MMP-2/MMP-9 proteins in esophageal squamous cell carcinoma (ESCC). We detected Nanog and MMP-2/MMP-9 protein expressions in 127 ESCC tissues and 82 adjacent normal tissues using immunohistochemistry and explored their correlations with the clinicopathological parameters and prognosis of the patients. GEO database was utilized to analyze the pathways enriched with the stemness-related molecules including Nanog, and TIMER online tool was used to analyze the correlations among TβR1, MMP-2, and MMP-9 in esophageal cancer. Nanog and MMP-2/MMP-9 proteins were significantly upregulated in ESCC tissues and positively intercorrelated. Their expression levels were closely correlated with infiltration depth and lymph node metastasis of ESCC but not with age, gender, or tumor differentiation. The patients with high expressions of Nanog and MMP-2/MMP-9 had significantly shorter survival time. Bioinformatics analysis showed enrichment of stemness-associated molecules in the TGF-β signaling pathway, and the expressions of MMP-2/MMP-9 and TβR1 were positively correlated. In cultured ESCC cells, Nanog knockdown significantly decreased the expression of TβR1, p-Smad2/3, MMP-2, and MMP-9 and strongly inhibited cell migration. The high expressions of Nanog, MMP-2, and MMP-9, which are positively correlated, are closely related with invasion depth, lymph node metastasis, and prognosis of ESCC. Nanog regulates the expressions of MMP-2/MMP-9 proteins through the TGF-β signaling pathway, and its high expression promotes migration of ESCC cells.

Multi-omics analysis reveals NNMT as a master metabolic regulator of metastasis in esophageal squamous cell carcinoma

Metabolic reprogramming has been observed in cancer metastasis, whereas metabolic changes required for malignant cells during lymph node metastasis of esophageal squamous cell carcinoma (ESCC) are still poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of paired ESCC tumor tissues and lymph nodes to uncover the reprogramming of tumor microenvironment (TME) and metabolic pathways. By integrating analyses of scRNA-seq data with metabolomics of ESCC tumor tissues and plasma samples, we found nicotinate and nicotinamide metabolism pathway was dysregulated in ESCC patients with lymph node metastasis (LN+), exhibiting as significantly increased 1-methylnicotinamide (MNA) in both tumors and plasma. Further data indicated high expression of N-methyltransferase (NNMT), which converts active methyl groups from the universal methyl donor, S-adenosylmethionine (SAM), to stable MNA, contributed to the increased MNA in LN+ ESCC. NNMT promotes epithelial–mesenchymal transition (EMT) and metastasis of ESCC in vitro and in vivo by inhibiting E-cadherin expression. Mechanically, high NNMT expression consumed too much active methyl group and decreased H3K4me3 modification at E-cadherin promoter and inhibited m6A modification of E-cadherin mRNA, therefore inhibiting E-cadherin expression at both transcriptional and post-transcriptional level. Finally, a detection method of lymph node metastasis was build based on the dysregulated metabolites, which showed good performance among ESCC patients. For lymph node metastasis of ESCC, this work supports NNMT is a master regulator of the cross-talk between cellular metabolism and epigenetic modifications, which may be a therapeutic target.

Lymph node metastasis-related lncRNA GAS6-AS1 facilitates the progression of esophageal squamous cell carcinoma.

Lymph node metastasis is the main type of metastasis in esophageal squamous cell carcinoma (ESCC), especially when the primary tumor invasion depth reaches above the adventitia layer (T3 stage), the incidence of lymph node metastasis increases sharply. Abnormal expression of long non-coding RNAs (lncRNAs) has been confirmed in ESCC, but there are still many unknown connections between lncRNAs and lymph node metastasis. We used transcriptome sequencing (RNA-seq) to analyze 10 pairs of ESCC tissues with primary tumor stage T3 and their paired normal epithelium. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to further verify the sequencing results, and survival curve analysis, logistic regression analysis, and receiver operating characteristic (ROC) curve analysis were used to investigate its clinical application value. We investigated the growth and metastasis effects of lncRNA GAS6-AS1 on ESCC cell lines TE-1 and KYSE410 in vitro and in vivo. Other functional experiments included cell apoptosis and cell cycle experiments. Based on our RNA-seq data, lncRNA GAS6-AS1 is highly expressed in ESCC tissues, especially in cancer tissues with lymph node metastasis. The qRT-PCR experiment analysis showed that high expression of GAS6-AS1 was related to poor tumor differentiation and tumor stage. Logistic regression analysis showed that it was an independent risk factor for lymph node metastasis, and ROC analysis validated that it could predict lymph node metastasis. Further survival analysis suggested that high expression of GAS6-AS1 was associated with patients' poor prognosis. In vitro experiments, knocking down GAS6-AS1 inhibited the growth and metastasis of ESCC cells and inhibited tumor growth in vivo. In addition, knocking down GAS6-AS1 can inhibit cell cycle and promote cell apoptosis. Our results revealed that lncRNA GAS6-AS1 obtained from RNA-seq can be used as an independent risk factor for ESCC lymph node metastasis and an effective biomarker to predict, and that it was related to the growth and metastasis of ESCC. It may represent a new biomarker to aid in the assessment of the lymph node metastasis of ESCC.

RETRACTED ARTICLE: IL-1RA inhibits esophageal carcinogenesis and lymphangiogenesis via downregulating VEGF-C and MMP9

Interleukin-1 receptor antagonist (IL-1RA) has been shown to play an important role in cancer progression. However, its pathogenic effects and molecular mechanism in the malignant progression of esophageal squamous cell carcinoma (ESCC) remain largely unknown. This study was designed to explore the function of IL-1RA in ESCC and determine the relationship between IL-1RA and lymph node metastasis in ESCC patients. The clinical relevance of IL-1RA in relation to the clinicopathological features and prognosis of 100 ESCC patients was analyzed. The function and underlying mechanisms of IL-1RA in the growth, invasion, and lymphatic metastasis in ESCC were explored both in vitro and in vivo. The therapeutic effect of anakinra, an IL-1 receptor antagonist, on ESCC was also evaluated in animal experiments. Downregulation of IL-1RA was observed in ESCC tissues and cells and was found to be strongly correlated with pathological stage (P = 0.034) and lymphatic metastasis (P = 0.038). Functional assays demonstrated that upregulation of IL-1RA reduced cell proliferation, migration, and lymphangiogenesis both in vitro and in vivo. Mechanistic studies revealed that overexpression of IL-1RA activated the epithelial-to-mesenchymal transition (EMT) in the ESCC cells through activation of MMP9 and regulation of the expression and secretion of VEGF-C through the PI3K/NF-κB pathway. Anakinra treatment resulted in significant inhibition of tumor growth, lymphangiogenesis, and metastasis. IL-1RA inhibits lymph node metastasis of ESCC by regulating the EMT through activation of matrix metalloproteinase 9(MMP9) and lymphangiogenesis, driven by VEGF-C and the NF-κB signaling pathway. Anakinra may be an effective drug for the inhibition of ESCC tumor formation and lymph node metastasis.

Comparison of lymph node metastasis pattern from esophagogastric junction adenocarcinoma versus very low thoracic esophageal squamous cancer: a propensity-matched analysis.

The anatomical locations of esophagogastric junction adenocarcinoma (AEG) and very low thoracic esophageal squamous cell carcinoma (ESCC) are similar. This study aimed to evaluate the difference in lymph node metastasis (LNM) distribution between AEG and very low thoracic ESCC. Data from 156 Siewert I-II AEG patients and 120 ESCC patients with proximal edges located within 5 cm of the esophagogastric junction (EGJ) and underwent curative surgery from 2010 to 2015 were retrospectively analyzed using propensity score matching (PSM). Five or six baseline variables were included in PSM separately. All patients underwent curative transthoracic surgery and systematic lymphadenectomy. After PSM, LNM rates of major stations were compared using the chi-squared test or Fisher's exact test. After PSM was performed with covariates (age, sex, T stage, grade, tumor length), 60 pairs of patients were included. The lower mediastinal and total thoracic LNM rates of ESCC were significantly higher than those of AEG (18.3% vs. 3.3%, P=0.019; 25% vs. 3.3%, P=0.002). After further addition of the N stage as a variant to the previous PSM model, we found that the paracardial LNM distribution was significantly different between ESCC and AEG patients (36.1% vs. 19.7%, P=0.043). Among all tumor characteristics, only the T stage was positively correlated with paracardial LNM in ESCC (P=0.010), but not in AEG. In AEG, the median survival was poor for patients with thoracic LNM. Patients with very low thoracic ESCC exhibit stronger metastatic ability in the lower mediastinal and paracardial nodes than Siewert I-II AEG. However, the pathological metastasis of AEG in thoracic nodes was associated with poor survival outcomes.

196. RISK FACTORS OF CERVICAL LYMPH NODE METASTASIS IN ESCC AND ESTABILISHMENT OF PREDICTION MODEL

Abstract Cervical lymph node metastasis of esophageal squamous cell carcinoma (ESCC) is an important basis for lymph node dissection of ESCC. Now, there are still disputes about whether to perform two field lymph node dissection or three field lymph node dissection. Establishing prediction model by analysis big data could promote the accuracy of prediction of cervical lymph node metastasis in patients with ESCC which can help clinicians to select individualized surgical scheme for patients. The clinical data of 102 consecutive patients with ESCC who underwent radical esophagectomy and three-field lymph node dissection between December 2015 to December 2021 were retrospectively analyzed. They were divided into positive group and negative group according to cervical lymph node metastasis. The differences between the two groups were compared, and after the univariate analysis those who were statistically significant were included in multivariable logistic regression analysis to establish the risk prediction model and plot a nomogram by software R. Among the 102 patients, univariate analysis(P&amp;lt;0.2)indicated that cervical lymph node metastasis of ESCC was related to the distance from upper edge of the tumor to the incisors (P = 0.101), vascular invasion (P = 0.144) and recurrent laryngeal nerve lymph nodes metastasis (P = 0.159). Multivariate logistic regression analysis showed that the distance from upper edge of the tumor to the incisors (P = 0.032) and the vascular invasion (P = 0.147) were independent risk factors. The AUC of ROC curve is 0.6817 (95% confidence interval: 0.5775–0.7859) and the Youden index is 0.294, 95% CI (0.620, 0.742). The prediction model includes two variables: the distance from upper edge of the tumor to the incisors and the vascular invasion. It plays a certain role in predicting the risk of cervical lymph node metastasis of ESCC, and can assist doctors in individualized treatment for patients with ESCC.

513. RISK FACTORS OF CERVICAL LYMPH NODE METASTASIS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA AND ESTABLISHMENT OF RISK PREDICTION MODEL

Abstract Cervical lymph node metastasis of esophageal squamous cell carcinoma (ESCC) is an important basis for lymph node dissection of ESCC. Now, there are still disputes about whether to perform two field lymph node dissection or three field lymph node dissection. Establishing prediction model by analysis big data could promote the accuracy of prediction of cervical lymph node metastasis in patients with ESCC which can help clinicians to select individualized surgical scheme for patients The clinical data of 102 consecutive patients with ESCC who underwent radical esophagectomy and three-field lymph node dissection between September 2015 to December 2021 were retrospectively analyzed. They were divided into positive group and negative group according to cervical lymph node metastasis. The differences between the two groups were compared, and after the univariate analysis those who were statistically significant were included in multivariable logistic regression analysis to establish the risk prediction model and plot a nomogram by software R. Among the 102 patients, univariate analysis (P&amp;lt;0.2) indicated that cervical lymph node metastasis of ESCC was related to the distance from upper edge of the tumor to the incisors (P=0.085), vascular invasion (P= 0.141) and recurrent laryngeal nerve lymph nodes metastasis (P = 0.157). Multivariate logistic regression analysis showed that the distance from upper edge of the tumor to the incisors (P=0.153) and the vascular invasion (P=0.198) were independent risk factors. The AUC of ROC curve is 0.682 (95% confidence interval: 0.578-0.786) and the Youden index is 0.294, 95% CI (0.620, 0.742). The prediction model includes two variables: the distance from upper edge of the tumor to the incisors and the vascular invasion. It plays a certain role in predicting the risk of cervical lymph node metastasis of ESCC, and can assist doctors in individualized treatment for patients with ESCC.

201. PREDICTION OF LYMPH NODE METASTASIS IN PATIENTS WITH ESOPHAGEAL SQUAMOUS CELL CARCINOMA USING GENETIC SIGNATURES FROM LARGE-SCALE GENOMIC REPERTOIRES

Abstract The presence of lymph node metastasis puts patients with resectable esophageal squamous cell carcinoma (ESCC) at a high risk of disease recurrence, and these patients would benefit from neoadjuvant treatment. However, current clinical staging is not enough to capture all patients with high-risk disease, and novel diagnostic tools are required to improve the accuracy of treatment planning. We tried to explore a better prediction using genomic features from large-scale gene mutation data. The major public data repositories of whole-exome sequencing for ESCC were involved in this study. The absolute differences in gene mutation frequency were calculated using Fisher’s exact test in nodal negative versus nodal positive patients. The classification of gene mutation status was dichotomized as wild-type and mutant type. To find a reliable gene panel that could provide sufficient population coverage, we set the cut-off level above 85% in both nodal phenotypes. The genomic classifier for nodal metastasis risk prediction was further generated by LASSO logistic regression analysis with 10-fold cross-validation based on the topmost significantly different genes. Above the threshold values, a total of 120 differentially mutated genes were identified in the 561 ESCC genomic sequencing data. The population coverage rates were 85.97% in the nodal positive group (n = 335) and 88.05% in the nodal negative group (n = 226), respectively. According to the LASSO logistic regression approach, a panel of 112 topmost relevant genes (termed LNM112) was selected. The receiver operating characteristic curve of the LNM112 predictive model was plotted with an area under the curve value of 0.95 (95% CI: 0.93–0.96). The sensitivity and specificity of the LNM112 model were 96.72% (324/335) and 71.24% (161/226), respectively. We identified a genomic signature (112 genes) from the large-scale genomic repertoires capable of predicting lymph node metastasis in ESCC. It can be incorporated into the current staging modalities to help inform treatment decisions, and external validation is warranted in different populations.

Insulin receptor substrate 1(IRS1) is related with lymph node metastases and prognosis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer-related mortality globally with a high risk of lymph node metastasis (LNM). In this study, weighted gene co-expression network analysis (WGCNA) showed the identification of 10 modules among which the significant module (turquoise), including 1352 genes, was correlated with LNM. A group 52 overlapping differentially expressed genes (DEGs) was identified based on the comparison of turquoise module with GSE23400 and GSE20347 datasets. Using Ctyohubba plugin, we identified 7 hub genes (ACTG2, SORBS1, MYH11, CXCL12, CNN1, IRS1 and CXCL8). IRS1 displayed significant correlation with metastasis. The decreased expression of IRS1 was also a predictor of poor OS of ESCC patients whereas the hub genes namely ACTG2, MYH11, CXCL8, CXCL12, IRS1 and CNN1 were associated with RFS of ESCC patients. These findings suggest that the altered expression of these hub genes are associated with prognosis and thus can be used as potential biomarkers for ESCC. Moreover, immunohistochemical staining and cell functional experiments displayed that the overexpression of IRS1 was negatively associated with metastasis in ESCC. In general, our research revealed several novel genes in ESCC especially the association of IRS1 with LNM in ESCC, which could provide novel insights into the initiation and progression of ESCC.

Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway

BackgroundMetastasis is the leading cause of mortality in human cancers, including esophageal squamous cell carcinoma (ESCC). As a pro-inflammatory cytokine, IL-32 was reported to be a poor prognostic factor in many cancers. However, the role of IL-32 in ESCC metastasis remains unknown.MethodsESCC cells with ectopic expression or knockdown of IL-32 were established and their effects on cell motility were detected. Ultracentrifugation, Transmission electron microscopy and Western blot were used to verify the existence of extracellular vesicle IL-32 (EV-IL-32). Coculture assay, immunofluorescence, flow cytometry, and in vivo lung metastasis model were performed to identify how EV-IL-32 regulated the crosstalk between ESCC cells and macrophages.ResultsHere, we found that IL-32 was overexpressed and positively correlated to lymph node metastasis of ESCC. IL-32 was significantly higher in the tumor nest compared with the non-cancerous tissue. We found that IL-32β was the main isoform and loaded in EV derived from ESCC cells. The shuttling of EV-IL-32 derived from ESCC cells into macrophages could promote the polarization of M2 macrophages via FAK-STAT3 pathway. IL-32 overexpression facilitated lung metastasis and was positively correlated with the proportion of M2 macrophages in tumor microenvironment.ConclusionsTaken together, our results indicated that EV-IL-32 derived from ESCC cell line could be internalized by macrophages and lead to M2 macrophage polarization via FAK-STAT3 pathway, thus promoting the metastasis of ESCC. These findings indicated that IL-32 could serve as a potential therapeutic target in patients with ESCC.

Optimal criteria for predicting lymph node metastasis in esophageal squamous cell carcinoma by anatomical location using preoperative computed tomography: a retrospective cohort study.

Predicting lymph node metastasis (LNM) in esophageal squamous cell carcinoma (ESCC) is critical for selecting appropriate treatments despite the low accuracy of computed tomography (CT) for detecting LNM. Variation in potential nodal sizes among locations or patients' clinicopathological background factors may impact the diagnostic quality. This study explored the optimal criteria and diagnostic ability of CT by location. We retrospectively reviewed preoperative CT scans of 229 patients undergoing curative esophagectomy. We classified nodal stations into six groups: Cervical (C), Right-upper mediastinal (UR), Left-upper mediastinal (UL), Middle mediastinal (M), Lower mediastinal (L), and Abdominal (A). We then measured the short-axial diameter (SAD) of the largest lymph node in each area. We used receiver operating characteristics analyses to evaluate the CT diagnostic ability and determined the cut-off values for the SAD in all groups. Optimal cut-offs were 6.5mm (M), 6mm (C, L, and A), and 5mm (UR and UL). Diagnostic abilities differed among locations, and UR had the highest sensitivity. A multivariate analysis showed poor differentiation to be an independent risk factor for a false-negative diagnosis (p = 0.044). Optimal criteria and diagnostic abilities for predicting LNM in ESCC varied among locations, and poor differentiation might contribute to failure to detect LNM.

Cystathionine-β-synthase (CBS)/H2S system promotes lymph node metastasis of esophageal squamous cell carcinoma (ESCC) by activating SIRT1.

Lymph node metastasis is a key factor of death and prognosis in patients with esophageal squamous cell carcinoma (ESCC). Previous studies have demonstrated that Cystathionine-β-synthase (CBS)/H2S system plays important roles in progression of various cancer. However, the function and mechanism of CBS/H2S system in lymph node metastasis of ESCC remains unclear. Here, we found that CBS was highly expressed in human ESCC tissues and closely associated with lymph node metastasis in ESCC patients. Functional studies demonstrated that CBS could significantly promote lymph node metastasis of ESCC tumor cells. In vitro, CBS knockdown inhibited tumor cell proliferation, migration and invasion, whereas CBS overexpression produced the opposite results. In vivo, downregulation of CBS distinctly inhibited ESCC tumor growth and lymphatic metastasis, as evidenced by the decreased size and weight of tumor and popliteal lymph node. Meanwhile, we also found high expression of CBS-induced ESCC angiogenesis and lymphangiogenesis in vitro and in vivo by upregulating VEGF, VEGF-C, and VEGF-D. Mechanistically, CBS up-regulated the expression of SIRT1 and thus interrupted the Notch1/Hes1 axis, which plays a crucial role in lymph node metastasis of ESCC. Moreover, it was demonstrated that H2S derived from CBS-activated SIRT1 via increasing the NAD+/NADH ratio and promoting the phosphorylation of SIRT1. In addition, H2S derived from CBS also enhanced SIRT1 protein stability. Taken together, these data show that the high expression of CBS/H2S system promotes ESCC lymph node metastasis via activating SIRT1 signaling pathway and CBS could serve as a potential therapeutic target for clinical intervention in ESCC.

Exosomal and intracellular miR-320b promotes lymphatic metastasis in esophageal squamous cell carcinoma

Cancer-cell-released exosomal microRNAs (miRNAs) are important mediators of cell-cell communication in the tumor microenvironment. In this study, we sequenced serum exosome miRNAs from esophageal squamous cell carcinoma (ESCC) patients and identified high expression of miR-320b to be closely associated with peritumoral lymphangiogenesis and lymph node (LN) metastasis. Functionally, miR-320b could be enriched and transferred by ESCC-released exosomes directly to human lymphatic endothelial cells (HLECs), promoting tube formation and migration in vitro and facilitating lymphangiogenesis and LN metastasis in vivo as assessed by gain- and loss-of-function experiments. Furthermore, we found programmed cell death 4 (PDCD4) as a direct target of miR-320b through bioinformatic prediction and luciferase reporter assay. Re-expression of PDCD4 could rescue the effects induced by exosomal miR-320b. Notably, the miR-320b-PDCD4 axis activates the AKT pathway in HLECs independent of vascular endothelial growth factor-C (VEGF-C). Moreover, overexpression of miR-320b promotes the proliferation, migration, invasion, and epithelial-mesenchymal transition progression of ESCC cells. Finally, we demonstrate that METTL3 could interact with DGCR8 protein and positively modulate pri-miR-320b maturation process in an N6-methyladenosine (m6A)-dependent manner. Therefore, our findings uncover a VEGF-C-independent mechanism of exosomal and intracellular miR-320b-mediated LN metastasis and identify miR-320b as a novel predictive marker and therapeutic target for LN metastasis in ESCC.

Overexpression of microRNA-19a-3p promotes lymph node metastasis of esophageal squamous cell carcinoma via the RAC1/CDC42-PAK1 pathway.

BackgroundA tendency towards extensive regional lymph node metastasis (LNM) is a typical clinical characteristic of esophageal squamous cell carcinoma (ESCC). Up-regulated microRNA (miR)-19a-3p was verified as a predictor of LNM in ESCC in previous microarray analyses, but the underlying mechanisms remain unclear. Here, in vitro experiments were performed to confirm the effect of miR-19a-3p on promoting LNM and to explore the underlying mechanisms.MethodsKYSE-150 and TE-1 cell lines were transfected with lentiviral vectors to inhibit miR-19a-3p (LV-miR-19a-3p-inhibition), and cell proliferation, invasion, and migration were assessed. Target genes of miR-19a-3p were identified by sequencing analysis and quantitative reverse transcription PCR (qRT-PCR); Western blotting was performed to confirm targets and explore the potential mechanisms underlying the effect of miR-19a-3p on LNM.ResultsmiR-19a-3p had no effect on ESCC cell proliferation, whereas miR-19a-3p overexpression promoted the invasion and migration of ESCC cells. qRT-PCR verification and western blot analysis showed that LV-miR-19a-3p-inhibition downregulated cell division cycle 42 (CDC42), Rac family small GTPase 1 (RAC1), and p21 activated kinase 1 (PAK1).ConclusionsOverexpression of miR-19a-3p increased the invasion and migration of ESCC cells via the RAC1/CDC42-PAK1 pathway, suggesting that this pathway mediates the effect of miR-19a-3p on promoting LNM in ESCC.

CircRNA_2646 functions as a ceRNA to promote progression of esophageal squamous cell carcinoma via inhibiting miR-124/PLP2 signaling pathway

MicroRNA-124 (miR-124) has been predicted as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). However, factors contributing to miR-124 reduction remain unclear. Circular RNAs (circRNAs) are a new family of non-coding RNAs with gene regulatory potential via interacting with miRNAs. We predicted three circRNAs, including CircRNA_14359, CircRNA_2646, and CircRNA_129, that could interact with miR-124 by bioinformatics analysis and determined their expressions in ESCC tissues and adjacent normal tissues. We found that CircRNA_2646 was up-regulated in ESCC, negatively correlated with the expression of miR-124 and positively associated with TNM stage and lymph node metastasis of ESCC. Luciferase reporter assay showed that CircRNA_2646 interacted with miR-124 in ESCC Eca109 and TE-1 cells. Moreover, ectopical overexpression of CircRNA_2646 accelerated cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT), but restoration of miR-124 abrogated these functions and promoted Bcl-2-dependent cell apoptosis. Furthermore, it was found that the oncogene Proteolipid Protein 2 (PLP2) was the target gene of miR-124. In Eca109 and TE-1 cells, restoration of miR-124 decreased the level of PLP2 and inhibited PLP2-induced cell proliferation, migration, invasion, and EMT, but enhanced cell apoptosis. The in vivo study confirmed that CircRNA_2646 promoted ESCC development by repressing miR-124 and activating PLP2. Taken together, we identified that CircRNA_2646 functioned as an inhibitor in miR-124 signaling pathway in ESCC for carcinogenesis and could be a promising target for ESCC therapy.

Long Noncoding RNA VESTAR Regulates Lymphangiogenesis and Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma by Enhancing VEGFC mRNA Stability.

Lymph node metastasis is one of the most malignant clinical features in patients with esophageal squamous cell carcinoma (ESCC). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with ESCC. Long noncoding RNAs (lncRNA) play a critical role in the development and progression of human cancers. However, the role and mechanism of lncRNAs in lymph node metastasis remain largely unknown. Here we show that VEGFC mRNA stability-associated long noncoding RNA (VESTAR) is involved in lymph node metastasis of ESCC. VESTAR was overexpressed in ESCC tissues and was predictive of poor prognosis in patients with ESCC. In ESCC, NXF1 and SRSF3 facilitated nuclear export of VESTAR to the cytoplasm, which was associated with lymph node metastasis. Depletion of VESTAR inhibited ESCC-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, VESTAR directly bound and stabilized VEGFC mRNA. VESTAR also interacted with HuR, a positive regulator of VEGFC mRNA stability, and increased HuR binding to VEGFC mRNA. Our study reveals a novel lncRNA-guided mechanism of lymph node metastasis in ESCC and may provide a potential target for treatment of ESCC lymphatic metastasis. SIGNIFICANCE: These findings illustrate the lncRNA-guided regulation of VEGFC mRNA stability via direct RNA-RNA interactions, highlighting a therapeutic target for patients with ESCC with lymphatic metastasis.

PTIP Inhibits Cell Invasion in Esophageal Squamous Cell Carcinoma via Modulation of EphA2 Expression.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy and treatment failure is largely due to metastasis and invasion. Aberrant tumor cell adhesion is often associated with tumor progression and metastasis. However, the exact details of cell adhesion in ESCC progression have yet to be determined. In our study, the clinical relevance of Pax2 transactivation domain-interacting protein (PTIP/PAXIP1) was analyzed by immunohistochemistry of ESCC tissues. We found that low expression of PTIP was associated with lymph node metastasis in ESCC, and loss-of-function approaches showed that depletion of PTIP promoted ESCC cell migration and invasion both in vitro and in vivo. Analysis integrating RNA-seq and ChIP-seq data revealed that PTIP directly regulated ephrin type-A receptor 2 (EphA2) expression in ESCC cells. Moreover, PTIP inhibited EphA2 expression by competing with Fosl2, which attenuated the invasion ability of ESCC cells. These results collectively suggest that PTIP regulates ESCC invasion through modulation of EphA2 expression and hence presents a potential therapeutic target for its treatment.

Predicting Lymph Node Metastasis Using Computed Tomography Radiomics Analysis in Patients With Resectable Esophageal Squamous Cell Carcinoma.

We investigated the value of radiomics data, extracted from pretreatment computed tomography images of the primary tumor (PT) and lymph node (LN) for predicting LN metastasis in esophageal squamous cell carcinoma (ESCC) patients. A total 338 ESCC patients were retrospectively assessed. Primary tumor, the largest short-axis diameter LN (LSLN), and PT and LSLN interaction term (IT) radiomic features were calculated. Subsequently, the radiomic signature was combined with clinical risk factors in multivariable logistic regression analysis to build various clinical-radiomic models. Model performance was evaluated with respect to the fit, overall performance, differentiation, and calibration. A clinical-radiomic model, which combined clinical and PT-LSLN-IT radiomic signature, showed favorable discrimination and calibration. The area under curve value was 0.865 and 0.841 in training and test set. A venous computed tomography radiomic model based on the PT, LSLN, and IT radiomic features represents a novel noninvasive tool for prediction LN metastasis in ESCC.

Microarray analysis of miRNA based on the regional lymph node metastasis status of esophageal squamous cell carcinoma.

BackgroundTendency toward extensive regional lymph node metastasis (LNM) is an important clinical characteristic of esophageal squamous cell carcinoma (ESCC) and differs greatly between patients. MicroRNAs (miRNAs) are involved in the invasion and metastasis of ESCC. We performed a microarray analysis of miRNAs based on LNM status to identify tumor-specific miRNAs for the prediction of LNM in ESCC.MethodsFour pairs of ESCC tumor tissues with or without LNM were selected for microarray analysis to identify differentially expressed miRNAs, then 50 tumor tissue samples were selected to verify the differences of the screened miRNAs with quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The relations between candidate miRNAs and clinicopathologic features were analyzed to confirm tumor specificity in the prediction of LNM in ESCC. Target gene prediction using miRWalk2.0 was used to analyze the potential mechanisms of the tumor-specific miRNAs.ResultsThe present microarray analysis identified significant differential expression in 62 miRNAs in the ESCC samples with LNM, compared to those without LNM. Of these, 19 miRNAs were selected for qRT-PCR verification, and three miRNAs were significantly upregulated in ESCC samples with LNM compared to those without LNM. The three miRNAs were not significantly associated with any other clinicopathologic features except for the TNM stage and could be regarded as tumor-specific miRNAs capable of predicting LNM in ESCC. Finally, 858 mRNAs were significantly associated with tumor-specific miRNAs and possibly involved in the regulation of LNM in ESCC.ConclusionsThe present microarray analysis based on LNM status identified three tumor-specific miRNAs for predicting regional LNM in ESCC, which provides valuable clues for potential mechanism research and guarantees further investigation.