KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Iron-Nitrogen Amendment Reduced Perfluoroalkyl Acids' Phyto-Uptake in the Wheat-Soil Ecosystem: Contributions of Dissolved Organic Matters in Soil Solution and Root Extracellular Polymeric Substances.

Understanding the mechanisms underlying perfluoroalkyl acids (PFAAs) translocation, distribution, and accumulation in wheat-soil ecosystems is essential for agricultural soil pollution control and crop ecological risk assessment. This study systematically investigated the translocation of 13 PFAAs under different iron and nitrogen fertilization conditions in a wheat-soil ecosystem. Short-chain PFAAs including PFBA, PFPeA, PFHxA, and PFBS mostly accumulated in soil solution (10.43-55.33%) and soluble extracellular polymeric substances (S-EPS) (11.39-14.77%) by the adsorption to amino- (-NH2) and hydroxyl (-OH) groups in dissolved organic matter (DOM). Other PFAAs with longer carbon chain lengths were mostly distributed on the soil particle surface by hydrophobic actions (74.63-94.24%). Iron-nitrogen amendments triggered (p < 0.05) soil iron-nitrogen cycling, rhizospheric reactive oxygen species fluctuations, and the concentration increases of -NH2 and -OH in the DOM structure. Thus, the accumulation capacity of PFAAs in soil solution and root EPS was increased. In sum, PFAAs' translocation from soil particles to wheat root was synergistically reduced by iron and nitrogen fertilization through increased adsorption of soil particles (p < 0.05) and the retention of soil solution and root EPSs. This study highlights the potential of iron-nitrogen amendments in decreasing the crop ecological risks to PFAAs' pollution.

Spatial analysis of stromal signatures identifies invasive front carcinoma-associated fibroblasts as suppressors of anti-tumor immune response in esophageal cancer.

BACKGROUND: Increasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC). METHODS: Herein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages. RESULTS: We demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy. CONCLUSIONS: Our newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Impacts of iron amendments and per-fluoroalkyl substances' bio-availability to the soil microbiome in wheat ecosystem.

Per-fluoroalkyl substances (PFASs) have become ubiquitous in farmland ecosystems and pose risks to agricultural safety, and iron is often applied to farmland soils to reduce the availability of pollutants. However, the effects of iron amendment on the availability of PFASs in the soil and on the soil microbiome are not well understood. Here, we investigated the responses of wheat soil containing PFASs to iron addition using a 21-day experiment. Our results showed that iron amendment enhanced PFAS availability (p < 0.05) and stimulated superoxide dismutase (SOD) activity in the wheat soil (p < 0.05), but iron amendment decreased the activities of soil catalase (CAT) and peroxidase (POD) (p < 0.05). Soil bacterial community was more structurally stable than fungal community in response to iron addition, while species' pools were more stable in fungi than in bacteria (p < 0.05). Finally, PFPeA's availability in the wheat soil was the most important abiotic factors driving community succession of iron-cycling bacteria (p < 0.05). These results highlighted the potential interactions among PFASs' availability and microbial iron cycling in wheat farmland soil ecosystems and provided guidance in farmland environmental conservation and management.

Prognostic impact of lymph node harvest for patients with node-negative esophageal squamous cell carcinoma: a large-scale multicenter study.

BACKGROUND: We examined the association between the number of resected lymph nodes and survival to determine the optimal lymphadenectomy for thoracic esophageal squamous cell carcinoma (ESCC) patients with negative lymph node. METHODS: We included 1,836 patients from Chinese three high-volumed hospitals with corresponding clinicopathological characters such as gender, age, tumor location, tumor grade and TNM stage of patients. The median follow-up of included patients was 45.7 months (range, 1.03-117.3 months). X-Tile plot was used to identify the lowest number of lymphadenectomy. The multivariate model's construction was in use of parameters with clinical significance for survival and a nomogram based on clinical variable with P<0.05 in Cox regression analysis. Both two models were validated using a cohort extracted from the Surveillance, Epidemiology, and End Results (SEER) 18 registries database between 1975 and 2016 (n=951). RESULTS: More lymphadenectomy numbers were significantly associated with better survival in patients both in training cohort [hazard ratio (HR) =0.980; 95% confidence interval (CI): 0.971-0.988; P<0.001] and validation cohort (HR =0.980; 95% CI: 0.968-0.991; P=0.001). Cut-off point analysis determined the lowest number of 9 for thoracic ESCC patients in N0 stage through training cohort (C-index: 0.623; sensitivity: 80.7%; 1 - specificity: 72.5%) when compared with 10 in validation cohort (C-index: 0.643; sensitivity: 78.2%; 1 - specificity: 63.0%). The cut-off points of 9 were examined in training cohort and validated in the divided cohort from validation cohort (all P<0.05). Meanwhile, nomograms for both cohorts were constructed and the calibration curves for both cohorts agreed well with the actual observations in terms of predicting 3- and 5-year survival, respectively. CONCLUSIONS: Larger number for lymphadenectomy was associated with better survival in thoracic ESCC patients in N0 stage. Nine was what we got as the lowest number for lymphadenectomy in pN0 ESCC patients through this study, and our result should be confirmed further.

P300/CBP-associated factor (PCAF)-mediated acetylation of Fascin at lysine 471 inhibits its actin-bundling activity and tumor metastasis in esophageal cancer.

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

Prognostic Role of Nodal Skip Metastasis in Thoracic Esophageal Squamous Cell Carcinoma: A Large-Scale Multicenter Study.

BACKGROUND: Nodal-skip metastasis (NSM) is found in esophageal squamous cell carcinoma (ESCC), but its prognostic role is controversial. This study aimed to investigate the prognostic value of NSM for thoracic ESCC patients. METHODS: Categorization of NSM was according to the N groupings of Japan Esophagus Society (JES) staging system, which is dependent on tumor location. Using the Kaplan-Meier method and Cox-regression analysis, this study retrospectively analyzed the overall survival (OS) for 2325 ESCC patients after radical esophagectomy at three high-volume esophageal cancer centers. Predictive models also were constructed. RESULTS: The overall NSM rate was 20% (229/1141): 37.4% in the in upper, 12.9% in the middle, and 22.2% in the lower thoracic ESCC. The patients with NSM always had a better prognosis than those without NSM. Furthermore, NSM was an independent prognostic factor for thoracic ESCC patients (hazard ratio [HR], 0.633; 95% confidence interval [CI], 0.499-0.803; P < 0.001). By integrating the prognostic values of NSM and N stage, the authors proposed the new N staging system. The categories defined by the new N staging system were more homogeneous in terms of OS than those defined by the current N system. Moreover, the new N system was shown to be an independent prognostic factor also for thoracic ESCC patients (HR, 1.607; 95% CI, 1.520-1.700; P < 0.001). Overall, the new N system had slightly better homogeneity, discriminatory ability, and monotonicity of gradient than the current N system. CONCLUSIONS: This study emphasized the prognostic power of NSM and developed a modified node-staging system to improve the efficiency of the current International Union for Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) N staging system.

Identification of key genes by integrating DNA methylation and next-generation transcriptome sequencing for esophageal squamous cell carcinoma.

Aberrant DNA methylation leads to abnormal gene expression, making it a significant regulator in the progression of cancer and leading to the requirement for integration of gene expression with DNA methylation. Here, we identified 120 genes demonstrating an inverse correlation between DNA methylation and mRNA expression in esophageal squamous cell carcinoma (ESCC). Sixteen key genes, such as SIX4, CRABP2, and EHD3, were obtained by filtering 10 datasets and verified in paired ESCC samples by qRT-PCR. 5-Aza-dC as a DNA methyltransferase (DNMT) inhibitor could recover their expression and inhibit clonal growth of cancer cells in seven ESCC cell lines. Furthermore, 11 of the 16 genes were correlated with OS (overall survival) and DFS (disease-free survival) in 125 ESCC patients. ChIP-Seq data and WGBS data showed that DNA methylation and H3K27ac histone modification of these key genes displayed inverse trends, suggesting that there was collaboration between DNA methylation and histone modification in ESCC. Our findings illustrate that the integrated multi-omics data (transcriptome and epigenomics) can accurately obtain potential prognostic biomarkers, which may provide important insight for the effective treatment of cancers.

A Novel Three-Gene Model Predicts Prognosis and Therapeutic Sensitivity in Esophageal Squamous Cell Carcinoma.

To precisely predict the clinical outcome and determine the optimal treatment options for patients with esophageal squamous cell carcinoma (ESCC) remains challenging. Prognostic models based on multiple molecular markers of tumors have been shown to have superiority over the use of single biomarkers. Our previous studies have identified the crucial role of ezrin in ESCC progression, which prompted us to hypothesize that ezrin-associated proteins contribute to the pathobiology of ESCC. Herein, we explored the clinical value of a molecular model constructed based on ezrin-associated proteins in ESCC patients. We revealed that the ezrin-associated proteins (MYC, PDIA3, and ITGA5B1) correlated with the overall survival (OS) and disease-free survival (DFS) of patients with ESCC. High expression of MYC was associated with advanced pTNM-stage (P=0.011), and PDIA3 and ITGA5B1 were correlated with both lymph node metastasis (PDIA3: P < 0.001; ITGA5B1: P=0.001) and pTNM-stage (PDIA3: P=0.001; ITGA5B1: P=0.009). Furthermore, we found that, compared with the current TNM staging system, the molecular model elicited from the expression of MYC, PDIA3, and ITGA5B1 shows higher accuracy in predicting OS (P < 0.001) or DFS (P < 0.001) in ESCC patients. Moreover, ROC and regression analysis demonstrated that this model was an independent predictor for OS and DFS, which could also help determine a subgroup of ESCC patients that may benefit from chemoradiotherapy. In conclusion, our study has identified a novel molecular prognosis model, which may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for ESCC treatment.

LOXL2 Upregulates Phosphorylation of Ezrin to Promote Cytoskeletal Reorganization and Tumor Cell Invasion.

Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family and its nonsecreted, catalytically dead spliced isoform L2Δ13, enhance cell migration and invasion, stimulate filopodia formation, modulate the expression of cytoskeletal genes, and promote tumor development and metastasis in vivo. We previously showed that LOXL2 reorganizes the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC) cells, however, the underlying molecular mechanisms were not identified. Here, using interactome analysis, we identified ezrin (EZR), fascin (FSCN1), heat shock protein beta-1 (HSPB1), and tropomodulin-3 (TMOD3) as actin-binding proteins that associate with cytoplasmic LOXL2, as well as with its L2Δ13 variant. High levels of LOXL2 and L2Δ13 and their cytoskeletal partners correlated with poor clinical outcome in patients with ESCC. To better understand the significance of these interactions, we focused on the interaction of LOXL2 with ezrin. Phosphorylation of ezrin at T567 was greatly reduced following depletion of LOXL2 and was enhanced following LOXL2/L2Δ13 reexpression. Furthermore, LOXL2 depletion inhibited the ability of ezrin to promote tumor progression. These results suggest that LOXL2-induced ezrin phosphorylation, which also requires PKCα, is critical for LOXL2-induced cytoskeletal reorganization that subsequently promotes tumor cell invasion and metastasis in ESCC. In summary, we have characterized a novel molecular mechanism that mediates, in part, the protumorigenic activity of LOXL2. These findings may enable the future development of therapeutic agents targeting cytoplasmic LOXL2. SIGNIFICANCE: LOXL2 and its spliced isoform L2Δ13 promote cytoskeletal reorganization and invasion of esophageal cancer cells by interacting with cytoplasmic actin-binding proteins such as ezrin.

Lymph Node Station-Based Nodal Staging System for Esophageal Squamous Cell Carcinoma: A Large-Scale Multicenter Study.

BACKGROUND: The American Joint Committee on Cancer (AJCC) nodal staging for esophageal squamous cell carcinoma (ESCC) has been defined by the number of metastatic lymph nodes (N system). However, the precise counting of individual positive lymph nodes is difficult and unreliable in some clinical settings, which calls for a more available and reliable system. This study examined the performance of a newly proposed nodal staging category, termed the S system, based on the number of metastatic lymph node stations. METHODS: Using the Kaplan-Meier method and Cox-regression analysis, this study retrospectively analyzed the overall survival (OS) of 2285 ESCC patients who underwent esophagectomy in three major China hospitals. Predictive models were constructed, and C-indices were computed to evaluate the discriminatory power of the S system, and to compare it with the N system. RESULTS: The categories defined by the S system were more homogeneous in terms of OS than those defined by the N system. Overall, the S system had a slightly better C-index (p = 0.659) than the N system ((p = 0.658). Subgroup analyses also showed that the C-index of the S system was slightly better than that of the N system for each subgroup of sex and age, but the two were comparable for each subgroup defined by the tumor location. CONCLUSION: The S system demonstrated a competing prognostic performance compared with the current AJCC N system. Due to the relatively easy accessibility of the number of metastatic lymph node stations, the S system may offer an easier option for cancer staging without a loss of discriminative power.

Diagnostic and prognostic value of serum L1-cell adhesion molecule in esophageal squamous cell carcinoma.

OBJECTIVE: L1 cell adhesion molecule (L1CAM) has been found to be dysregulated in several types of human cancers. Here, we aimed to determine the level of soluble L1CAM in serum of patients with esophageal squamous cell carcinoma (ESCC). METHODS: Serum levels of L1CAM were determined by an enzyme-linked immunosorbent assay (ELISA) in 191 patients with ESCC and 94 normal controls. Receiver operating characteristics (ROC) was employed to calculate diagnostic accuracy. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the logrank test. RESULTS: Levels of L1CAM were significantly lower in all ESCC patients than in normal controls (P < 0.001). Detection of serum L1CAM provided a sensitivity of 28.3%, a specificity of 90.4% and an area under the curve (AUC) of 0.644 (95% CI: 0.579-0.710) in diagnosing ESCC. Similar results were observed in the diagnosis of early-stage ESCC (26.2% sensitivity, 90.4% specificity, and an AUC of 0.629). Moreover, decreased level of L1CAM was correlated with depth of tumor invasion (P < 0.05). Kaplan-Meier analysis showed that lower serum L1CAM level was significantly related to shorter overall survival time (P = 0.036) and disease-free survival time (P = 0.021) of ESCC patients. CONCLUSIONS: Our study demonstrated that serum L1CAM might serve as a potential biomarker for the diagnosis and prognosis of ESCC.

MASAN: a novel staging system for prognosis of patients with oesophageal squamous cell carcinoma.

BACKGROUND: Oesophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers worldwide. Treatment of ESCC is in progress through accurate staging and risk assessment of patients. The emergence of potential molecular markers inspired us to construct novel staging systems with better accuracy by incorporating molecular markers. METHODS: We measured H scores of 23 protein markers and analysed eight clinical factors of 77 ESCC patients in a training set, from which we identified an optimal MASAN (MYC, ANO1, SLC52A3, Age and N-stage) signature. We constructed MASAN models using Cox PH models, and created MASAN-staging systems based on k-means clustering and minimum-distance classifier. MASAN was validated in a test set (n = 77) and an independent validation set (n = 150). RESULTS: MASAN possessed high predictive accuracies and stratified ESCC patients into three prognostic groups that were more accurate than the current pTNM-staging system for both overall survival and disease-free survival. To facilitate clinical utilisation, we also constructed MASAN-SI staging systems based on staining indices (SI) of protein markers, which possessed similar prognostic performance as MASAN. CONCLUSION: MASAN provides a good alternative staging system for ESCC prognosis with a high precision using a simple model.

Protein-coding genes combined with long noncoding RNA as a novel transcriptome molecular staging model to predict the survival of patients with esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal carcinoma in China. This study was to develop a staging model to predict outcomes of patients with ESCC. METHODS: Using Cox regression analysis, principal component analysis (PCA), partitioning clustering, Kaplan-Meier analysis, receiver operating characteristic (ROC) curve analysis, and classification and regression tree (CART) analysis, we mined the Gene Expression Omnibus database to determine the expression profiles of genes in 179 patients with ESCC from GSE63624 and GSE63622 dataset. RESULTS: Univariate cox regression analysis of the GSE63624 dataset revealed that 2404 protein-coding genes (PCGs) and 635 long non-coding RNAs (lncRNAs) were associated with the survival of patients with ESCC. PCA categorized these PCGs and lncRNAs into three principal components (PCs), which were used to cluster the patients into three groups. ROC analysis demonstrated that the predictive ability of PCG-lncRNA PCs when applied to new patients was better than that of the tumor-node-metastasis staging (area under ROC curve [AUC]: 0.69 vs. 0.65, P < 0.05). Accordingly, we constructed a molecular disaggregated model comprising one lncRNA and two PCGs, which we designated as the LSB staging model using CART analysis in the GSE63624 dataset. This LSB staging model classified the GSE63622 dataset of patients into three different groups, and its effectiveness was validated by analysis of another cohort of 105 patients. CONCLUSIONS: The LSB staging model has clinical significance for the prognosis prediction of patients with ESCC and may serve as a three-gene staging microarray.

Association between 5-lipoxygenase expression, and malignant behaviors and poor prognosis in esophageal squamous cell carcinoma.

5-lipoxygenase (5-LO) catalyzes the first step of arachidonic acid metabolism to inflammatory mediator leukotrienes. The present study assessed 5-LO expression in esophageal squamous cell carcinoma (ESCC) tissue specimens for associations with clinicopathological and survival data from patients, then explored 5-LO activity in ESCC cells in vitro. 5-LO expression was detected in tissue microarrays containing 297 ESCC samples using immunohistochemistry. Kaplan-Meier curves were used to analyze the survival significance of 5-LO expression and relative risk was evaluated using the multivariate Cox proportional hazards model. Cultured tumor cells were subjected to gene transfection, western blotting, and cell migration and proliferation assays. 5-LO protein was primarily expressed in normal cell cytoplasm and/or membrane, and never in the whole cytoplasm, whereas 5-LO was expressed diffusely in ESCC tissues with nearly homogeneous whole-cytoplasm staining. 5-LO expression was significantly associated with tumor regional lymph node metastasis (P=0.013) and pTNM stage (P=0.004). 5-LO expression was associated with poor overall survival (P=0.029). Multivariate analysis demonstrated that 5-LO overexpression was an independent prognostic factor for ESCC patients (P=0.041). Furthermore, the inhibition of 5-LO expression reduced ESCC cell viability and migration in vitro. These data provide further evidence that the upregulation of 5-LO expression is associated with advanced stages of disease and poor ESCC prognosis, and that 5-LO expression may independently predict overall survival in patients with ESCC. The inhibition of 5-LO expression reduced ESCC malignant behavior in vitro.

Super-Enhancer-Driven Long Non-Coding RNA LINC01503, Regulated by TP63, Is Over-Expressed and Oncogenic in Squamous Cell Carcinoma.

BACKGROUND & AIMS: Long non-coding RNAs (lncRNAs) are expressed in tissue-specific pattern, but it is not clear how these are regulated. We aimed to identify squamous cell carcinoma (SCC)-specific lncRNAs and investigate mechanisms that control their expression and function. METHODS: We studied expression patterns and functions of 4 SCC-specific lncRNAs. We obtained 113 esophageal SCC (ESCC) and matched non-tumor esophageal tissues from a hospital in Shantou City, China, and performed quantitative reverse transcription polymerase chain reaction assays to measure expression levels of LINC01503. We collected clinical data from patients and compared expression levels with survival times. LINC01503 was knocked down using small interfering RNAs and oligonucleotides in TE7, TE5, and KYSE510 cell lines and overexpressed in KYSE30 cells. Cells were analyzed by chromatin immunoprecipitation sequencing, luciferase reporter assays, colony formation, migration and invasion, and mass spectrometry analyses. Cells were injected into nude mice and growth of xenograft tumors was measured. LINC01503 interaction with proteins was studied using fluorescence in situ hybridization, RNA pulldown, and RNA immunoprecipitation analyses. RESULTS: We identified a lncRNA, LINC01503, which is regulated by a super enhancer and is expressed at significantly higher levels in esophageal and head and neck SCCs than in non-tumor tissues. High levels in SCCs correlated with shorter survival times of patients. The transcription factor TP63 bound to the super enhancer at the LINC01503 locus and activated its transcription. Expression of LINC01503 in ESCC cell lines increased their proliferation, colony formation, migration, and invasion. Knockdown of LINC01503 in SCC cells reduced their proliferation, colony formation, migration, and invasion, and the growth of xenograft tumors in nude mice. Expression of LINC01503 in ESCC cell lines reduced ERK2 dephosphorylation by DUSP6, leading to activation of ERK signaling via MAPK. LINC01503 disrupted the interaction between EBP1 and the p85 subunit of PI3K, increasing AKT signaling. CONCLUSIONS: We identified an lncRNA, LINC01503, which is increased in SCC cells compared with non-tumor cells. Increased expression of LINC01503 promotes ESCC cell proliferation, migration, invasion, and growth of xenograft tumors. It might be developed as a biomarker of aggressive SCCs in patients.

A three-lncRNA signature predicts overall survival and disease-free survival in patients with esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence shows that dysregulated long non-coding RNAs (lncRNAs) can serve as potential biomarkers for cancer prognosis. However, lncRNA signatures, as potential prognostic biomarkers for esophageal squamous cell carcinoma (ESCC), have been seldom reported. METHODS: Based on our previous transcriptome RNA sequencing analysis from 15 paired ESCC tissues and adjacent normal tissues, we selected 10 lncRNAs with high score rank and characterized the expression of those lncRNAs, by qRT-PCR, in 138 ESCC and paired adjacent normal samples. These 138 patients were divided randomly into training (n = 77) and test (n = 59) groups. A prognostic signature of lncRNAs was identified in the training group and validated in the test group and in an independent cohort (n = 119). Multivariable Cox regression analysis evaluated the independence of the signature in overall survival (OS) and disease-free survival (DFS) prediction. GO and KEGG pathway analysis, combined with cell transwell and proliferation assays, are applied to explore the function of the three lncRNAs. RESULTS: A novel three-lncRNA signature, comprised of RP11-366H4.1.1 (ENSG00000248370), LINC00460 (ENSG00000233532) and AC093850.2 (ENSG00000230838), was identified. The signature classified patients into high-risk and low-risk groups with different overall survival (OS) and disease-free survival (DFS). For the training group, median OS: 23.1 months vs. 39.1 months, P < 0.001; median DFS: 15.2 months vs. 33.3 months, P < 0.001. For the test group, median OS: 23 months vs. 59 months, P < 0.001; median DFS: 16.4 months vs. 50.8 months, P < 0.001. For the independent cohort, median OS: 22.4 months vs. 60.4 months, P < 0.001). The signature indicates that patients in the high-risk group show poor OS and DFS, whereas patients with a low-risk group show significantly better outcome. The independence of the signature was validated by multivariable Cox regression analysis. GO and KEGG pathway analysis for 588 protein-coding genes-associated with the three lncRNAs indicated that the three lncRNAs were involved in tumorigenesis. In vitro assays further demonstrated that the three lncRNAs promoted the migration and proliferation of ESCC cells. CONCLUSIONS: The three-lncRNA signature is a novel and potential predictor of OS and DFS for patients with ESCC.

SLC52A3 expression is activated by NF-κB p65/Rel-B and serves as a prognostic biomarker in esophageal cancer.

The human riboflavin transporter-3 (encoded by SLC52A3) plays a prominent role in riboflavin absorption. Interestingly, abnormal expression patterns of SLC52A3 in multiple types of human cancers have been recently noted. However, the molecular mechanisms underlying its dysregulation remain unclear. In this study, we find that SLC52A3 has two transcript variants that differ in the transcriptional start site, and encode different proteins: SLC52A3a and SLC52A3b. Importantly, aberrant expressions of SLC52A3 are associated with stepwise development of esophageal squamous cell carcinoma (ESCC) as well as the survival rates of ESCC patients. Functionally, SLC52A3a, but not SLC52A3b, strongly promotes the proliferation and colony formation of ESCC cells. Furthermore, SLC52A3 5'-flanking regions contain NF-κB p65/Rel-B-binding sites, which are crucial for mediating SLC52A3 transcriptional activity in ESCC cells. Chromatin immunoprecipitation and electrophoretic mobility shift assay reveal that p65/Rel-B bind to 5'-flanking regions of SLC52A3. Accordingly, NF-κB signaling upregulates SLC52A3 transcription upon TNFα stimulation. Taken together, these results elucidate the mechanisms underlying SLC52A3 overexpression in ESCC. More importantly, our findings identify SLC52A3 as both a predictive and prognostic biomarker for this deadly cancer.

Overexpression of Stathmin 1 correlates with poor prognosis and promotes cell migration and proliferation in oesophageal squamous cell carcinoma.

Stathmin 1 (STMN1) is a microtubule-regulated protein that plays an important role in tumour cell proliferation and migration. Overexpression of STMN1 is associated with clinicopathological characteristics in many human cancers. The aim of the present study was to investigate STMN1 expression, its correlation with clinicopathological characteristics, and its exact biological function in oesophageal squamous cell carcinoma (ESCC). STMN1 levels were measured in the ESCC tissue specimens of 276 patients by immunohistochemistry (IHC) to assess the prognostic efficacy of STMN1. IHC showed that patients with overexpression of STMN1 had a poorer prognosis compared with those with low expression, both in regards to 5-year overall survival (OS; 21.2 vs. 53.7%, P<0.001) and disease-free survival (DFS; 20.6 vs. 50.9%, P<0.001). STMN1 overexpression was associated with lower cell differentiation in tumour grade (correlation coefficient: 0.127, P=0.037). In multivariate analysis, STMN1 expression was found to be an independent prognostic factor for both OS (P<0.001; 95% CI, 1.555-2.970) and DFS (P=0.001; 95% CI, 1.978-2.444). Compared with the control, STMN1 downregulation significantly decreased cell migration, invasion and proliferation, whereas these were increased by STMN1 upregulation. STMN1 expression was significantly associated with prognosis and tumour differentiation in ESCC, indicating that STMN1 expression is an independent prognostic factor for ESCC and could be a potential biomarker. Regulating the expression of STMN1 could influence tumour cell motility, invasion and proliferation.

L1CAM drives oncogenicity in esophageal squamous cell carcinoma by stimulation of ezrin transcription.

L1 cell adhesion molecule (L1CAM) is highly expressed in various types of human cancers, displaying yet unknown molecular mechanisms underlying their oncogenic potential. Here, we found that L1CAM expression was significantly increased in esophageal squamous cell carcinoma (ESCC; n = 157) lesions compared with non-cancerous tissues. High tumorous L1CAM expression significantly correlated with reduced overall survival. Experimentally, L1CAM knockdown led to decreased cell growth, migration, and invasiveness in vitro, whereas overexpression of L1CAM showed the opposite effect. In nude mice, L1CAM depletion attenuated tumorigenesis and ability to penetrate the tissues surrounding ESCC cells. Gene set enrichment analysis (GSEA) and SubpathwayMiner analysis on gene expression profiles (microarray data on ESCC tissues, GSE53625; cDNA microarray data on L1CAM-knockdown ESCC cell line, GSE86268) suggested that L1CAM-co-expression genes were related to cell motility, cell proliferation, and regulation of actin cytoskeleton, validating the above experimental findings. Further mechanistical analysis showed that L1CAM upregulated the expression of the cytoskeletal protein ezrin via activating integrin ß1/MAPK/ERK/AP1 signaling and thus led to the malignant phenotypes of ESCC cells. Together, our findings suggest that L1CAM may be employed as a valuable prognosis marker and a therapeutic target for ESCC patients and that L1CAM promotes ESCC tumorigenicity by upregulating ezrin expression. KEY MESSAGES: L1CAM promotes growth and invasiveness of ESCC cells in vitro and in vivo. L1CAM upregulates the expression of ezrin by integrin α5ß1/MAPK/ERK/AP1 pathway. Ezrin is a key downstream effector in the L1CAM-promoted malignant phenotypes. High expression levels of both L1CAM and ezrin significantly correlated with reduced overall survival. Nuclear L1CAM is an independent prognosis marker for esophageal squamous cell carcinoma.