Esophageal Squamous Cell Carcinoma Clinical Samples Research Articles

Identification and Characterization of Metastasis-Initiating Cells in ESCC in a Multi-Timepoint Pulmonary Metastasis Mouse Model.

Metastasis is the biggest obstacle to esophageal squamous cell carcinoma (ESCC) treatment. Single-cell RNA sequencing analyses are applied to investigate lung metastatic ESCC cells isolated from pulmonary metastasis mouse model at multiple timepoints to characterize early metastatic microenvironment. A small population of parental KYSE30 cell line (Cluster S) resembling metastasis-initiating cells (MICs) is identified because they survive and colonize at lung metastatic sites. Differential expression profile comparisons between Cluster S and other subpopulations identified a panel of 7 metastasis-initiating signature genes (MIS), including CD44 and TACSTD2, to represent MICs in ESCC. Functional studies demonstrated MICs (CD44high) exhibited significantly enhanced cell survival (resistances to oxidative stress and apoptosis), migration, invasion, stemness, and in vivo lung metastasis capabilities, while bioinformatics analyses revealed enhanced organ development, stress responses, and neuron development, potentially remodel early metastasis microenvironment. Meanwhile, early metastasizing cells demonstrate quasi-epithelial-mesenchymal phenotype to support both invasion and anchorage. Multiplex immunohistochemistry (mIHC) staining of 4 MISs (CD44, S100A14, RHOD, and TACSTD2) in ESCC clinical samples demonstrated differential MIS expression scores (dMISs) predict lymph node metastasis, overall survival, and risk of carcinothrombosis.

LncRNA SNHG1 acts as a ceRNA for miR-216a-3p to regulate TMBIM6 expression in esophageal squamous cell carcinoma.

Background: The long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) has been demonstrated to play a crucial role in the progression of esophageal squamous cell carcinoma (ESCC). The current study aims to explore the deeper molecular mechanisms of SNHG1 in ESCC. Methods: Fifty patients with ESCC were enrolled to assess overall survival. Quantitative real-time PCR was performed to measure the levels of SNHG1, miR-216a-3p, and TMBIM6 in ESCC cells. Functional assessments of SNHG1 on ESCC cells were conducted using CCK-8 assay, flow cytometry, and Transwell assays. Western blot was conducted to detect the protein levels of TMBIM6 and proapoptotic proteins (Calpain and Caspase-12). The interaction among SNHG1, miR-216a-3p, and TMBIM6 was assessed with luciferase reporter assays. Results: Our study revealed that SNHG1 was notably increased in both clinical ESCC samples and cellular lines. Upregulation of SNHG1 in ESCC tissues was indicative of poor overall survival. Functionally, SNHG1 knockdown significantly inhibited the proliferation, migration, and invasion while promoting apoptosis in ESCC cells. Mechanistically, SNHG1 functioned as a competing endogenous RNA by sequestering miR-216a-3p to modulate TMBIM6 levels in ESCC cells. Notably, inhibiting miR-216a-3p or restoring TMBIM6 reversed the inhibitory effect induced by SNHG1 knockdown in ESCC cells. Conclusions: We demonstrate for the first time that SNHG1 may act as a competing endogenous RNA and promote ESCC progression through the miR-216a-3p/TMBIM6 axis. This highlights the potential of SNHG1 as a target for ESCC treatment.

Homeobox A7 promotes esophageal squamous cell carcinoma progression through C-C motif chemokine ligand 2-mediated tumor-associated macrophage recruitment.

Homeobox A7 (HOXA7) plays essential roles in multiple malignancies and was reported to be overexpressed in esophageal squamous cell carcinoma (ESCC). However, its functions in the ESCC tumor microenvironment remain to be explored. In this study, we showed that HOXA7 was overexpressed in ESCC among HOXA family members and correlated with tumor-associated macrophage (TAM) infiltration both in The Cancer Genome Atlas database and ESCC clinical samples. Moreover, transactivation of C-C motif chemokine ligand 2 (CCL2) by HOXA7 was identified (real-time quantitative PCR [RT-qPCR], western blot analysis, ELISA, and ChIP-qPCR), which was detected to drive chemotaxis and M2 polarization of macrophages both in vitro (Transwell assay) and in vivo (xenograft tumors models). In addition, CCL2 triggers macrophage expression of epidermal growth factor (EGF) (RT-qPCR and ELISA), which promotes tumor proliferation and metastasis by activating its receptor EGFR. In addition, EGF-induced ESCC cell proliferation and migration can be abrogated by HOXA7 knockdown (CCK-8 proliferation assay, EdU fluorescence, and Transwell assay). These results indicate a novel mechanistic role of HOXA7 in the cross-talk between ESCC and TAMs, which could be an underlying therapeutic target for ESCC.

Long noncoding RNAs with peptide-encoding potential identified in esophageal squamous cell carcinoma: KDM4A-AS1-encoded peptide weakens cancer cell viability and migratory capacity.

Currently, the knowledge of long noncoding RNA (lncRNA)-encoded peptides is quite lacking in esophageal squamous cell carcinoma (ESCC). In this study, we simultaneously identified six lncRNA open reading frames (ORFs) with peptide-coding abilities including lysine-specific demethylase 4A antisense RNA 1 (KDM4A-AS1) ORF by combining weighted gene co-expression network analysis (WGCNA) for ESCC clinical samples, ribosome footprints, ORF prediction, mass spectrometry (MS) identification, and western blotting. KDM4A-AS1 ORF-encoded peptide reduced ESCC cell viability and migratory ability. Co-immunoprecipitation and MS analysis revealed that KDM4A-AS1-encoded peptide specifically bound with 103 proteins in ESCC cells, and enrichment analysis suggested that peptide-bound proteins were related to fatty acid metabolism and redox process. Cell and molecular experiments demonstrated that KDM4A-AS1-encoded peptide inhibited stearoyl-CoA desaturase and fatty acid synthase expression, increased reactive oxygen species level, and reduced mitochondrial membrane potential in ESCC cells. In summary, multiple lncRNAs with translation potential were simultaneously identified by combining multiple approaches in ESCC, providing novel identificationstrategies for lncRNA-encoded peptides. Moreover, lncRNA KDM4A-AS1-encoded peptide weakened ESCC cell viability and migratory capacity and functioned in fatty acid metabolism and redox process.

PES1 reduces CD8+ T cell infiltration and immunotherapy sensitivity via interrupting ILF3-IL15 complex in esophageal squamous cell carcinoma

BackgroundAlthough immune checkpoint blockade (ICB) therapy has brought survival benefits to patients with specific cancer types, most of cancer patients remain refractory to the ICB therapy, which is largely attributed to the immunosuppressive tumor microenvironment. Thereby, it is urgent to profile key molecules and signal pathways responsible for modification of tumor microenvironment.MethodsMultiple databases of esophageal squamous cell carcinoma (ESCC) were integratively analyzed to screen candidate genes responsible for infiltration of CD8+ T cells. Expression of pescadillo ribosomal biogenesis factor 1 (PES1) in clinical ESCC samples was examined by qRT-PCR, western blotting, and immunohistochemistry. The mechanisms of PES1 were investigated via RNA sequencing and mass spectrometry followed by immunoprecipitation and proximity ligation assay. The clinical and therapeutic significance of PES1 in ESCC was comprehensively investigated using ESCC cells and mouse model.ResultsPES1 was significantly upregulated and correlated with poor prognosis in ESCC patients. PES1 knockdown decreased ESCC cell growth in vitro and in vivo and enhanced the efficacy of ICB therapy in mouse model, which was established through subcutaneous inoculation with ESCC cells. Analyses on RNA sequencing and mass spectrometry suggested that PES1 expression was negatively correlated with IL15 and ILF3 was one of the PES1-associated proteins. It has been known that ILF3 interacts with and stabilizes IL15 mRNA to increase IL15 protein level. Our data further indicated that PES1 interfered with the interaction between ILF3 and IL15 mRNA and impaired ILF3-mediated stabilization of IL15 mRNA, which eventually reduced the protein level of IL15. Interestingly, the inhibitory effect of ICB therapy boosted by PES1 knockdown dramatically antagonized by knockdown of IL15, which suppressed the tumor-infiltrated CD8+ T cells in ESCC. Finally, we confirmed the relationships among PES1, IL15, and CD8+ T cell infiltration in 10 locally advanced ESCC patients receiving ICB neoadjuvant therapy and demonstrated that ICB therapy would be more effective in those with low expression of PES1.ConclusionsAltogether, our findings herein provided novel insights on biological function and clinical significance of PES1 and suggested that high expression of PES1 could suppress ILF3-IL15 axis-mediated immunosurveillance and promote resistance to ICB through restraining tumor-infiltrated CD8+ T cells.

Src heterodimerically activates Lyn or Fyn to serve as targets for the diagnosis and treatment of esophageal squamous cell carcinoma.

Although Src is one of the oldest and most investigated oncoproteins, its function in tumor malignancy remains to be defined further. In this study, we demonstrated that the inhibition of Src activity by ponatinib effectively suppressed several malignant phenotypes of esophageal squamous cell carcinoma (ESCC) both in vitro and in vivo, whereas it did not produce growth-inhibitory effects on normal esophageal epithelial cells (NEECs). Importantly, we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src interacted with the members of Src-family kinases (SFKs), such as Fyn or Lyn, to form heterodimers. Src interactions with Fyn and Lyn phosphorylated the tyrosine sites in SH2 (Fyn Tyr185 or Lyn Tyr183) and kinase domains (Fyn Tyr420 or Lyn Tyr397), which critically contributed to ESCC development. By contrast, Src could not form heterodimers with Fyn or Lyn in NEECs. We used RNA sequencing to comprehensively demonstrate that the inhibition of Src activity effectively blocked several critical tumor-promoting pathways, such as JAK/STAT, mTOR, stemness-related, and metabolism-related pathways. Results of the real-time polymerase chain reaction (RT-PCR) assay confirmed that Lyn and Fyn were critical effectors for the Src-mediated expression of tumor growth or metastasis-related molecules. Furthermore, results of the clinical ESCC samples showed that the hyperactivation of pSrc Tyr419, Fyn Tyr185 or Tyr420, and Lyn Tyr183 or Tyr397 could be biomarkers of ESCC prognosis. This study illustrates that Src/Fyn and Src/Lyn heterodimers serve as targets for the treatment of ESCC.

Long noncoding RNA GK-IT1 promotes esophageal squamous cell carcinoma by regulating MAPK1 phosphorylation.

Long noncoding RNAs (lncRNAs) are implicated in the oncogenesis and metastasis of multiple human cancers. Nonetheless, the precise molecular mechanisms underlying the oncogenic role of lncRNA in esophageal squamous cell carcinoma (ESCC) remains to be clarified. The expression of GK intronic transcript 1 (GK-IT1) was analyzed using ESCC RNA-seq data from The Cancer Genome Atlas database. Quantitative real-time PCR was used to measure the expression of GK-IT1 in ESCC clinical samples and cells. The correlation between GK-IT1 expression and clinicopathological variables was examined using chi-squared tests. Kaplan-Meier survival and Cox regression analyses were employed to generate the survival curve and assess the prognostic value of GK-IT1. Functional experiments were utilized to explore the role of GK-IT1 in promoting cell migration, invasion, proliferation, and suppressing apoptosis and autophagy in ESCC. To understand the mechanism, an RNA pulldown assay, RNA immunoprecipitation, agarose gel electrophoresis, immunofluorescence, and co-immunoprecipitation assays were used. In this study we identified an unreported lncRNA, termed GK-IT1 that was aberrantly overexpressed in ESCC tissues and cells. GK-IT1 was closely associated with advanced clinical stage, and it was an independent prognostic indicator of ESCC. Functional assays verified that GK-IT1 significantly promoted ESCC proliferation, invasion, and migration, and suppressed ESCC apoptosis and autophagy. Furthermore, tumorigenesis experiments in nude mice indicated that GK-IT1 promoted ESCC tumor growth and metastasis. Mechanistically, GK-IT1 competitively bound to mitogen-activated protein kinase 1 (MAPK1) to prevent the interaction between dual specificity phosphatase 6 (DUSP6) and MAPK1, thereby controlling the phosphorylation of MAPK1 and promoting ESCC progression. Our study revealed that GK-IT1 competed with DUSP6 to attenuate the interaction between DUSP6 and MAPK1, leading to activation of the ERK/MAPK pathway, thereby promoting progression of ESCC. Our research indicated that GK-IT1 served as a novel potential target for the diagnosis and treatment of ESCC.

A Transcriptomic Liquid Biopsy Assay for Predicting Resistance to Neoadjuvant Therapy in Esophageal Squamous Cell Carcinoma.

The aim of this study was to establish a liquid-biopsy assay to predict response to neoadjuvant therapy (NAT) in esophageal squamous cell carcinoma (ESCC) patients. Pretreatment prediction of resistance to NAT is of great significance for the selection of treatment options in ESCC patients. In this study, we comprehensively translated tissue-based microRNA (miRNA) and messenger RNA (mRNA) expression biomarkers into a liquid biopsy assay. We analyzed 186 clinical ESCC samples, which included 128 formalin-fixed paraffin-embedded and a matched subset of 58 serum samples, from 2 independent institutions. We performed quantitative reverse-transcription polymerase chain reaction, and developed a resistance-prediction model using the logistic regression analyses. We first evaluated the potential of 4-miRNAs and 3-mRNAs panel, which robustly predicted resistance to NAT [area under the curve (AUC): 0.85]. Moreover, addition of tumor size to this panel increased predictive potential to establish a combination signature (AUC: 0.92). We successfully validated this signature performance in independent cohort, and our model was more accurate when the signature was combined with clinical predictors (AUC: 0.81) to establish a NAT resistance risk (NATRR) model. Finally, we successfully translated our NATRR model into a liquid biopsy assay (AUC: 0.78), and a multivariate regression analysis revealed this model as an independent predictor for response to NAT (odds ratio: 6.10; P < 0.01). We successfully developed a liquid biopsy-based assay that allows robust prediction of response to NAT in ESCC patients, and our assay provides fundamentals of developing precision-medicine.

FGFR2 maintains cancer cell differentiation via AKT signaling in esophageal squamous cell carcinoma

ABSTRACT Fibroblast growth factors (FGFs) and their receptors (FGFRs) are important for signaling to maintain cancer stem-like cells (CSCs) in esophageal squamous cell carcinoma (ESCC). However, which FGF receptor, 1, 2, 3, 4, and L1, is essential or whether FGFRs have distinct different roles in ESCC-CSCs is still in question. This study shows that FGFR2, particularly the IIIb isoform, is highly expressed in non-CSCs. Non-CSCs have an epithelial phenotype, and such cells are more differentiated in ESCC. Further, FGFR2 induces keratinocyte differentiation through AKT but not MAPK signaling and diminishes CSC populations. Conversely, knockdown of FGFR2 induces epithelial–mesenchymal transition (EMT) and enriches CSC populations in ESCC. Finally, data analysis using The Cancer Genome Atlas (TCGA) dataset shows that expression of FGFR2 significantly correlated with cancer cell differentiation in clinical ESCC samples. The present study shows that each FGFR has a distinct role and FGFR2-AKT signaling is a key driver of keratinocyte differentiation in ESCC. Activation of FGFR2-AKT signaling could be a future therapeutic option targeting CSC in ESCC.

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.

Autocrine Leukemia Inhibitory Factor Promotes Esophageal Squamous Cell Carcinoma Progression via Src Family Kinase-Dependent Yes-Associated Protein Activation.

The IL6 family of cytokines, including IL6 and leukemia-inhibitory factor (LIF), are induced during inflammation and are also expressed in many types of cancer where they play an important role in tumor development. IL6 family cytokines mainly activate the JAK-STAT3 pathway via the coreceptor, gp130, and IL6 is known to activate the Src family kinase (SFK)-Yes-associated protein (YAP) pathway. The current study investigated the role of autocrine LIF in human esophageal squamous cell carcinoma (ESCC) that highly expresses LIF. LIF knockdown had various effects on cancer cells, including profound changes in gene expression, suppression of cell proliferation, migration/invasion and sphere formation, and induction of apoptosis. Similar to IL6, LIF activated the SFK-YAP pathway as well as the JAK-STAT3 pathway. LIF-induced YAP activation was more important for cancer cell proliferation than LIF-induced STAT3 activation, and concomitant YAP and STAT3 activation completely compensated for the role of LIF in human ESCC growth. We also confirmed that SFK activation and LIF expression were correlated with YAP activation in human ESCC clinical samples. Furthermore, simultaneous inhibition of the SFK-YAP and JAK-STAT3 pathways in human ESCC cells was more effective at suppressing cell proliferation than single inhibition, and autocrine LIF signaling promoted human ESCC growth in vivo. Therefore, the LIF-SFK-YAP axis may represent a new therapeutic target for human ESCC. IMPLICATIONS: Autocrine LIF signaling promotes human ESCC progression via SFK-dependent YAP activation and is a new potential target of treatment for human ESCC.

EIF3H promotes aggressiveness of esophageal squamous cell carcinoma by modulating Snail stability

BackgroundOverexpression of eukaryotic translation initiation factor 3H (EIF3H) predicts cancer progression and poor prognosis, but the mechanism underlying EIF3H as an oncogene remains unclear in esophageal squamous cell carcinoma (ESCC).MethodsTCGA database and the immunohistochemistry (IHC) staining of ESCC samples were used and determined the upregulation of EIF3H in ESCC. CCK8 assay, colony formation assay and transwell assay were performed to examine the ability of cell proliferation and mobility in KYSE150 and KYSE510 cell lines with EIF3H overexpression or knockdown. Xenograft and tail-vein lung metastatic mouse models of KYSE150 cells with or without EIF3H knockdown were also used to confirm the function of EIF3H on tumor growth and metastasis in vivo. A potential substrate of EIF3H was screened by co-immunoprecipitation assay (co-IP) combined with mass spectrometry in HEK293T cells. Their interaction and co-localization were confirmed using reciprocal co-IP and immunofluorescence staining assay. The function of EIF3H on Snail ubiquitination and stability was demonstrated by the cycloheximide (CHX) pulse-chase assay and ubiquitination assay. The correlation of EIF3H and Snail in clinical ESCC samples was verified by IHC.ResultsWe found that EIF3H is significantly upregulated in esophageal cancer and ectopic expression of EIF3H in ESCC cell lines promotes cell proliferation, colony formation, migration and invasion. Conversely, genetic inhibition of EIF3H represses ESCC tumor growth and metastasis in vitro and in vivo. Moreover, we identified EIF3H as a novel deubiquitinating enzyme of Snail. We demonstrated that EIF3H interacts with and stabilizes Snail through deubiquitination. Therefore, EIF3H could promote Snail-mediated EMT process in ESCC. In clinical ESCC samples, there is also a positive correlation between EIF3H and Snail expression.ConclusionsOur study reveals a critical EIF3H-Snail signaling axis in tumor aggressiveness in ESCC and provides EIF3H as a promising biomarker for ESCC treatment.

Ubiquitin-specific peptidase 46 promotes tumor metastasis through stabilizing ENO1 in human esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) has high aggressiveness and poor prognosis, and is the major histological subtype of esophageal cancer in East Asia and East Africa. In this study, we found that USP46, a deubiquitinating enzyme, is overexpressed in clinical ESCC samples, especially in patients with positive lymph node metastasis. Moreover, USP46 enhances the migration and invasion of ESCC cells by mediating the EMT process in vitro, and promotes lymph nodes and lung metastasis of ESCC in vivo. In addition, we found that USP46 is a bona fide deubiquitinating enzyme to stabilize the protein level of ENO1 through deubiquitination. ENO1 protein level was also positively correlated with USP46 in the ESCC samples. In summary, these findings reveal the functional role of USP46 as a deubiquitinating enzyme on ESCC metastasis, providing us a potential therapeutic target for the treatment of ESCC.

&lt;p&gt;HSP90 Inhibitor Ganetespib (STA-9090) Inhibits Tumor Growth in c-Myc-Dependent Esophageal Squamous Cell Carcinoma&lt;/p&gt;

PurposeCurrently, the paucity of classical effective pharmacological drugs to treat esophageal squamous cell carcinoma (ESCC) is a major problem. The c-Myc (MYC) protein is a promising target as it is overexpressed in ESCC. MYC is a sensitive client protein of the heat shock protein 90 (HSP90) and, therefore, targeting the HSP90-MYC axis by inhibition of HSP90 is a potential therapeutic strategy for ESCC. Here, we evaluated the clinical application value of the HSP90 inhibitor (Ganetespib, STA-9090) as an anti-cancer agent for MYC-positive ESCC.Materials and MethodsWe first analyzed ESCC tissue microarrays and clinical tissue samples to determine MYC expression. The relationship between MYC and HSP90 was analyzed by co-immunoprecipitation assays and immunofluorescence. In in vitro cell models, cell growth was analyzed using the CCK-8 kit, and MYC protein expression was analyzed by Western blot. The in vivo antitumor activity of STA-9090 was assessed in two xenograft animal models.ResultsWe demonstrated that MYC-overexpressing ESCC cells were highly sensitive to STA-9090 treatment through suppressing ESCC cell proliferation, cell cycle progression and survival. Moreover, STA-9090 treatment decreased MYC expression, reducing the half-life of the MYC protein. We further established two xenograft mouse models using ESCC cells and clinical ESCC samples to validate the effectiveness of STA-9090 in vivo. In both xenograft models, STA-9090 substantially inhibited the growth of MYC-positive ESCC tumors in vivo. In contrast, STA-9090 treatment demonstrated no beneficial effects in mice with low-MYC expressing ESCC tumors.ConclusionIn conclusion, our data support that the HSP90 inhibitor, STA-9090, suppresses the expression of the MYC protein and interferes with HSP90-MYC protein–protein interaction. This, in turn, leads to inhibition of ESCC cell proliferation and promotion of apoptosis in ESCC cells in vitro and reduction of ESCC tumors in vivo. We propose, based on our findings, that STA-9090 is a potential novel therapeutic target for MYC-positive ESCC.

High TLR4 expression predicts a poor prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma.

Poor oral health is an independent risk factor for upper aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC). The pattern recognition receptor Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide in the cell walls of Gram-negative periodontal pathogens associated with the development and progression of ESCC. It is, therefore, plausible that TLR4 plays a crucial role in the pathogenesis of ESCC. We used an ESCC tissue microarray to confirm expression of TLR4 in patients with ESCC and to determine whether TLR4 expression status correlates with the clinicopathological features of these patients or their prognosis after esophagectomy. We also tested whether the combined expression statuses of TLR4 and TLR3 better correlate with prognosis in these patients than either parameter alone. Clinical ESCC samples from all 177 patients tested showed expression of TLR4. Moreover, high TLR4 expression (3 + and 2 +) correlated with poorer 5-year overall survival after esophagectomy than lower TLR4 expression (1 +) (p = 0.0491). Patients showing high TLR4 expression tended to have a poorer prognosis whether treated with surgery alone or with surgery and adjuvant chemotherapy. Univariate and multivariate analyses showed TLR4 expression status to be an independent prognostic factor affecting 5-year overall survival. Patients exhibiting high TLR4 expression with low TLR3 expression had a much poorer prognosis than other patients (p = < 0.0001). High TLR4 expression predicts a poor prognosis in advanced thoracic ESCC patients after esophagectomy.

MTA1 promotes tumorigenesis and development of esophageal squamous cell carcinoma via activating the MEK/ERK/p90RSK signaling pathway.

Metastasis-associated protein 1 (MTA1) is upregulated in multiple malignancies and promotes cancer proliferation and metastasis, but whether and how MTA1 promotes esophageal squamous cell carcinoma (ESCC) tumorigenesis remain unanswered. Here, we established an ESCC model in MTA1 transgenic mice induced by the chemical carcinogen 4-nitroquinoline 1-oxide (4-NQO) and found that MTA1 promotes ESCC tumorigenesis in mice. MTA1 overexpression was observed in ESCC cells and clinical ESCC samples. Overexpressed MTA1 increased colony formation and the invasiveness and migration of ESCC cells, whereas knock down of MTA1 in ESCC cells significantly decreased colony formation, invasion and migration in vitro and inhibited the growth of xenograft tumors in vivo. RNA sequencing (RNA-seq) analysis combined with western blot assays revealed that MTA1 promotes carcinogenesis by enhancing MEK/ERK/p90RSK signaling. The phosphorylation of MEK, ERK and their downstream target p90RSK was significantly decreased after MTA1 knockdown in ESCC cells and was increased in MTA1-overexpressing cells. Moreover, colony formation, invasion and migration potential were dramatically suppressed when cells overexpressing MTA1 were treated with MEK (PD0325901) or ERK (SCH772948) inhibitors. In conclusion, MTA1 plays a pivotal oncogenic role in ESCC tumorigenesis and development through activating the MEK/ERK/p90RSK pathway.

MiR-92b-3p Functions As A Key Gene In Esophageal Squamous Cell Cancer As Determined By Co-Expression Analysis.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy. The aims of the present study were to screen the critical miRNA and corresponding target genes that related to development of ESCC by weighted gene correlation network analysis (WGCNA) and investigate the functions by experimental validation.MethodsDatasets of mRNA and miRNA expression data were downloaded from GEO. The R software was used for data preprocessing and differential expression gene analysis. The differentially expressed protein-coding genes (DEGs) and miRNAs (DEMs) were selected (FDR <0.05 or |Fold Change (FC)| >1.5). Meanwhile, 81 expression data of ESCC patients in TCGA combined with clinic information were applied by WGCNA to create networks. The correlational analyses between each module and clinical parameters were conducted, and enrichment analyses of GO and KEGG were subsequently performed. Then, a series of experiments were conducted in ESCC cells by use of miRNA mimics.ResultsIn total, 4,023 DEGs and 328 DEMs were screened. After checking good genes and samples, 3,841 genes (3,696 DEGs and 145 DEMs) were used for WGCNA. As a consequence, altogether 11 gene modules were found. Among them, the brown modules were found to be strongly inversely associated with pathological grade. Meanwhile, has-mir-92b, the only miRNA in brown module, had a positive correlation with grade and negatively correlated with potential target gene (KFL4 and DCS2) in the same module. Furthermore, an increased expression of miR-92b-3p and down-regulated KLF4 and DSC2 protein was detected in the ESCC clinical samples. Up-regulated miR-92b-3p shortened G0/G1 phase and promote ESCC cells invasion and migration. Furthermore, we verified that DSC2 and KFL4 was target genes of miR-92b-3p by luciferase report assay.ConclusionWGCNA is an efficient approach to system biology. By this procedure, miR-92b-3p was identified as an ESCC-promoting gene by target KLF4 and DCS2.

SREBP2 is upregulated in esophageal squamous cell carcinoma and co‑operates with c‑Myc to regulate HMGCR expression.

Dysregulations of the mevalonate pathway (MVA) have been previously identified. Our previous study demonstrated that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the MVA pathway, was upregulated in esophageal squamous cell carcinoma (ESCC) and statin-inhibited ESCC tumorigenesis. However, the underlying mechanism of HMGCR regulation in ESCC remains unknown. In the present study, western blotting and immunohistochemistry analysis demonstrated that sterol regulatory element-binding protein 2 (SREBP2), the master regulator for HMGCR, was upregulated in ESCC clinical samples. Overexpression of SREBP2 expression in ESCC cell lines promoted the growth, migration and colony formation of cancer cells in the MTT, Boyden chamber and soft agar assays, respectively, which was inhibited by lovastatin. Downregulation of SREBP2 expression in ESCC cell lines inhibited the viability, and migration and colony formation abilities of cancer cells. Assessment of the molecular mechanism demonstrated that SREBP2 interacted with c-Myc and cooperated with c-Myc to activate HMGCR expression. Collectively, the present study identified SREBP2 as an oncogene associated with the tumorigenesis of ESCC and further demonstrated the therapeutic effects of statins in ESCC.

Upregulation of long noncoding RNA LINC00152 promotes proliferation and metastasis of esophageal squamous cell carcinoma.

Purpose: Esophageal cancer is a major cause of cancer-related mortality worldwide. The long noncoding RNA LINC00152 has been confirmed to play an oncogenic role in many cancers. However, the expression pattern and function of LINC00152 in human esophageal squamous cell carcinoma (ESCC) remain unclear. Materials and methods: We evaluated LINC00152 expression in ESCC by qPCR and in situ hybridization. Proliferation, apoptosis, cell cycle, migration and invasion were examined in ESCC cells knocked down for LINC00152 knockdown by siRNA. Furthermore, an mRNA microarray was performed in ESCC cells with LINC00152 knockdown. Results: LINC00152 was significantly upregulated in human ESCC clinical samples (P<0.001) and cell lines (P=0.008), and LINC00152 overexpression was related to lymphatic metastasis (P=0.03) and advanced pTNM classification (P=0.005). Furthermore, ESCC patients with LINC00152 overexpression had significantly shorter overall survival (P=0.007), and LINC00152 overexpression was an independent risk factor for overall survival of ESCC patients. LINC00152 knockdown inhibited the proliferation, migration and invasion of ESCC cells in vitro. In addition, mechanistic investigations through mRNA array and immunoblot analyses demonstrated that LINC00152 regulated the expression of several cell cycle-related proteins and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) interactions in vesicular transport pathway proteins. Conclusion: Our research indicated that LINC00152 exhibits oncogenic functions in ESCC and may represent a potential new target for ESCC therapy.

REV7 confers radioresistance of esophagus squamous cell carcinoma by recruiting PRDX2.

Radiotherapy has been widely used for the clinical management of esophageal squamous cell carcinoma. However, radioresistance remains a serious concern that prevents the efficacy of esophageal squamous cell carcinoma (ESCC) radiotherapy. REV7, the structural subunit of eukaryotic DNA polymerase ζ, has multiple functions in bypassing DNA damage and modulating mitotic arrest in human cell lines. However, the expression and molecular function of REV7 in ESCC progression remains unclear. In this study, we first examined the expression of REV7 in clinical ESCC samples, and we found higher expression of REV7 in ESCC tissues compared to matched adjacent or normal tissues. Knockdown of REV7 resulted in decreased colony formation and increased apoptosis in irradiated Eca‐109 and TE‐1 cells coupled with decreased tumor weight in a xenograft nude mouse model postirradiation. Conversely, overexpression of REV7 resulted in radioresistance in vitro and in vivo. Moreover, silencing of REV7 induced increased reactive oxygen species levels postirradiation. Proteomic analysis of REV7‐interacting proteins revealed that REV7 interacted with peroxiredoxin 2 (PRDX2), a well‐known antioxidant protein. Existence of REV7‐PRDX2 complex and its augmentation postirradiation were further validated by immunoprecipitation and immunofluorescence assays. REV7 knockdown significantly disrupted the presence of nuclear PRDX2 postirradiation, which resulted in oxidative stress. REV7‐PRDX2 complex also assembled onto DNA double‐strand breaks, whereas REV7 knockdown evidently increased double‐strand breaks that were unmerged by PRDX2. Taken together, the present study sheds light on REV7‐modulated radiosensitivity through interacting with PRDX2, which provides a novel target for ESCC radiotherapy.