Esophageal Squamous Cell Carcinoma Tumor Tissues Research Articles

BTN3A1 promotes tumor progression and radiation resistance in esophageal squamous cell carcinoma by regulating ULK1-mediated autophagy

Radiotherapy is one of the most effective treatments for esophageal squamous cell carcinoma (ESCC); however, radioresistance is a clinical problem that must urgently be solved. Here, we found that butyrophilin subfamily 3 member A1 (BTN3A1) is upregulated in ESCC tumor tissues compared with nontumor tissues. We also evaluated BTN3A1 expression in patients with ESCC receiving adjuvant radiotherapy. The results demonstrated that BTN3A1 upregulation predicts a poor prognosis for ESCC patients. BTN3A1 overexpression promotes ESCC cell proliferation in vitro and in vivo. Moreover, BTN3A1 knockdown sensitized ESCC cells to radiation. We further explored the mode of death involved in BTN3A1-mediated radioresistance. Previous studies have shown that apoptosis, autophagy, necrosis, pyroptosis and ferroptosis are important for the survival of ESCC cells. We performed an RT-PCR array and western blotting (WB) to identify the mode of death and revealed for the first time that BTN3A1 promotes cell radioresistance by activating autophagy. In addition, by performing immunoprecipitation and mass spectrometry analyses, we found that BTN3A1 regulated the expression of UNC-51-like autophagy activating kinase 1(ULK1) and promoted its phosphorylation to subsequently initiate autophagy. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay results indicated that BTN3A1 is a novel direct target of hypoxia inducible factor-1α (HIF-1α). HIF-1α, a transcription factor, promotes BTN3A1 transcription upon irradiation. Overall, the present study is the first to show that BTN3A1 plays a key role in radioresistance and that targeting BTN3A1 might be a promising strategy to improve radiotherapy efficacy in patients with ESCC.

Identification of an IGF2BP2-Targeted Peptide for Near-Infrared Imaging of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies globally. Peptide-based tumor-targeted imaging is critical for ESCC imaging. In this study, we aim to identify a peptide-targeting IGF2BP2 that specifically binds to human ESCC for near-infrared imaging of esophageal cancer. Applying phage display techniques, we identified a peptide target for IGF2BP2 which was confirmed to be highly expressed in ESCC cell lines or tumor tissue and may serve as an imaging target for ESCC. We conjugated the peptide to the NIRF group, Cy5, and further evaluated the targeting efficacy of the probe at a cellular level and in animal tumor models. The Cy5 conjugated peptide (P12-Cy5) showed a high binding affinity to human ESCC cells in vitro. In vivo, optical imaging also validated the tumor-targeting ability of P12-Cy5 in KYSE-30-bearing subcutaneous ESCC tumor models. Furthermore, the results of biodistribution showed a significantly higher fluorescence intensity in tumors compared to scrambled peptide, which is consistent with in vivo observations. In summary, an IGF2BP2-targeted peptide was successfully identified. In vitro and in vivo experiments confirmed that P12-Cy5 has high affinity, specificity and tumor-targeting properties. Thus, P12-Cy5 is a prospective NIR probe for the imaging of ESCC.

Cysteinyl-tRNA Synthetase 1 Promotes Ferroptosis-Induced Cell Death via Regulating GPX4 Expression.

Esophageal squamous cell carcinoma (ESCC) has still been considered to be the most common malignant tumors in China. Emerging evidence indicates that cysteinyl-tRNA synthetase 1 (CARS1) has been considered as a ferroptosis-related gene in ESCC. However, the roles and molecular mechanisms of CARS1 in ferroptosis-induced cell death of ESCC are still largely unknown. In our study, we investigated an aberrantly upregulated gene in ESCC tumor tissues CARS1 significantly inhibited cell proliferation, and the ability of migration and invasion promoted the relative level of MDA and ROS and decreased GPX4 expression level in two ESCC cell lines. Mechanistically, both the ferroptosis inhibitor ferrostatin-1 and its inducer erastin were further used and indicated that CARS1 participated in the ferroptosis-induced cell death. Together, these results revealed that CARS1 has a critical function in the progression of ESCC by promoting ferroptosis-induced cell death.

Forming cytoplasmic stress granules PURα suppresses mRNA translation initiation of IGFBP3 to promote esophageal squamous cell carcinoma progression

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

Interleukin 13 participates in terminal differentiation of esophageal squamous cell carcinoma cells.

BackgroundIn China, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of all esophageal cancer cases. Interleukin 13 (IL-13) was widely reported to play a key role in tumor progression. Our previous study reported that IL-13 was a favorable predictive marker for the overall survival of esophageal squamous cell carcinoma (ESCC) patients, but how IL-13 contributes to ESCC progression remains unknown. This study aims to explore the role of IL-13 and its underlying downstream molecular mechanisms in ESCC progression.MethodsTissue microarrays including 262 primary ESCC tumor tissues were collected and analyzed. The expression of IL-13 in ESCC tumor tissue was detected with immunohistochemistry staining (IHC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to qualify the expressions of KRT13, KRT4 and 15-lipoxygenase-1 (15-LOX-1) in cultured ESCC cell lines with recombinant IL-13 treatment.ResultsIL-13 was expressed in the esophageal epithelium cells and ESCC tumor cells. High IL-13 expression in ESCC tumor cells predicted a good prognosis for patients. Recombinant human IL-13 raised KRT13 and 15-LOX-1 mRNA levels, but lowered KRT4 mRNA level 15-LOX-1 in ESCC cells in vitro.ConclusionsIn summary, our study suggests that IL-13 might improve the prognosis of ESCC by promoting the terminal differentiation of ESCC cells. This may offer potential new therapeutic target for early treatment of ESCC.

Characteristics and interplay of esophageal microbiota in esophageal squamous cell carcinoma

BackgroundEsophageal microbiota may influence esophageal squamous cell carcinoma (ESCC) pathobiology. Therefore, we investigated the characteristics and interplay of the esophageal microbiota in ESCC.MethodsWe performed 16S ribosomal RNA sequencing on paired esophageal tumor and tumor-adjacent samples obtained from 120 primarily ESCC patients. Analyses were performed using quantitative insights into microbial 2 (QIIME2) and phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2). Species found to be associated with ESCC were validated using quantitative PCR.ResultsThe microbial diversity and composition of ESCC tumor tissues significantly differed from tumor-adjacent tissues; this variation between subjects beta diversity is mainly explained by regions and sampling seasons. A total of 56 taxa were detected with differential abundance between the two groups, such as R. mucilaginosa, P. endodontalis, N. subflava, H. Pylori, A. Parahaemolyticus, and A. Rhizosphaerae. Quantitative PCR confirmed the enrichment of the species P. endodontalis and the reduction of H. Pylori in tumor-adjacent tissues. Compared with tumor tissue, a denser and more complex association network was formed in tumor-adjacent tissue. The above differential taxa, such as H. Pylori, an unclassified species in the genera Sphingomonas, Haemophilus, Phyllobacterium, and Campylobacter, also participated in both co-occurrence networks but played quite different roles. Most of the differentially abundant taxa in tumor-adjacent tissues were negatively associated with the epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2), erb-b2 receptor tyrosine kinase 4 (ERBB4), and fibroblast growth factor receptor 1 (FGFR1) signaling pathways, and positively associated with the MET proto-oncogene, receptor tyrosine kinase (MET) and phosphatase and tensin homolog (PTEN) signaling pathways in tumors.ConclusionAlterations in the microbial co-occurrence network and functional pathways in ESCC tissues may be involved in carcinogenesis and the maintenance of the local microenvironment for ESCC.

Exosomal miR-301a-3p from esophageal squamous cell carcinoma cells promotes angiogenesis by inducing M2 polarization of macrophages via the PTEN/PI3K/AKT signaling pathway

BackgroundGrowing evidence has indicated that tumor-associated macrophages (TAMs) promote tumor angiogenesis. However, the mechanisms underlying the pro-angiogenic switch of TAMs remains unclear. Here, we examined how exosomal miR-301a-3p secreted by esophageal squamous cell carcinoma (ESCC) cells triggers the pro-angiogenic switch of TAMs.MethodsWe quantified miR-301a-3p levels in ESCC tumors using qRT-PCR. Macrophage phenotypes were identified using flow cytometry and qRT-PCR. The pro-angiogenic ability of TAMs was measured using the CCK-8 assay, scratch assay, Transwell migration and invasion assay, and tube formation assay. The mechanism by which exosomal miR-301a-3p secreted by ESCC cells triggers the pro-angiogenic switch of TAMs was elucidated using western blots, qRT-PCR, and a dual-luciferase reporter assay.ResultsWe observed anomalous miR-301a-3p overexpression in ESCC tumor tissues and cell lines. Then, we verified that ESCC-derived exosomes promoted angiogenesis by inducing macrophage polarization into M2 type, and exosomal miR-301a-3p secreted by ESCC cells was responsible for this effect. Finally, we discovered that exosomal miR-301a-3p promoted M2 macrophage polarization via the inhibition of PTEN and activation of the PI3K/AKT signaling pathway, subsequently promoting angiogenesis via the secretion of VEGFA and MMP9.ConclusionThe pro-angiogenic switch of TAMs is triggered by exosomal miR-301a-3p secreted from ESCC cells via the PTEN/PI3K/AKT signaling pathway. Although tumor angiogenesis can be regulated by a wide range of factors, exosomal miR-301a-3p could hold promise as a novel anti-angiogenesis target for ESCC treatment.

GPx8 regulates apoptosis and autophagy in esophageal squamous cell carcinoma through the IRE1/JNK pathway

Glutathione peroxidase 8 (GPx8) belongs to a family of enzymes that have a critical role in controlling levels of reactive oxygen species (ROS). GPX family members have been associated with several cancers. Here, we examined the role of GPx8 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical staining and western blot analysis were used to study the clinical significance of GPx8 in ESCC tissue. GPx8 was further evaluated in cells by MTT assay and colony formation. RT-PCR, western blot, immunofluorescence staining, TUNEL assay, TEM, and flow cytometry were used to assess the molecular mechanism underlying endoplasmic reticulum (ER) stress associated with GPx8 in ESCC cells. Xenografted tumor growth was used to assess the in vivo role of GPx8. We found that GPx8 was overexpressed in both ESCC cell lines and tumor tissue. GPx8 knockdown significantly suppressed ESCC proliferation and induced autophagy and apoptosis in ESCC cell lines, whereas GPx8 overexpression led to increased proliferation and inhibition of apoptosis. GPx8-mediated inhibition of apoptosis was associated with the ER stress pathway through inositol-requiring enzyme 1 (IRE1) and Jun N-terminal kinase (JNK). Knockdown of GPx8 in xenograft models of ESCC resulted in a significant reduction in tumor weight and volume, which was further reduced with IRE1 or JNK inhibitors. Our study suggests that GPx8 regulates apoptosis and autophagy in ESCC through the IRE1/JNK pathway in response to ER stress. Targeting this pathway might be a potential therapeutic strategy for ESCC.

Long Noncoding RNA PCAT6 Regulates Cell Proliferation and Migration in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the sixth most common cancer type in East Asian countries. Mounting evidences illustrated that long noncoding RNAs (lncRNAs) play important roles in a variety of human cancers, including ESCC. LncRNA PCAT6 has been identified as a tumor promoter in multiple cancers. However, the roles and underlying mechanism of PCAT6 in ESCC remain largely unclear. In the current study, we discovered that lncRNA PCAT6, which was aberrantly upregulated in ESCC tumor tissues, significantly promoted cell proliferation and migration in ESCC cell lines Eca-109 and Kyse-30 cells. Flow cytometry assays showed that PCAT6 knockdown promoted the apoptosis of ESCC cells. Mechanistically, RNA-seq and Gene Ontology analyses indicated that PCAT6 knockdown influenced the expression of genes that participated in cell proliferation and migration. Furthermore, real-time PCR and western blot assays validated that knockdown of PCAT6 could increase the levels of GDF15 and DUSP4 in Eca-109 and Kyse-30 cells. In brief, our findings reveal that lncRNA PCAT6 plays an oncogenic role in the progression of ESCC by inhibiting the expression of genes related to cell proliferation and migration.

Sh-HNF1A-AS1 reduces the epithelial-mesenchymal transition and stemness of esophageal cancer cells

HNF1A-AS1 is a cancer-related lncRNA. Elevated expression of HNF1A-AS1 has been observed in various malignancies including esophageal squamous cell carcinoma (ESCC). However, the role of HNF1A-AS1 in ESCC has not been fully understood. This study aimed to investigate the potential role of HNF1A-AS1 in ESCC. Expression of HNF1A-AS1, miRNA (miR)-298, and transcription factor 4 (TCF4) was detected using qRT-PCR. The interactions between HNF1A-AS1 and miR-298 or miR-298 and TCF4 were evaluated. Short hairpin RNAs (shRNAs) were used to knock down HNF1A-AS1 (sh-HNF1A-AS1). The EMT and stemness of ESCC cells were detected. HNF1A-AS1 was overexpressed in ESCC tumor tissues and cells. miR-298 was validated as a direct target of HNF1A-AS1. sh-HNF1A-AS1 significantly inhibited EMT and stemness of ESCC cells. The high expression of miR-298 significantly inhibited the expression of TCF4, and further inhibited the expression of N-cadherin and stemness-related genes. Animal experiments showed that sh-HNF1A-AS1 significantly inhibited tumor growth and increased the level of miR-298 in tissues. In conclusion, knockdown of HNF1A-AS1 could inhibit EMT and stemness by regulating the miR-298/TCF4 axis.

PD-1H Expression Associated With CD68 Macrophage Marker Confers an Immune-Activated Microenvironment and Favorable Overall Survival in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma (EC) in China. Although the PD-1 inhibitor pembrolizumab has been approved to treat patients with EC, its therapeutic efficacy is limited. Thus, additional immunotherapeutic targets for EC treatment are needed. Programmed Death-1 Homolog (PD-1H) is a negative checkpoint regulator that inhibits antitumor immune responses. Here, PD-1H expression in 114 patients with ESCC was evaluated by immunohistochemistry. Next, 12 randomly selected tumor tissue sections were used to assess the colocalization of PD-1H protein and multiple immune markers by multiplex immunohistochemistry. Our results demonstrated that PD-1H was expressed at high frequency in ESCC tumor tissues (85.1%). PD-1H protein was predominantly expressed in CD68+ tumor-associated macrophages and expressed at low levels in CD4+ T cells and CD8+ T cells in ESCC tumor tissues. Furthermore, based on ESCC data in The Cancer Genome Atlas (TCGA), the gene expression levels of PD-1H were positively associated with the infiltration levels of immune-activated cells especially CD8+ cytotoxic T cells. In contrast, the gene expression levels of PD-1H were negatively correlated with myeloid-derived suppressor cells (MDSCs). Importantly, PD-1H expression in tumor sites was significantly correlated with favorable overall survival in patients with ESCC. Collectively, our findings first provided direct information on the PD-1H expression pattern and distribution in ESCC, and positive correlation of PD-1H expression with overall survival suggested PD-1H expression levels could be a significant prognostic indicator for patients with ESCC. Future studies need to explore the immunoregulatory of PD-1H in the tumor microenvironment of ESCC.

Immuno-oncological role of 20S proteasome alpha-subunit 3 in aggravating the progression of esophageal squamous cell carcinoma.

PSMA3, a member of the proteasome subunit, has been shown to play a major player in protein degradation. Reportedly, PSMA3 functions as a negative regulator in various cancers including colon, pancreatic and gastric cancers. However, the contributions of PSMA3 to the progression of esophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. Therefore, in this study, we investigated whether PSMA3 is involved in ESCC progression and the potential underlying mechanism. The results revealed that PSMA3 was highly expressed in the ESCC tumor tissues and functioned as a negative indicator according to the data from The Cancer Genome Atlas (TCGA)/Gene Expression Omnibus (GEO) datasets and clinical patients' samples. Pathway enrichment analysis showed that PSMA3 was closely correlated with ESCC cancer stemness and the inflammatory response; however, this correlation was absent after knockdown of PSMA3 in vitro. We further demonstrated that PSMA3 suppressed CD8+ T-cells infiltration depending on the C-C motif chemokine ligand 3 (CCL3)/C-C motif chemokine receptor 5 (CCR5) axis. Collectively, these results demonstrate the role of PSMA3 in ESCC cancer stemness and the negative regulation of CD8 T-cells infiltration mediated by PSMA3. The results of this study may provide a potential target for the immuno-oncology effect of PSMA3 in ESCC therapy.

Analyzing the Expression Level and Associated Clinical Characteristics of miRNA-138 in Esophageal Squamous Cell Carcinoma in Iranian Patients

Background: Esophageal Squamous Cell Carcinoma (ESCC) is the seventh most common and lethal malignancy worldwide with a high incidence in Iran. Esophageal cancer has a poor prognosis and low 5-years survival rate. Despite several studies on esophageal cancer, its underlying mechanism remains unclear. Therefore, it is significant to analyze the molecular factors involved in disease for introducing effective diagnosis and therapeutic approaches. Accordingly, the aberrant expression of miRNA was demonstrated in the etiology of esophageal cancer, and applying them as detecting biomarkers was further considered by researchers. This study aimed to evaluate the expression level of miRNA-138 in the tumor tissues of ESCC and normal adjacent tissues. Objective: In total, 35 samples of tumor and normal tissues of the esophagus were collected from ESCC patients. Methods: Then, RNA extraction was conducted and the expression level of miRNA-138 was assessed in the collected tissue samples, applying real-time RT-PCR. Moreover, the relationship between the miRNA-138 expression level and the patient’s clinical characteristics was assessed. Results: According to the obtained results, the expression of miR-138 was significantly downregulated in the tumor tissues of ESCC, compared to normal adjacent tissues (P<0.05). Additionally, the level of miRNA-138 downregulation was significantly correlated with tumor differentiation degree and metastasis status (P<0.05). Conclusion: The significant downregulation of miRNA-138 in the tumor samples of ESCC indicated the role of this epigenetic alteration in esophageal carcinogenesis. Therefore, applying the miRNA-138 could be considered as a potential molecular biomarker for ESCC diagnosis and screening.

Aberrant MCM10 SUMOylation induces genomic instability mediated by a genetic variant associated with survival of esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the common gastrointestinal malignancy with an inferior prognosis outcome. DNA replication licensing aberration induced by dysregulation of minichromosome maintenance proteins (MCMs) causes genomic instability and cancer metastasis. SUMOylation modification plays a pivotal role in regulation of genomic integrity, while its dysregulation fueled by preexisting germline variants in cancers remains poorly understood.MethodsFirstly, we conducted two‐stage survival analysis consisting of an exome‐wide association study in 904 ESCC samples and another independent 503 ESCC samples. Then, multipronged functional experiments were performed to illuminate the potential biological mechanisms underlying the promising variants, and MCM10 influences the ESCC progression. Finally, we tested the effects of MCM10 inhibitors on ESCC cells.ResultsA germline variant rs2274110 located at the exon 15 of MCM10 was identified to be significantly associated with the prognosis of ESCC patients. Individuals carrying rs2274110‐AA genotypes confer a poor survival (hazard ratio = 1.61, 95% confidence interval = 1.35–1.93, p = 1.35 × 10−7), compared with subjects carrying rs2274110‐AG/GG genotypes. Furthermore, we interestingly found that the variant can increase SUMOylation levels at K669 site (Lys[K]699Arg[R]) of MCM10 protein mediated by SUMO2/3 enzymes, which resulted in an aberrant overexpression of MCM10. Mechanistically, aberrant overexpression of MCM10 facilitated the proliferation and metastasis abilities of ESCC cells in vitro and in vivo by inducing DNA over‐replication and genomic instability, providing functional evidence to support our population finding that high expression of MCM10 is extensively presented in tumor tissues of ESCC and correlated with inferior survival outcomes of multiple cancer types, including ESCC. Finally, MCM10 inhibitors Suramin and its analogues were revealed to effectively block the metastasis of ESCC cells.ConclusionsThese findings not only demonstrate a potential biological mechanism between aberrant SUMOylation, genomic instability and cancer metastasis, but also provide a promising biomarker aiding in stratifying ESCC individuals with different prognosis, as well as a potential therapeutic target MCM10.

Long non-coding RNA small nucleolar RNA host gene 7 facilitates the proliferation, migration, and invasion of esophageal cancer cells by regulating microRNA-625.

Esophageal cancer (EC) is a highly aggressive malignant tumor, of which esophageal squamous cell carcinoma (ESCC) constitutes the main subtype. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) has been extensively studied in many tumors and has been confirmed to be an oncogene; however, it has yet to be investigated in an ESCC study. Therefore, this study intended to uncover the role of SNHG7 in ESCC. Quantitative real-time polymerase chain reaction was applied to measure the expression levels of SNHG7 and miR-625 in ESCC tumor tissues and cell lines. Cell Counting Kit-8 assay, 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and Transwell assay were conducted to assess the proliferation, migration, and invasion ESCC cell. We verified the interaction between SNHG7 and miR-625 by performing the dual luciferase reporter gene experiment. Compared to that in adjacent normal tissues and HET1A cell lines, the expression level of SNHG7 in ESCC tumor tissues and ESCC cell lines was up-regulated, while the expression level of miR-625 was down-regulated. ESCC cell proliferation, migration, and invasion were significantly promoted by SNHG7 overexpression but inhibited by silencing of SNHG7. Further, luciferase reporter gene experiments confirmed that SNHG7 interacted with miR-625, and rescue experiments showed that SNHG7 promoted the malignant phenotype by inhibiting miR-625. SNHG7 is up-regulated in ESCC tumor tissues and cell lines, while miR-625 is expressed at a low level. SNHG7 is able to facilitate the proliferation, migration, and invasion of ESCC cells by targeting miR-625.

RNA sequencing reveals the expression profiles of circRNA and identifies a four-circRNA signature acts as a prognostic marker in esophageal squamous cell carcinoma

BackgroundCircRNAs with tissue-specific expression and stable structure may be good tumor prognostic markers. However, the expression of circRNAs in esophageal squamous cell carcinoma (ESCC) remain unknown. We aim to identify prognostic circRNAs and construct a circRNA-related signature in ESCC.MethodsRNA sequencing was used to test the circRNA expression profiles of 73 paired ESCC tumor and normal tissues after RNase R enrichment. Bioinformatics methods, such as principal component analysis (PCA), t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm, unsupervised clustering and hierarchical clustering were performed to analyze the circRNA expression characteristics. Univariate cox regression analysis, random survival forests-variable hunting (RSFVH), Kaplan–Meier analysis, multivariable Cox regression and ROC (receiver operating characteristic) curve analysis were used to screen the prognostic circRNA signature. Real-time quantitative PCR (qPCR) and fluorescence in situ hybridization(FISH) in 125 ESCC tissues were performed.ResultsCompared with normal tissues, there were 11651 differentially expressed circRNAs in cancer tissues. A total of 1202 circRNAs associated with ESCC prognosis (P < 0.05) were identified. Through bioinformatics analysis, we screened a circRNA signature including four circRNAs (hsa_circ_0000005, hsa_circ_0007541, hsa_circ_0008199, hsa_circ_0077536) which can classify the ESCC patients into two groups with significantly different survival (log rank P < 0.001), and found its predictive performance was better than that of the TNM stage(0.84 vs. 0.66; 0.65 vs. 0.62). Through qPCR and FISH experiment, we validated the existence of the screened circRNAs and the predictive power of the circRNA signature.ConclusionThe prognostic four-circRNA signature could be a new prognostic biomarker for ESCC, which has high clinical application value.

Heterogeneity Analysis of Esophageal Squamous Cell Carcinoma in Cell Lines, Tumor Tissues and Patient-Derived Xenografts.

Esophageal Squamous Cell Carcinoma (ESCC) is the predominant type of Esophageal Cancer (EC), accounting for nearly 88% of EC incidents worldwide. Importantly, it is also a life-threatening cancer for patients diagnosed in advanced stages, with only a 20% 5-year survival rate due to a limited number of actionable targets and therapeutic options. Increasing evidence has shown that inter-tumor and intra-tumor heterogeneity are widely distributed across ESCC tumor tissues. In our work, multi-omics data from ESCC cell lines, tumor tissue, normal tissue and Patient-Derived Xenograft (PDX) tissues were analyzed to investigate the heterogeneity among ESCC samples at the DNA, RNA, and protein level. We identified enrichment of ECM-receptor interaction and Focal adhesion pathways from the subset of protein-coding genes with non-silent mutations in ESCC patients. We also found that TP53, TTN, KMT2D, CSMD3, DNAH5, MUC16 and DST are the most frequently mutated genes in ESCC patient samples. Out of the identified genes, TP53 is the most frequently mutated, with 84 distinct non-silent mutation variants. We observed that p.R248Q, p.R175G/H, and p.R273C/H are the most common TP53 mutation variants. The diversity of TP53 mutations reveal its importance in ESCC progression and may also provide promising targets for precision therapeutics. Additionally, we identified the Olfactory transduction as the top signaling pathway, enriched from genes uniquely expressed in The Cancer Genome Atlas (TCGA)-ESCC patient tumor tissues, which may provide implications for the exact roles of the corresponding genes in ESCC. Cyclic nucleotide-gated channel subunit beta 1(CNGB1), a gene belonging to the Olfactory transduction pathway, was found exclusively overexpressed in ESCC. Expression of CNGB1 could serve as a marker, indicating potential diagnostic or therapeutic value. Finally, we investigated heterogeneity in the context of the ESCC PDX model, which is an emerging tool used to predict drug response and recapitulate tumor behavior in vivo. We observed trans-species heterogeneity in as high as 75% of the identified proteins, indicating that the ambiguity of proteins should be addressed by specific strategies to avoid drawing false conclusions. The identification and characterization of gene mutation and expression heterogeneity across different ESCC datasets, including various novel TP53 mutations, ECM-receptor interaction, Focal adhesion, and Olfactory transduction pathways (CNGB1), provide researchers with evidence and implications for accurate research and precision therapeutic development.

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.

Identification of miR-515-3p and its targets, vimentin and MMP3, as a key regulatory mechanism in esophageal cancer metastasis: functional and clinical significance

Metastasis is the main factor of treatment failure in cancer patients, but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed. This study aims to explore novel key metastasis-related microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). By comparing miRNA profiles of the highly metastatic ESCC cell sublines, we established through serial in vivo selection with the parental cells, we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues, further decreased in metastatic tumors, and moreover, markedly associated with advanced stage, metastasis and patient survival. The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers, and more importantly, suppress invasion and metastasis of ESCC cells. Mechanistically, we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3 (MMP3) expression by binding to the coding sequence and 3′untranslated region, respectively. In addition, the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues, which may lead to deregulation of miR-515-3p expression in ESCC. Furthermore, our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice. Taken together, this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

MEX3A is upregulated in esophageal squamous cell carcinoma (ESCC) and promotes development and progression of ESCC through targeting CDK6.

Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed malignant tumors worldwide and identified as a serious threat to human health. The role of MEX3A in ESCC remains unclear. In this study, we found that MEX3A was upregulated in tumor tissues of ESCC and positively associated with more advanced tumor stage, higher risk of lymphatic metastasis and poor prognosis. The downregulation of MEX3A in ESCC cell lines could induce inhibition of cell proliferation, colony formation, cell migration, and the promotion of cell apoptosis, while MEX3A overexpression exhibited opposite effects. In vivo experiments also verified the inhibition of ESCC induced by MEX3A knockdown. Moreover, we identified CDK6 as a potential target of MEX3A, which was also upregulated in ESCC. Further studies demonstrated that knockdown of CDK6 showed similar effects on the development of ESCC with MEX3A. More importantly, it was illustrated that CDK6 knockdown could alleviate the promotion effects of MEX3A overexpression on ESCC. In conclusion, MEX3A was identified as a tumor promotor in the development and progression of ESCC by targeting CDK6, which may be considered as a novel prognostic indicator and therapeutic target in treatment of ESCC.