Invasion Of ESCC Cells Research Articles

Corynoxine exerts the anti-tumor effect on esophageal squamous cell carcinoma principally via the EZH2-DUSP5-ERK1/2-mediated cell growth inhibition

BackgroundEsophageal cancer is one of the most prevalent malignant tumors and the sixth largest cause of tumor-associated death worldwide. Squamous cell carcinoma (ESCC) accounts for 85 % of all esophageal cancer cases. ESCC treatment remains to be significantly difficult. Corynoxine (Cory) is a tetracyclic hydroxyindole alkaloid isolated from Uncaria macrophylla. It is unclear whether Cory has an anti-tumor effect on ESCC. PurposeTo determine the anti-tumor activity of Cory and the associated mechanisms in ESCC. Study designCory's effects on proliferation, apoptosis, migration, and invasion, as well as the underlying molecular causes were assessed using two ESCC cell lines, KYSE150 and TE-1. A xenograft mouse model was then applied to evaluate the anti-tumor activity of Cory in vivo. MethodsWestern blot, assays including CCK-8, colony formation, EdU staining, TUNEL staining, cell scratch and Transwell, and a xenograft mouse model were used in this study. ResultsCory suppressed cell growth, provoked cell apoptosis, and hindered cell migration and invasion of ESCC cells. DUSP5 knockdown reduced the Cory-induced cell death and restored cell migration and invasion through ERK1/2 activation. Further analyses showed that Cory promoted DUSP5 expression via inhibiting EZH2 expression, leading to inactivation of ERK1/2 signaling and the subsequent cell growth inhibition of ESCC. In vivo experiments disclosed that Cory suppressed tumor growth of ESCC through upregulating DUSP5 expression. ConclusionsCory plays an anti-tumor role in ESCC by regulating EZH2-DUSP5-ERK1/2 signaling pathway. Cory may be promising to be a novel therapy for treating ESCC.

Prognostic modeling and Emerging therapeutic targets Unveiled through single-cell sequencing in esophageal squamous Cell carcinoma

ESCC presents a significant global health challenge due to its high mortality rates and varying responses to treatment. This underscores the critical need for novel diagnostic and predictive biomarkers to improve treatment outcomes.Initially, we conducted single-cell transcriptome sequencing on a total of 128,688 cells obtained from 10 patients as part of our research. Utilizing machine learning and cross-validation techniques, we developed a model incorporating 12 genes that distinguish malignant cells from non-malignant ones. In vitro, we explored the effects of IGFBP2 knockdown on the proliferation, invasion, and migration of ESCC cells. The clinical relevance of IGFBP2 was confirmed through IHC and Kaplan-Meier survival analyses. Furthermore, using bioinformatics tools such as GSVA and xCell on public databases, we discovered that high expression of IGFBP2 is associated with an immunosuppressive tumor microenvironment in ESCC, characterized by reduced CD8+ T cell infiltration. This was validated then through IHC.In summary, our study integrates single-cell sequencing and sophisticated computational techniques to highlight IGFBP2 as a promising biomarker and therapeutic target in ESCC.

NEK2 promotes the migration, invasion, proliferation of ESCC and mediates ESCC immunotherapy

PurposeEsophageal squamous cell carcinoma (ESCC) is a disease with a high incidence rate and high mortality worldwide. The Never in Mitosis A (NIMA) family member NIMA-related kinase 2 (NEK2) plays an important role in mitosis. However, the role of NEK2 in the pathogenesis of ESCC remains unclear. Patients and methodsThe expression and function of NEK2 in TCGA and GEO data sets were analyzed by bioinformatics. We verified the expression of NEK2 in ESCC tissues and cell lines by Western blotting and immunohistochemical methods and further explored the relationship between tumor stage and NEK2 expression. The differences in NEK2 expression and survival in patients with EC were verified by bioinformatics analysis. ESCC cell lines with stable knockdown of NEK2 were established by lentivirus-mediated shRNA delivery. The effects of NEK2 on ESCC cells were analyzed on the cytological level with assays including CCK-8, EdU, cell scratch, Transwell migration and invasion, colony formation, flow cytometry and apoptosis assays. Tumor growth was measured in a mouse xenograft model. ResultsWe found that NEK2 is highly expressed in ESCC tissues and ESCC cells and that the high expression of NEK2 is associated with poor tumor healing. Knockdown of the NEK2 gene inhibits the migration, proliferation, invasion and cell cycle of ESCC cells. Biologic analysis shows that NEK2 is involved in biological processes such as progression and apoptosis of esophageal cancer, and is related to E2F.Mechanistically, NEK2 knockdown decreases the expression levels of E2F1 and IGF2. NEK2 competes with the transcription factor E2F1 to bind CDC20, resulting in decreased degradation and increased expression of E2F1. IGF2 expression is also increased, which promotes the expression of thymidylate synthase, further promoting the drug resistance of ESCC cells. NEK2 is associated with immune infiltration in esophageal cancer. ConclusionNEK2 is highly expressed in ESCC and can promote the migration, proliferation and invasion of ESCC cells. NEK2 mediates ESCC immunotherapy.

NUPR1 induces autophagy and promotes the progression of Esophageal squamous cell carcinoma via the MAPK-mTOR pathway

PurposeEsophageal squamous cell carcinoma (ESCC) is a dominant pathological type in China. NUPR1 is a complex molecule implicated in various physiological and biological functions whose expression is upregulated in response to stress. Furthermore, autophagy is a vital physiological mechanism in the onset and metastasis of malignancies. This study aims to uncover the influence of NUPR1 on ESCC occurrence and development by regulating autophagy while also exploring its association with the MAPK signaling pathway. MethodsFirst, the differences in NUPR1 between ESCC and normal tissues were analyzed through online databases. Subsequently, the pathological tissues of clinical samples were stained and scored using immunohistochemistry. And NUPR1 expression in ESCC cells was investigated, as was the function of NUPR1 in the modulation of ESCC’s malignant behavior. Furthermore, a nude mouse ESCC xenograft model was developed. Finally, RNA sequencing was performed on NUPR1-downregulated ESCC cells, which was verified using WB. ResultsOur findings initially uncovered differences in the expression of NUPR1 in ESCC and normal tissues. In vitro experiments demonstrated that NUPR1 downregulation significantly inhibited ESCC cell proliferation, invasion, and migration, as well as promoted their apoptosis. Our xenograft model exhibited significant inhibition of ESCC tumors upon NUPR1 downregulation. Subsequently, RNA sequencing uncovered that NUPR1 regulates its malignant biological behavior through MAPK-mTOR signaling pathway. Finally, we found that NUPR1 downregulation can inhibit autophagic flux in ESCC. ConclusionCollectively, our findings show that NUPR1 enhances the progression of ESCC by triggering autophagy and is associated with the MAPK-mTOR signaling pathway.

SIRT2-mediated deacetylation of ACLY promotes the progression of oesophageal squamous cell carcinoma.

ATP citrate lyase (ACLY), as a key enzyme in lipid metabolism, plays an important role in energy metabolism and lipid biosynthesis of a variety of tumours. Many studies have shown that ACLY is highly expressed in various tumours, and its pharmacological or gene inhibition significantly inhibits tumour growth and progression. However, the roles of ACLY in oesophageal squamous cell carcinoma (ESCC) remain unclear. Here, our data showed that ACLY inhibitor significantly attenuated cell proliferation, migration, invasion and lipid synthesis in different ESCC cell lines, whereas the proliferation, migration, invasion and lipid synthesis of ESCC cells were enhanced after ACLY overexpression. Furthermore, ACLY inhibitor dramatically suppressed tumour growth and lipid metabolism in ESCC cells xenografted tumour model, whereas ACLY overexpression displayed the opposite effect. Mechanistically, ACLY protein harboured acetylated modification and interacted with SIRT2 protein in ESCC cells. The SIRT2 inhibitor AGK2 significantly increased the acetylation level of ACLY protein and inhibited the proliferation and migration of ESCC cells, while overexpression of ACLY partially reversed the inhibitory effect of AGK2 on ESCC cells. Overall, these results suggest that targeting the SIRT2/ACLY signalling axis may be a potential therapeutic strategy for ESCC patients.

Miltirone suppresses ESCC cell viability and invasion by promoting E3 ligase mediated‐FZD2 ubiquitination and degradation

AbstractEsophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer and lacks effective treatment. A growing number of studies have confirmed that Frizzled class receptor 2 (FZD2), a receptor for Wnts, regulates cancer metastasis in cancers. This study aims to reveal the anti‐metastatic activity of miltirone on ESCC cells and its mechanism on FZD2 in vitro. Cell‐counting‐kit‐8 (CCK‐8) was used to detect the effect of miltirone on cell growth in ESCC cells. The invasion of ESCC cells was detected by wound healing and transwell assay. Immunofluorescence staining was used to detect the expressions of epithelial‐mesenchymal transition (EMT) markers and the location of FZD2. Western blot and quantitative polymerase chain reaction (q‐PCR) were used to detect levels of target genes, and FZD2 ubiquitination was detected by the co‐immunoprecipitation method. The levels of roof plate‐specific spondins (RSPO1‐4) in cells were determined by ELISA, q‐PCR, and western blot assays. Miltirone exhibits its strongest cytotoxicity with IC50 values of 6.80 μM and 8.43 μM for K30 and K150 cells, respectively. Miltirone inhibited the cell viability and expression of EMT markers, affecting the invasion ability of ESCC, attenuated the Wnt2/FZD2 pathway, and down‐regulated the level of FZD2, which is regulated by the ubiquitination of E3 ubiquitin ligase. Furthermore, we found that miltirone could reduce the expression of RSPO1, and RSPO2, increasing E3 protein levels, and promoting FZD2 ubiquitination, leading to the ubiquitination and subsequent degradation of FZD2. Collectively, our results revealed that miltirone might be a promising drug for the treatment of ESCC, at least partly through regulation of the FZD2 ubiquitination.

Involvement of circRNA Regulators MBNL1 and QKI in the Progression of Esophageal Squamous Cell Carcinoma.

To investigate the role of circRNA regulators MBNL1 and QKI in the progression of esophageal squamous cell carcinoma. MBNL1 and QKI are pivotal regulators of pre-mRNA alternative splicing, crucial for controlling circRNA production - an emerging biomarker and functional regulator of tumor progression. Despite their recognized roles, their involvement in ESCC progression remains unexplored. The expression levels of MBNL1 and QKI were examined in 28 tissue pairs from ESCC and adjacent normal tissues using data from the GEO database. Additionally, a total of 151 ESCC tissue samples, from stage T1 to T4, consisting of 13, 43, 87, and 8 cases per stage, respectively, were utilized for immunohistochemical (IHC) analysis. RNA sequencing was utilized to examine the expression profiles of circRNAs, lncRNAs, and mRNAs across 3 normal tissues, 3 ESCC tissues, and 3 pairs of KYSE150 cells in both wildtype (WT) and those with MBNL1 or QKI knockouts. Transwell, colony formation, and subcutaneous tumorigenesis assays assessed the impact of MBNL1 or QKI knockout on ESCC cell migration, invasion, and proliferation. ESCC onset significantly altered MBNL1 and QKI expression levels, influencing diverse RNA species. Elevated MBNL1 or QKI expression correlated with patient age or tumor invasion depth, respectively. MBNL1 or QKI knockout markedly enhanced cancer cell migration, invasion, proliferation, and tumor growth. Moreover, the absence of either MBNL1 or QKI modulated the expression profiles of multiple circRNAs, causing extensive downstream alterations in the expression of numerous lncRNAs and mRNAs. While the functions of circRNA and lncRNA among the top 20 differentially expressed genes remain unclear, mRNAs like SLCO4C1, TMPRSS15, and MAGEB2 have reported associations with tumor progression. This study underscores the tumor-suppressive roles of MBNL1 and QKI in ESCC, proposing them as potential biomarkers and therapeutic targets for ESCC diagnosis and treatment.

Vitamin D receptor induces oxidative stress to promote esophageal squamous cell carcinoma proliferation via the p53 signaling pathway

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). ObjectiveWe investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. MethodsESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and Western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. ResultsHigh VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. ConclusionsVDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.

Abstract A033: Secretogranin V as a Potential Biomarker for Esophageal Squamous Cell Carcinoma

Abstract Background: Oesophageal squamous cell carcinoma (ESCC) remains one of the most poorly diagnosed and deadly cancers worldwide. Identification of biomarkers to accurately predict the risk of recurrence and survival after surgery is therefore crucial to improving patient outcomes. This study examined the expression of Secretogranin V (SCG5) and its correlation with the prognosis of patients with ESCC. Methods: Transcription levels of SCG5 were evaluated in 22 cell lines of ESCC. The biological roles of SCG5 in cell invasion, proliferation, and migration were verified by small interfering RNA- mediated knockdown experiments. The expression of SCG5 was measured in 165 ESCC tissues by using quantitative reverse-transcription (qRT)-PCR, and its association with clinicopathological parameters was analyzed. Results: SCG5 mRNA expression levels varied widely in ESCC cell lines. Knockdown of SCG5 expression significantly suppressed cell invasion, proliferation, and migration of ESCC cells in vitro. Analysis of clinical specimens revealed that the expression of SCG5 mRNA was overexpressed in the ESCC compared to the adjacent normal oesophageal tissues. The high SCG5 expression group had significantly shorter overall and disease-free survival times. In the multivariable analysis, the high expression of SCG5 was determined to be an independent poor prognostic factor. Conclusion: SCG5 may have a significant role as a diagnostic and prognostic biomarker for ESCC. Citation Format: Mohammad Hussain Hamrah. Secretogranin V as a Potential Biomarker for Esophageal Squamous Cell Carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A033.

188. VITAMIN D RECEPTOR INDUCES OXIDATIVE STRESS TO PROMOTE ESOPHAGEAL SQUAMOUS CELL CARCINOMA PROLIFERATION VIA THE P53 SIGNALING PATHWAY

Abstract Background Esophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). We investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. Methods ESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. Results High VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. Conclusions VDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.

Downregulation of Claudin5 promotes malignant progression and radioresistance through Beclin1-mediated autophagy in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly prevalent and aggressive cancer with poor treatment outcomes. Despite the critical role of tight junction proteins in tumorigenesis, the involvement of Claudin5 in ESCC remains poorly understood. Thus, this study aimed to investigate the role of Claudin5 in ESCC malignant progression and radioresistance, as well as the underlying regulatory mechanisms.MethodsThe expression of Claudin5 was evaluated in esophageal cancer tissue using both public databases and 123 clinical samples. CCK-8, transwell invasion, wound healing and clonogenic survival assays were used to examine the proliferation, invasion, migration and radiosensitivity of ESCC cells in vitro. Xenograft and animal lung metastasis experiments were conducted to examine the impact of Claudin5 on tumor growth and lung metastasis in vivo. The effect of Claudin5 on autophagy was detected via transmission electron microscopy, western blotting and autophagy flux. Immunohistochemical staining was used to detect Claudin5 expression in ESCC patient samples. The statistical difference was assessed with Student t test or one-way ANOVA. The correlation between Claudin5 expression and radiotherapy response rate was performed by the Chi-square test. The significance of Kaplan–Meier curves was evaluated by the Logrank test.ResultsClaudin5 expression was downregulated in ESCC tissues. Downregulation of Claudin5 promoted ESCC cell proliferation, invasion, and migration both in vitro and in vivo. Downregulation of Claudin5 decreased the radiosensitivity of ESCC cells. Moreover, downregulation of Claudin5 promoted autophagy and the expression of Beclin1. Beclin1 knockdown reversed the effect of Claudin5 downregulation on autophagy induction and the promotion of ESCC cell malignant progression and radioresistance. Additionally, low expression of Claudin5 in ESCC cancer tissues was associated with poor radiotherapy response and prognosis.ConclusionsIn summary, these findings suggest that downregulation of Claudin5 promotes ESCC malignant progression and radioresistance via Beclin1-autophagy activation and may serve as a promising biomarker for predicting radiotherapy response and patient outcome in ESCC.

SCARA5 induced ferroptosis to effect ESCC proliferation and metastasis by combining with Ferritin light chain

BackgroundEsophageal squamous cell carcinoma (ESCC) remains one of the most lethal cancers worldwide accompany with an extremely poor prognosis. Therefore, this study aims to screen for new molecules affecting ESCC and explore their mechanisms of action to provide ideas for targeted therapies for ESCC.MethodsFirstly, we screened out the membrane protein SCARA5 by high-throughput sequencing of the ESCC patient tissues, and RT-qPCR and WB were used to verify the differential expression of SCARA5 in esophageal cell lines, and IHC analyzed the expression localization of SCARA5 in ESCC tissue. Then, flow cytometry, wound healing assay, Transwell assay and CCK-8 assay were used to explore the effects of SCARA5 on cell cycle, migration and invasion as well as cell proliferation activity of esophageal squamous carcinoma cells. Meanwhile, transmission electron microscopy was used to detect changes in cellular mitochondrial morphology, and flow cytometry were used to detect changes in intracellular reactive oxygen metabolism, and immunofluorescence and flow cytometry were used to detect changes in intracellular Fe2+. Mechanistically, co-immunoprecipitation was used to detect whether SCARA5 binds to ferritin light chain, and ferroptosis-related protein expression was detected by WB. Finally, the tumor xenograft model was applied to validation the role of SCARA5 tumor growth inhibition in vivo.ResultsWe found that SCARA5 was aberrantly decreased in ESCC tissues and cell lines. Furthermore, we confirmed that SCARA5 suppressed the cell cycle, metastasis and invasion of ESCC cells. Meanwhile, we also found that overexpression of SCARA5 caused changes in mitochondrial morphology, accumulation of intracellular reactive oxygen species and increased intracellular Fe2+ in ESCC cells, which induced ferroptosis in ESCC cells. Mechanically, we validated that SCARA5 combined with ferritin light chain and increased intracellular Fe2+. As well as, overexpression SCARA5 induced ferroptosis by increasing ferritin light chain in nude mice subcutaneous tumors and inhibited the growth of nude mice subcutaneous tumors.ConclusionCollectively, our findings demonstrated that SCARA5 suppressed the proliferation and metastasis of ESCC by triggering ferroptosis through combining with ferritin light chain.

CircFNDC3B knockdown restrains the progression of oesophageal squamous cell carcinoma through miR-214-3p/CDC25A axis.

Circular RNA (circRNAs) Fibronectin Type III Domain Containing 3B (FNDC3B) (circFNDC3B) has been revealed to be involved in the progression of oesophageal squamous cell carcinoma (ESCC). Hence, the potential regulatory network of circFNDC3B in ESCC was further investigated. Levels of genes and proteins were examined by qRT-PCR and Western blot. In vitro assays were performed using colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, wound healing assay, and transwell assay. The target relationship between miR-214-3p and circFNDC3B or cell division cycle 25 homologue A (CDC25A) was verified by dual-luciferase reporter and RIP assays. In vivo assay was carried out using the xenograft nude mice model. CircFNDC3B was highly expressed in ESCC, and high circFNDC3B expression was tightly associated with poor prognosis in ESCC patients. Functionally, circFNDC3B knockdown not only suppressed ESCC cell growth, migration and invasion in vitro, but hindered ESCC tumour growth in vivo. Mechanistically, circFNDC3B acted as a sponge for miR-214-3p to up-regulate the expression of its target CDC25A. Rescue experiments showed that miR-214-3p inhibitor reversed the anticancer effects of circFNDC3B knockdown. Moreover, forced expression of miR-214-3p suppressed the malignant phenotypes mentioned above, while this condition was abolished by CDC25A overexpression. CircFNDC3B silencing restrains the tumorigenesis of oesophageal squamous cell carcinoma through miR-214-3p/CDC25A axis, which opens a new window to the development of novel therapeutic strategy for ESCC patients.

Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common invasive malignancy worldwide with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been reported to be involved in cancer development. However, lncRNAs that are functional in ESCC and the underlying molecular mechanisms remain largely unknown.MethodsTranscriptomic analysis was performed to identify dysregulated lncRNAs in ESCC tissue samples. The high expression of LINC00680 in ESCC was validated by RT-qPCR, and the oncogenic functions of LINC00680 was investigated by cell proliferation, colony formation, migration and invasion assays in ESCC cells in vitro and xenografts derived from ESCC cells in mice. RNA-seq, competitive endogenous RNA (ceRNA) network analysis, and luciferase reporter assays were carried out to identify LINC00680 target genes and the microRNAs (miRNAs) bound to LINC00680. Antisense oligonucleotides (ASOs) were used for in vivo treatment.ResultsTranscriptome profiling revealed that a large number of lncRNAs was dysregulated in ESCC tissues. Notably, LINC00680 was highly expressed, and upregulation of LINC00680 was associated with large tumor size, advanced tumor stage, and poor prognosis. Functionally, knockdown of LINC00680 restrained ESCC cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, LINC00680 was found to act as a ceRNA by sponging miR-423-5p to regulate PAK6 (p21-activated kinase 6) expression in ESCC cells. The cell viability and motility inhibition induced by LINC00680 knockdown was significantly reversed upon PAK6 restoration and miR-423-5p inhibition. Furthermore, ASO targeting LINC00680 substantially suppressed ESCC both in vitro and in vivo.ConclusionsAn oncogenic lncRNA, LINC00680, was identified in ESCC, which functions as a ceRNA by sponging miR-423-5p to promote PAK6 expression and ESCC. LINC00680/miR-423-5p/PAK6 axis may serve as promising diagnostic and prognostic biomarkers and therapeutic targets for ESCC.

TGF-\u03b2-induced PLEK2 promotes metastasis and chemoresistance in oesophageal squamous cell carcinoma by regulating LCN2

Oesophageal squamous cell carcinoma (ESCC) has a relatively unfavourable prognosis due to metastasis and chemoresistance. Our previous research established a comprehensive ESCC database (GSE53625). After analysing data from TCGA database and GSE53625, we found that PLEK2 predicted poor prognosis in ESCC. Moreover, PLEK2 expression was also related to the overall survival of ESCC patients undergoing chemotherapy. Repression of PLEK2 decreased the proliferation, migration, invasion and chemoresistance of ESCC cells in vitro and decreased tumorigenicity and distant metastasis in vivo. Mechanistically, luciferase reporter assay and chromatin immunoprecipitation assay suggested that TGF-β stimulated the process that Smad2/3 binds to the promoter sequences of PLEK2 and induced its expression. RNA-seq suggested LCN2 might a key molecular regulated by PLEK2. LCN2 overexpression in PLEK2 knockdown ESCC cells reversed the effects of decreased migration and invasion. In addition, TGF-β induced the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. Moreover, AKT was phosphorylated in all regulatory processes. This study detected the major role of PLEK2 in driving metastasis and chemoresistance in ESCC by regulating LCN2, which indicates the potential use of PLEK2 as a biomarker to predict prognosis and as a therapeutic target for ESCC.

Abstract 3149: Investigation of the potential therapeutic value of CAF-derived milk fat globule-EGF factor 8 protein (MFG-E8) in esophageal squamous cell carcinoma

Abstract The physiological state of the tumor microenvironment (TME) is closely linked with tumorigenesis. RNA sequencing data from our lab showed a broad range of dysregulated genes between ESCC isolated CAFs and paired peripheral blood isolated Fbcs. Among the 2347 up-regulated genes, we found the GO analysis is many focused in extracellular matrix and the top 4 GO terms enriched in cell development and adhesion. Due to this, enriched CAF-secreted extarcellular proteins attracts our attention. MFG-E8 is highly expressed secreted protein in CAFs but low expressed in tumor cells. TCGA database revealed high expression of MFG-E8 is associated with poor overall survival and disease-free survival. In addition, ESCC cell line k410 and K180 with MFG-E8 protein showed a strong ability in cell migration, cell invasion and apoptosis inhibition compared with the PBS group. The further western blot demonstrated MFG-E8 promoted ESCC cell migration and invasion by triggering EMT, and activated an altered akt/stat3 signaling pathway. Moreover, Anti-MFG-E8 antibody effectively reduced tumor size, especially combined with cisplatin. Therefore, MFG-E8 may act as a potential target effective therapeutic strategies. Citation Format: Beilei Liu. Investigation of the potential therapeutic value of CAF-derived milk fat globule-EGF factor 8 protein (MFG-E8) in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3149.

High-mobility group AT-hook 2 promotes growth and metastasis and is regulated by miR-204-5p in oesophageal squamous cell carcinoma.

To investigate the expression of high-mobility group AT-hook 2 (HMGA2) and miR-204-5p in oesophageal squamous cell carcinoma (ESCC) and their biological roles in ESCC development and progression. HMGA2 and miR-204-5p expression levels in ESCC tissues and cell lines were detected by qRT-PCR, Western blotting and immunohistochemical staining. ESCC cell lines were transfected with a small interfering RNA for HMGA2 and miR-204-5p mimic to downregulate and upregulate the expression levels of HMGA2 and miR-204-5p, respectively. The growth, migration and invasion abilities of ESCC cells were assessed by MTT, colony formation, wound-healing and Transwell assays, respectively. A luciferase reporter gene assay was used to determine whether the 3'-untranslated coding regions of HMGA2 could be directly bound by miR-204-5p. HMGA2 expression was markedly upregulated (P<.001), while miR-204-5p expression was markedly downregulated (P=.003) in ESCC tissues compared with adjacent normal tissues. HMGA2 expression was correlated with tumour size, invasion depth, lymph node metastasis and tumour-node-metastasis stage (all P<.05) and was identified as an independent prognostic factor for ESCC patients. The expression levels of HMGA2 and miR-204-5p were negatively correlated (r2 =0.609, P<.001). HMGA2 knockdown or miR-204-5p overexpression markedly inhibited ESCC cell growth, migration and invasion (P<.05). In addition, restoration of HMGA2 expression partly reversed the inhibitory effects of miR-204-5p overexpression on migration and invasion (P<.05). The luciferase reporter gene assay suggested that HMGA2 is a direct downstream target of miR-204-5p. HMGA2 functions as an oncogene in the growth and metastasis of ESCC and is negatively regulated by miR-204-5p.

Downregulation of long non-coding RNA LOC101928477 correlates with tumor progression by regulating the epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the deadliest malignancies. There is a growing body of evidence showing that long non‐coding RNAs (lncRNAs) play critical roles in ESCC oncogenesis. The present study aimed to explore the role of LOC101928477, a newly discovered lncRNA, in the development and metastasis of ESCC.MethodsIn this study, real‐time PCR, western blotting, cell counting kit‐8 (CCK‐8), flow cytometry, colony formation, wound healing, transwell migration/invasion assay, immunofluorescence, and immunohistochemistry were used. We also applied an in situ xenograft mouse model and a lung metastasis mouse model to verify our findings.ResultsWe determined that LOC101928477 expression was inhibited in ESCC tissue and ESCC cell lines when compared with controls. Moreover, forced expression of LOC101928477 effectively inhibited ESCC cell proliferation, colony formation, migration, and invasion via suppression of epithelial‐mesenchymal transition (EMT). Furthermore, LOC101928477 overexpression inhibited in situ tumor growth and lung metastasis in a mouse model.ConclusionsTogether, our results suggested that LOC101928477 could be a novel suppressor gene involved in ESCC progression.

CTCF-silenced miR-137 contributes to EMT and radioresistance in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in gastrointestinal system. MicroRNAs (miRNAs) have been reported to be implicated in cancer development. However, the role of miR-137 has not been fully revealed in ESCC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses were separately used to examine RNA level and protein level. 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assays and flow cytometry analyses were conducted to assess biological behaviors of ESCC cells. Additionally, the interaction between genes were analyzed via Chromatin Immunoprecipitation (ChIP) assay, RNA Binding Protein Immunoprecipitation (RIP) assay, RNA pull down assay and luciferase reporter assay.ResultsMiR-137 was down-regulated in ESCC cells. Upregulation of miR-137 hindered ESCC cell proliferation, migration, invasion and epithelial mesenchymal transition (EMT). Besides, miR-137 enhanced the sensitivity of ESCC cells to irradiation. Moreover, CCCTC-binding factor (CTCF) inactivated miR-137 transcription in ESCC cells. Furthermore, we revealed enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and paxillin (PXN) as the downstream targets of miR-137. In turn, EZH2 was recruited by CTCF and induced methylation in miR-137 promoter.ConclusionCTCF/Suz12/EZH2 complex-silenced miR-137 facilitates ESCC progression and radioresistance by targeting EZH2 and PXN.

SNHG5 inhibits the progression of EMT through the ubiquitin-degradation of MTA2 in oesophageal cancer.

A substantial fraction of transcripts are known as long noncoding RNAs (lncRNAs), and these transcripts play pivotal roles in the development of cancer. However, little information has been published regarding the functions of lncRNAs in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanisms. In our previous studies, we demonstrated that small nucleolar RNA host gene 5 (SNHG5), a known lncRNA, is dysregulated in gastric cancer (GC). In this study, we explored the expression and function of SNHG5 in development of ESCC. SNHG5 was found to be downregulated in human ESCC tissues and cell lines, and this downregulation was associated with cancer progression, clinical outcomes and survival rates of ESCC patients. Furthermore, we also found that overexpression of SNHG5 significantly inhibited the proliferation, migration and invasion of ESCC cells in vivo and in vitro. Notably, we found that metastasis-associated protein 2 (MTA2) was pulled down by SNHG5 in ESCC cells using RNA pulldown assay. We also found that SNHG5 reversed the epithelial-mesenchymal transition by interacting with MTA2. In addition, overexpression of SNHG5 downregulated the transcription of MTA2 and caused its ubiquitin-mediated degradation. Thus, overexpression of MTA2 partially abrogated the effect of SNHG5 in ESCC cell lines. Furthermore, we found that MTA2 mRNA expression was significantly elevated in ESCC specimens, and a negative correlation between SNHG5 and MTA2 expression was detected. Overall, this study demonstrated, for the first time, that SNHG5-regulated MTA2 functions as an important player in the progression of ESCC and provide a new potential therapeutic strategy for ESCC.