Esophageal Carcinoma Cell Lines Research Articles

Oxymatrine inhibits migration and invasion of esophageal squamous cell carcinoma cell lines via the MEK1/ERK/β-catenin pathway

Esophageal, cancer is a prevalent malignant tumour of the digestive system in China, and esophageal squamous cell carcinoma (ESCC) accounts for 90 % of all esophageal cancer cases. Currently, the primary treatment involves surgical resection combined with postoperative radiotherapy. In this study, we used two ESCC cell lines to determine whether oxymatrine (OMT) inhibits ESCC, whether the mechanism involves the MEK1/ERK/β-catenin pathway, and how OMT modulates this pathway to affect the development of ESCC. The effects of OMT treatment were monitored with Cell Counting Kit-8 (CCK-8) assays as well as with clony formation, migration and invasion, wound healing, Hoechst 33258, and Western blot analyses. The relationship between OMT and the target was also evaluated by molecular docking and cell stability experiments. These findings suggest that ESCC development and metastasis may be inhibited by OMT and that OMT targets MEK1 through the ERK/β-catenin/EMT pathway to suppress ESCC cell migration and invasion. In addition, in vivo studies confirmed that OMT can inhibit the growth of ESCC cell lines in NOG mice without causing damage to other organs. In conclusion, in vitro experiments, revealed that OMT prevents the migration and invasiveness of ESCC cells by inhibiting the ERK/β-catenin/EMT pathway and thus targeting MAP2K1 (MEK1) in ESCC.

The mechanism of MMP14-positive tumor-associated fibroblast subsets in inhibiting PD-1 immunotherapy for esophageal cancer through exosomal tsRNA-10522

Esophageal cancer (EC) continues to pose a significant health risk. Cancer-associated fibroblasts (CAFs), an essential part of the tumor microenvironment (TME), are viewed as potential therapeutic targets. However, their role in tumor mechanisms specific to esophageal cancer remains to be elucidated. This study identified MMP14+ CAFs and MMP14- CAFs using immunofluorescence staining. The cytotoxic activity of CD8 T cells was assessed via western blot and ELISA. Using a transwell test, the migratory potential of MMP14+ CAFs was evaluated. Using flow cytometry, apoptosis was found in the esophageal squamous cell carcinoma cell line KYSE30. To determine the important tsRNAs released by MMP14+ CAFs, tsRNA-seq was used. Two subgroups of EC receiving PD-1 immunotherapy were identified by our research: MMP14+ CAFs and MMP14- CAFs. MMP14+ CAFs showed improved migratory capacity and released more inflammatory factors linked to cancer. Through exosomes, these CAFs may prevent anti-PD-1-treated CD8 T cells from being cytotoxic. Furthermore, exosomal tsRNA from MMP14+ CAFs primarily targeted signaling pathways connected with cancer. Notably, it was discovered that tsRNA-10522 plays a critical role within inhibiting CD8 T cell tumor cell death. The tumor cell killing of CD8 T cells by exosomal tsRNA-10522 is inhibited by a subgroup of cells called MMP14+ CAFs inside the EC microenvironment during PD-1 immunotherapy. This reduces the effectiveness of PD-1 immunotherapy for EC. Our findings demonstrate the inhibitory function of MMP14+ CAFs within EC receiving PD-1 immunotherapy, raising the prospect that MMP14+ CAFs might serve as predictive indicators in EC receiving PD-1 immunotherapy.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

Anti-tumour activity of Panobinostat in oesophageal adenocarcinoma and squamous cell carcinoma cell lines

BackgroundOesophageal cancer remains a challenging disease with high mortality rates and few therapeutic options. In view of these difficulties, epigenetic drugs have emerged as potential alternatives for patient care. The goal of this study was to evaluate the effect and biological consequences of Panobinostat treatment, an HDAC (histone deacetylase) inhibitor already approved for treatment of patients with multiple myeloma, in oesophageal cell lines of normal and malignant origin, with the latter being representative of the two main histological subtypes: adenocarcinoma and squamous cell carcinoma.ResultsPanobinostat treatment inhibited growth and hindered proliferation, colony formation and invasion of oesophageal cancer cells. Considering HDAC tissue expression, HDAC1 was significantly upregulated in normal oesophageal epithelium in comparison with tumour tissue, whereas HDAC3 was overexpressed in oesophageal cancer compared to non-malignant mucosa. No differences between normal and tumour tissue were observed for HDAC2 and HDAC8 expression.ConclusionsPanobinostat exposure effectively impaired malignant features of oesophageal cancer cells. Because HDAC3 was shown to be overexpressed in oesophageal tumour samples, this epigenetic drug may represent an alternative therapeutic option for oesophageal cancer patients.

Synthesis and biological evaluation of 4-phenyl-5-quinolinyl substituted isoxazole analogues as potent cytotoxic and tubulin polymerization inhibitors against ESCC

The identification of chemically different inhibitors that target the colchicine site of tubulin is still of great value for cancer treatment. Combretastatin A-4(CA-4), a naturally occurring colchicine-site binder characterized by its structural simplicity and biological activity, has served as a structural blueprint for the development of novel analogues with improved safety and therapeutic efficacy. In this study, a library of forty-eight 4-phenyl-5-quinolinyl substituted triazole, pyrazole or isoxazole analouges of CA-4, were synthesized and evaluated for their cytotoxicity against Esophageal Squamous Cell Carcinoma (ESCC) cell lines. Compound C11, which features a 2-methyl substitution at the quinoline and carries an isoxazole ring, emerged as the most promising, with 48 h IC50s of less than 20 nmol/L against two ESCC cell lines. The findings from EBI competitive assay, CETA, and in vitro tubulin polymerization assay of C11 are consistent with those of the positive control colchicine, demonstrating the clear affinity of compound C11 to the colchicine binding site. The subsequent cellular-based mechanism studies revealed that C11 significantly inhibited ESCC cell proliferation, arrested cell cycle at the M phase, induced apoptosis, and impeded migration. Experiments conducted in vivo further confirmed that C11 effectively suppressed the growth of ESCC without showing any toxicity towards the selected animal species. Overall, our research suggests that the tubulin polymerization inhibitor incorporating quinoline and the isoxazole ring may deserve consideration for cancer therapy.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

The role of tumor-associated macrophages in the radioresistance of esophageal cancer cells via regulation of the VEGF-mediated angiogenic pathway.

Tumor-associated macrophages (TAMs) are known to promote tumor growth, invasion, metastasis, and protumor angiogenesis, but the role of TAMs in evading radiotherapy in esophagus cancer remains unclear. In this study, we first induced TAMs from human monocytes (THP-1) and identified using immunofluorescence and Western blotting assays. We then co-cultured them with human esophageal cancer cell lines. CCK-8, colony formation, Transwell, scratch test, and TUNEL assays showed that TAMs could promote proliferation, survival rate, invasion, migration, and radioresistance and could inhibit apoptosis of the esophageal squamous carcinoma cell lines KYSE-150 and TE-1 before and after radiotherapy both in vivo and in vitro. Using LV-VEGFA-RNAi lentiviral vectors, we also found that TAMs could increase the expression of VEGFA and that inhibition of VEGFA could inhibit the biological function caused by TAMs. Finally, a Western blotting assay was used to evaluate the expression of various factors underlying the mechanism of TAMs. VEGFA, MAPK, P-MAPK, BCL-2, and Snail proteins were found to be overexpressed in co-cultured groups, whereas after VEGFA inhibition, MAPK, P-MAPK, BCL-2, and Snail proteins were found to be significantly downregulated in the radiotherapy group. These study results offer important information regarding the mechanism of radioresistance in esophageal cancer.

Epigenetic silencing schlafen-11 sensitizes esophageal cancer to ATM inhibitor.

Targeting DNA damage response (DDR) pathway is a cutting-edge strategy. It has been reported that Schlafen-11 (SLFN11) contributes to increase chemosensitivity by participating in DDR. However, the detailed mechanism is unclear. To investigate the role of SLFN11 in DDR and the application of synthetic lethal in esophageal cancer with SLFN11 defects. To reach the purpose, eight esophageal squamous carcinoma cell lines, 142 esophageal dysplasia (ED) and 1007 primary esophageal squamous cell carcinoma (ESCC) samples and various techniques were utilized, including methylation-specific polymerase chain reaction, CRISPR/Cas9 technique, Western blot, colony formation assay, and xenograft mouse model. Methylation of SLFN11 was exhibited in 9.15% of (13/142) ED and 25.62% of primary (258/1007) ESCC cases, and its expression was regulated by promoter region methylation. SLFN11 methylation was significantly associated with tumor differentiation and tumor size (both P < 0.05). However, no significant associations were observed between promoter region methylation and age, gender, smoking, alcohol consumption, TNM stage, or lymph node metastasis. Utilizing DNA damaged model induced by low dose cisplatin, SLFN11 was found to activate non-homologous end-joining and ATR/CHK1 signaling pathways, while inhibiting the ATM/CHK2 signaling pathway. Epigenetic silencing of SLFN11 was found to sensitize the ESCC cells to ATM inhibitor (AZD0156), both in vitro and in vivo. SLFN11 is frequently methylated in human ESCC. Methylation of SLFN11 is sensitive marker of ATM inhibitor in ESCC.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma

BackgroundIncreasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins.MethodsThe GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms.ResultsPDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription.ConclusionsOur research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Cell-SELEX and application research of a DNA aptamer against esophageal squamous cell carcinoma (ESCC) cell line TE-1.

Esophageal cancer is a common cancer with high morbidity and mortality that severely threatens the safety and quality of human life. The strong metastatic nature of esophageal cancer enables it to metastasize more quickly and covertly, making it difficult for current diagnostic and treatment methods to achieve efficient early screening, as well as timely and effective treatment. As a promising solution, nucleic acid aptamers, a kind of special single-stranded DNA or RNA oligonucleotide selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, can specifically bind with different molecular targets. In this paper, random DNA single-stranded oligonucleotides were used as the initial library. Using TE-1 cells and HEEC cells as targets, specific binding sequences were selected by 15 rounds of the cell-SELEX method, and the aptamer sequence that binds to TE-1 cells with the most specificity was obtained and named Te4. The Te4 aptamer was further validated for binding specificity, binding affinity, type of target, in vitro cytotoxicity when conjugated with DOX(Te4-DOX), and in vivo distribution. Results of in vitro validation showed that Te4 has outstanding binding specificity with a Kd value of 51.16 ± 5.52 nM, and the target type of Te4 was preliminarily identified as a membrane protein. Furthermore, the cytotoxicity experiment showed that Te4-DOX has specific cytotoxicity towards cultured TE-1 cells. Finally, the results of the in vivo distribution experiment showed that the Te4 aptamer is able to specifically target tumor regions in nude mice, showing great potential to be applied in future diagnosis and targeted therapy of esophageal cancer.

Circ_0003855 involvement of esophageal cancer progression through miR-622/FLOT1.

To confirm the relationship between Circ_0003855 and EC, we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC, and the expression levels of Circ_0003855, miR-622, and FLOT1 were detected. The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells, while miR-622 was lowly expressed (p < 0.05). Subsequently, Circ_0003855 small interfering RNA (si-Circ_0003855) and its negative control (si-NC) were used to detect changes in cellular biological behaviors. We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855, and miR-622 expression was elevated, while FLOT1 was decreased (p < 0.05). Additionally, si-Circ_0003855 and miR-622 inhibitor sequence (miR-622-inhibition) were co-transfected into cells with miR-622-inhibition alone, and untreated Eca109 cells were used as a control to detect the expression of FLOT1. Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells (p > 0.05). Synthesizing the results of these experiments above, we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells, and its mechanism is related to miR-622 and FLOT1.

Abstract B148: Novel low-atomic cluster Ag5 induced oxidative stress and antiproliferation in Esophageal Squamous Cell Carcinoma Cells

Abstract Background: Accumulation of reactive oxygen species (ROS) intensively activates cellular stress responses leading to cell death, thus ROS levels are strictly controlled by various antioxidants in normal cells. Esophageal cancer (ESCA), the sixth most common cause of cancer deaths, highly accumulates ROS under uncontrolled redox balances to activate oxidative-stress response pathways. Arjuna Therapeutics has developed a novel low-atomic cluster Ag5 as a ROS inducer, which strongly inhibits antioxidant pathways of glutathione and thioredoxin. In this study, we pharmacologically evaluate Ag5 on its therapeutic potential in preclinical ESCA models. Materials and Methods: Gene enrichment analyses were performed with 6,327 RNA-seq data of 14 cancer types in TCGA database. Cell panel studies of ATP-based cell viability was performed in vehicle control, Ag5 (3 uM), N-acetylcysteine (NAC, 5mM), and combination treatments in 31 esophageal squamous cell carcinoma (ESCC) cell lines and 5 non-ESCC cell lines. NAC) treatment was used as a rescue control of an antioxidant. We also conducted time-lapse imaging analysis with DCFDA (ROS indicator) and SYTOX (cell death marker). The morphology of mitochondria and ER were determined by electron microscope (EM). Results: Gene enrichment analyses revealed ROS-related genes were upregulated in ESCA compared to other cancer types. In the cell panel studies, Ag5 also exhibited potent antiproliferation in large numbers of ESCC cell lines, and NAC profoundly attenuated its antiproliferative activity. Time-lapse microscopy in ESCC cell line KYSE190 demonstrated that Ag5 increased DCFDA in 20 min and SYTOX in 60 min after the treatment, respectively, while combination treatment with NAC less detected these markers. Ag5-treated KYSE190 also exhibited necrosis-like morphology of swollen- and round-shape cells. In EM, damaged mitochondria and expanded endoplasmic reticulum were frequently observed in Ag5-treated KYSE190 cells. These data indicated that Ag5 induced ROS-mediated antiproliferation in ESCC cell lines. Conclusions: Ag5 exhibited ROS-mediated antiproliferative activities in large numbers of ESCC cell lines. Our preclinical results suggest the therapeutic potential of Ag5 as a novel cancer drug candidate for ESCC. Conflict of interest disclosure: AO is a part-time employee of Astellas Pharma Inc. from 2023 as a cross-appointment system. AO was an employee of Takeda Pharmaceutical Company, Ltd. AO reported paid consulting or advisory roles for Ono Pharmaceutical Company Ltd., Craif Inc., and GEXVal Inc. out of this study. AO receives research fundings from Astellas Pharma Inc., Astellas Pharma Global Development Inc., and Daiichi Sankyo Company, Ltd. out of this study. RB and MT are board members of Arjuna Therapeutics. Citation Format: Ryo Kamata, Toyohiro Yamauchi, Yuta Sakae, Ross Breckenridge, Martin Treder, Akihiro Ohashi. Novel low-atomic cluster Ag5 induced oxidative stress and antiproliferation in Esophageal Squamous Cell Carcinoma Cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B148.

The expression of annexin-A1 in esophageal squamous cell carcinoma and its association with the biological behavior of the primary human esophageal squamous carcinoma cell line.

This study aimed to observe the differential expression of Annexin-A1 in esophageal squamous cell carcinoma (ESCC) and explored the effect of small interfering ribonucleic acid (RNAi)-Annexin-A1 on the biological behavior of CE81T-0 cells. An immunohistochemical approach was used to detect the expression of Annexin-A1 in 86 pairs of ESCC samples. Quantitative reverse transcription polymerase chain reaction was used to detect the expression of Annexin-A1 in CE81T-0 and CE81T-4 cells, and the expression of Annexin-A1 in CE81T-0 cells was knocked out by RNAi. A methyl-thiazolyl-tetrazolium assay was used to observe the effect of Annexin-A1 on cell proliferation, and flow cytometry was conducted to analyze its effect on cell cycles and apoptosis. A scratch assay and a Transwell chamber were used to detect changes in cell migration and invasion. From the results, compared with the Annexin-A1 expression rate of 59.3% in para-carcinoma tissues, the expression of Annexin-A1 in cancer was reduced to only 32.6% in ESCC cells. Annexin-A1 was strongly expressed in highly differentiated ESCC cells without lymphatic metastasis and highly expressed in the CE81T-0 cell group with low metastasis. Annexin-A1 gene silencing promoted cell proliferation and inhibited apoptosis, blocked cells in the S-phase, and increased cell migration, leading to an increase in the number of invaded cells. Above all, Annexin-A1 could reflect the differentiation degree and lymph node metastasis of ESCC cells to some extent and was involved in the invasion, metastasis, proliferation, and other biological behaviors of ESCC cells, indicating an experimental basis for Annexin-A1 as a molecular marker in the early diagnosis of ESCC and the prediction of cell metastasis, invasion, and differentiation degree.

Mammalian esophageal stratified tissue homeostasis is maintained distinctively by the epithelial pluripotent p63+Sox2+ and p63-Sox2+ cell populations.

Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.

Effects of irradiation with different fractionations on radioresistant esophageal cancer cells and lung tissues

BackgroundLocal recurrence is the main cause of treatment failure for esophageal cancer and a large part of them require re-irradiation which needs to face the two major challenges of radioresistance and radiation-induced lung injury (RILI). This study aimed to evaluate the effects of irradiation with different fractionations on inhibition in radioresistant esophageal cancer cells in vitro and RILI in vivo. MethodsEC109R was selected from the human esophageal squamous carcinoma cell line EC109 by multiple exposures and verified to be radioresistant through colony forming assay and cell proliferation assay. EC109R was irradiated with different fractionations (conventional radiotherapy, CR: 2 Gy/d, 5 d; pulsed low dose-rate radiotherapy, PLDR: 0.2 Gy x 10 pulses with 3-min intervals/d, 5 d; hypo-fractionated radiotherapy, HR: 5 Gy/d, 2 d) to verify their inhibition by CCK-8. The protection of PLDR on RILI was assessed by H&E-staining paraffin sections and mice serum TGF-β1 levels. ResultsEC109R exhibited much better radioresistance (p < 0.0001) than EC109 in colony forming and cell proliferation assays. For EC109R, PLDR showed slightly lower inhibition by detection of optical density than HR (p = 0.0025) but similar to CR (p = 0.1155). In addition, pathological sections of lung tissues from PLDR group showed less alveolar structural damage and inflammatory cells infiltration, and serum TGF-β1 levels were significantly lower than those in the other two fractionations (vs CR: p = 0.0092; vs HR: p = 0.0045). ConclusionPLDR could effectively inhibit the proliferation of radioresistant esophageal cancer cells in vitro and mitigate RILI in vivo.

MiR-129-2-3p Inhibits Esophageal Carcinoma Cell Proliferation, Migration, and Invasion via Targeting DNMT3B.

The study aims to explore the regulatory mechanism of miR-129-2-3p underlying esophageal carcinoma (EC) cell progression and generate new ideas for targeted treatment of EC. Mature miRNA expression data and total RNA sequencing data of EC in the TCGAESCA dataset were utilized to explore differentially expressed miRNAs (DEmiRNAs). StarBase database was then utilized to predict targets of miRNA. MiR-129-2-3p and DNMT3B expression in EC cell lines was assayed through qRT-PCR and Western blot. CCK-8, scratch healing, and transwell assays were conducted to assess the impact of miR-129-2-3p on EC cell phenotypes. In addition, a dual-luciferase assay was completed to identify the binding relationship between DNMT3B and miR-129-2-3p. MiR-129-2-3p was noticeably less expressed in EC cell lines, while DNMT3B was highly expressed. MiR-129-2-3p could bind to DNMT3B. Furthermore, in vitro functional experiments uncovered that overexpressed miR-129-2-3p repressed EC cell progression while further overexpressing DNMT3B would restore the above inhibitory effect. MiR-129-2-3p is a cancer repressor in EC cells, and it could target DNMT3B, thus hampering the progression of EC cells.

Clinical and Prognostic Impact of the Warburg Effect in Esophageal Carcinoma: Monocarboxylate Transporters as Candidates for Therapeutic Targeting

Introduction: Esophageal cancer (EC) seems to display increased glycolytic activity, but clinical studies on the expression/prognostic significance of glycometabolism-related proteins, as well as functional assays, are missing. Methods: Expression of 10 glycolytic biomarkers was evaluated by immunohistochemistry in tissue sections from 95 patients. Two esophageal squamous cell carcinoma (ESCC) cell lines were used to assess the effect of monocarboxylate transporter (MCT) downregulation on cell viability and extracellular lactate/glucose accumulation. Results: Expression of MCT1, MCT4, CD147, and GLUT1 was significantly associated with an ESCC histopathology, while a poor clinicopathological profile was seen in GLUT1- and LDHA-positive EC cases. In the ESCC group, MCT1 immunoreactivity is associated with high TNM stage and metastasis. The 3-year overall survival (OS) rate was significantly influenced by MCT4 and CAIX positivity and HKII negativity. Those biomarkers were considered independent prognostic factors of OS in multivariate analysis. Dual inhibition of MCT1/4 expression decreased cell viability and extracellular lactate accumulation in ESCC cells. Conclusion: Elevated glycolytic rates correlate with a poor clinicopathological profile in EC patients. MCT4 and CAIX positivity independently predict a worse prognosis. Due to the lack of information on treatment modalities, we could not further infer the role of these biomarkers in predicting response to therapy, which needs to be assessed in future studies. In addition, MCT1/4 targeting should be performed both “in vitro” and “in vivo” to further explore its impact on tumor growth and response to classical therapies. HKII expression and function, particularly in the tumor stroma, should be investigated.

Abstract B013: Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas

Abstract The tumor microenvironment (TME), at both the primary tumor and sites of metastases, is a key determinant in the success of metastatic colonization. Cancer-associated fibroblasts (CAFs) are a predominant cell type in the TME that, through deposition and remodeling of extracellular matrix (ECM) components, can mediate the TME and promote cancer progression. In addition to cancer progression and metastasis, CAFs have also been shown to mediate resistance to chemotherapy and radiotherapy. Therefore, there is a need to identify the factors which activate and recruit CAFs to solid tumors. Though cytokine arrays, we identified CC motif chemokine ligand 5 (CCL5) as a secreted factor which is increased upon co-culture of esophageal cancer cell lines and CAFs. Using CRISPR/Cas9 technology, we determined that CCL5 is predominantly tumor-cell derived and loss of tumor-cell derived CCL5 reduced esophageal carcinoma cell proliferation in vitro and in vivo. We propose that this reduction in cell proliferation is mediated by ERK1/2 signaling as loss of tumor-cell derived CCL5 reduces expression of p-ERK1/2 and cyclin D1, an ERK1/2 target gene which is essential for cell cycle progression. Interestingly, the loss of tumor-derived CCL5 also reduced the percentage of CAFs, identified by αSMA positivity, recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists called Maraviroc. We treated xenografts generated from esophageal carcinoma cell lines or patient derived tumor fragments with Maraviroc. Maraviroc treated xenografts had reduced tumor volume, CAF recruitment and ERK1/2 signaling. Thus, mimicking the effects of the genetic loss of CCL5. Analysis of the TCGA database, identified that high CCL5 or CCR5 expression is associated with poorer prognosis in low grade esophageal carcinomas. Together, we propose that this data highlights the importance of CCL5 in esophageal carcinomas with regards to CAF recruitment and tumor cell proliferation, and that the maraviroc in vivo experiments suggest that targeting the CCL5-CCR5 axis may have therapeutic potential in esophageal carcinomas. Citation Format: Karen J. Dunbar, Qiaosi Tang, Tatiana A. Karakasheva, Anil K. Rustgi. Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B013.

LncRNA PVT1 Confers Cisplatin Resistance of Esophageal Cancer Cells through Modulating the miR-181a-5p-Glutaminase (GLS) Axis

Esophageal carcinoma (ESCA) is one of the prevalent malignancies worldwide. Cisplatin (CDDP) is a conventional chemotherapy drug. However, the acquired cisplatin resistance limits its extensively clinical applications. In this study, the roles and underlying mechanisms of lncRNA PVT1 in cisplatin-resistant ESCA are investigated. PVT1 was significantly upregulated in ESCA patient specimens and cell lines. Higher PVT1 level was associated with a poor survival rate of ESCA patients. Silencing PVT1 effectively increased cisplatin sensitivity of ESCA cells. We established cisplatin-resistant ESCA cell line (EC109 CDDP Res) and detected that PVT1 and glutamine metabolism were remarkedly elevated in CDDP-resistant esophageal cancer cells. Bioinformatical analysis and luciferase assay illustrated that PVT1 sponged miR-181a-5p to form a ceRNA network, resulting in the downregulation of miR-181a-5p expression in ESCA cells. Glutaminase (GLS), which is a key enzyme in the glutamine metabolism, was identified and validated as a direct target of miR-181-5p in ESCA cells. Inhibiting glutamine metabolism effectively re-sensitized CDDP-resistant cells. Rescue experiments demonstrated that restoration of miR-181a-5p in PVT1-overexpressing CDDP-resistant ESCA cells successfully overcame the PVT1-promoted cisplatin resistance through targeting GLS. Summarily, our study revealed molecular mechanisms of the lncRNA PVT1-promoted cisplatin resistance in ESCA by modulating the miR-181a-5p-GLS axis.

The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells.

To investigate the role and mechanism of β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.