Human Esophageal Adenocarcinoma Cell Lines Research Articles

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4559.

Generation and multiomic profiling of a TP53/CDKN2A double-knockout gastroesophageal junction organoid model.

Inactivation of the tumor suppressor genes tumor protein p53 (TP53) and cyclin-dependent kinase inhibitor 2A (CDKN2A) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated TP53 and CDKN2A knockout (TP53/CDKN2AKO) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2AKO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

The Cytotoxic Effect of Iraqi Rumex Acetosella against Breast and Esophagus Cancer Cells

Background: cell lines derived from cancer cells are frequently used in research, including use as a model to understand cancer and to identify potential new treatments. The aim of this article has been prepared to evaluate the anticancer effect of Rumex Acetosella that has been studied for their anticancer activity on esophagus and breast cancer cell line. Method: AMJ13 (new breast cancer cell line (AMJ13) has been established from an Iraqi breast cancer patient and SK-GT-4 cells (Human esophageal adenocarcinoma cell line) were treated with whole methanolic extract of the leaves of Rumex acetosella. the MTT assay to determine the anticancer activity was done using 96-well plates where cell lines were seeded at 1 × 104cells/well after 24 hrs.m the cells were treated with tested compounds at different concentrations. Cell viability was measured after 72 hrs of treatment by removing the medium, adding 28 μL of 2 mg/mL solution of MTT, and incubating the cells for 2.5 h at 37 °C. After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 μL of DMSO (Dimethyl Sulphoxide) followed by 37 °C incubation for 15 min with shaking. The absorbency was determined on a microplate reader at 492 nm. Conclusion: Plant extract from Rumex Acetosella showed particularly strong anticancer capabilities since it inhibited actual tumor progression in a breast adenocarcinoma mouse model. Our results suggest that whole plant extracts are promising anticancer reagents.

Combination effect of lapatinib with foretinib in HER2 and MET co-activated experimental esophageal adenocarcinoma

Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors lapatinib and foretinib, respectively, both in in-vitro and in-vivo models of experimental EAC. We characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of foretinib and lapatinib. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. The OE33 EAC cell line with strong expression of phosphorylated both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, enhanced inhibition of cell proliferation and xenograft tumor growth by inducing apoptosis, and significantly enhanced mouse overall survival, overcoming single agent resistance. In the OE19 EAC cell line with mainly HER2 phosphorylation, and the ESO51 EAC cell line with mainly MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC, and could be a novel treatment strategy for specific EAC patients.

Abstract 289: MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma

Abstract Introduction: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). Unfortunately, patients treated with lapatinib, a dual EGFR and HER2 inhibitor, frequently develop resistance. Lapatinib fails to improve patient survival in HER2-postive EAC, and the mechanisms contributing to resistance remain largely unknown. Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset EAC. In this study, we therefore studied the role of MET activation in lapatinib resistance mechanisms in experimental EAC. Methods: We first characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of MET and HER2 inhibitions by foretinib and lapatinib, respectively. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. We also tested MET and HER activation status with sensitivity to their inhibitors in newly generated lapatinib resistant OE19 EAC cells (OE19-LPR). Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as single agents. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell proliferation and xenograft tumor growth, induced apoptosis, and significantly enhanced overall mouse survival, thus overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. Interestingly, OE19-LPR cells showed significant upregulated expression of phosphorylated MET compared to parent OE19 cells, detected by both Activesignal assay and Western blot analysis. In addition, OE19-LPR cells showed significantly reduced sensitivity to lapatinib compared to parent OE19 cells, and the co-administration of foretinib and lapatinib synergistically inhibited cell proliferation in OE19-LPR cells. Conclusion: These data suggest that resistance to HER2 targeted therapies in HER2 and MET overexpressed EAC cells can be due to MET activation. Thus MET and HER2 targeted therapy appears to be a sensible treatment strategy for HER2 positive MET-overexpressing EAC. Citation Format: Md Sazzad Hassan, Fiona Williams, Lucia Petrova, Niranjan Awasthi, Margaret Schwarz, Roderich Schwarz, Urs von Holzen. MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 289.

Abstract 4826: Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma

Abstract Introduction: Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors foretinib and lapatinib both in-vitro and in-vivo models of experimental EAC. Methods: In this study we first characterized MET and HER2 activation in a panel of human EAC cell lines and the differential susceptibility of these EAC cell lines to single agents or combinations of foretinib, a multi-kinase MET inhibitor, with HER2 targeted agent lapatinib. We evaluated the levels of phosphorylation status of MET and HER2 proteins using western blot in EAC cell lines. Foretinib and lapatinib, as single agent or in combination were tested for effect on cell growth as detected by WST-1 assay and on cell apoptosis as detected by western blot of cleavage of caspase 3 and poly ADP ribose polymerase (PARP). In addition, we explored the antitumor efficacy with survival advantage following foretinib and lapatinib mono and combination therapies for two weeks in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell growth and induced apoptosis, overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent foretinib and lapatinib, respectively, with lack of enhanced growth inhibition when the two drugs were combined. Foretinib in combination with lapatinib treatment resulted in significantly higher antitumor efficacy and survival benefit compared with foretinib or lapatinib treatment alone. In subcutaneous xenografts using OE33 cells, average net tumor growth after two weeks in different therapy groups was 247.83 mm3 in control, 216.71 mm3 after foretinib (p=0.49), 239.68 mm3 after lapatinib (p=0.74), and 108.06 mm3 after foretinib plus lapatinib (p=0.0011). In the OE33 survival model there was a significant increase in median animal survival after two weeks foretinib plus lapatinib treatment (71 days) compared to control (60 days, p=0.0021), to foretinib therapy (63 days, p=0.0019) or to lapatinib (61 days, p=0.0019) therapy. Conclusion: These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC. Therefore, this combination therapy could be a novel treatment strategy for EAC with MET and HER co-activation. Citation Format: Md Sazzad Hassan, Fiona Williams, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs von Holzen. Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4826.

Hypoxia imaging with 18F-FAZA PET/CT predicts radiotherapy response in esophageal adenocarcinoma xenografts

BackgroundEsophageal cancer is an aggressive disease with poor survival rates. A more patient-tailored approach based on predictive biomarkers could improve outcome. We aimed to predict radiotherapy (RT) response by imaging tumor hypoxia with 18F-FAZA PET/CT in an esophageal adenocarcinoma (EAC) mouse model. Additionally, we investigated the radiosensitizing effect of the hypoxia modifier nimorazole in vitro and in vivo.MethodsIn vitro MTS cell proliferation assays (OACM5 1.C SC1, human EAC cell line) were performed under normoxic and hypoxic (< 1%) conditions: control (100 μL PBS), nimorazole, irradiation (5, 10 or 20 Gy) with or without nimorazole. In vivo, subcutaneous xenografts were induced in nude mice (OACM5 1.C SC1). Treatment was given daily for 5 consecutive days: (A) control (600 μl NaCl 0.9% intraperitoneally (IP)) (N = 5, n = 7), (B) RT (5 Gy/d) (N = 11, n = 20), (C) combination (nimorazole (200 mg/kg/d IP) 30 min before RT) (N = 13, n = 21). N = number of mice, n = number of tumors. 18F-FAZA PET/CT was performed before treatment and tumor to background (T/B) ratios were calculated. Relative tumor growth was calculated and tumor sections were examined histologically (hypoxia, proliferation).ResultsA T/B ≥ 3.59 on pre-treatment 18F-FAZA PET/CT was predictive for worse RT response (sensitivity 92.3%, specificity 71.4%). Radiation was less effective in hypoxic tumors (T/B ≥ 3.59) compared to normoxic tumors (T/B < 3.59) (P = 0.0025). In vitro, pre-treatment with nimorazole significantly decreased hypoxic radioresistance (P < 0.01) while in vivo, nimorazole enhanced the efficacy of RT to suppress cancer cell proliferation in hypoxic tumor areas (Ki67, P = 0.064), but did not affect macroscopic tumor growth.ConclusionsTumor tissue hypoxia as measured with 18F-FAZA PET/CT is predictive for RT response in an EAC xenograft model. The radiosensitizing effect of nimorazole was questionable and requires further investigation.

Animal Model: Xenograft Mouse Models in Esophageal Adenocarcinoma.

Researchers often use murine models of esophageal cancer to evaluate novel therapies prior to clinical protocol treatment. Subcutaneous xenograft models are often used for testing the efficacy of anticancer agents in many cancers including esophageal adenocarcinoma. However, mice subcutaneous esophageal adenocarcinoma models only represent local tumor growth and do not provide any information about a survival benefit for a particular anticancer regimen, which is very crucial for experimental treatment efficacy. In addition, anticancer agents may well inhibit subcutaneous tumor growth without effecting overall animal survival. Herein, we describe a peritoneal dissemination mouse xenograft model for survival outcome analysis with intraperitoneal injection of human esophageal adenocarcinoma cell lines.

Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells

Walnuts contain many components including specific fatty acids, which could be active against cancer. Even though the anticarcinogenic effect of some of the individual fatty acids in walnut oil has been described, the effect of walnut oil itself on esophageal cancer cells hasn't yet been investigated. We aimed to investigate whether walnut oil affects tumor growth and metastatic potential in esophageal cancer cells. The human esophageal adenocarcinoma cell line, OE19, was treated with different doses of walnut oil and cell viability, apoptosis/necrosis and cell cycle analyses were performed using WST-1 assay and flow cytometry respectively. Adhesion, colony formation and wound healing assays were performed to assess the antimetastatic effects of walnut oil. NFkB expression was evaluated with western blot analysis. Walnut oil decreased the cell viability of esophageal cancer cells in a dose-dependent manner. 20mg/mL walnut oil reduced cell viability by ∼50% when compared with control. The analysis revealed that necrosis and accumulation of cells in G0/G1 phase was induced in the cells treated with high doses of walnut oil. It also down-regulated the protein levels of NFkB. Walnut oil suppressed the adhesion, migration and colony formation of the cells. High-dose short-term administration of walnut oil reduces the cell viability and metastatic ability of esophageal cancer cells, while exhibiting anticarcinogenic effect by inducing necrosis and cell cycle arrest at the G0/G1 phase, probably through suppression of the NFkB pathway. These data indicate that walnut oil, and by extension walnut consumption, may have beneficial effects in esophageal cancer in humans. This should be tested by clinical trials in the future.

Abstract 2826: A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Improvement of esophageal adenocarcinoma outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of esophageal adenocarcinoma that would support survival outcome analyses. Methods: To find the best candidate cell line from 7 esophageal adenocarcinoma cell lines of various origin, we injected them intraperitoneally and subcutaneously into severe combined immunodeficiency (SCID) mice and examined the tumor progression and survival outcomes. Human esophageal adenocarcinoma cell lines of Caucasian/Hispanic origin ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 originating from gastroesophageal junction, distal esophagus and gastric cardia/fundus were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups. Results: All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p=0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p=0.023). Conclusion: Peritoneal esophageal adenocarcinoma xenografts were successfully established after intraperitoneal injection of OE19 cells. This animal model of peritoneal dissemination for survival outcome will provide a useful survival outcome assessment model for the preclinical evaluation of cancer therapeutics in esophageal adenocarcinoma. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2826. doi:10.1158/1538-7445.AM2017-2826

A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. The 5-year survival rate of EAC is below 20% and most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. Improvement of EAC patient outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of EAC that would support survival outcome analyses. To find the best candidate cell line from 7 human EAC cell lines of different origin named ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups. All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p = 0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p = 0.00011). Thus peritoneal dissemination mouse xenograft model for survival outcome assessment after intraperitoneal injection of OE19 cells will be very useful for the evaluation of cancer therapeutics.

Interleukin-17A promotes esophageal adenocarcinoma cell invasiveness through ROS-dependent, NF-κB-mediated MMP-2/9 activation.

Interleukin-17A (IL-17A), a pro-inflammatory cytokine secreted primarily by Th17 cells, has been proved to be involved in the microenvironment of certain inflammation-related tumors. However, the role of IL-17A in cancer development has always been controversial. In this study, we investigated the effect of IL-17A on the regulation of esophageal adenocarcinoma (EAC) cell invasiveness and related molecular mechanism. Surface IL-17 receptor (IL-17R) expression on human EAC cell line OE19 was examined using flow cytometry. The effect of IL-17A on cell proliferation was measured by MTT assay. Cell migration and invasive ability invitro were assessed by wound-healing and Matrigel-coated Transwell invasion assay. Intracellular reactive oxygen species (ROS) levels were determined by flow cytometry and fluorescence microscope. The protein expression levels of MMP-2, MMP-9, NF-κB and p-IκB-α were detected by western blotting. Our results showed that IL-17A promoted migration and invasion of OE19 cells in a dose-dependent manner, however it had less effect on OE19 cell proliferation. Furthermore, IL-17A treatment significantly upregulated the expression of MMP-2 and MMP-9, stimulated intracellular ROS production, increased IκB-α phosphorylation and NF-κB nuclear translocation. Nevertheless, IL-17A-induced expression of MMP-2/9 and OE19 cell invasiveness were both inhibited by pretreatment with N-acetyl-L-cysteine (NAC, a ROS scavenger) or pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor). In conclusion, these findings demonstrate that IL-17A can promote the migration and invasiveness of EAC cells through ROS/NF-κB/MMP-2/9 signaling pathway activation, indicating that IL-17A may be a potential therapeutic target for EAC.

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines

Purpose: To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure.Materials and methods: Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells.Results: We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1 Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5 Gy.Conclusion: These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.

Cranberry proanthocyanidins inhibit esophageal adenocarcinoma in vitro and in vivo through pleiotropic cell death induction and PI3K/AKT/mTOR inactivation.

Cranberries are rich in bioactive constituents known to improve urinary tract health and more recent evidence supports cranberries possess cancer inhibitory properties. However, mechanisms of cancer inhibition by cranberries remain to be elucidated, particularly in vivo. Properties of a purified cranberry-derived proanthocyanidin extract (C-PAC) were investigated utilizing acid-sensitive and acid-resistant human esophageal adenocarcinoma (EAC) cell lines and esophageal tumor xenografts in athymic NU/NU mice. C-PAC induced caspase-independent cell death mainly via autophagy and low levels of apoptosis in acid-sensitive JHAD1 and OE33 cells, but resulted in cellular necrosis in acid-resistant OE19 cells. Similarly, C-PAC induced necrosis in JHAD1 cells pushed to acid-resistance via repeated exposures to an acidified bile cocktail. C-PAC associated cell death involved PI3K/AKT/mTOR inactivation, pro-apoptotic protein induction (BAX, BAK1, deamidated BCL-xL, Cytochrome C, PARP), modulation of MAPKs (P-P38/P-JNK) and G2-M cell cycle arrest in vitro. Importantly, oral delivery of C-PAC significantly inhibited OE19 tumor xenograft growth via modulation of AKT/mTOR/MAPK signaling and induction of the autophagic form of LC3B supporting in vivo efficacy against EAC for the first time. C-PAC is a potent inducer of EAC cell death and is efficacious in vivo at non-toxic behaviorally achievable concentrations, holding promise for preventive or therapeutic interventions in cohorts at increased risk for EAC, a rapidly rising and extremely deadly malignancy.

Translational research on esophageal adenocarcinoma: from cell line to clinic

Human esophageal adenocarcinoma (EAC) cell lines have made a substantial contribution to elucidating mechanisms of carcinogenesis and drug discovery. Model research on EAC relies almost entirely on a relatively small set of established tumor cell lines because appropriate animal models are lacking. Nowadays, more than 20% of all fundamental translational research studies regarding EAC are partially or entirely based on these cell lines. The ready availability of these cell lines to investigators worldwide have resulted in more than 250 publications, including many examples of important biomedical discoveries. The high genomic similarities (but certainly not completely identical) between the EAC cell lines and their original tumors provide rational for their use. Recently, in a collaborative effort all available EAC cell lines have been verified resulting in the establishment of a reliable panel of 10 EAC cell lines. It could be expected that the value of these cell lines increases as unlimited source of tumor material because new biomedical techniques require more tumor cells and the supply of viable tumor cells is diminishing because of neoadjuvant chemo(radio)therapy of patients with EAC. Here, we review the history of the EAC cell lines and their utility in translational research and biomedical discovery.

Identification and characterization of stemlike cells in human esophageal adenocarcinoma and normal epithelial cell lines

Recent studies have suggested that human solid tumors may contain subpopulations of cancer stem cells with the capacity for self-renewal and the potential to initiate and maintain tumor growth. The aim of this study was to use human esophageal cell lines to identify and characterize putative esophageal cancer stem cell populations. To enrich stemlike cells, Het-1A (derived from immortalized normal esophageal epithelium), OE33, and JH-EsoAd1 (each derived from primary esophageal adenocarcinomas) were cultured using serum-free media to form spheres. A comprehensive analysis of parent and spheroid cells was performed by flow cytometry, Western blot analysis, immunohistochemistry and polymerase chain reaction array to study cancer stem cell-related genes, colony formation assays to assess clonogenicity, xenotransplantation to assess tumorigenicity, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays to assess chemosensitivity to 5-fluorouracil and cisplatin. For all cell lines, clonogenicity, tumorigenicity, and chemoresistance to 5-fluorouracil and cisplatin were significantly higher than for spheroid cells compared with parent cells. Spheroids exhibited an increased frequency of cells expressing integrin α6(bri)/CD71(dim), and Achaete-scute complex homolog 2 messenger RNA and protein were also significantly overexpressed in spheroid cells compared with parent cells. The higher clonogenicity, tumorigenicity, and drug resistance exhibited by spheroids derived from Het-1A, OE33, and JH-EsoAd1 reflects an enrichment of stemlike cell populations within each esophageal cell line. Esophageal cells enriched for integrin α6(bri)/CD71(dim) and/or overexpressing Achaete-scute complex homolog 2 would appear to represent at least a subpopulation of stemlike cells in Het-1A, OE33, and JH-EsoAd1.

Targeting the intrinsic inflammatory pathway: honokiol exerts proapoptotic effects through STAT3 inhibition in transformed Barrett's cells

One way to link chronic inflammation with cancer is through the intrinsic inflammatory pathway, in which genetic alterations that induce malignant transformation also produce a cancer-promoting, inflammatory microenvironment. Signal transducer and activator of transcription 3 (STAT3) contributes to the intrinsic inflammatory pathway in Barrett's esophagus. In human tumors, honokiol (a polyphenol in herbal teas) has growth-inhibitory and proapoptotic effects associated with suppressed activation of STAT3. We used human Barrett's epithelial and esophageal adenocarcinoma cell lines to determine effects of honokiol on cell number, necrosis, apoptosis, and anchorage-independent growth and to explore STAT3's role in those effects. We determined Ras activity and expression of phosphorylated ERK1/2, phosphorylated Akt, and phosphorylated STAT3 in the presence or absence of honokiol. Cells were infected with constitutively active Stat3-C to assess effects of honokiol-induced STAT3 inhibition on apoptosis. Honokiol decreased cell number and increased necrosis and apoptosis in transformed Barrett's cells, but not in nontransformed cells. In adenocarcinoma cells, honokiol also increased necrosis and apoptosis and decreased anchorage-independent growth. Within 30 min of honokiol treatment, transformed Barrett's cells decreased expression of phosphorylated STAT3; decreases in Ras activity and phosphorylated ERK1/2 expression were detected at 24 h. Infection with Stat3-C significantly reduced apoptosis after honokiol treatment. Honokiol causes necrosis and apoptosis in transformed Barrett's and esophageal adenocarcinoma cells, but not in nontransformed Barrett's cells, and the proapoptotic effects of honokiol are mediated by its inhibition of STAT3 signaling. These findings suggest a potential role for targeting the intrinsic inflammatory pathways as a therapeutic strategy to prevent Barrett's carcinogenesis.

Abstract 190: Long noncoding RNA XY11-10-11 regulates proliferation and migration of esophageal adenocarcinoma cells

Abstract Esophageal carcinoma is one of the most common types of cancer, ranked among the top eight causes of cancer death worldwide. The molecular mechanisms underlying esophageal cancer are very complicated and have not yet been completely clarified. In recent years, long noncoding RNAs (lncRNAs) have been shown to play critical regulatory roles in various cancer biology, yet their potential involvement in esophageal cancer is not well understood. Here, we identified a set of lncRNAs that are differentially expressed in esophageal cancer tissues in comparison to the normal tissues using quantitative real-time polymerase chain reaction. A new lncRNA, which we name XY11-10-11, is significanty increased in expression in human esophageal adenocarcinoma tissues and cell lines. XY11-10-11 is an intergenic lncRNA expressed from a locus located on chromosome 14. Importantly, knockdown of XY11-10-11 by siRNA resulted in inhibition of proliferation and migration in esophageal adenocarcinoma cells (EACs). Moreover, patients with high XY11-10-11 expression levels had a relatively poorer prognosis. Our findings indicate that XY11-10-11 may play a significant role in the development or progression of esophageal adenocarcinoma and may be an potential biomarker for diagnosis and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 190. doi:1538-7445.AM2012-190

Mapping of homozygous deletions in verified esophageal adenocarcinoma cell lines and xenografts

Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 1-6 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development.

Deciphering the role of paclitaxel in the SKGT4 human esophageal adenocarcinoma cell line

Paclitaxel (taxol) has been used for the treatment of various human tumors and is an exceedingly efficient chemotherapy agent against esophageal cancer. However, the precise molecular mechanisms of paclitaxel effects on human esophageal adenocarcinoma cells are not well understood. MTT assay and cell cycle analysis were performed to examine the mechanism of antiproliferative and cell viability effects of paclitaxel in human esophageal adenocarcinoma cancer cells. Western blotting was also used to examine the cell cycle- and apoptosis-related proteins. Paclitaxel inhibited the proliferation of SKGT4 cells in a dose- and time-dependent manner with G2/M arrest. In addition, paclitaxel induced apoptosis through the activation of caspase-3 followed by PARP degradation. In conclusion, our results suggest that paclitaxel leads to mitotic cell cycle arrest following G2/M arrest and induces apoptosis via a caspase-3 pathway in SKGT4 cells.