Abstract 6968: Targeting overexpressed NEK2 kinase in esophageal adenocarcinoma

Objective: The incidence of esophageal adenocarcinoma (EAC) has increased over 300% in the past decades in western countries. EAC is characterized by an early metastasis, with a 5-year survival rate of only 19%, making the identification of clinically-targetable proteins critical for improving patient outcome. Osteopontin (SPP1/OPN) is a secreted glycosylated phosphoprotein that is found overexpressed in many cancer types including EAC. OPN protein functions in cell adhesion and cell migration by regulating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. However, little is known about the role of OPN in EAC invasion and metastasis. Methods: We analyzed the OPN gene expression in a series of 46 esophageal samples including esophageal Barrett's, low-grade dysplasia, high-grade dysplasia and adenocarcinomas mRNA using Affymetrix U133A arrays. Real-time RT-PCR was performed to validate array results and examine a larger cohort of 130 EAC samples. Tissue microarrays (TMA) were made containing 64 EACs, eight lymph node metastases, eight dysplastic and 11 none dysplastic Barrett's mucosa sections from 59 patients. Primers flanking the potential gene alternative splice regions were designed and the in-frame transcript variants were sequenced. Expression of OPN in EAC cell lines was analyzed using RT-PCR and western blot analysis. Ectopic OPN expression of splice variants and OPN knockdown in EAC cell lines have been performed to examine its roles in tumor invasion and EMT assays. Results: OPN is highly overexpressed in EAC and overexpression of the gene is associated with poorer survival in patients. Three OPN splice variants were identified with their expression either proportionally increased or differentially expressed in the primary EAC and EAC-derived cells. Highly overexpressed OPN is a transcriptional event as no OPN gene amplification was found in the corresponding EACs. In addition, abundant OPN protein was found primarily in tumor cells in the tissue sections using immunohistochemical (IHC)-TMA assays. OPN variants differ in their N-terminal thrombin fragment with either exon 6 or 5 being absent in OPN-b or OPN-c, respectively. We found that EAC cells treated with the medium containing different OPN variants demonstrated distinctive morphological phenotypes. We further identified that OPN variants played different roles in tumor cell proliferation, epithelial-mesenchymal transition (EMT), and chemosensitivity. Conclusion: Upregulation of OPN is predominant event in primary EACs and associated with poorer survival in patients. The alternative splice variants demonstrate differential tumor invasion, EMT transition and chemosensitivity in EAC cells. Citation Format: Zhuwen Wang, Kimmy Leverenz, Dafydd G. Thomas, Amy L. Myers, Andrew C. Chang, Mark B. Orringer, Thomas J. Giordano, Jules Lin, David G. Beer, Lin Lin. Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3879. doi:10.1158/1538-7445.AM2013-3879

Background: The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in Western countries. Most EACs originate from Barrett’s esophagus (BE), a precancerous lesion initiated by chronic reflux of gastrointestinal contents, especially acidic bile salts (ABS). Several chemotherapeutics induce high reactive oxygen species (ROS) levels to eliminate tumor growth. Ferroptosis is a recently identified cell death mode triggered by ROS and subsequent lipid peroxidation. However, cancer cells develop adaptive abilities to recalibrate redox balance via hijacking the antioxidant systems, resulting in drug resistance. PRDX2 protein is a member of the peroxiredoxin family of antioxidant enzymes. We hypothesized that PRDX2 may be a critical mediator in redox-associated chemoresistance under reflux conditions. Methods: We analyzed RNA sequencing data and public databases to unveil the aberrant overexpression and potential chemoresistance function of PRDX2 in EAC. To simulate both acute and chronic clinical conditions of gastroesophageal reflux disease (GERD) in vitro, we employed transient and repeated ABS exposure (rABS). EAC cells and 3D organotypic culture (OTC) served as platforms to explore the anti-ferroptosis function and regulatory mechanisms of PRDX2. Our initial findings were validated in patient-derived xenografts (PDX), and human EAC tissue microarrays (TMA). Results: Aberrant expression of PRDX2 was detected in both human EAC tissues and cell lines exhibiting resistance to oxaliplatin treatment. Knockdown of PRDX2 impaired the recovery of EAC cells from ABS-induced ROS-dependent lipid peroxidation. Moreover, silencing PRDX2 sensitized the chemo-resistant EAC cells to oxaliplatin. Chromatin immunoprecipitation (ChIP) assay identified that NF-kB activated PRDX2 transcription by directly binding to its promoter region. We also found that APE1-redox-mediated NF-kB activation is crucial for ABS-induced PRDX2. We further discovered that PRDX2 contributes to chemoresistance by upregulating GPX4 and suppressing ferroptosis. Furthermore, immunofluorescent staining in the 3D model (OTC) and immunohistochemistry staining in human TMA confirmed co-overexpression of APE1 and PRDX2. In addition, APX2009, an APE1-redox-specific inhibitor, significantly sensitized EAC cells to oxaliplatin by inhibiting PRDX2 expression and inducing ferroptosis. Notably, we observed synergistic effects of the combination of Oxaliplatin and APX2009 in the xenografts derived from a chemo-resistant EAC cell line. Conclusion: Our findings revealed a novel link between reflux-induced redox unbalance and ferroptosis-related chemoresistance in EAC via APE1/redox/NF-kB/PRDX2/GPX4 axis. Targeting PRDX2 by APE1-redox-specific inhibitors is a potential novel strategy for drug combination in refractory EAC. Citation Format: Lei Chen, Heng Lu, Farah Ballout, Dunfa Peng, Zheng Chen, Jianwen Que, Oliver G. McDonald, Alexander Zaika, Wael El-Rifai. Chronic gastroesophageal reflux promotes chemoresistance in esophageal adenocarcinoma through APE1-redox-dependent PRDX2 activation and ferroptosis inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 542.

68 Background: EACs often carry amplifications in MYC (19%) and mutations in the SWI/SNF complex (13%) which act as oncogenic drivers. Targeting of MYC-amplified tumors via BET inhibition, as well as HDAC2 or EZH2 inhibition in SWI/SNF-mutated tumors, show promise in hematological cancers, and solid tumors, respectively. Herein, we investigate the effects of BET, HDAC2, and EZH2 inhibition on EAC cell line proliferation. Methods: MYC-wild type (n = 5), MYC-amplified (n = 2), SWI/SNF-wild type (n = 6) and SWI/SNF-mutant (n = 1) EAC cell lines that have undergone whole genome sequencing (WGS) were plated at 10% confluency and treated with BET (JQ1), pan-HDAC (SAHA), and EZH2 (EPZ-6438) inhibitors at various concentrations. The CellTiter-Glo cell proliferation assay was used to compare the effects of drug treatment on proliferation between different cell lines and drug concentrations before and after treatment. Results: MYC-amplified EAC cell lines were more sensitive to BET inhibition (Table); GI50 concentrations for JQ1 are comparable to those achieved in melanoma and lymphoma cell lines, cancer types in which clinical activity has been demonstrated. ERBB2 amplified or ERBB2 mutant EAC cells were also sensitive to BET inhibition. HDAC2 inhibition reduced proliferation of both SWI/SNF-wild type and mutant cell lines at high concentrations, whilst all cell lines were resistant to EZH2 inhibition, even at the highest concentrations used. Conclusions: MYC amplified EAC tumors appear to be sensitive to BET inhibition; these results warrant further evaluation in pre-clinical models and clinical trials. HDAC2 and EZH2 inhibition were ineffective in both the SWI/SNF-wild type and mutant type EAC cell lines; these therapies are less likely to provide benefit for EAC patients. [Table: see text]

Introduction: Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in the United States, and prognosis of EAC remains poor despite modern combination therapies. Therefore, new therapeutic approaches are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to EAC aggressiveness and poor clinical prognosis. Typically, EAC cell lines are used in two-dimensional (2D) cultures for screening of anticancer agents, and they do not represent the hypoxic tumor microenvironment. In this study, we established a novel three dimensional (3D) spheroid co-culture model of EAC. We then observed treatment response of chemo and hypoxia-targeting therapies in 2D, 3D and in vivo models of experimental EAC. Methods: EAC 3D spheroids were generated from co-culturing human EAC and fibroblast cell lines. NanoCulture® plates and dishes were used for 3D spheroid cultures. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Paclitaxel (PT), carboplatin (CP) and nanoparticle albumin-bound paclitaxel (NPT) were used as chemotherapeutic agents, whereas acriflavin was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 assay, in vivo tumor growth was detected by measuring subcutaneous xenograft using OE19 EAC cells, apoptosis was detected by analysis of cleaved caspase 3 and PARP expressions, hypoxia-targeting was detected by analysis of HIF-1α expression and stem cell phenotype was detected by flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity. Results: The results demonstrated that the 3D culture was more resistant to antiproliferative/proapoptotic effects of chemotherapeutic agents PT, CP, NPT and their combinations over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavin showed stronger antiproliferative/proapoptotic effects in 3D culture than in 2D culture. We observed strong expression of hypoxia inducible factor-1α (HIF-1α) in 3D culture with no expression of HIF-1α in 2D culture, and acriflavin treatment completely abolished the HIF-1α expression in 3D culture. We also observed hypoxia inside the 3D culture spheroids, but not in cells grown in 2D culture. Interestingly, we observed a reduced number of ALDH positive cells in 3D culture after acriflavin treatment compared to that after NPT treatment, indicating the preferential antiproliferative effect of acriflavin over NPT on cancer stem cells. In addition, acriflavin showed significant antitumor efficacy both as monotherapy and in combination with NPT. The xenograft tumor growth inhibition rate after a 2-week treatment with acriflavin, NPT and their combination was 44.97, 58.35 and 67.29 percent respectively (p Conclusion: Thus 3D cultures may be better than 2D cultures in simulating the important in vivo tumor characteristic of hypoxia, and HIF-targeting therapy acriflavin could be a novel treatment strategy for EAC. Citation Format: Md Sazzad Hassan, Marisa Lenga, Lucia Petrova, Urs von Holzen. Therapeutic targeting of hypoxia 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 1230.

Introduction: Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in the United States, and prognosis of EAC remains poor despite modern combination therapies. Therefore, new therapeutic approaches are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to EAC aggressiveness and poor clinical prognosis. Typically, EAC cell lines are used in two-dimensional (2D) cultures for screening of anticancer agents, and they do not represent the hypoxic tumor microenvironment. In this study, we established a novel three dimensional (3D) spheroid co-culture model of EAC. We then observed treatment response of chemo and hypoxia-targeting therapies in 2D, 3D and in vivo models of experimental EAC. Methods: EAC 3D spheroids were generated from co-culturing human EAC and fibroblast cell lines. NanoCulture® plates and dishes were used for 3D spheroid cultures. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Paclitaxel (PT), carboplatin (CP) and nanoparticle albumin-bound paclitaxel (NPT) were used as chemotherapeutic agents, whereas acriflavin was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 assay, in vivo tumor growth was detected by measuring subcutaneous xenograft using OE19 EAC cells, apoptosis was detected by analysis of cleaved caspase 3 and PARP expressions, hypoxia-targeting was detected by analysis of HIF-1α expression and stem cell phenotype was detected by flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity. Results: The results demonstrated that the 3D culture was more resistant to antiproliferative/proapoptotic effects of chemotherapeutic agents PT, CP, NPT and their combinations over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavin showed stronger antiproliferative/proapoptotic effects in 3D culture than in 2D culture. We observed strong expression of hypoxia inducible factor-1α (HIF-1α) in 3D culture with no expression of HIF-1α in 2D culture, and acriflavin treatment completely abolished the HIF-1α expression in 3D culture. We also observed hypoxia inside the 3D culture spheroids, but not in cells grown in 2D culture. Interestingly, we observed a reduced number of ALDH positive cells in 3D culture after acriflavin treatment compared to that after NPT treatment, indicating the preferential antiproliferative effect of acriflavin over NPT on cancer stem cells. In addition, acriflavin showed significant antitumor efficacy both as monotherapy and in combination with NPT. The xenograft tumor growth inhibition rate after a 2-week treatment with acriflavin, NPT and their combination was 44.97, 58.35 and 67.29 percent respectively (p<0.05). Conclusion: Thus 3D cultures may be better than 2D cultures in simulating the important in vivo tumor characteristic of hypoxia, and HIF-targeting therapy acriflavin could be a novel treatment strategy for EAC. Citation Format: Md Sazzad Hassan, Marisa Lenga, Lucia Petrova, Urs von Holzen. Therapeutic targeting of hypoxia 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 1230.

We compared whole genomic changes in cell lines generated from esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). To do so, we investigated chromosomal DNA copy number changes in four EAC cell lines and three ESCC cell lines using comparative genomic hybridization (CGH). Frequent gains of chromosome 5p, 8q, and 20q occurred in both ESCC and EAC cell lines, but gains of 3q, 5q, and 9q were mainly seen in ESCC cell lines; gain of chromosome 10q25-qtel was mainly seen in EAC cell lines. It was noticeable that 18q12 loss existed in 2 EAC and 1 ESCC cell lines in our study. The chromosomal abnormalities common to all of the cell lines may help to identify candidate genes related to both EAC and ESCC. The chromosome aberrations mainly seen in either EAC or ESCC cell lines are in keeping with their known different etiology and may lead to the identification of genes important for disease specific pathogenesis.

Esophageal adenocarcinoma cells do not traffic Fas Ligand to the cell surface: Evidence against the “Counterattack” hypothesis

Background: Esophageal adenocarcinoma (EAC) is one the deadliest cancers in United States (US) and the only major cancer in the US with increasing incidence. Gastroesophageal reflux disease (GERD) can cause Barrett’s esophagus (BE), a columnar metaplasia of the esophagus strongly associated with the development of EAC. GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal esophageal cells and found high expression of tripartite motif-containing protein 31 (TRIM31) in BE and EAC compared to normal esophagus (NE) by transcriptomic profiling of RNA extracted from human esophageal tissues. E3 ubiquitin ligase TRIM 31 is a new member of TRIM family and it has been reported to have oncogenic potentials in some cancers. However, its role in EAC pathogenesis is yet to be understood. Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot analysis. The Het-1A cell line, after exposure to acidic pH and bile acids, was assessed for variable TRIM31 expression. Cell morphology and viability analyses of NE and EAC cell lines after exposure to acidic pH and bile acids either alone or in combinations were observed by microscopy and WST-1 assay respectively. Results: Transcriptomic analysis showed that BE and EAC are similar in their differential gene expression with upregulation of the TRIM31 compared to that in NE. Concordant with their columnar phenotype, BE and EAC groups displayed a multitude of differentially expressed genes relative to NE samples; however, less differentially expressed genes were found comparing BE and EAC groups. Western blot demonstrated frequent overexpression of TRIM31 in EAC cell lines with very low expression of TRIM31 in NE epithelial cell line. When NE epithelial cell line Het-1A was exposed to acidic pH as well as bile acids in acidic pH, we saw a change in cell morphology that may correlate with the metaplasia of these cells to more resilient forms, as seen in human BE and EAC. Interestingly, bile acids in acidic pH increased TRIM31 expression in NE cell line Het-1A. Bile acids had a greater effect on decreasing cell viability at high concentrations and that effect is enhanced when bile acids were added in acidic pH. WST-1 cell viability assay revealed that EAC cells were more resistant to bile acids as well as bile acids in acidic pH induced decreased in cell viability compared to that in NE cell line Het-1A. The sustained cell viability of EAC cell lines under these harsh conditions correlates with the upregulation of TRIM31. Conclusions: Thus, TRIM31 may be an important oncogenic factor in GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC. Citation Format: Md Sazzad Hassan, Jesse Mast, Annie Ritter, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. Role of TRIM31 in the pathogenesis of 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 555.

Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett's Esophagus and Esophageal Adenocarcinoma

70 Background: There is a pressing need to identify new therapeutic targets for esophageal adenocarcinoma (EAC). Hence, we utilized siRNA-screening libraries to identify genes impacting on EAC cancer cell growth to identify potential therapeutic targets. Methods: A “druggable genome” library (6,022 individual siRNAs) was utilized to examine EAC cell survival using the MTT assay. Statistical analysis combined the use of Z-factor, t-test and SSMD. EAC cell lines GohTRT, SKGT4 and OE33 were utilized. Functional validation of the resulting siRNAs utilized RT-PCR, Western Blot, ELISA and TOPFLASH assays. Results: siRNA library screening resulted in positive quality metrics (Z-factor>0.5) confirming its validity and further identifying 118 high confidence gene targets affecting EAC cell growth. Verification of these siRNA targets in multiple cell lines indicated a good level of concordance with the primary screening data. Bioinformatic and pathway mapping approaches of these targets emphasized links between EAC cell proliferation and regulators of inflammation and “immune cell processes” (LIF, C1Qa, C1r, C1s, GDF15, IL9R and TREM2). Pathological and transcriptomic studies demonstrated that LIF (FC=96.7; P<0.0001), GDF15 (EAC: FC=74; P<0.0001), C1Qa and TREM2 may be up-regulated in EAC biopsies. In functional work, exposure of EAC cell lines to recombinant or native proteins of C1q, LIF and GDF15 rescued the observed effects of their respective silencing in EAC cells (>90%,p0.001) and acted as potential growth promoters (>40%, p0.01). Auto-regulatory feedback loops were discovered in response to treatment with exogenous C1q and LIF in EAC cells. Signal transduction could be induced through b-catenin stabilization and STAT3 pathways in response to C1q and LIF treatment respectively. GDF15 was observed to act in a similar fashion to TGFb in scratch wound assays and additionally regulate Th17 type T-cell differentiation. Conclusions: Genes regulating EAC proliferation have been defined by siRNA library screening. We have identified secreted immune factors, not previously associated with EAC biology, capable of regulating EAC cell survival.

BackgroundVisceral obesity has a strong association with both the incidence and mortality of esophageal adenocarcinoma (EAC). Alterations in mitochondrial function and energy metabolism is an emerging hallmark of cancer, however, the potential role that obesity plays in driving these alterations in EAC is currently unknown.MethodsAdipose conditioned media (ACM) was prepared from visceral adipose tissue taken from computed tomography-determined viscerally-obese and non-obese EAC patients. Mitochondrial function in EAC cell lines was assessed using fluorescent probes, mitochondrial gene expression was assessed using qPCR-based gene arrays and intracellular ATP levels were determined using a luminescence-based kit. Glycolysis and oxidative phosphophorylation was measured using Seahorse XF technology and metabolomic analysis was performed using 1H NMR. Expression of metabolic markers was assessed in EAC tumor biopsies by qPCR.ResultsACM from obese EAC patients significantly increased mitochondrial mass and mitochondrial membrane potential in EAC cells, which was significantly associated with visceral fat area, and was coupled with a significant decrease in reactive oxygen species. This mitochondrial dysfunction was accompanied by altered expression of 19 mitochondrial-associated genes and significantly reduced intracellular ATP levels. ACM from obese EAC patients induced a metabolic shift to glycolysis in EAC cells, which was coupled with significantly increased sensitivity to the glycolytic inhibitor 2-deoxyglucose. Metabolomic profiling demonstrated an altered glycolysis and amino acid-related signature in ACM from obese patients. In EAC tumors, expression of the glycolytic marker PKM2 was significantly positively associated with obesity.ConclusionThis study demonstrates for the first time that ACM from viscerally-obese EAC patients elicits an altered metabolic profile and can drive mitochondrial dysfunction and altered energy metabolism in EAC cells in vitro. In vivo, in EAC patient tumors, expression of the glycolytic enzyme PKM2 is positively associated with obesity.

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.

251 A Functional Interplay Between TGF- and Notch Signaling Determines Esophageal Cancer Cell Fates

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling. 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 3243. doi:1538-7445.AM2012-3243

252 CD44 Defines Highly Plastic Esophageal Squamous Cancer Cells With Tumor Initiating Capabilities