Cell Lines OE33 Research Articles

557. POTENTIAL THERAPEUTIC TARGETS OF TRPV2 AND SLC12A2 IN ESOPHAGEAL ADENOCARCINOMA CANCER STEM CELLS

Abstract Background Cancer stem cells (CSCs) are resistant to current anticancer drugs and radiation. The membrane transporters have recently been reported to play crucial roles for Esophageal adenocarcinoma (EA) CSCs. The aim of our study was to investigate ion channel expression profiles in esophageal adenocarcinoma CSCs (EACSCs) and to examine these potential as therapeutic targets. Methods Using the EA cell line OE33, cells with strong ALDH1A1 activity, a CSC marker, were isolated by flow cytometry. The characteristics of CSCs for the separated cells were verified by sphere formation assay, the expression of CSC markers on real-time reverse transcription-polymerase chain reaction and western blotting, and anti-cancer agent sensitivity assay. Gene expression profiles in EACSCs were examined by a microarray analysis. The transporters, high expression in CSC, were examined the anti-tumor effect of these inhibitors. Results Separated cells formed spheres in low-adhesion plates. The expressions of CSC markers were higher in CSCs than in non-CSCs. The CSCs also exhibited resistance to cisplatin and redifferentiation potential. The microarray analysis showed that the expression of transient receptor potential vanilloid 2 (TRPV2) and solute carrier family 12 member 2 (SLC12A2), which related to ion channels, were upregulated in CSCs. The cytotoxicities of tranilast and furosemide, which were TRPV2 and SLC12A2 inhibitors respectively, were added to cisplatin. Conclusions We revealed that TRPV2 and SLC12A2 are highly expressed in EACSCs and have potential as therapeutic targets for EA.

Characterization of MET Alterations in 37 Gastroesophageal Cancer Cell Lines for MET-Targeted Therapy.

Capmatinib and savolitinib, selective MET inhibitors, are widely used to treat various MET-positive cancers. In this study, we aimed to determine the effects of these inhibitors on MET-amplified gastric cancer (GC) cells. Methods: After screening 37 GC cell lines, the following cell lines were found to be MET-positive with copy number variation >10: SNU-620, ESO51, MKN-45, SNU-5, and OE33 cell lines. Next, we assessed the cytotoxic response of these cell lines to capmatinib or savolitinib alone using cell counting kit-8 and clonogenic cell survival assays. Western blotting was performed to assess the effects of capmatinib and savolitinib on the MET signaling pathway. Xenograft studies were performed to evaluate the in vivo therapeutic efficacy of savolitinib in MKN-45 cells. Savolitinib and capmatinib exerted anti-proliferative effects on MET-amplified GC cell lines in a dose-dependent manner. Savolitinib inhibited the phosphorylation of MET and downstream signaling pathways, such as the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways, in MET-amplified GC cells. Additionally, savolitinib significantly decreased the number of colonies formed on the soft agar and exerted dose-dependent anti-tumor effects in an MKN-45 GC cell xenograft model. Furthermore, a combination of trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in human epidermal growth factor receptor-2 (HER2)- and MET-positive OE33 cells. Targeting MET with savolitinib and capmatinib efficiently suppressed the growth of MET-amplified GC cells. Moreover, these MET inhibitors exerted synergistic effects with trastuzumab on HER2- and MET-amplified GC cells.

Abstract 6700: Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma

Abstract Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p<0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p<0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6700.

Abstract 375: Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma

Abstract Esophageal Adenocarcinoma (EAC) is the most common subtype of Esophageal Cancer in Western countries, and its incidence rate has increased over the past few decades. The overall five-year survival rate of EAC is less than 20%. Smoking is a major risk factor for EAC development. WEE1 kinase plays a crucial role in the cell cycle by regulating the G2/M checkpoint, providing a time frame for the cells to repair DNA damage. Thus, targeting WEE1 using an inhibitor could potentiate the effect of chemotherapeutic drugs. This study investigated how smoking induces WEE1 protein expression, promoting docetaxel resistance in EAC. IHC staining for WEE1 in the normal esophagus and EAC tissue sections revealed significant over-expression of WEE1 protein in EAC. Nicotine, Nicotine derived Nitrosamine Ketone (NNK), and 2% cigarette smoke extract (CSE) treatment induced WEE1 expression with a concomitant increase in CDC2 phosphorylation (p-CDC2, Y15), a well-established read-out of WEE1 activity in EAC cells. In addition, we found that smoking upregulates WEE1 expression by decreasing miR-195-5p expression levels in EAC. Based on the differential gene expression signatures in EAC cell lines FLO1 and OE33 following WEE1 knockdown, drug and small molecule induced gene expression signatures analysis, L1000 fireworks display (L1000FWD), predicted that docetaxel is one of the best drug candidates which can synergize with the WEE1 inhibitor MK1775. Smoking-induced docetaxel resistance in EAC cells measured by cell viability assay. Down-regulating WEE1 expression with siRNA or pharmacological inhibition using MK1775 significantly sensitized EAC cells to docetaxel treatment, as evidenced by a remarkable decrease in the IC50 value of docetaxel. Inhibition of WEE1 combined with docetaxel can be considered an ideal therapeutic strategy due to its superior anti-tumor efficacy compared to the standard single-agent treatment. In conclusion, our data provide a firm rationale for the clinical combination of docetaxel with MK1775 in EAC patients. Citation Format: Krishnapriya Thangaretnam, Islam MD Obaidul, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Zheng Chen. Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 375.

OGC P21 EZH2: A novel therapeutic target in oesophageal adenocarcinoma

Abstract Background Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of the histone lysine methyltransferase Polycomb Repressor Complex 2 (PRC2), mediates gene silencing of target genes epigenetically. It is over-expressed and associated with poor outcomes in numerous malignancies. EZH2 inhibitors are currently used in the treatment of follicular lymphomas and epithelioid sarcomas. Oesophageal adenocarcinoma (OAC) has predicted sensitivity to EZH2 inhibition due to the high prevalence of potentially sensitizing mutations. This in vitro study aimed to assess the efficacy of EZH2 inhibitors in the treatment of OAC. Methods Bioinformatical analysis of publicly available bulk RNA transcriptomic data from OAC samples included in the Cancer Genome Atlas (TCGA) database was carried out. OAC cell lines OE33, FLO1 and MFD1 cultured in 2D were treated with a panel of EZH2 inhibitors at a range of clinically relevant concentrations either as monotherapies or in combination with OAC standard of care (SOC) chemotherapies. Cell viability was assessed via colorimetric MTS assay at 72hrs and 6 days post-treatment. Results Median EZH2 mRNA expression levels calculated from OAC TCGA data were significantly greater than EZH2 mRNA expression in normal oesophageal tissue (p<0.01). OAC patients with high EZH2 expression had reduced median overall survival compared to patients with low EZH2 expression (13.1 months vs 32 months, p= 0.03). EZH2 inhibitors demonstrated efficacy as monotherapies in all 3 cell lines. EZH2 inhibitors in combination with 5-fluorouracil (5-FU) showed greater efficacy than 5-FU alone, demonstrated by reductions in 5-FU half-maximal inhibitory concentration (IC50) values. Conclusions These early in vitro results demonstrate the potential of EZH2 inhibition as a novel therapy in OAC, particularly in combination with current chemotherapy agents. Further in vitro work is focused on their efficacy in patient derived tumour organoid models.

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transitionof ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.

Abstract 2624: Methadone hydrochloride blocks epinephrine-induced proliferation and enhances apoptotic effects of nab-paclitaxel in esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is an aggressive form of cancer with 5-year survival rates under 20%. The low survival rate is due largely to advanced stage upon diagnosis. Improved treatment options remain essential in combating the disease and bolstering patient outcomes. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is a chemotherapeutic receiving attention for its potential in EAC treatment. The taxane has exhibited greater specificity for targeting cancer cells while decreasing cytotoxic effects to adjacent healthy populations. Unfortunately, chemoresistance remains an issue surrounding adequate treatment regimes. Epinephrine and its receptor, β2 adrenergic, have been shown to dysregulate various oncogenic pathways and reduce chemotherapeutics efficacy. Typically associated with pain management and addiction treatment, Methadone hydrochloride has been investigated for its potential to block the oncogenic effects of epinephrine and strengthen the impact of chemotherapeutics. In this study, we evaluated the enhancement of apoptosis by combination of nab-paclitaxel and methadone, and we demonstrated the ability of methadone to block epinephrine-induced chemoresistance. Method: We utilized PCR to elucidate EAC cell lines with the greatest expression of Mu opioid and β2 adrenergic receptors. Next, we investigated adequate dosages of methadone and epinephrine for the EAC cell lines by WST-1 assay. WST-1 assays were also utilized to determine cell growth under co-administration of nab-paclitaxel and methadone as well as methadone and epinephrine. Propidium iodine cell viability flow cytometry assay was used to detect cell death. Annexin V staining was utilized to further distinguish between apoptotic and necroptotic cells. Result: OE19 and OE33 cell lines showed increased expression of Mu and β2 adrenergic receptors, ensuring the cells were capable of binding to methadone or epinephrine. WST-1 assays established 100 nM of epinephrine and 200 nM of methadone as optimal dosages for both cell lines. Our previous studies demonstrated that about 5.85 uM of nab-paclitaxel (5ug/mL) induces apoptosis in OE19 and OE33. Propidium iodide staining showed the enhancement of cell death under combination treatment of nab-paclitaxel and methadone. Annexin V staining confirmed that the effect was via apoptosis. Conclusion: The current study demonstrated methadone’s ability to block epinephrine-induced cell proliferation and showed an enhanced effect on apoptosis when combining nab-paclitaxel with methadone in EAC treatment. Citation Format: Nicholas Cwidak, Sazzad Hassan, Chloe Johnson, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. Methadone hydrochloride blocks epinephrine-induced proliferation and enhances apoptotic effects of nab-paclitaxel in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2624.

Insights into the anticancer activity of chiral alkylidene-β-lactams and alkylidene-γ-lactams: Synthesis and biological investigation

Chiral alkylidene-β-lactams and alkylidene-γ-lactams were synthesized and screened for their in vitro activity against four human cancer cell lines (melanoma, esophageal, lung and fibrosarcoma carcinoma). Alkylidene-β-lactams were synthesized via Wittig reaction of diverse phosphorus ylides with benzhydryl 6‐oxopenicillanate, derived from 6-aminopenicillanic acid. Moreover, novel chiral alkylidene-γ-lactams were synthesized through a multistep strategy starting from a chiral substrate (d-penicillamine). The in vitro assays allowed the identification of four compounds with IC50 values < 10 μM for A375 cell line, and three compounds with IC50 values < 10 μM for OE19 cell line. The effect of the most promising compounds on cell death mechanism, reactive oxygen species generation as well as the evaluation of their ability to act as MMP-9 inhibitors were studied. The reported results unveil the potential of alkylidene-β-lactams as anticancer agents.

Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models.

The molecular processes that predispose the development of Barrett’s esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 μM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment.

A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer

BackgroundGastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 13–23% of all GC cases and patients with HER2 overexpression exhibit a poor prognosis. Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, is an effective agent to treat HER2-amplified breast cancer but it failed in gastric cancer (GC) clinical trials. However, the molecular mechanism of lapatinib resistance in HER2-amplified GC is not well studied.MethodsWe employed an unbiased, genome-scale screening with pooled CRISPR library on HER2-amplified GC cell lines to identify genes that are associated with resistance to lapatinib. To validate the candidate genes, we applied in vitro and in vivo pharmacological tests to confirm the function of the target genes.ResultsWe found that loss of function of CSK or PTEN conferred lapatinib resistance in HER2-amplified GC cell lines NCI-N87 and OE19, respectively. Moreover, PI3K and MAPK signaling was significantly increased in CSK or PTEN null cells. Furthermore, in vitro and in vivo pharmacological study has shown that lapatinib resistance by the loss of function of CSK or PTEN, could be overcome by lapatinib combined with the PI3K inhibitor copanlisib and MEK inhibitor trametinib.ConclusionsOur study suggests that loss-of-function mutations of CSK and PTEN cause lapatinib resistance by re-activating MAPK and PI3K pathways, and further proved these two pathways are druggable targets. Inhibiting the two pathways synergistically are effective to overcome lapatinib resistance in HER2-amplified GC. This study provides insights for understanding the resistant mechanism of HER2 targeted therapy and novel strategies that may ultimately overcome resistance or limited efficacy of lapatinib treatment for subset of HER2 amplified GC.

Identification of Phospho-Tyrosine Targets as a Strategy for the Treatment of Esophageal Adenocarcinoma Cells.

IntroductionEsophageal cancer (EC) is an aggressive cancer type that is increasing at a high rate in the US and worldwide. Extensive sequencing of EC specimens has shown that there are no consistent driver mutations that can impact treatment strategies. The goal of this study was to identify activated tyrosine kinase receptors (TKRs) in EC samples as potential targets in the treatment of EC.MethodsActivated tyrosine kinase receptors were detected using a dot-blot array for human TK receptors. Human esophageal cancer cell lines were transplanted into immunocompromised mice, and tumor xenografts were subjected to tyrosine kinase inhibitors based on the dot-blot array data.ResultsUsing the OE33 esophageal cancer cell line, we identified activated EGF receptor (EGFR), as well as ErbB2 and ErbB3. Treatment of this cell line with erlotinib, a specific inhibitor of EGFR, did not impact the growth of this tumor cell line. Treating the OE33 cell line with afatinib, a pan-EGFR family inhibitor resulted in the growth inhibition of OE33, indicating that the ErbB2 and ErbB3 receptors were contributing to tumor cell proliferation. Afatinib treatment of mice growing OE33 tumors inhibited growth of the OE33 tumor cells.DiscussionActivated tyrosine kinase receptors were readily detected in both cancer cell lines and human esophageal cancer samples. By identifying the activated receptors and then using the appropriate tyrosine kinase inhibitors, we can block tumor growth in vitro and in animal xenografts. We propose that identifying and targeting activated TKRs can be used as a personalized EC tumor treatment strategy.

Enhanced Antitumor Effect of Trastuzumab and Duligotuzumab or Ipatasertib Combination in HER-2 Positive Gastric Cancer Cells.

Simple SummaryThe ToGA trial has demonstrated, in HER2-expressing patients, that unresectable and advanced gastric cancer, chemotherapy and trastuzumab in combination increase overall survival, even if it is still unclear why after one year the same patients are non-responsive to trastuzumab treatment. Here, we have demonstrated that in HER2-positive gastric cancer cell lines, the addition of duligotuzumab, targeting HER3 receptor, or ipatasertib, targeting AKT protein, enhances the antitumor effect of trastuzumab in vitro through a full inhibition of the membrane signals, on HER2 and HER3, and of downstream signaling, including AKT, and MAPK pathways. Hence, this study suggests a novel and biomarker-driven therapeutic strategy supporting further evaluation of the anti-tumor efficacy of these combinations in HER2 human gastric cancer patients.The anti-HER2 monoclonal antibody trastuzumab is a key drug for the treatment of HER2-positive gastric cancer (GC); however, its activity is often limited by the onset of resistance and mechanisms of resistance are still poorly understood. Several targeted agents showed synergistic activity by concomitant use with trastuzumab in vitro and are under clinical investigation. The aim of this study was to assess the antitumor activity of duligotuzumab, an anti HER3/EGFR antibody or ipatasertib, an AKT inhibitor, combined with trastuzumab in a panel of HER2-positive human gastric cancer cells (GCC), and the efficacy of such combinations in HER2-resistant cells. We have assessed the efficacy of duligotuzumab or ipatasertib and trastuzumab in combination, analyzing proliferation, migration and apoptosis and downstream intracellular signaling in vitro on human HER2-positive GCC (NCI-N87, OE33, OE19) and in negative HER2 GCC (MKN28). We observed a reduction of proliferation, migration and apoptotic rate in HER2-positive OE33, OE19 and N87 cell lines with the combination of duligotuzumab or ipatasertib plus trastuzumab. In particular, in OE33 and OE19 cell lines, the same combined treatment inhibited the activation of proteins downstream of HER2, HER3, AKT and MAPK pathways. Targeting both HER2 and HER3, or HER2 and AKT, results in an improved antitumor effect on HER2-positive GCC.

Cellular molecular and proteomic profiling deciphers the SIRT1 controlled cell death pathways in esophageal adenocarcinoma cells

Worldwide prevalence of esophageal adenocarcinomas with high rates of mortality coupled with increased mutations in esophageal cells warrants investigation to understand deregulation of cell signaling pathways leading to cancer. To this end, the current study was undertaken to unravel the cell death signatures using the model human esophageal adenocarcinoma cell line-OE33. The strategy involved targeting the key epigenetic modulator SIRT1, a histone deacetylase by a small molecule inhibitor - sirtinol. Sirtinol induced a dose-dependent inhibition of cell viability under both normoxic and hypoxic conditions with long term impact on proliferation as shown by clonogenic assays. Signature apoptotic signaling pathways including caspase activation and decreased Bcl-2 were observed. Proteomic analysis highlighted an array of entities affected including molecules involved in replication, transcription, protein synthesis, cell division control, stress-related proteins, spliceosome components, protein processing and cell detoxification/degradation systems. Importantly, the stoichiometry of the fold changes of the affected proteins per se could govern the cell death phenotype by sirtinol. Sirtinol could also potentially curb resistant and recurrent tumors that reside in hypoxic environments. Overall, in addition to unraveling the cellular, molecular and proteomics basis of SIRT1 inhibition, the findings open up avenues for designing novel strategies against esophageal adenocarcinoma.

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

274 ASSESSMENT OF THE ANTI-PROLIFERATIVE EFFECTS OF STANDARD CHEMOTHERAPY AND PAN-HER FAMILY TYROSINE KINASE INHIBITORS IN ESOPHAGEAL CANCER CELL LINE MODELS.

Abstract Esophageal cancer is the 9th most common cause of cancer globally with a mortality:incidence ratio of 0.88 and a 5 year mortality rate of 19%. Herceptin, a HER2 targeted monoclonal antibody, is approved for use in metastatic HER2+ gastroesophageal carcinoma. To further examine the benefits of HER family inhibition in esophageal cancer, the effects of pan-HER family TKIs and standard chemotherapy in HER2+ and HER2-low cell line models were examined. Methods Two cell lines, OE19 (HER2+) and FLO-1 (HER2-low) were used to examine the effect of the standard FLOT chemotherapy regimen (5 flurouracil (5FU), oxaliplatin and docetaxel) and pan-HER TKIs neratinib and poziotinib. All compounds were from commercial sources. 2D acid phosphatase-based proliferation assays were utilised to assess the anti-proliferative effects of these agents. Triplicates of each assay were performed and standard deviations were determined. IC50 values were calculated using Calcusyn ® software. Results The OE19 cell line demonstrated a high sensitivity to neratinib and poziotinib with IC50 values of 3.426+/−0.187 nM and 0.307 +/−0.051 nM), respectively. IC50 values for neratinib (0.147+/−0.080 μM) and poziotinib (2.4+/−0.471 μM) were significantly higher (p &amp;lt; 0.05) in the FLO-1 cell line compared to OE19. The IC50 values for oxaliplatin and docetaxel were comparable between the OE19 and FLO-1. However, both cell lines were insensitive to 5FU with IC50 values greater than 10 μM. Conclusion The HER2+ cell line was significantly more sensitive to the pan-HER family TKIs neratinib and poziotinib, compared to the HER2-low cell line. This may indicate a potential use for pan-HER family TKIs in HER2+ esophageal cancer. Further work is being conducted to assess potential synergy between pan-HER family TKIs and chemotherapy in these models.

Abstract 2218: High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells

Abstract Introduction: When targeting tumors with monoclonal antibodies, natural killer (NK) cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. Celularity has developed a novel proprietary technology platform that enables production of an allogeneic, off-the-shelf, placental CD34+-derived NK cell therapy for the treatment of hematologic and solid tumor malignancies. Here we report the genetic modification of CD16 in NK cells for high binding affinity to IgG and resistance to protease cleavage, together with functional evaluation of anti-tumor ADCC activity against epidermal growth factor receptor 2 (HER2) positive gastric cancer cells in combination with Trastuzumab (anti-Her2). Methods: Human placental CD34+ cells were transduced with lentivirus expressing a high binding affinity (158Val) and protease resistant (197Pro) CD16 variant (CD16VP) and cultured for 35 days in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, to generate CD16VP cells. The tumorigenic activity of CD16VP cells against HER2-positive gastric cancer cell lines NCI-N87 and OE19 was assessed in combination with Trastuzumab. ADCC activity was measured by real-time xCELLigence, and cytokine secretion by Luminex. The kinase inhibitors PD98059 (MEK/ERK), SB203580 (p38 MAPK), SP600125 (JNK), and Wortmannin (PI3K) were used to study the molecular mechanisms underlying cytotoxicity of CD16VP cells against gastric cancer. Results and conclusion: High transduction efficiency was achieved in multiple placental CD34+ donors with an average of 64.6 ± 10.3% (n=8) CD16 expression on NK cells compared to 12.1 ± 3.3% (n=8) on non-transduced cells. These cells expanded [7095 ± 2998-fold (n=8)] and differentiated into NK cells with &amp;gt;90% purity (CD56+CD3-). While treatment of PMA/ionomycin for 4 hours resulted in &amp;gt;89% CD16 cleavage on non-transduced cells, &amp;lt;11% cleavage was observed on CD16VP cells, demonstrating shedding resistance. CD16VP cells in the presence of Trastuzumab compared to IgG control, showed enhanced lysis of NCI-N87 and OE19 targets (97 ± 7% and 95 ± 2% vs. 55 ± 19% and 42 ± 14% respectively, p&amp;lt;0.01), secreted higher levels of GM-CSF, IFN-γ, and TNF-α, and demonstrated increased degranulation against OE19. The ADCC mediated cytotoxicity and degranulation were PI3K and JNK pathway-dependent as Wortmannin decreased ADCC by 82.2 ± 17.7% and degranulation by 43.9 ± 1.9%, and SP600125 decreased degranulation by 72.3 ± 1.2%. Our results demonstrate that CD16VP cells have enhanced Trastuzumab-mediated ADCC activity against gastric cancer tumor cell lines with resistance to CD16 cleavage. Development of CD16VP cells provides a combination therapeutic option by harnessing the anti-tumor activities of both targeted biologics and innate cytotoxicity of NK cells. Citation Format: Irene Raitman, Salvatore Rotondo, Tanel Mahlakõiv, Xuan Guo, Andrea DiFiglia, Hemlata Rana, Qian Ye, Srinivas Somanchi, William van der Touw, Lin Kang, Robert Hariri, Xiaokui Zhang. High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2218.

MiRNA-181a-5p Enhances the Sensitivity of Cells to Cisplatin in Esophageal Adenocarcinoma by Targeting CBLB.

BackgroundCisplatin (CDDP) is extensively used for esophageal adenocarcinoma (EAC) chemotherapy, while cisplatin resistance is getting worse. microRNA-181a-5p (miR-181a-5p) has been reported to play an important role in various human cancers. However, the effect and underlying mechanism of miR-181a-5p in cisplatin resistance of EAC remain unclear.MethodsCisplatin-resistant EAC cells OE19/CDDP and parental sensitive OE19 cells were applied for experiments in vitro. The expressions of miR-181a-5p and CBLB were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. The cisplatin resistance of cells was expressed by cell viability, IC50 and apoptosis rate by using CCK-8 assay or flow cytometry. The interaction between miR-181a-5p and CBLB was evaluated by luciferase reporter assay and RIP assay. In vivo experiments were conducted via the murine xenograft model.ResultsmiR-181a-5p was highly expressed while CBLB was lowly expressed in OE19 cell lines compared with OE19/CDDP cells. In cisplatin-resistant OE19/CDDP cells, miR-181a-5p up-regulation or CBLB knockdown inhibited cell viability and inducted apoptosis. In cisplatin-sensitive OE19 cells, miR-181a-5p inhibition or CBLB overexpression promoted cell viability and suppressed apoptosis. CBLB was confirmed to be a target of miR-181a-5p, and rescue assay showed CBLB overexpression reversed the suppression of OE19/CDDP cell viability induced by miR-181a-5p up-regulation, and its down-regulation attenuated miR-181a-5p-inhibition-mediated enhancement of OE19 cell viability. In addition, miR-181a-5p up-regulation enhanced the cytotoxicity of cisplatin in EAC in vivo.ConclusionmiR-181a-5p enhanced the sensitivity of cells to cisplatin in EAC by targeting CBLB, indicating a promising sensitizer of cisplatin therapy in clinical esophageal cancer.

Disruption of Cancer Metabolic SREBP1/miR-142-5p Suppresses Epithelial-Mesenchymal Transition and Stemness in Esophageal Carcinoma.

Elevated activity of sterol regulatory element-binding protein 1 (SREBP1) has been implicated in the tumorigenesis of different cancer types. However, the functional roles of SREBP1 in esophageal cancer are not well appreciated. Here, we aimed to investigate the therapeutic potential of SREBP1 and associated signaling in esophageal cancer. Our initial bioinformatics analyses showed that SREBP1 expression was overexpressed in esophageal tumors and correlated with a significantly lower overall survival rate in patients. Additionally, tumor suppressor miR-142-5p was predicted to target SREBP1/ZEB1 and a lower miR-142-5p was correlated with poor prognosis. We then performed in vitro experiments and showed that overexpressing SREBP1 in OE33 cell line led to increased abilities of colony formation, migration, and invasion; the opposite was observed in SREBP1-silenced OE21cells and SREBP1-silencing was accompanied by the reduced mesenchymal markers, including vimentin (Vim) and ZEB1, while E-cadherin and tumor suppressor miR-142-5p were increased. Subsequently, we first demonstrated that both SREBP1 and ZEB1 were potential targets of miR-142-5p, followed by the examination of the regulatory circuit of miR-142-5p and SREBP1/ZEB1. We observed that increased miR-142-5p level led to the reduced tumorigenic properties, such as migration and tumor sphere formation, and both observations were accompanied by the reduction of ZEB1 and SREBP1, and increase of E-cadherin. We then explored the potential therapeutic agent targeting SREBP1-associated signaling by testing fatostatin (4-hydroxytamoxifen, an active metabolite of tamoxifen). We found that fatostatin suppressed the cell viability of OE21 and OE33 cells and tumor spheres. Interestingly, fatostatin treatment reduced CD133+ population in both OE21 and OE33 cells in concert of increased miR-142-5p level. Finally, we evaluated the efficacy of fatostatin using a xenograft mouse model. Mice treated with fatostatin showed a significantly lower tumor burden and better survival rate as compared to their control counterparts. The treatment of fatostatin resulted in the reduced staining of SREBP1, ZEB1, and Vim, while E-cadherin and miR-142-5p were increased. In summary, we showed that increased SREBP1 and reduced miR-142-5p were associated with increased tumorigenic properties of esophageal cancer cells and poor prognosis. Preclinical tests showed that suppression of SREBP1 using fatostatin led to the reduced malignant phenotype of esophageal cancer via the reduction of EMT markers and increased tumor suppressor, miR-142-5p. Further investigation is warranted for the clinical use of fatostatin for the treatment of esophageal malignancy.

The Cytotoxicity and Synergistic Potential of Aspirin and Aspirin Analogues Towards Oesophageal and Colorectal Cancer.

BackgroundOesophageal cancer (OC) is a deadly cancer because of its aggressive nature with survival rates that have barely improved in decades. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of OC.MethodsThe toxicity of aspirin and aspirin derivatives to OC and a CRC cell line were investigated in the presence and absence of platins.ResultsThe data in this study show the effects of a number of aspirin analogues and aspirin on OC cell lines that originally presented as squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues fumaryldiaspirin (PN517) and the benzoylsalicylates (PN524, PN528 and PN529), were observed to be more toxic against the OC cell lines than aspirin. Both quantitative and qualitative apoptosis experiments reveal that these compounds largely induce apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The OE21 (SSC) and OE33 (ADC) cell lines were more sensitive to the aspirin analogues compared to the Flo-1 cell line (ADC). A non-cancerous oesophageal primary cells NOK2101, was used to determine the specificity of the aspirin analogues and cytotoxicity assays revealed that analogues PN528 and PN529 were selectively toxic to cancer cell lines, whereas PN508, PN517 and PN524 also induced cell death in NOK2101. In combination index testing synergistic interactions of the most promising compounds, including aspirin, with cisplatin, oxaliplatin and carboplatin against the OE33 cell line and the SW480 colorectal cancer (CRC) cell line were investigated. Compounds PN517 and PN524, and to a lesser extent PN528, synergised with cisplatin against OE33 cells. Cisplatin and oxaliplatin synergised with aspirin and PN517 when tested against the SW480 cell line.ConclusionThese findings indicate the potential and limitations of aspirin and aspirin analogues as chemotherapeutic agents against OC and CRC when combined with platins