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RNAse Esc2 TXNIP

In 2017, an estimated 17,000 individuals were diagnosed with esophageal adenocarcinoma (EAC), and less than 20% will survive 5 years. Positron emission tomography avidity is indicative of high glucose utilization and is nearly universal in EAC. TXNIP blocks glucose uptake and exhibits proapoptotic functions. Higher expression in EAC has been associated with improved disease-specific survival, lack of lymph node involvement, reduced perineural invasion, and increased tumor differentiation. We hypothesized that TXNIP may act as a tumor suppressor that sensitizes EAC cells to standard chemotherapeutics. EAC cell lines and a Barrett epithelial cell line were used. qRT-PCR, immunoblot, and immunofluorescence techniques evaluated gene expression. TXNIP was stably overexpressed or knocked down using lentiviral RNA transduction techniques. Murine xenograft methods examined growth following overexpression of TXNIP. Apoptosis and DNA damage were measured by annexin V and γH2AX assays. Activation of the intrinsic apoptosis was quantitated with green fluorescence protein-caspase 3 reporter assay. In cultured cells and an esophageal tissue array, TXNIP expression was higher in Barrett epithelia and normal tissue compared with EAC. Constitutive overexpression of TXNIP decreased proliferation, clonogenicity, and tumor xenograft growth. TXNIP overexpression increased, whereas knockdown abrogated, DNA damage and apoptosis following cisplatin treatment. An HDAC inhibitor, entinostat (currently in clinical trials), upregulated TXNIP and synergistically increased cisplatin-mediated DNA damage and apoptosis. TXNIP is a tumor suppressor that is downregulated in EACC. Its reexpression dramatically sensitizes these cells to cisplatin. Our findings support phase I/II evaluation of “priming” strategies to enhance the efficacy of conventional chemotherapeutics in EAC. Mol Cancer Ther; 17(9); 2013–23. ©2018 AACR.

Cells and Reagents

Supplemental Figure 1: Chromatin immunoprecipitation was performed to assess the histone modification marks, H3K27Me3, H3K9Ac2, H3K4Me3, and H3K79Me2 in Flo-1 and Esc2 after glutamine withdrawal; Supplemental Figure 2: qRT-PCR normalized to b-actin measured TXNIP mRNA expression after Flo-1, Esc2, OE33, and CP-C were treated for 72 hours with L-Glutamic acid γ-(p-nitroanilide) hydrochloride (GPNA) and grown in 96-well plates; Supplemental Figure 3: Realtime Seahorse extracellular flux analysis of extracellular acidification rate (ECAR) measures glycolytic activity based on sequential glucose, oligomycin (oligo), and 2-DG injections at the time points indicated; Supplemental Figure 4: YSI analyzer measured glucose uptake over a 5-day period in Flo-1 and Esc2 with overexpression of TXNIP; Supplemental Figure 5: qRT-PCR normalized to b-actin measured mRNA levels of GLUT1, GLUT4, HK2, and LDH-A in Flo-1 and Esc2 overexpressing TXNIP versus controls; Supplemental Figure 6: Oxidative consumption ratio (OCR) measured mitochondrial function with sequential injections of oligomycin (oligo), FCCP, and rotenone/antimycin a (R/A) at the time points indicated in Flo-1, Esc2, and OE33 overexpressing TXNIP versus controls; Supplemental Figure 7: Oxidative consumption ratio (OCR) measured mitochondrial function with sequential injections of oligomycin (oligo), FCCP, and rotenone/antimycin a (R/A) at the time points indicated in Esc2 cells with lentiviral shRNA knockdown of TXNIP; Supplemental Figure 8: Proliferation was measured using Cyquant DNA quantification kits over 5 days in Esc2 cells with lentiviral shRNA knockdown of TXNIP versus controls; Supplemental Figure 9: Esc2 cells overexpressing TXNIP versus controls were grown in 96-well plates with or without N-acetyl-cysteine (NAC); Supplemental Figure 10: Esc2 cells overexpressing TXNIP were grown in 96-well plates at normoxic conditions (20%) or hypoxic conditions (5%); Supplemental Figure 11: Colonies of Flo-1 overexpressing TXNIP versus controls were grown at normoxic conditions (20%) or hypoxic conditions (5%) in soft agar and were stained with crystal violet, photographed, and counted; Supplemental Figure 12: YSI analyzer measured lactate secretion at 24 and 48 hours in Flo-1 overexpressing TXNIP; Supplemental Figure 13: In Flo-1 and Esc2 cells treated with cisplatin, entinostat, or the combination, quantification of Annexin V apoptosis assay based on stage of apoptosis; Supplemental Figure 14: Representative pictures from Flow Cytometry results of Esc2 cells with lentiviral shRNA knockdown of TXNIP versus controls after treatment with cisplatin, entinostat, or the combination; Supplemental Figure 15: Chromatin immunoprecipitation was performed to assess the histone modification mark, H3K9Ac2, in Flo-1 after treatment with entinostat, cisplatin, or the combination of both.

PDF file - 57K, SuperArray Analysis of Oncogenes, Tumor Suppressors, and Stem Cell-Related Genes in MPM Lines Following Selective PRC2 Knockdown or 5 �M DZNep.

PDF file - 661K, TaqMan Primers, shRNA, and Antibodies.

PDF file - 102K, Effects of DZNep treatment on cultured mesothelioma cell lines relative to normal mesothelia cells. Pyrosequencing analysis of NBL2 DNA repeat in normal mesothelia (LP9) and MPM cell lines cultured in the presence or absence of DZNep.

PDf file - 63K, qRT-PCR and immunoblot analysis of EZH2 and EED levels in MPM lines following transduction with shRNA targeting EZH2, EED, or sham sequences. Effects of knock-down of EZH2 and EED on proliferation of MPM lines.

PDF file - 63K, Genes Simultaneously Modulated by DZNep in MES1 and H28.

PDF file - 661K, Photomicrographs of NMES1 and NMES2 normal pleura cultures, and MES1 through MES4 mesothelioma cell lines established at the NCI.

PDF file - 97K, Supplementary Materials and Methods.

PDF file - 62K, Demographic Data Pertaining to the Patients from whom NCI-SB-MES 1-4 lines Were Established.

PDF file - 245K, EZH2 expression in tumors correlates with cell line expression.

Purpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).Experimental Design: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.Results:EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy. Clin Cancer Res; 18(1); 77–90. ©2011 AACR.

PDF file - 65K, Top 20 Gene Sets Enriched in MES1 and H28 Cells following DZNep Treatment.

PDF file - 54K, Top 20 Gene Ontologies Modulated by DZNep in MES1 and H28 Cells.

PDF file - 66K, Summary of percentages of mesothelioma or normal mesothelia specimens expressing EZH2 in figure 2D.

PDF file - 52K, ABCG2 Expression in Side Populations (sp) and Non-side Populations (nsp) of A549 and Calu-6 Cells Cultured with or without CSC

PDF file - 87K, Bar graph depicting total number of differentially expressed genes, as well as relative numbers induced or repressed under various treatment conditions

PDF file - 116K, qRT-PCR analysis of ABCG2 expression in untreated NSCLC and SCLC lines relative to SAEC, and in NSCLC and SCLC lines following CSC exposure

PDF file - 111K

Supplementary Tables 1-6 from Inhibition of Histone Lysine Methylation Enhances Cancer–Testis Antigen Expression in Lung Cancer Cells: Implications for Adoptive Immunotherapy of Cancer

PDF file - 363K, Ingenuity pathway analysis demonstrating two common networks targeted by mithramycin in lung cancer cells in vitro and in vivo

PDF file - 76K, TaqMan and ChIP Primers, and Antibodies

PDF file - 346K, Ingenuity pathway analysis depicting representative intracellular networks modulated by mithramycin lung cancer xenografts

Context Aaptamine is a potent ocean-derived non-traditional drug candidate against human cancers. However, the underlying molecular mechanisms governing aaptamine-mediated repression of lung cancer cells remain largely undefined. Objective To examine the inhibitory effect of aaptamine on proliferation and progression of non-small cell lung carcinoma (NSCLC) and dissect the potential mechanisms involved in its anticancer functions. Materials and methods In vitro assays of cell proliferation, cell cycle analysis, clonal formation, apoptosis and migration were performed to examine the inhibitory effects of aaptamine (8, 16 and 32 μg/mL) on NSCLC cells. The expression levels of proteins were analysed using western blotting analysis when cells were treated with a single drug or a combination treatment for 48 h. Results Aaptamine significantly inhibited A549 and H1299 cells proliferation with IC50 values of 13.91 and 10.47 μg/mL. At the concentrations of 16 and 32 μg/mL, aaptamine significantly reduced capacities in clonogenicity, enhanced cellular apoptosis and decreased the motile and invasive cellular phenotype. In addition, aaptamine arrested cell cycle at G1 phase via selectively abating cell cycle regulation drivers (CDK2/4 and Cyclin D1/E). Western blotting results showed that aaptamine attenuated the protein expression of MMP-7, MMP-9 and upregulated the expression of cleaved-PARP and cleaved-caspase 3. Moreover, aaptamine inhibited PI3K/AKT/GSK3β signalling cascades through specifically degrading the phosphorylated AKT and GSK3β. Discussion and conclusions Aaptamine retarded the proliferation and invasion of NSCLC cells by selectively targeting the pathway PI3K/AKT/GSK3β suggesting it as a potential chemotherapeutic agent for repressing tumorigenesis and progression of NSCLC in humans.

Esophageal cancer (EsC) is the sixth leading cause of cancer-related mortality worldwide. Esophageal squamous cell cancers (ESCC) typically arise in the upper and mid-esophagus, whereas esophageal adenocarcinomas (EAC) arise in the distal esophagus and gastroesophageal junction. Presently, epigenetic mechanisms contributing to initiation, progression, and dissemination of EsC have not been fully elucidated. The present study was undertaken to determine if induced pluripotent stem cells (iPSCs) derived from normal esophageal epithelial cells (Eso-iPSCs), EsC cell lines cultured in stemness enriched conditions, and cancer stem-like cells isolated from primary EsC specimens could be used to identify unique primitive stem-like transcription factor networks associated with epigenetic plasticity, chemoresistance, and metastatic potential of EsC cells. RNA-seq analysis demonstrated that genes differentially expressed in Eso-iPSC overlapped with transcriptome signatures in lung-iPSC (Lu-iPSC) which we previously generated to identify novel therapeutic targets in lung cancers; similar to Lu-iPSC, Eso-iPSC transcriptome signatures overlapped considerably more with small cell lung cancers (SCLC) compared to non-small cell lung cancers (NSCLC), possibly reflecting greater stemness in SCLC. We next compared transcriptome signatures of Eso-iPSC with stem-like-EAC enrichment models (cell lines and PDX). 6% and 8% of differentially expressed genes in Eso-iPSC uniquely overlapped with EAC or ESCC lines, respectively, whereas 24% were common to Eso-iPSC, EAC cells, and ESCC cells. Culturing of EAC cells on matrigel or propagation of EAC as primary patient derived xenografts increased commonalities of Eso-iPSC and EAC transcriptome signatures. Lastly, we compared transcriptomes of Eso-iPSC with Barrett’s esophagus (BE; a premalignant condition in which the squamous epithelium of the distal esophagus is replaced by metaplastic columnar epithelial cells) and EAC samples. By combining and stringently filtering transcriptome, ATAC-seq, DNA methylome, and ChIP-seq datasets for tissue- and disease-specific signatures, we identified several potentially druggable, master TFs mediating stemness/plasticity, chemoresistance, and metastatic potential which are shared between EsC lines and primary tumor specimens. Enrichment techniques promoted acquisition of more stem-like states in cultured EAC cells as reflected in multi-omic signatures of these cells relative to Eso-iPSC and primary EAC specimens. Collectively, these findings establish proof of principal for our experimental approach which is now being used to further characterize epigenetic mechanisms contributing to esophageal carcinogenesis and to identify novel targets for EsC therapy. Citation Format: Haitao Wang, Ruihong Wang, Katherine P. Prothro, Sichuan Xi, Weilong Hou, Xinwei Wu, Tuana Tolunay, Vivek Shukla, Mary R. Zhang, Stephanie J. Shiffka, Sudheer Gara, David S. Schrump. Induced pluripotent stem cells derived from normal human esophageal epithelial cells identify transcription factor networks contributing to esophageal carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A013.

Introduction Although communal smoking of hookah by means of water pipes is perceived to be a safe alternative to cigarette smoking, the effects of hookah smoke in respiratory epithelia have not been well characterized. This study evaluated epigenomic and transcriptomic effects of hookah smoke relative to cigarette smoke in human respiratory epithelial cells. Methods Primary normal human small airway epithelial cells from three donors and cdk4 and hTERT-immortalized small airway epithelial cells and human bronchial epithelial cells were cultured for 5 days in normal media with or without cigarette smoke condensates (CSCs) or water pipe condensates (WPCs). Cell count, immunoblot, RNA sequencing, quantitative real-time reverse-transcriptase polymerase chain reaction, methylation-specific polymerase chain reaction, and quantitative chromatin immunoprecipitation techniques were used to compare effects of hookah and cigarette smoke on cell proliferation, global histone marks, gene expression, and promoter-related chromatin structure. Results CSC and WPC decreased global H4K16ac and H4K20me3 histone marks and mediated distinct and overlapping cancer-associated transcriptome signatures and pathway modulations that were cell line dependent and stratified across lung cancer cells in a histology-specific manner. Epiregulin encoding a master regulator of EGFR signaling that is overexpressed in lung cancers was up-regulated, whereas FILIP1L and ABI3BP encoding mediators of senescence that are repressed in lung cancers were down-regulated by CSC and WPC. Induction of epiregulin and repression of FILIP1L and ABI3BP by these condensates coincided with unique epigenetic alterations within the respective promoters. Conclusions These findings support translational studies to ascertain if hookah-mediated epigenomic and transcriptomic alterations in cultured respiratory epithelia are detectable and clinically relevant in hookah smokers.

Barrett’s esophagus (BE) is associated with reflux and is implicated the development of esophageal adenocarcinoma (EAC). Apoptosis induces cell death through mitochondrial outer membrane permeabilization (MOMP), which is considered an irreversible step in apoptosis. Activation of MOMP to levels that fail to reach the apoptotic threshold may paradoxically promote cancer—a phenomenon called “Minority MOMP.” We asked whether reflux-induced esophageal carcinogenesis occurred via minority MOMP and whether compensatory resistance mechanisms prevented cell death during this process. We exposed preneoplastic, hTERT-immortalized Barrett’s cell, CP-C and CP-A, to the oncogenic bile acid, deoxycholic acid (DCA), for 1 year. Induction of minority MOMP was tested via comet assay, CyQuant, annexin V, JC-1, cytochrome C subcellular localization, caspase 3 activation, and immunoblots. We used bcl-2 homology domain-3 (BH3) profiling to test the mitochondrial apoptotic threshold. One-year exposure of Barrett’s cells to DCA induced a malignant phenotype noted by clone and tumor formation. DCA promoted minority MOMP noted by minimal release of cytochrome C and limited caspase 3 activation, which resulted in DNA damage without apoptosis. Upregulation of the antiapoptotic protein, Mcl-1, ROS generation, and NF-κB activation occurred in conjunction with minority MOMP. Inhibition of ROS blocked minority MOMP and Mcl-1 upregulation. Knockdown of Mcl-1 shifted minority MOMP to complete MOMP as noted by dynamic BH3 profiling and increased apoptosis. Minority MOMP contributes to DCA induced carcinogenesis in preneoplastic BE. Mcl-1 provided a balance within the mitochondria that induced resistance complete MOMP and cell death. Targeting Mcl-1 may be a therapeutic strategy in EAC.

Deregulated Sonic Hedgehog (SHH) pathway facilitates the initiation, progression, and metastasis of Non-small cell lung cancer (NSCLC), confers drug resistance and renders a therapeutic interference option to lung cancer patients with poor prognosis. In this study, we screened and evaluated the specificity of a Chinese herb Scutellariabarbata D. Don extraction (SBE) in repressing SHH signaling pathway to block NSCLC progression. Our study confirmed that aberrant activation of the SHH signal pathway conferred more proliferative and invasive phenotypes to human lung cancer cells. This study revealed that SBE specifically repressed SHH signaling pathway to interfere the SHH-mediated NSCLC progression and metastasis via arresting cell cycle progression. We also found that SBE significantly sensitized lung cancer cells to chemotherapeutic agent DDP via repressing SHH components in vitro and in vivo. Mechanistic investigations indicated that SBE transcriptionally and specifically downregulated SMO and consequently attenuated the activities of GLI1 and its downstream targets in SHH signaling pathway, which interacted with cell cycle checkpoint enzymes to arrest cell cycle progression and lead to cellular growth inhibition and migration blockade. Collectively, our results suggest SBE as a novel drug candidate for NSCLC which specifically and sensitively targets SHH signaling pathway.

Introduction Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. Methods Microarray, real-time quantitative reverse transcription–polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. Results UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. Conclusions UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

Ethnopharmacological relevance Scutellariabarbata D. Don extraction (SBE), a traditional Chinese medicine, has been proved effective against various malignant disorders in clinics with tolerable side-effects when administered alone or in combination with conventional chemotherapeutic regimens. Aim of this study Multi-drug resistance of cancer is attributed to existence of cancer stemness-prone cells that harbor aberrantly high activation of Sonic Hedgehog (SHH) cascade. Our previous study has demonstrated that SBE sensitized non-small cell lung cancer (NSCLC) cells to Cisplatin (DDP) treatment by downregulating SHH pathway. Yet, whether SBE could prohibit proliferation of cancer stemness-prone cells and its underlying molecular mechanisms remain to be investigated. In this article, we further investigated intervention of SBE on NSCLC cell stemness-associated phenotypes and its potential mode of action. Materials and methods CCK-8 and clonal formation detection were used to measure the anti-proliferative potency of SBE against NSCLC and normal epithelial cells. Sphere formation assay and RQ-PCR were used to detect proliferation of cancer stemness cells and associated marker expression upon SBE incubation. Mechanistically, DARTS-WB and SPR were used to unveil binding target of SBE. Immunodeficient mice were implanted with patient derived tumor bulk for in vivo validation of anti-cancer effect of SBE. Results SBE selectively attenuated proliferation and stemness-like phenotypes of NSCLC cells rather than bronchial normal epithelial cells. Drug-protein interaction analysis revealed that SBE could directly bind with stem cell-specific transcription factor sex determining region Y-box 2 (SOX2) and interfere with the SOX2/SMO/GLI1 positive loop. In vivo assay using patient-derived xenografts (PDXs) model further proved that SBE diminished tumor growth and SOX2 expression in vivo. Conclusion Our data indicate that SBE represses stemness-related features of NSCLC cells via targeting SOX2 and may serve as an alternative therapeutic option for clinic treatment.

Cisplatin (DDP) resistance has become the leading cause of mortality in non-small cell lung cancer (NSCLC). miRNA dysregulation significantly contributes to tumor progression. In this study, we found that miR-495 was significantly downregulated in lung cancer tissue specimens. This study aimed to elucidate the functions, direct target genes, and molecular mechanisms of miR-495 in lung cancer. miR-495 downregulated its substrate UBE2C through direct interaction with UBE2C 3′- untranslated region. UBE2C is a proto-oncogene activated in lung cancer; however, its role in chemotherapeutic resistance is unclear. Herein, UBE2C expression levels were higher in DDP-resistant NSCLC cells; this was associated with the proliferation, invasion, and DDP resistance in induced cisplatin-resistant NSCLC cells. Furthermore, epithelial–mesenchymal transitions (EMT) contributed to DDP resistance. Moreover, UBE2C knockdown downregulated vimentin. In contrast, E-cadherin was upregulated. Importantly, miR-495 and UBE2C were associated with cisplatin resistance. We attempted to evaluate their effects on cell proliferation and cisplatin resistance. We also performed EMT, cell migration, and invasion assays in DDP-resistant NSCLC cells overexpressing miR-495 and under-expressing UBE2C. Furthermore, in silico assays coupled with western blotting and luciferase assays revealed that UBE2C directly binds to the 5′-UTR of the drug-resistance genes ABCG2 and ERCC1. Furthermore, miR-495 downregulated ABCG2 and ERCC1 via regulation of UBE2C. Together, the present results indicate that the miR495-UBE2C-ABCG2/ERCC1 axis reverses DDP resistance via downregulation of anti-drug genes and reducing EMT in DDP-resistant NSCLC cells.

In 2017, an estimated 17,000 individuals were diagnosed with esophageal adenocarcinoma (EAC), and less than 20% will survive 5 years. Positron emission tomography avidity is indicative of high glucose utilization and is nearly universal in EAC. TXNIP blocks glucose uptake and exhibits proapoptotic functions. Higher expression in EAC has been associated with improved disease-specific survival, lack of lymph node involvement, reduced perineural invasion, and increased tumor differentiation. We hypothesized that TXNIP may act as a tumor suppressor that sensitizes EAC cells to standard chemotherapeutics. EAC cell lines and a Barrett epithelial cell line were used. qRT-PCR, immunoblot, and immunofluorescence techniques evaluated gene expression. TXNIP was stably overexpressed or knocked down using lentiviral RNA transduction techniques. Murine xenograft methods examined growth following overexpression of TXNIP. Apoptosis and DNA damage were measured by annexin V and γH2AX assays. Activation of the intrinsic apoptosis was quantitated with green fluorescence protein-caspase 3 reporter assay. In cultured cells and an esophageal tissue array, TXNIP expression was higher in Barrett epithelia and normal tissue compared with EAC. Constitutive overexpression of TXNIP decreased proliferation, clonogenicity, and tumor xenograft growth. TXNIP overexpression increased, whereas knockdown abrogated, DNA damage and apoptosis following cisplatin treatment. An HDAC inhibitor, entinostat (currently in clinical trials), upregulated TXNIP and synergistically increased cisplatin-mediated DNA damage and apoptosis. TXNIP is a tumor suppressor that is downregulated in EACC. Its reexpression dramatically sensitizes these cells to cisplatin. Our findings support phase I/II evaluation of “priming” strategies to enhance the efficacy of conventional chemotherapeutics in EAC. Mol Cancer Ther; 17(9); 2013–23. ©2018 AACR.

Limited information is available regarding mechanisms that link the known carcinogenic risk factors of gastro-esophageal reflux and cigarette smoking to metabolic alterations in esophageal adenocarcinoma (EAC). In the present study, we utilized a novel in-vitro model to examine whether bile acid and cigarette smoke increase the aggressiveness of EAC and whether these changes are associated with metabolic changes. EAC cells (EACC) were exposed to 10 μg/ml cigarette smoke condensate (CSC) and/or 100 μM of the oncogenic bile acid, deoxycholic acid (DCA), for 5 days. These exposure conditions were chosen given their lack of effect on proliferation or viability. DCA and CSC increased invasion, migration, and clonogenicity in EAC cells. These changes were associated with concomitant increases in ATP, ROS, and lactate production indicative of increased mitochondrial respiration as well as glycolytic activity. DCA and CSC exposure significantly decreased expression of uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein implicated in regulation of the proton gradient. Knockdown of UCP2 in EACC phenocopied DCA and CSC exposure as evidenced by increased cell migration, invasion, and clonogenicity, whereas over-expression of UCP2 had an inverse effect. Furthermore, over-expression of UCP2 abrogated DCA and CSC-mediated increases in lactate and ATP production in EACC. DCA and CSC promote the aggressive phenotype of EACC with concomitant metabolic changes occurring via downregulation of UCP2. These results indicate that UCP2 is integral to the aggressive phenotype of EACC. This mechanism suggests that targeting alterations in cellular energetics may be a novel strategy for EAC therapy.

DNA methylation is an important epigenetic modification as a hallmark in cancer. Conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) family enzymes plays an important biological role in embryonic stem cells, development, aging and disease. Lymphoid specific helicase (LSH), a chromatin remodeling factor, is regarded as a reader of 5-hmC. Recent reports show that the level of 5-hmC is altered in various types of cancers. However, the change in 5-hmC levels in cancer and associated metastasis is not well defined. We report that the level of 5-hmC was decreased in metastatic tissues of nasopharyngeal carcinoma, breast cancer, and colon cancer relative to that in non-metastasis tumor tissues. Furthermore, our data show that TET2, but not TET3, interacted with LSH, whereas LSH increased TET2 expression through silencing miR-26b-5p and miR-29c-5p. Finally, LSH promoted genome stability by silencing satellite expression by affecting 5-hmC levels in pericentromeric satellite repeats, and LSH was resistant to cisplatin-induced DNA damage. Our data indicate that 5-hmC might serve as a metastasis marker for cancer and that the decreased expression of LSH is likely one of the mechanisms of genome instability underlying 5-hmC loss in cancer.