GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's (BE) progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL - gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and, stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for Hsp40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain, or a cell permeable peptide (Pep-J) encoding the above 10 amino acids, can bind and inhibit DNAJ-Hsp70 co-chaperone activity thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic BE and EAC cells and inhibit growth of patient-derived dysplastic BE organoids (PDOs) in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J is comparable to simvastatin, a cholesterol lowering drug, that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase independent, chaperone regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Isoform alterations in the ubiquitination machinery impacting gastrointestinal malignancies.

The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.

Improved prediction of radiation pneumonitis by combining biological and radiobiological parameters using a data-driven Bayesian network analysis.

Grade 2 and higher radiation pneumonitis (RP2) is a potentially fatal toxicity that limits efficacy of radiation therapy (RT). We wished to identify a combined biomarker signature of circulating miRNAs and cytokines which, along with radiobiological and clinical parameters, may better predict a targetable RP2 pathway. In a prospective clinical trial of response-adapted RT for patients (n = 39) with locally advanced non-small cell lung cancer, we analyzed patients' plasma, collected pre- and during RT, for microRNAs (miRNAs) and cytokines using array and multiplex enzyme linked immunosorbent assay (ELISA), respectively. Interactions between candidate biomarkers, radiobiological, and clinical parameters were analyzed using data-driven Bayesian network (DD-BN) analysis. We identified alterations in specific miRNAs (miR-532, -99b and -495, let-7c, -451 and -139-3p) correlating with lung toxicity. High levels of soluble tumor necrosis factor alpha receptor 1 (sTNFR1) were detected in a majority of lung cancer patients. However, among RP patients, within 2 weeks of RT initiation, we noted a trend of temporary decline in sTNFR1 (a physiological scavenger of TNFα) and ADAM17 (a shedding protease that cleaves both membrane-bound TNFα and TNFR1) levels. Cytokine signature identified activation of inflammatory pathway. Using DD-BN we combined miRNA and cytokine data along with generalized equivalent uniform dose (gEUD) to identify pathways with better accuracy of predicting RP2 as compared to either miRNA or cytokines alone. This signature suggests that activation of the TNFα-NFκB inflammatory pathway plays a key role in RP which could be specifically ameliorated by etanercept rather than current therapy of non-specific leukotoxic corticosteroids.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Chaperones and Ubiquitin Ligases Balance Mutant p53 Protein Stability in Esophageal and Other Digestive Cancers.

The incidence of esophageal adenocarcinoma (EAC) and other gastrointestinal (GI) cancers have risen dramatically, thus defining the oncogenic drivers to develop effective therapies are necessary. Patients with Barrett's Esophagus (BE), have an elevated risk of developing EAC. Around 70%-80% of BE cases that progress to dysplasia and cancer have detectable TP53 mutations. Similarly, in other GI cancers higher rates of TP53 mutation are reported, which provide a significant survival advantage to dysplastic/cancer cells. Targeting molecular chaperones that mediate mutant p53 stability may effectively induce mutant p53 degradation and improve cancer outcomes. Statins can achieve this via disrupting the interaction between mutant p53 and the chaperone DNAJA1, promoting CHIP-mediated degradation of mutant p53, and statins are reported to significantly reduce the risk of BE progression to EAC. However, statins demonstrated sub-optimal efficacy depending on cancer types and TP53 mutation specificity. Besides the well-established role of MDM2 in p53 stability, we reported that individual isoforms of the E3 ubiquitin ligase GRAIL (RNF128) are critical, tissue-specific regulators of mutant p53 stability in BE progression to EAC, and targeting the interaction of mutant p53 with these isoforms may help mitigate EAC development. In this review, we discuss the critical ubiquitin-proteasome and chaperone regulation of mutant p53 stability in EAC and other GI cancers with future insights as to how to affect mutant p53 stability, further noting how the precise p53 mutation may influence the efficacy of treatment strategies and identifying necessary directions for further research in this field.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Isoforms of RNF128 Regulate the Stability of Mutant P53 in Barrett's Esophageal Cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53. METHODS: We obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53. RESULTS: Progression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3ß-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice. CONCLUSIONS: We found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

Association of Schizophrenia Risk With Disordered Niacin Metabolism in an Indian Genome-wide Association Study.

Importance: Genome-wide association studies (GWASs) in European populations have identified more than 100 schizophrenia-associated loci. A schizophrenia GWAS in a unique Indian population offers novel findings. Objective: To discover and functionally evaluate genetic loci for schizophrenia in a GWAS of a unique Indian population. Design, Setting, and Participants: This GWAS included a sample of affected individuals, family members, and unrelated cases and controls. Three thousand ninety-two individuals were recruited and diagnostically ascertained via medical records, hospitals, clinics, and clinical networks in Chennai and surrounding regions. Affected participants fulfilled DSM-IV diagnostic criteria for schizophrenia. Unrelated control participants had no personal or family history of psychotic disorder. Recruitment, genotyping, and analysis occurred in consecutive phases beginning January 1, 2001. Recruitment was completed on February 28, 2018, and genotyping and analysis are ongoing. Main Outcomes and Measures: Associations of single-nucleotide polymorphisms and gene expression with schizophrenia. Results: The study population included 1321 participants with schizophrenia, 885 family controls, and 886 unrelated controls. Among participants with schizophrenia, mean (SD) age was 39.1 (11.4) years, and 52.7% were male. This sample demonstrated uniform ethnicity, a degree of inbreeding, and negligible rates of substance abuse. A novel genome-wide significant association was observed between schizophrenia and a chromosome 8q24.3 locus (rs10866912, allele A; odds ratio [OR], 1.27 [95% CI, 1.17-1.38]; P = 4.35 × 10-8) that attracted support in the schizophrenia Psychiatric Genomics Consortium 2 data (rs10866912, allele A; OR, 1.04 [95% CI, 1.02-1.06]; P = 7.56 × 10-4). This locus has undergone natural selection, with the risk allele A declining in frequency from India (approximately 72%) to Europe (approximately 43%). rs10866912 directly modifies the abundance of the nicotinate phosphoribosyltransferase gene (NAPRT1) transcript in brain cortex (normalized effect size, 0.79; 95% CI, 0.6-1.0; P = 5.8 × 10-13). NAPRT1 encodes a key enzyme for niacin metabolism. In Indian lymphoblastoid cell lines, (risk) allele A of rs10866912 was associated with NAPRT1 downregulation (AA: 0.74, n = 21; CC: 1.56, n = 17; P = .004). Preliminary zebrafish data further suggest that partial loss of function of NAPRT1 leads to abnormal brain development. Conclusions and Relevance: Bioinformatic analyses and cellular and zebrafish gene expression studies implicate NAPRT1 as a novel susceptibility gene. Given this gene's role in niacin metabolism and the evidence for niacin deficiency provoking schizophrenialike symptoms in neuropsychiatric diseases such as pellagra and Hartnup disease, these results suggest that the rs10866912 genotype and niacin status may have implications for schizophrenia susceptibility and treatment.

Identification of Tumor Specific Peptide as EpCAM Ligand and Its Potential Diagnostic and Therapeutic Clinical Application.

Tumor targeting agents are being developed for early tumor detection and therapeutics. We previously identified the peptide SNFYMPL (SNF*) and demonstrated its specific binding to human esophageal specimens of high-grade dysplasia (HGD) and adenocarcinoma with imaging ex vivo. Here, we aim to identify the target for this peptide and investigate its potential applications in imaging and drug delivery. With SNF* conjugated affinity chromatography, mass spectrum, Western blot, enzyme-linked immunosorbent assay (ELISA), and molecular docking, we found that the epithelial cell adhesion molecule (EpCAM) was the potential target of SNF*. Next, we showed that FITC-labeled SNF* (SNF*-FITC) colocalized with EpCAM antibody on the surface of esophageal adenocarcinoma cells OE33, and SNF*-FITC binding patterns significantly changed after EpCAM knockdown or exogenous EpCAM transfection. With the data from TCGA, we demonstrated that EpCAM was overexpressed in 17 types of cancers. Using colon and gastric adenocarcinoma cells and tissues as examples, we found that SNF*-FITC bound in a pattern was colocalized with EpCAM antibody, and the SNF* binding did not upregulate the EpCAM downstream Wnt signals. Subsequently, we conjugated SNF* with our previously constructed poly(histidine)-PEG/DSPE copolymer micelles. SNF* labeling significantly improved the micelle binding with colon and gastric adenocarcinoma cells in vitro, and enhanced the antitumor effects and decreased the toxicities of the micelles in vivo. In conclusion, we identified and validated SNF* as a specific peptide for EpCAM. The future potential use of SNF* peptide in multiple tumor surveillance and tumor-targeted therapeutics was demonstrated.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Positron Emission Tomography 18F-Fluorodeoxyglucose Uptake Correlates with KRAS and EMT Gene Signatures in Operable Esophageal Adenocarcinoma.

BACKGROUND: 18F-fluorodeoxyglucose positron emission tomography is an imaging modality critical to the diagnosis and staging of esophageal cancer. Despite this, the genetic abnormalities associated with increased 18F-fluorodeoxyglucose (FDG)-maximum standardized uptake value (SUVmax) have not been previously explored in esophageal adenocarcinoma. MATERIALS AND METHODS: Treatment-naïve patients, for whom frozen tissue and 18F-fluorodeoxyglucose positron emission tomography data were available, undergoing esophagectomy from 2003 to 2012, were identified. Primary tumor FDG-uptake (SUVmax) was quantified as low (<5), moderate, or high (>10). Genome-wide expression analyses (e.g., microarray) were used to examine gene expression differences associated with FDG-uptake. RESULTS: Eighteen patients with stored positron emission tomography data and tissue were reviewed. Overall survival was similar between patients with high (n = 9) and low (n = 6) FDG-uptake tumors (P = 0.71). Differences in gene expression between tumors with high and low FDG-uptake included enriched expression of various matrix metalloproteinases, extracellular-matrix components, oncogenic signaling members, and PD-L1 (fold-change>2.0, P < 0.05) among the high-FDG tumors. Glycolytic gene expression and pathway involvement were similar between the high- and low-FDG tumor subsets (P = 0.126). Gene ontology analysis of the most differentially expressed genes demonstrated significant upregulation of gene sets associated with extracellular matrix organization and vascular development (P < 0.005). Gene set enrichment analysis further demonstrated associations between FDG-uptake intensity and canonical oncogenic processes, including hypoxia, angiogenesis, KRAS signaling, and epithelial-to-mesenchymal transition (P < 0.001). Interestingly, KRAS expression did not predict worse survival in a larger cohort (n = 104) of esophageal adenocarcinomas (P = 0.64). CONCLUSIONS: These results suggest that elevated FDG-uptake is associated with a variety of oncogenic alterations in operable esophageal adenocarcinoma. These pathways present potential therapeutic targets among tumors exhibiting high FDG-uptake.

Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma.

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.

Glyco-centric lectin magnetic bead array (LeMBA) - proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals.

This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA) - mass spectrometry techniques, "Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma" [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE) and esophageal adenocarcinoma (EAC) individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉) using search key "jn7qafftux". The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS) data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Genomic similarity between gastroesophageal junction and esophageal Barrett's adenocarcinomas.

The current high mortality rate of esophageal adenocarcinoma (EAC) reflects frequent presentation at an advanced stage. Recent efforts utilizing fluorescent peptides have identified overexpressed cell surface targets for endoscopic detection of early stage Barrett's-derived EAC. Unfortunately, 30% of EAC patients present with gastroesophageal junction adenocarcinomas (GEJAC) and lack premalignant Barrett's metaplasia, limiting this early detection strategy. We compared mRNA profiles from 52 EACs (tubular EAC; tEAC) collected above the gastroesophageal junction with 70 GEJACs, 8 normal esophageal and 5 normal gastric mucosa samples. We also analyzed our previously published whole-exome sequencing data in a large cohort of these tumors. Principal component analysis, hierarchical clustering and survival-based analyses demonstrated that GEJAC and tEAC were highly similar, with only modest differences in expression and mutation profiles. The combined expression cohort allowed identification of 49 genes coding cell surface targets overexpressed in both GEJAC and tEAC. We confirmed that three of these candidates (CDH11, ICAM1 and CLDN3) were overexpressed in tumors when compared to normal esophagus, normal gastric and non-dysplastic Barrett's, and localized to the surface of tumor cells. Molecular profiling of tEAC and GEJAC tumors indicated extensive similarity and related molecular processes. Identified genes that encode cell surface proteins overexpressed in both Barrett's-derived EAC and those that arise without Barrett's metaplasia will allow simultaneous detection strategies.

Identification of the CIMP-like subtype and aberrant methylation of members of the chromosomal segregation and spindle assembly pathways in esophageal adenocarcinoma.

The incidence of esophageal adenocarcinoma (EAC) has risen significantly over recent decades. Although survival has improved, cure rates remain poor, with <20% of patients surviving 5 years. This is the first study to explore methylome, transcriptome and ENCODE data to characterize the role of methylation in EAC. We investigate the genome-wide methylation profile of 250 samples including 125 EAC, 19 Barrett's esophagus (BE), 85 squamous esophagus and 21 normal stomach. Transcriptome data of 70 samples (48 EAC, 4 BE and 18 squamous esophagus) were used to identify changes in methylation associated with gene expression. BE and EAC showed similar methylation profiles, which differed from squamous tissue. Hypermethylated sites in EAC and BE were mainly located in CpG-rich promoters. A total of 18575 CpG sites associated with 5538 genes were differentially methylated, 63% of these genes showed significant correlation between methylation and mRNA expression levels. Pathways involved in tumorigenesis including cell adhesion, TGF and WNT signaling showed enrichment for genes aberrantly methylated. Genes involved in chromosomal segregation and spindle formation were aberrantly methylated. Given the recent evidence that chromothripsis may be a driver mechanism in EAC, the role of epigenetic perturbation of these pathways should be further investigated. The methylation profiles revealed two EAC subtypes, one associated with widespread CpG island hypermethylation overlapping H3K27me3 marks and binding sites of the Polycomb proteins. These subtypes were supported by an independent set of 89 esophageal cancer samples. The most hypermethylated tumors showed worse patient survival.

Serum Glycoprotein Biomarker Discovery and Qualification Pipeline Reveals Novel Diagnostic Biomarker Candidates for Esophageal Adenocarcinoma.

We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett's esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.

IGFBP2 modulates the chemoresistant phenotype in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) patients commonly present with advanced stage disease and demonstrate resistance to therapy, with response rates below 40%. Understanding the molecular mechanisms of resistance is crucial for improvement of clinical outcomes. IGFBP2 is a member of the IGFBP family of proteins that has been reported to modulate both IGF and integrin signaling and is a mediator of cell growth, invasion and resistance in other tumor types. In this study, high IGFBP2 expression was observed in a subset of primary EACs and was found to be significantly higher in patients with shorter disease-free intervals as well as in treatment-resistant EACs as compared to chemonaive EACs. Modulation of IGFBP2 expression in EAC cell lines promoted cell proliferation, migration and invasion, implicating a role in the metastatic potential of these cells. Additionally, knockdown of IGFBP2 sensitized EAC cells to cisplatin in a serum-dependent manner. Further in vitro exploration into this chemosensitization implicated both the AKT and ERK pathways. Silencing of IGFBP2 enhanced IGF1-induced immediate activation of AKT and reduced cisplatin-induced ERK activation. Addition of MEK1/2 (selumetinib or trametinib) or AKT (AKT Inhibitor VIII) inhibitors enhanced siIGFBP2-induced sensitization of EAC cells to cisplatin. These results suggest that targeted inhibition of IGFBP2 alone or together with either the MAPK or PI3K/AKT signaling pathway in IGFBP2-overexpressing EAC tumors may be an effective approach for sensitizing resistant EACs to standard neoadjuvant chemotherapy.