Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

<div><p>Background</p><p>Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis.</p> <p>Methodology/Principal Findings</p><p>We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption.</p> <p>Conclusions/Significance</p><p>Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.</p> </div

CP-C and CP-D display a lower ECAR increase after mitochondrial uncoupling than other cell lines.

<p>Following addition of 50 µM 2,4-DNP, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) of statistically significant differences from CP-A are shown.</p

Barrett's esophagus progression to esophageal adenocarcinoma involves an intermediate metabolic stage with increased glycolysis and functional mitochondria.

<p>Early-stage BE cells (e.g. CP-A), rely mainly on mitochondrial oxidative phosphorylation for energy needs prior to the glycolytic increase which occurs in late-stage BE cells (CP-B, CP-C and CP-D), which demonstrate elevated ECAR (all) and the Crabtree effect (CP-C and CP-D). Finally in esophageal adenocarcinoma (OE-33) mitochondrial uncoupling occurs with increased OCR and glycolysis.</p

CP-D displays a greater OCR and ECAR response to glycolytic inhibition than other cell lines.

<p>Following addition of 50 mM 2-DG, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) of statistically significant differences from CP-A are shown.</p

CP-C and CP-D demonstrate a stronger Crabtree effect response than other cell lines.

<p>Following the increase of glucose concentration in media from 0 mM to 5 mM, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) indicating significant differences from CP-A are shown.</p

Cohort characteristics.

<p>Cohort characteristics.</p

Examples of p16 mutations in Barrett's esophagus.

<p>A) C to T transition at basepair 247. B) 1 basepair deletion at nucleotide 289.</p

Mutations in p16/CDKN2a detected in esophagectomy patients.

<p>Headings are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003809#pone-0003809-t002" target="_blank">Table 2</a>. Diagnosis is at the time of the esophagectomy.</p

High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett’s Esophagus

<div><p>Purpose</p><p>Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients.</p><p>Experimental Design</p><p>Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with ≥ 1 GC, and the proportion of crypts with ≥1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length.</p><p>Results</p><p>High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with ≥1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively).</p><p>Conclusions</p><p>The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities.</p></div

<i>Streptococcus</i> to <i>Prevotella</i> species ratio corresponds to phylogenetic distance sample clustering and correlates with Barrett’s esophagus risk factors.

<p>(A) Cluster analysis of KR distances between microbial communities of individual study samples. Pyroseq. <i>Strep</i>:<i>Prev</i> ratio was calculated using relative abundance of mapped reads for all <i>Streptococcus</i> and <i>Prevotella</i> species as determined by pyrosequencing. ddPCR <i>Strep</i>:<i>Prev</i> ratio was calculated using copies/μl of a <i>Streptococcus</i> or <i>Prevotella</i>-specific 16s rRNA gene segment as determined by droplet digital PCR. Samples color-coded based on the majority of calculated Pyroseq. <i>Strep</i>:<i>Prev</i> ratios in a group being <0.5 (blue), 0.5–1.5 (green), 1.5–4.0 (magenta) or >4.0 (red). (B) Boxplots comparing <i>Streptococcus</i> to <i>Prevotella</i> ratio as determined by pyrosequencing and ddPCR. The central line within each box represents the median of the data, the whiskers represent the 5^th and 95^th percentiles and data outside that range are plotted as individual points. (C) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratio (measured by ddPCR) and waist-hip ratio of all male participants segregated by anatomic site. Strength of association between these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated. (D) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratios (measured by ddPCR) and hiatal hernia length in all participants segregated by anatomic site. Strength of association tween these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated.</p