Katerina Dvorak

Hydrophobic bile acids, genomic instability, Darwinian selection, and colon carcinogenesis

Sporadic colon cancer is caused predominantly by dietary factors. We have selected bile acids as a focus of this review since high levels of hydrophobic bile acids accompany a Western-style diet, and play a key role in colon carcinogenesis. We describe how bile acid-induced stresses cause cell death in susceptible cells, contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression to colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells (DNA damage, endoplasmic reticulum stress, mitochondrial damage) are described. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability are discussed, and include oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy. Since bile acids and oxidative stress decrease DNA repair proteins, an increase in DNA damage and increased genomic instability through this mechanism is also described. This review provides a mechanistic explanation for the important link between a Western-style diet and associated increased levels of colon cancer.

Crypt-restricted loss and decreased protein expression of cytochrome C oxidase subunit I as potential hypothesis-driven biomarkers of colon cancer risk.

There is an increasing demand for the development of intermediate biomarkers to assess colon cancer risk. We previously determined that a live cell bioassay, which assesses apoptosis resistance in the nonneoplastic colonic mucosa, detects ∼50% of patients with colon cancer. A hypothesis-driven biomarker that reflects apoptosis resistance in routine formalin-fixed, paraffin-embedded tissue would be easier to use. Cytochrome c oxidase is a critical enzyme that controls mitochondrial respiration and is central to apoptosis. We did an immunohistochemical study of cytochrome c oxidase subunit I expression in 46 colonic mucosal samples from 16 patients who had undergone a colonic resection. These included five patients without evidence of colonic neoplasia (three normal and two diverticulitis), three patients with tubulovillous adenomas, and eight patients with colonic adenocarcinomas. Analysis of aberrancies in expression of cytochrome c oxidase subunit I showed that, compared with nonneoplasia, the patients with neoplasia had a higher mean incidence of crypts having decreased expression (1.7 versus 22.8, P = 0.03) and a higher mean incidence having crypt-restricted loss (0.6 versus 3.2, P = 0.06). The percentage with segmented loss was low and was similar in the two groups. Combining these results, the mean % normal (i.e., with none of the three types of abnormality) was 96.7 in nonneoplasia versus only 73.2 in patients with neoplasia (P = 0.02). It should be noted that a defect in cytochrome c oxidase subunit I immunostaining was not detected in all biopsy samples from each patient for whom some abnormality was found, indicating a “patchiness” in the cytochrome c oxidase subunit I field defect. As a result of this “patchiness,” the increased variability in the incidence of crypt-restricted loss of cytochrome c oxidase subunit I expression was a statistically significant feature of the neoplasia group. Crypt-restricted loss of cytochrome c oxidase subunit I has not been previously reported in colonic mucosa and is presumably the result of a crypt-restricted stem cell mutation. Decreased cytochrome c oxidase subunit I expression also significantly correlated with apoptosis resistance, a factor known to contribute to carcinogenesis. The results suggest, however, that aberrant cytochrome c oxidase subunit I expression may be a better biomarker than loss of apoptosis competence for increased colon cancer risk.

Abnormal Expression of Biomarkers in Incompletely Ablated Barrett’s Esophagus

Objective:The aim of this study was to evaluate expression of cancer risk-associated biomarkers in columnar epithelium at squamocolumnar junctions produced by an ablation procedure and proton pump inhibitors in incompletely ablated Barrett’s esophagus (BE) patients that were nondysplastic prior to ablation. Summary Background Data:Ablation of BE to squamous epithelium is achievable by combining a re-injury method with acid suppression. We previously reported that, when there is complete ablation, the neo-squamous epithelium is normal histologically and in biomarker expression. However, squamous islands observed after prolonged use of PPIs were associated with abnormalities in p53 expression and Ki-67 labeling. Methods:Twenty-one nondysplastic BE cases with incomplete ablation were evaluated for the expression of Ki-67 (proliferation), cyclooxygenase-2 (COX-2), and p53 by immunohistochemistry. Results:Pre-ablation biopsies showed the normal staining patterns in columnar epithelium, ie, normal Ki-67 labeling, rare positive COX-2 staining of interstitial cells, and negative or mild staining for p53 in the majority of patients’ biopsies. However, post-ablation biopsies demonstrated abnormal staining patterns in the glandular area at the new squamocolumnar junctions. In 13 of 21 post-ablation cases (62%), increased Ki-67 staining was seen in BE glands. In 8 of 21 patients (38%), increased COX-2 expression was seen in columnar epithelium. Similarly, in 8 of 21 post-ablation junctions (38%), there was increased p53 staining. Conclusions:Our findings of increased expression of cancer-associated biomarkers in incompletely ablated BE patients raise a cautionary note regarding this procedure. We hypothesize that newly formed junctions contain cells undergoing replication, differentiation, etc, and are thus more susceptible to genomic damage.

Deoxycholate, an Endogenous Cytotoxin/Genotoxin, Induces the Autophagic Stress-Survival Pathway: Implications for Colon Carcinogenesis

We report that deoxycholate (DOC), a hydrophobic bile acid associated with a high-fat diet, activates the autophagic pathway in non-cancer colon epithelial cells (NCM-460), and that this activation contributes to cell survival. The DOC-induced increase in autophagy was documented by an increase in autophagic vacuoles (detected using transmission electron microscopy, increased levels of LC3-I and LC3-II (western blotting), an increase in acidic vesicles (fluorescence spectroscopy of monodansycadaverine and lysotracker red probes), and increased expression of the autophagic protein, beclin-1 (immunohistochemistry/western blotting). The DOC-induced increase in beclin-1 expression was ROS-dependent. Rapamycin (activator of autophagy) pre-treatment of NCM-460 cells significantly (P < .05) decreased, and 3-MA (inhibitor of autophagy) significantly (P < .05) increased the cell loss caused by DOC treatment, alone. Rapamycin pre-treatment of the apoptosis-resistant colon cancer cell line, HCT-116RC (developed in our laboratory), resulted in a significant decrease in DOC-induced cell death. Bafilomycin A1 and hydroxychloroquine (inhibitors of the autophagic process) increased the DOC-induced percentage of apoptotic cells in HCT-116RC cells. It was concluded that the activation of autophagy by DOC has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents.

Role of interleukin-6 in Barrett’s esophagus pathogenesis

Barretts esophagus (BE) is a metaplastic lesion of the distal esophagus arising as a consequence of chronic gastroesophageal reflux disease. Multiple studies show that BE is associated with increased risk of esophageal adenocarcinoma (EAC). Epidemiological studies and animal models demonstrate that chronic inflammation triggered by repeated exposure to refluxate predisposes to the development of BE and EAC. The chronic inflammation is associated with cytokine alterations. Interleukin 6 (IL-6) is a cytokine that stimulates cell proliferation and apoptosis resistance is frequently increased in different cancers. Importantly, IL-6 and transcriptional factor signal transducer and activator of transcription 3 (STAT3) that is activated by IL-6 are also increased in BE and EAC. This review critically appraises the role of IL-6/STAT3 pathway in progression of BE to EAC from the published evidence currently available.

Barrett's esophagus: proton pump inhibitors and chemoprevention I: Barrett's esophagus: proton pump inhibitors and chemoprevention

The following on proton pump inhibitors and chemoprevention in Barretts esophagus includes commentaries on normalization of esophageal refluxate; the effects of 5‐HT4 agonists on EGF secretion and of lubripristone on chloride channels agents; the role of Campylobacter toxin production; the deleterious effects of unconjugated bile acids; the role of baclofen in nonacid reflux; the threshold for adequate esophageal acid exposure; the effects of proton pump inhibitor (PPI) therapy on normalization of esophageal pH and on cell proliferation; the role of the phenotype of cellular proliferation on the effects of PPI therapy; and the value of Symptom Index and Symptom Association Probability in the evaluation of potential response to treatment.

Abstract 544: Lung cancer classification using new immunohistochemical assay with anti-p40 (BC28) mouse monoclonal antibody: Comparison with the p40 Echelon assay and anti-p63 (4A4) antibody

Background: The precise differentiation of lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC) is essential for the determination of appropriate cancer therapy. Currently, the anti-p63 (4A4) antibody is the most frequently used marker for the lung SCC classification. A major limitation of this antibody is low specificity due to its reactivity in a substantial proportion of lung ADCs. The p63 antibody recognizes two p63 isoforms - 1) long TAp63 isoform and 2) a truncated variant designated as p40. In contrast to the anti-p63 antibody, the anti-p40 (BC28) antibody recognizes only the p40 isoform. The goal of this study was to compare a newly optimized IHC assay using the anti-p40 (BC28) antibody and OptiView IHC DAB Detection Kit with 1) the commercially available p40 Echelon assay, and with 2) an IHC assay using the anti-p63 (4A4) antibody in various lung tumors. Methods: Lung tumors from 271 patients were evaluated for p40 and p63 expression using IHC on VENTANA BenchMark XT platform with OptiView detection. The p40 expression was also evaluated by the commercially available anti-p40 (BC28) Echelon assay (Biocare Medical Inc.) using the manufacturer9s recommended protocol. After the initial classification of these tumors by HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 544. doi:10.1158/1538-7445.AM2015-544

Su1163 The Loss of Beclin-1 Expression Correlates With Progression to Esophageal Adenocarcinoma and Autophagy Resistance: the Role of Bile Acids

Background and Aims: Autophagy is a normal physiological mechanism for the degradation of cellular proteins and organelles. Excessive autophagy induced by cellular stress leads to cell death. However, the exact role of autophagy in cancer is not completely clear. Beclin1 plays a central role in the regulation of autophagy. The major goal of our study was to evaluate the role of Beclin-1 and autophagy and the effect of bile acids in the esophageal carcinogenesis. Barretts esophagus (BE) is a premalignant lesion of distal esophagus associated with an increased risk for the development of esophageal adenocarcinoma (EAC). Methods: Beclin-1 expression was evaluated using immunohistochemistry, immunoblotting, or RTPCR in (1) biopsies obtained from 62 patients with BE or EAC, (2) tissues from a rat model of BE and EAC, and (3) esophageal cell lines. Since reflux of bile acids is important in esophageal carcinogenesis, the effect of acute and chronic exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression was evaluated. Autophagy was assessed by electron microscopy or by transfection with GFP-LC3 plasmid and calculating the percentage of GFP-LC3 positive cells with punctuate pattern. Results: Our studies using human biopsies and rat tissues showed that Beclin-1 expression was high in squamous epithelium, while its expression was low in BE and EAC. A similar pattern was observed in esophageal cell lines. HET-1A cells (derived from normal squamous epithelium) showed high levels of Beclin-1, but lower levels of Beclin-1 were found in BE cells (CP-A, CP-C) and EAC cells (OE33 cells). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy in CP-A cells. In contrast, chronic exposure to DCA did not result in any alteration of Beclin-1 or autophagy. To demonstrate that Beclin-1 plays central role in autophagy we used siRNA to decrease Beclin-1 expression and then autophagy was evaluated after treatment with DCA. No increase in autophagy was detected compared to untreated control CP-A cells. Conclusions: In summary, our data suggests that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance. We propose that autophagy resistance in combination with apoptosis resistance induced by chronic exposure to bile acids contributes to EAC development.

Abstract 3793: Bile acids and autophagy resistance in Barrett's esophagus

Background: Barrett9s esophagus (BE) is a premalignant lesion of the distal esophagus that is associated with increased risk of esophageal cancer (EAC). Bile acids are implicated in BE development. Autophagy is a tightly-regulated process involving degradation and recycling of cellular proteins and organelles. The genetic links between autophagy defects and cancer suggest that autophagy is involved in tumor suppression. We have previously shown that acute exposure to bile acids induces autophagy. In this study we tested the hypothesis that chronic exposure to bile acids leads to loss of capability to undergo autophagy and that BE is resistant to autophagy. Methods: Tissue biopsies from sixty patients with different grades of BE dysplasia, EAC and control tissues were evaluated for the expression of Beclin, p-mTOR, p-AKT by immunohistochemistry. Transmission electron microscopy (TEM) was used to evaluate autophagy in tissues starved for 4 hours in Hank9s balanced salt solution (HBSS, autophagy inducer). To study chronic effects of DCA in vitro we developed CP-A cells resistant to DCA. These new CP-AR cells are able to survive and proliferate in medium containing 0.2mM DCA. Both control CP-A cells and CP-AR cells were treated with DCA and then evaluated for autophagic markers, Beclin 1 and p62. Autophagy was also assessed by quantification of cells with the punctuate pattern of GFP-LC3. Results: Our data indicate that tissue biopsies from patients with BE dysplasia and esophageal adenocarcinoma: (1) are resistant to autophagy induced by HBSS, (2) express increased levels of proteins associated with autophagy resistance [phosphorylated Akt (p-Akt) and p-mTOR] and (3) express decreased levels of the key pro-autophagic protein Beclin-1. In addition, the levels of Beclin-1 in non-dysplastic tissue or control tissues (duodenum, squamous epithelium) were high, while p-mTOR and p-Akt were low. CP-AR cells treated with DCA expressed same levels of p62 and Beclin 1 as untreated cells. In contrast, the levels of these proteins were increased in parental CP-A cells exposed to DCA indicating an increase in autophagy. Furthermore, there was no significant difference in number of GFP-LC3 positive cells with punctuate pattern between untreated and DCA treated resistant CP-AR cells. However, 103% increase in GFP-LC3 positive cells with punctuate pattern was observed after DCA treatment in sensitive CP-A cells compared to untreated CP-A cells. Conclusion: Dysplastic BE and EAC have decreased capability to undergo autophagy. In vitro studies show that chronic exposure to bile acids may be crucial factor in the development of autophagy resistance in BE esophagus. These alterations may cause a consequent increase in DNA damage leading to cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3793. doi:10.1158/1538-7445.AM2011-3793

Barrett's esophagus: endoscopic treatments II: Barrett's esophagus: endoscopic treatments II

The following on endoscopic treatments of Barretts esophagus includes commentaries on indications for endoscopic treatments; endo‐luminal plication procedures; the cellular modifications induced by the endoscopic ablation therapies; eradication by banding without resection; the evaluation of complete ablation; recurrence after ablation; association of antireflux surgery; radiofrequency ablation; and nondysplastic Barretts esophagus.