Winkel, A. van de

Barrett's Esophagus: New insights in the genetic patchwork of transdifferentiation and malignant transformation (Doctoral Thesis)

2011-12-15T00:00:00Z

De behandeling en vooruitzichten voor patiënten met een slokdarm carcinoom zijn weinig hoopgevend. Vroegtijdige detectie en preventie zijn noodzakelijk, en daarom richt veel onderzoek zich op de premaligne Barrett slokdarm, waarin gezond slokdarm epitheel vervangen is door darmachtig epitheel. De precieze ontstaanswijze en progressie van een Barrett slokdarm is onduidelijk. Daarom heeft dit proefschrift zich gericht op genen die van belang kunnen zijn hierin. Allereerst vonden wij dat de nucleaire receptor Pregnane X Receptor (PXR) niet aanwezig is in gezond slokdarm epitheel, maar wel in het darmachtige epitheel van Barrett slokdarm en adenocarcinomen. In adenocarcinomen werd PXR na blootstelling aan galzuren, in de celkernen gevonden waar het actief andere genen reguleert. Wij concluderen dat PXR mogelijk een voorspeller is van progressie in Barrett slokdarm. In een histologische studie hebben wij deze hypothese verder onderzocht en hierin aangetoond dat de combinatie van de eerder bestudeerde galzuur receptor Farnesoid X Receptor (FXR) en PXR, klinische waarde hebben voor de diagnostiek van dysplasie. Verder vonden wij dat bepaalde DNA mutaties in het regulerende deel van het Vitamine D receptor (VDR) gen geassocieerd zijn met een verminderd expressie van het VDR gen in Barrett slokdarm middels de binding van transcriptie factor GATA1. Het haplotype dat deze mutaties omvat is bovendien geassocieerd met een twee maal lager risico op het manifesteren van een Barrett slokdarm (OR 0.44; 95%CI 0.23-0.85) of adenocarcinoom in de slokdarm (OR 0.50; 95%CI 0.27-0.96). Het identificeren van dragers van dit haplotype is een eerste stap in het ontwikkelen van persoonspecifieke behandelingen voor slokdarmkanker. Ten slotte zochten we genen die verantwoordelijk kunnen zijn voor Barrett’s slokdarm. Een proces vergelijkbaar met Barrett’s metaplasie vindt plaats tijdens de embryonale ontwikkeling van het maag-darmkanaal, waarin Homeobox (HOX) genen een cruciale rol spelen. Wij toonden het belang van HOXA genen aan voor het epitheel van het volwassen maag-darmkanaal en identificeerden HOXA7 en A11 als belangrijke galgereguleerde spelers in Barrett metaplasie. Aanvullende studies zijn nodig om de regulerende rol van HOX genen te bestuderen in het ontstaan van Barrett slokdarm.

Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma (Article)

2011-10-06T00:00:00Z

Background: The continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma.Methods: PXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender.Results: PXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P < 0.001), and RE patients (P = 0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P = 0.037).Conclusions: PXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease.

Differential expression of the nuclear receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR) for grading dysplasia in patients with Barrett's oesophagus (Article)

2011-01-01T00:00:00Z

Aims: To investigate expression of nuclear receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR) as a diagnostic tool to improve grading of dysplasia in Barrett's oesophagus patients. Methods and results: Immunostaining was analysed on a total of 192 biopsy samples of 22 Barrett's patients with no dysplasia (ND), 17 with low-grade dysplasia (LGD), 20 high-grade dysplasia (HGD) and 24 with adenocarcinoma (AC). Nuclear FXR expression was observed in 15 of 22 (68%) ND cases versus none of 19 HGD; 3 of 17 (18%); LGD; 5 of 60 (8%) patients with AC (P<0.001). FXR expression was highly specific for non-dysplastic tissue. Nuclear PXR was expressed in 16 of 20 (80%) HGD cases versus two of 16 (13%) LGD cases (PPV 89%). Upon examining adjacent tissue taken from HGD and AC patients, PXR expression was high in samples of all tissue types. Conclusions: Nuclear receptors are expressed differentially during neoplastic progression, with FXR positivity being useful to distinguish ND from dysplasia and AC. PXR nuclear expression is able to separate HGD from LGD and ND. The combination of FXR and PXR also appears to have diagnostic and possibly prognostic value, but future prospective studies are required to investigate their predictive power for neoplastic progression in Barret's oesophagus.

Bile acid-stimulated expression of the farnesoid X receptor enhances the immune response in Barrett esophagus (Article)

2008-06-01T00:00:00Z

OBJECTIVES: Barrett's esophagus (BE) is a premalignant condition of the esophagus. It is a consequence of mucosal injury from chronic gastroesophageal reflux in which bile acids are an important toxic component. The farnesoid X receptor (FXR) is a nuclear receptor involved in the regulation of bile acid synthesis, transport, and absorption. FXR activation is also involved in the induction of the innate immune response. This suggests that FXR is involved in the pathogenesis and the inflammation seen in BE. METHODS: mRNA levels of FXR and the FXR-regulated genes, ileal bile acid-binding protein (IBABP), small heterodimer partner (SHP), and chemokines interleukin (IL)-8 and macrophage inflammatory protein 3α (MIP3α), were determined by real time-polymerase chain reaction (RT-PCR). Protein expression was determined by immunohistochemistry. RESULTS: FXR was not expressed in squamous epithelium of healthy subjects (N = 7), but was present in both squamous and columnar epithelium of BE patients. Compared to the squamous epithelium of BE patients, their columnar epithelium displayed a 2.3-fold (P = 0.02) increase in FXR mRNA. Also, IBABP (2.2-fold; P = 0.0029), SHP (2.7-fold; P = 0.007), IL-8 (1.5-fold; P = 0.04), and MIP3α (1.7-fold; P = 0.019) transcription levels were increased. Exposure of esophageal cell line TE7 to deoxycholic acid (DCA) resulted in a similar induction. The induction was abolished by the FXR antagonist guggulsterone. CONCLUSIONS: Expression levels of the bile acid receptor FXR, the bile acid metabolism genes IBABP and SHP, and the chemokines IL-8 and MIP3α are increased in Barrett's epithelium. The in vitro induction of FXR by DCA suggests that bile acids can actively induce the inflammatory response in BE by recruiting immune cells.