DysregulaRon of redox enzymes in BarreT's ...

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AGR2, a PDI homolog, is strongly associated with BarreB's and other adenocarcinomas, and has been suggested to support tumour growth (1). Our experiments ...
Dysregula,on of redox enzymes in Barre:'s oesophagus and gastro-intes,nal cancer

Lee Simpson, Danielle M. Ba:le, Sanjika Dias-Gunasekara, Y.K.S Viswanath* and Adam M. Benham

School of Biological and Biomedical Sciences, Durham University, Durham, UK and *Department of General Surgery, James Cook University Hospital, Middlesbrough, UK

1. Introduc,on

3. Induc,on of Ero1α in oesophageal cell lines



Oesophageal cancer o.en occurs a.er the appearance of Barre3’s epithelium, a metaplasia of the oesophageal lining. However, it is unclear how and why some pa>ents maintain a stable Barre3's epithelium whereas others go on to develop oesophageal cancer (Figure 1). We are interested in understanding the role of protein disulfide isomerases (PDIs), redox enzymes and other folding factors in Barre3's oesophagus. AGR2, a PDI homolog, is strongly associated with Barre3’s and other adenocarcinomas, and has been suggested to support tumour growth (1). Our experiments lead us to propose that dysregula>on of oxida>ve protein folding in the endoplasmic re>culum (ER) may be involved in the transi>on of Barre3's to adenocarcinoma. Disulfide bond forma>on in the Endoplasmic Re>culum is chiefly controlled by electron exchange reac>ons between Endoplasmic Re>culum Oxidoreduc>ns (Eros) and PDIs (Figure 2). This pathway creates disulfide bonds from oxygen and generates hydrogen peroxide, which can be u>lised by peroxiredoxin IV (and glutaredoxins) and coupled to further disulfide bond forma>on (2). In mammals, there are two Ero1 proteins, Ero1α and Ero1β, that are upregulated by hypoxia and the unfolded protein response, respec>vely. Ero1α is strongly expressed in the oesophageal epithelium and Ero1β is strongly expressed in the stomach, but the func>onal significance of this in health and disease is largely unknown (3,4). S

O2

H2O2

Figure 1: Barre3’s oesophagus may remain stable or become cancerous.

S

S

S

S

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Ero1

PDI

client protein

HS SH

HS SH

HS SH

Ero1

PDI

client protein

To understand how Ero1α responds to hypoxia in oesophageal cells, the expression of Ero1α protein was analysed in OE21 cells (derived from a squamous carcinoma) and OE33 cells (derived from a Barre3’s adenocarcinoma). Ero1α was strongly expressed in OE33 cells but not in OE21 cells (Figure 4). However, Ero1α could be induced by hypoxia (lane 6) but not by ER stress (lane 8) in OE21 cells. Ero1β was not expressed in either cell line (not shown). Both Ero1α expression (Figure 4) and ac,vity (not shown) can be modulated by the hypoxic and redox environment. Figure 4: Western blot showing Ero1α (A), PDI (B) and ac>n (C, control) expression in oesophageal cell lines OE33 (lanes 1-4) and OE21 (lanes 5-8) a.er exposure of the cells to 200 µM deferrioxamine (DF), 10 µM cobalt chloride (Co) or 1 µg/ml tunicamycin.

4. Ero1 expression is altered in Barre:'s oesophagus and dysplasia Ero1α (but not Ero1β) is expressed in normal oesophagus, whereas Ero1β is expressed strongly in the stomach (4,7). To determine the expression of Ero1α/β in Gl disease, >ssue sec>ons represen>ng Barre3's oesophagus (Figure 5), and dysplasia (Figure 6) were immunohistochemically stained with an>bodies to detect the Ero proteins. A representa>ve example of the results obtained are shown here. Ero1α expression becomes elevated in Barre:’s oesophagus, whereas Ero1β expression becomes elevated in dysplas,c ,ssue.



Figure 2: Protein disulfide bond catalysis and peroxide forma>on in the Endoplasmic Re>culum.

2. A role for the Endoplasmic Re,culum as an oxida,ve stress inducer in Barre:’s?

In pathological situa>ons such as Barre3’s (Figure 3), the peroxide and free radicals produced during oxida>ve protein folding could lead to inappropriate expression or ac>va>on of Ero1α and hence the uncontrolled produc>on of peroxide, which may result in bystander DNA and protein damage. Taken together with findings that the ER resident glutathione peroxidase GPx7 is epigene>cally silenced in Barre3’s (5), a “two-hit” mechanism may occur in which peroxide genera>on by oxida>ve folding could lead to bystander damage when ER detoxifying enzymes are downregulated. High expression of oxida>ve folding enzymes may also help support cell growth and differen>a>on in the hypoxic tumor environment. Figure 3: Possible associa>on between reflux, inflamma>on and the development of Barre3's; adapted from ref 6.

Figure 5 (leN): Barre3’s >ssue stained with preimmune serum (A,B), an>-Ero1β (C,D) or an>-Ero1α (E,F). Magnifica>ons: 5x (A,C,E) and 20x (B,D,F). Figure 6 (right): Dysplas>c >ssue was stained with pre-immune serum (A,B), TBS only (C) an>-Ero1β (D) or an>-Ero1α (E,F). Brown= posi>ve staining, blue= haematoxylin counterstain. Columnar epithelium is indicated by >, mul>nuclear cells by arrows, infiltra>ng cells by arrowheads; the dysplas>c epithelium in Fig 6A (right) is shown by a double-headed arrow. Scale bar = 200 µm

5. Conclusion

Ero1α and Ero1β have contras>ng expression pa3erns in Barre3’s oesophagus. Ero1α is strongly expressed in Barre3’s epithelium and in dysplas>c >ssue, whereas Ero1β is not expressed in Barre3’s epithelium and is strongly expressed in dysplas>c >ssue. The control of other redox enzymes and folding factors in Barre3’s is under inves>ga>on. By understanding how redox regula>on and disulfide bonds are controlled in the GI tract, it may be possible to find novel targets for drug development and new biomarkers for Barre3’s Oesophagus.

References: 1) Dong et al JBC 286:18301-10 (2011). 2) Bulleid and Ellgaard TIBS 36:485-92 (2011). 3) Benham et al Philos Trans R Soc Lond B Biol Sci. 368:20110403 (2013) 4) Ba3le et al An>oxid. Redox. Signal. 19:24-35 (2013). 5) Wild and Hardie Nat. Rev. Cancer 3, 676-84 (2003); 6) Peng et al Gut 61:1250-60 (2012); 7) Dias-Gunasekara et al JBC 280: 33066-75 (2005).