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Opinion
Is Campylobacter to esophageal adenocarcinoma as Helicobacter is to gastric adenocarcinoma? Nadeem O. Kaakoush1, Natalia Castan˜o-Rodrı´guez1, Si Ming Man1,2, and Hazel M. Mitchell1 1 2
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, NSW, Australia Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
Esophageal adenocarcinoma develops through a cascade of cellular changes that shares similarities to the etiology of Helicobacter pylori-associated intestinal-type gastric adenocarcinoma. While host genetics and immune response have been implicated in the progression to esophageal adenocarcinoma, studies investigating esophageal microbial communities suggest that bacteria may also play an important role in driving the inflammation that leads to disease. Of these, emerging Campylobacter species have been found to be more prevalent and abundant in patients progressing through the esophageal adenocarcinoma cascade compared to controls. Given that these bacteria possess several virulence mechanisms such as toxin production, cellular invasion, and intracellular survival, emerging Campylobacter species should be investigated as etiological agents of the chronic esophageal inflammation that leads to cancer. The esophageal adenocarcinoma cascade Esophageal cancer is the 6th leading cause of cancerrelated deaths worldwide, resulting in an estimated 456 000 new cases and 400 000 deaths in 2012 (age-standardized incidence rate: 5.9 per 100 000 person-years; ratio of mortality to incidence: 0.88) (http://globocan.iarc.fr). Esophageal cancer comprises two major histological types, squamous cell carcinoma and adenocarcinoma (EAC), with incidence rates of EAC markedly increasing in both males and females across most ethnicities over the past several decades [1]. For example, in the USA alone the incidence of EAC has risen from 0.4 to over 3 per 100 000 person-years since the 1970s (http://www.seer.cancer.gov). The development of EAC occurs through a cascade of events starting with gastro-esophageal reflux disease (GERD), which then progresses to Barrett’s esophagus (BE) [2], dysplasia, and finally adenocarcinoma (Figure 1). GERD, a condition believed to affect 20–30% of the population in developed countries [3], is characterized Corresponding author: Kaakoush, N.O. (
[email protected]). Keywords: gastro-esophageal reflux diseaseBarrett’s esophagus; esophageal adenocarcinoma; Campylobacter concisus; Helicobacter pylori; gastric adenocarcinoma. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.03.009
by chronic reflux of acid, bile, and other stomach contents which induce inflammation in the squamous epithelium of the esophagus. In some GERD patients, particularly those with increased duration and/or frequency of reflux [4], the injured squamous epithelium is replaced by columnar epithelial cells similar to those found in the intestine. This metaplastic process culminates in the development of BE, a recognized premalignant condition that dramatically increases the risk (relative risk, 11.3; 95% CI, 8.8–14.4) of developing EAC [5,6]. Perhaps the most alarming aspect of BE is not that approximately 1–5% of the population of developed countries have this condition [7,8], or that the incidence of BE is on the rise [9,10], but instead the fact that BE can be present in a high percentage of asymptomatic patients [11]. In this Opinion article, the role of esophageal microbial species in the etiology of EAC is discussed, with particular emphasis on the involvement of Campylobacter and Fusobacterium species, including discussion of the mechanisms through which these species may contribute to the progression of the EAC cascade. Factors associated with the EAC cascade Several risk factors have been recognized to predispose an individual to developing GERD, and thus increase their risk of developing BE and EAC. These include smoking, male gender, age, presence of a hiatus hernia, high bodymass index (BMI), and lack of Helicobacter pylori infection. For example, in a recent study by Thrift and colleagues which analyzed 999 patients with EAC, 2061 patients with BE, and 2169 population controls, higher BMIs were significantly associated with both BE and EAC [12]. Indeed, a recent analysis by Olsen and colleagues estimates that high BMI (30) accounts for approximately 23% of EAC cases [13]. These results are supported by studies that have investigated the role of BMI in the development of esophageal metaplasia and neoplasia [14,15]. Interestingly, a genetic predisposition to obesity has also been shown to be associated with BE and EAC, with patients more predisposed to developing obesity being at higher risk [12]. By contrast, while alcohol consumption is generally accepted as a risk factor for squamous cell carcinoma, a large pooled analysis found no evidence that alcohol consumption increases the risk of BE or EAC [16]. Trends in Microbiology xx (2015) 1–8
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EAC cascade Gastro-esophageal reflux disease
Barre’s esophagus
Low-grade dysplasia
High-grade dysplasia
Esophageal adenocarcinoma TRENDS in Microbiology
Figure 1. The esophageal adenocarcinoma cascade. Esophageal adenocarcinoma develops through a cascade of events starting with gastro-esophageal reflux disease, and then progressing to Barrett’s esophagus, dysplasia, and finally adenocarcinoma.
Perhaps the most intriguing of these associations is the inverse correlation between EAC and H. pylori infection of the stomach. While eradication of H. pylori does not appear to be associated with the development of new cases of GERD [17], an inverse association exists between cytotoxin-associated gene A (CagA)-positive [odds ratio (OR), 0.41; 95% CI, 0.28–0.62] but not CagA-negative (OR, 1.08; 95% CI, 0.76–1.53) H. pylori colonization and risk of EAC development [18]. CagA is a protein encoded on the Cag pathogenicity island of H. pylori, which has been associated with perturbation of host signaling pathways, increased levels of inflammation, and a greater risk of gastric cancer [19]. In support of these findings, a retrospective study by Fassan and colleagues showed H. pylori to be present in 14.8% of BE patients, 17.4% of EAC patients, and 41.0% of controls [20]. Furthermore, the authors reported a significantly lower prevalence of severe atrophic gastritis and non-cardia gastric neoplasia in BE patients compared to controls [20]. Several mechanisms to explain the protective nature of H. pylori infection have been proposed; these include reduction in gastric acidity, regulation of gastric secretion of leptin and ghrelin, dysregulation of the immune response, modulation of the esophageal microbiota, and alteration of esophageal–gastric motility [21]. Given that gastric atrophy (associated with low acidity) resulting 2
from H. pylori infection is negatively correlated with GERD and BE [22], and that retrospective analysis of patient data [23] shows that patients with EAC are more likely to have had a peptic ulcer (associated with high acidity) compared to controls (EAC, 21%; controls, 13%; P=0.0766), reduction of gastric acidity by H. pylori is one promising area that should be investigated further in relation to GERD. While this may initially appear contradictory, given that infection with CagA-positive H. pylori is also associated with peptic ulcer disease, it is plausible that only infected patients progressing through Correa’s cascade, and not towards peptic ulcer disease, are protected against EAC. Analysis of the presence of H. pylori dupA (duodenal ulcer promoting gene) in the context of EAC may shed light on this matter. Furthermore, regulation of the immune response by H. pylori infection is also a likely contributor to lack of disease, given that the presence of this bacterium has been inversely correlated with diseases that have a strong immune component – including asthma and inflammatory bowel diseases [24,25]. The role of host genetics and the immune response in the EAC cascade Similarly to gastric adenocarcinoma, the development of EAC is a multifactorial process where host genetics and the immune response play a very important role. Analysis of genome wide association study (GWAS) data in relation to the involvement of germline genetic variations in the EAC cascade has found a strong correlation between genes that influence the risk of developing BE and EAC, while this correlation was absent for GERD [26]. This suggested that although germline genetic variations in the host are important for the transition of GERD to BE, and then to EAC, they are not important for the development of GERD per se. Consistently, several studies have identified genes that contain or are in proximity to germline mutations associated with BE or EAC [27–29]. Genes identified to be involved in EAC using different omics approaches are summarized in a recent review by Weaver and colleagues [30]. Host germline mutations are not the only important genetic factors involved in the EAC cascade because genomic instability is also associated with the development of EAC. In an analysis of the mutational load of microdissected targets from esophageal biopsies of BE patients, increasing mutational load correlated with the severity of histology in biopsies, suggesting that BE patients with a high mutational load may be at greater risk of developing EAC [31]. Further, loss of normal function of the tumor suppressor p53 is one of the strongest predictive factors for progression to BE [6]. Inflammation is now considered to be one of the hallmarks of carcinogenesis. Thus, it is no surprise that several immune signaling pathways such as transforming growth factor b1 (TGF-b1) [32], Notch [32], insulin-like growth factor (ILGF) [33], and interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) [34], and proinflammatory molecules such as protease-activated receptor 2 (PAR2) [35] and cyclooxygenase 2 (COX-2) [36], are involved in the EAC cascade. Furthermore, the transcription factor nuclear factor kB (NF-kB) is heavily involved in the EAC cascade [34], likely through activation of Toll-like
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Cyclin E
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Molity, differenaon and other processes
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MTMR9
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MMP-3 MMP-1
146a
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EPHB3
CNTNAP5
29c let-7c
21
miRNA TRENDS in Microbiology
Figure 2. Genes and miRNA involved in the esophageal adenocarcinoma cascade. Molecules highlighted in pink have been found to be significantly regulated by macrophages during Campylobacter concisus infection [64].
receptors such as TLR4, TLR5, and TLR9 [37], and through antioxidant enzymes such as the glutathione peroxidase GPX7 [38]. The immune response is not limited to inflammation arising from the innate immune responses because T cell composition suggests similarities between the immune response of BE tissue and the duodenum [39]. Other molecules, such as the transcription factors CDX1 and CDX2 (caudal-related homeobox transcription factors 1/2, that are important for intestinal development) [29], cyclin E [40], the vitamin D receptor (VDR) [41], and several microRNAs [42,43], have documented roles in the progression of the EAC cascade. For example, CDX1 activation, influenced by NF-kB [44], may be involved in intestinal metaplasia through induction of stemness-associated reprogramming factors SALL4 (spalt-like transcription factor 4) and KLF5 (Kruppel-like factor 5), and subsequent trans-differentiation of esophageal epithelial cells to intestinal epithelial cells [45]. Of particular interest, another signaling pathway that has been suggested to be involved in the EAC cascade, is the sonic hedgehog (SHH)–bone morphogenetic protein 4 (BMP4)–sex determining region Y-box 9 (SOX9) signaling axis [43,46], the
activation of which provides mechanistic insights into the transition from squamous to intestinal-like columnar epithelium. Further details on this signaling axis can be found in Gibson et al. [46]. Overall, at least 67 genes/proteins and 48 miRNAs involved in different cellular functions, including the immune response, have been associated with the EAC cascade (Figure 2). A role for the esophageal microbiota in the EAC cascade In addition to environmental factors, and a person’s own genetic susceptibility and immune response, microbial species colonizing the esophageal mucosa have been suggested to play an important role in the EAC cascade. For example, exposure to lipopolysaccharide may influence gastric emptying and the relaxation of the lower esophageal sphincter leading to reflux [47], or, alternatively, an increase in immunogenic species within the microbiota may drive inflammatory processes. Early studies into the esophageal microbiota identified the genus Streptococcus, and to a lesser extent Prevotella and Veillonella, to be the dominant taxa within the healthy 3
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(B)
50
C. concisus (Log CFU/ml)
C. concisus prevalence (%)
(A) 60
40 30 20 10 0
6 5 4 3 2 1 0
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EAC
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1.2
Key:
Campylobacter
9
Fusobacterium
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Dialister
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IL-18 expression
1.4
Abundance
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1 0.8 0.6 0.4
P