Clinical Pathogenesis, Molecular Mechanisms of Gastric Cancer Development

Abstract

The human pathogen Helicobacter pylori is the strongest known risk factor for gastric disease and cancer, and gastric cancer remains a leading cause of cancer-related death across the globe. Carcinogenic mechanisms associated with H. pylori are multifactorial and are driven by bacterial virulence constituents, host immune responses, environmental factors such as iron and salt, and the microbiota. Infection with strains that harbor the cytotoxin-associated genes (cag) pathogenicity island, which encodes a type IV secretion system (T4SS) confer increased risk for developing more severe gastric diseases. Other important H. pylori virulence factors that augment disease progression include vacuolating cytotoxin A (VacA), specifically type s1m1 vacA alleles, serine protease HtrA, and the outer-membrane adhesins HopQ, BabA, SabA and OipA. Additional risk factors for gastric cancer include dietary factors such as diets that are high in salt or low in iron, H. pylori-induced perturbations of the gastric microbiome, host genetic polymorphisms, and infection with Epstein-Barr virus. This chapter discusses in detail host factors and how H. pylori virulence factors augment the risk of developing gastric cancer in human patients as well as how the Mongolian gerbil model has been used to define mechanisms of H. pylori-induced inflammation and cancer.

1. Introduction

In the majority of countries across the globe the overall incidence of gastric cancer (GC) is declining, however, it is still a serious condition and GC remains the fourth leading cause of cancer-related mortality worldwide behind lung, colorectal, and liver cancers (Sung et al. 2021). Adenocarcinoma is the most common type of cancer that affects the stomach, but other types of cancer can also arise, including lymphoma and leiomyosarcoma. The Cancer Genome Atlas Network has identified four molecular subtypes of GC: (i) Chromosomal instability with intestinal-type histology, (ii) Microsatellite instability, (iii) Genomically stable, and (iv) Epstein-Barr virus (EBV)-related (Cancer Genome Atlas Research 2014). However, in all anatomic regions of the stomach, chromosomal instability tumors with intestinal-type histology predominate. Intestinal-type adenocarcinoma is initiated by the transition from normal mucosa to chronic superficial gastritis; this is followed by the development of atrophic gastritis (loss of acid secreting parietal cells), SPEM (spasmolytic polypeptide-expressing metaplasia) and/or intestinal metaplasia, finally leading to dysplasia and adenocarcinoma (Fig. 1) (Correa 1996; Sipponen and Marshall 2000; Song et al. 2015). GC is a multifactorial disease, and the interplay between many different factors including, but not limited to, bacterial virulence factors, host genetics, diet, and the gastric microbiota can influence disease progression and outcome (Fig. 2). In this chapter, we discuss mechanisms driving the development of GC in humans and the use of the Mongolian gerbil as a model that recapitulates GC development in humans.

Fig. 1. The sequential stages of gastric carcinoma modified from the Correa cascade.

H. pylori-induced inflammation is the strongest known risk factor for gastric carcinoma. H. pylori colonize the gastric mucosa of approximately 50% of the world’s population. Almost all infected individuals develop gastritis and 1–3% of infected individuals will develop gastric cancer. In the gerbil model, almost all colonized gerbils develop gastritis and the majority develop gastric carcinoma

Fig. 2. Risk factors for developing gastric cancer in the context of chronic H. pylori infection.

Host diet, the gastric microbiome, host cell factors and infection with Epstein-Barr virus may all influence disease outcome in the context of H. pylori infection

2. Epidemiology and Risk Factors for Gastric Cancer Development

Each year, approximately 1 million people worldwide are diagnosed with GC, of whom approximately 800,000 will die (Pilleron et al. 2019). Although the overall incidence of GC has been decreasing in most parts of the world (Ferlay et al. 2021) the incidence and mortality rates of GC are increasing in certain populations. Overall, males are two to three times more likely to develop GC than females, and there are significantly more GC cases in less developed regions of the world compared to more developed regions. Approximately half of the total GC burden is found in East Asia, especially China and Japan. GC incidence and mortality rates are also higher in central and eastern Europe, South America, and Central America (Ang and Fock 2014). In the United States, GC rates are increasing specifically among young female and Hispanic male populations (Siegel et al. 2015; Anderson et al. 2018).

2.1. Helicobacter pylori

Helicobacter pylori infects more than 4.4 billion individuals and is the strongest known risk for GC, prompting its designation in 1994 as a WHO Group I carcinogen and a high-priority pathogen for which new therapies are urgently needed (Savoldi et al. 2018; Hooi et al. 2017). H. pylori selectively colonizes the gastric epithelium and has colonized humans for more than 60,000 years. Infection is usually acquired in childhood and in the absence of combined antibiotic therapy, can persist for the lifetime of the host (Wroblewski et al. 2010). Most H. pylori-infected individuals remain asymptomatic, but approximately 1–3% will develop gastric adenocarcinoma, and 0.1% develop MALT lymphoma (Hooi et al. 2017). In addition to H. pylori, other components of the gastric microbiota may also influence gastric disease progression (see Sect. 2.6 Human microbiome and chapter “Gastric Cancer: The Microbiome Beyond Helicobacter pylori” of this book).

2.2. H. pylori Virulence Factors

Colonization with H. pylori is the strongest known risk factor for developing GC, however, most infected individuals do not develop GC. H. pylori virulence factors including urease, adhesins, vacuolating cytotoxin A and the cag pathogenicity island (cagPAI), play key roles in determining disease outcome (Cover and Blanke 2005; Franco et al. 2008; Wroblewski et al. 2010; Koch et al. 2015; Pachathundikandi et al. 2015) (Fig. 3). The best-studied factor involved in pathogenicity is the cagPAI, which contains genes that encode for proteins that form a bacterial type IV secretion system (T4SS) and the oncoprotein CagA (Fischer et al. 2020; for more details see Chapter “Impact of the Helicobacter Pylori Oncoprotein CagA in Gastric Carcinogenesis” of this book). CagA is translocated through the cagT4SS by adherent H. pylori across the bacterial and epithelial membranes into host cells. Approximately 60% of H. pylori isolates from Western countries contain the cagPAI and almost all strains from East Asia harbor this locus (Odenbreit et al. 2000; Fischer et al. 2001; Kwok et al. 2007; Shaffer et al. 2011). Infection with cagA-positive H. pylori strains is associated with developing intestinal and diffuse gastric adenocarcinoma at 2–3 times the frequency compared to persons infected with H. pylori strains that are cagA-negative (Parsonnet et al. 1997; Huang et al. 2003).

Fig. 3. H. pylori virulence factors involved in the development of gastric cancer.

H. pylori is genetically highly variable, and some strains are more strongly associated with the development of gastric disease. This schematic summarizes some of the host-cell alterations that H. pylori virulence factors elicit

The carboxyl-terminal part of CagA contains repeat phosphorylation glutamate-proline-isoleucine-tyrosine-alanine (EPIYA) motifs, which may also be used as indicators of pathologic outcome (Hatakeyama 2004; Higashi et al. 2005; Naito et al. 2006). Four different EPIYA motifs (EPIYA-A, -B, -C, or –D) have been identified (Hatakeyama 2004; Higashi et al. 2005; Naito et al. 2006; Lind et al. 2014, 2016). EPIYA-A and EPIYA-B motifs are found in most strains, while the EPIYA-C motif is predominately found in Western strains and the number of EPIYA-C sites is associated with an elevated risk of developing GC in multiple populations (Basso et al. 2008; Estaji et al. 2022; Rodriguez Gomez et al. 2020). Strains that contain the EPIYA-D motif are typically East Asian strains and are associated with increased pathogenesis compared to strains harboring C-type CagA motifs (Hatakeyama 2004; Argent et al. 2008).

Following translocation, CagA becomes tyrosine-phosphorylated at EPIYA motifs and can induce cellular responses with carcinogenic potential. The primary phosphorylation site is the tyrosine of the EPIYA-C or -D motif and phosphorylation of these motifs is required for binding to the Src homology region 2 domain-containing phosphatase (SHP2) and increasing pathogenicity through mechanisms such as cytoskeletal rearrangements, cell proliferation and inflammatory responses (Mueller et al. 2012; Backert and Tegtmeyer 2017; Hatakeyama 2014; Higashi et al. 2004, 2002; Li et al. 2018).

CagA also exerts phosphorylation-independent effects within host cells that contribute to pathogenesis. Non-phosphorylated CagA targets many cellular effectors including apical-junctional components, the hepatocyte growth factor receptor c-Met, the phospholipase PLC-γ, the adaptor protein Grb2, and the kinase PAR1b/MARK2, leading to pro-inflammatory and mitogenic responses, disruption of cell–cell junctions, and loss of cellular polarity (Mimuro et al. 2002; Saadat et al. 2007; Murata-Kamiya et al. 2007; Churin et al. 2003; Amieva et al. 2003; Franco et al. 2005; Bagnoli et al. 2005; Suzuki et al. 2005; Takahashi-Kanemitsu et al. 2020). Independent of CagA, H. pylori can also induce mislocalization of the tight junction proteins occludin and claudin-7 and alter barrier function (Wroblewski et al. 2009, 2015).

The protease high temperature requirement A (HtrA) protein family are serine proteases (Tegtmeyer et al. 2016). HtrA from H. pylori is a secreted virulence factor involved in the disruption of cellular junctions by cleaving occludin, claudin-8 and E-cadherin (Hoy et al. 2010; Schmidt et al. 2016; Tegtmeyer et al. 2017), and permits H. pylori to colonize and persist under harsh conditions (Zarzecka et al. 2019). Recently, H. pylori isolated from humans was found to contain natural mutations in the htrA gene, which resulted in an amino acid change at position 171 that was associated with protein trimer stability (Zarzecka et al. 2023). Further analysis identified a single nucleotide polymorphism (SNP) in HtrA at serine/leucine 171 that significantly correlated with GC. In vitro, the 171S-to-171L mutation activated HtrA trimer formation, which resulted in disruption of the junctional proteins occludin and E-cadherin, increased translocation of CagA, increased accumulation of β-catenin in cell nuclei, and augmented host DNA double-strand breaks, all pre-malignant changes (Sharafutdinov et al. 2023) (Fig. 3).

Another widely studied H. pylori virulence factor is the cytotoxin VacA, which elicits multiple effects on host cells including vacuolation, altered plasma and mitochondrial membrane permeability, autophagy, and apoptosis (Cover and Blanke 2005; Boquet and Ricci 2012; Caso et al. 2021) (Fig. 3). The vacA gene is found in all strains of H. pylori and contains a number of variable loci in the 5’ region termed s, i and m regions. This 5’ terminus encodes the signal sequence and amino-terminus of the secreted toxin (allele types s1a, s1b, s1c, or s2), an intermediate region (allele types i1 or i2), and a mid-region (allele types m1 or m2) (Atherton et al. 1995; Rhead et al. 2007). Strains containing type s1, i1, or m1 alleles are more strongly associated with GC (Atherton et al. 1995, 1997; Miehlke et al. 2000). Infection with H. pylori VacA s1m1 strains is associated with increased risk for developing peptic ulcer disease (Matos et al. 2013). Similarly, Latin American strains with s1 and m1 genotypes increase the risk for developing gastric cancer and peptic ulcers. African strains with the s1 or m1 genotypes also augment the risk of peptic ulcers and gastric cancer (Sugimoto and Yamaoka 2009). In middle eastern populations, the s1m1 genotypes significantly increased the risk of gastric cancer and peptic ulcers (Sugimoto et al. 2009b).

In human gastric organoids, VacA m1 and m2 alleles have been shown to have similar vacuolating activity (Caston et al. 2020). VacA and CagA may also counter-regulate each other’s actions to manipulate host cell responses (Backert and Tegtmeyer 2010;Barker et al. 2010; Tsugawa et al. 2012). VacA is secreted from H. pylori as an 88 kDa monomer (p88) and assembles into multiple water-soluble oligomeric structures, including hexamers, heptamers, dodecamers, and tetradecamers (Chambers et al. 2013; Lupetti et al. 1996; Adrian et al. 2002). Oligomerization appears to be essential for VacA activity (Ivie et al. 2008; Genisset et al. 2006), and recent studies using Cryo-EM have provided structural insights into the process of VacA oligomerization (Su et al. 2019).

H. pylori genomes contain approximately 30 different hop genes, which encode outer membrane proteins. HopQ binds to the human Carcinoembryonic Antigenrelated Cell Adhesion Molecule (CEACAM) receptor on the host cell surface and the interaction between HopQ and CEACAM has been shown to facilitate CagA translocation via the T4SS (Javaheri et al. 2016; Zhao et al. 2018; Nguyen et al. 2023; Belogolova et al. 2013; Moonens et al. 2018). There are two different families of hopQ alleles: type I and type II. Type I hopQ alleles were found significantly more frequently in cag-positive/type s1-vacA allele strains from patients with peptic ulcer disease, whereas type II hopQ alleles were more frequently observed in cagnegative/type s2-vacA strains from patients without peptic ulcer disease (Cao and Cover 2002).

2.3. Dietary Factors (Salt/Iron)

The risk of developing gastric adenocarcinoma is also influenced by environmental factors such as diet. Diets that are high in salt, pickled, smoked or poorly preserved foods, those with a high meat content, and those with low fruit and vegetable content are most commonly associated with an increased risk for developing GC. Conversely, diets plentiful in fruits and vegetables are associated with less risk for developing GC (Tsugane and Sasazuki 2007; Epplein et al. 2008; Gonzalez et al. 2006, 2012; Kim et al. 2010, 2004; Ren et al. 2012).

In the context of H. pylori infection, high dietary salt intake and low iron levels are highly associated with an increased risk for developing GC (Lee et al. 2003; Shikata et al. 2006; Noto et al. 2013a, 2022, 2015; Noto and Peek 2015; Loh et al. 2023). Iron deficiency is the most common nutritional disorder in the world and can be a result of a diet deficient in iron, blood loss, or colonization by certain H. pylori strains, which have been associated with hemorrhagic gastritis (Yip et al. 1997). Chronic H. pylori infection can further exacerbate iron deficiency through the development of gastric atrophy which leads to decreased acid secretion, reduced ascorbic acid levels and decreased iron absorption (Yip et al. 1997). Iron deficiency in H. pylori-infected individuals accelerates the development of gastric adenocarcinoma by increasing the virulence potential of H. pylori, and this has been further studied in the Mongolian gerbil model (see Sect. 3 for further discussion) (Noto et al. 2013a, 2022) and mouse models. Further, iron deficiency has been associated with increased levels of secondary bile acids, such as deoxycholic acid, in H. pylori-infected animals. A retrospective cohort analysis of the association between the use of bile acid sequestrant medications and the incidence of GC in 416,885 patients suggests that bile acid sequestrants are protective against GC (Noto et al. 2022). Furthermore, expression of the bile acid receptor, TGR5, paralleled the severity of gastric disease in humans. Expression of TGR5 increased in the progression from atrophic gastritis to intestinal metaplasia, dysplasia, and finally GC (Noto et al. 2022).

In addition to host iron levels, high salt diets have also been found to modulate the virulence potential of H. pylori. The association between high dietary salt intake and increased GC risk has been reported in both humans and Mongolian gerbil models (discussed in Sect. 3) of H. pylori infection (Gaddy et al. 2013; Bergin et al. 2003). A cross-sectional study performed in Japan identified a significant correlation between the amount of salt excreted in urine and GC mortality rates in both men and women (Tsugane et al. 1992). In animal models and in vitro models, high salt diets have been reported to increase the expression of the H. pylori virulence factors CagA, VacA and UreA, in addition to selecting fur mutations that confer a selective advantage to H. pylori in high-salt conditions (discussed further in Sect. 3.4) (Loh et al. 2007, 2012, 2023, 2018; Gancz et al. 2008; Voss et al. 2015; Caston et al. 2019).

2.4. Host Constituents

H. pylori induces a robust inflammatory response in its host and specific host gene polymorphisms are associated with further increases in the risk of developing GC (Usui et al. 2023). H. pylori infection increases gastric mucosal expression of the T helper (Th) type 1 (Th1) pro-inflammatory cytokine and inhibitor of gastric acid secretion, IL-1ß (Noach et al. 1994). In the context of H. pylori infection, individuals who harbor polymorphisms in IL-1ß that result in high expression levels of IL-1ß are at significantly higher risk for developing distal gastric adenocarcinoma compared to those with genotypes that limit IL-1ß expression (El-Omar et al. 2000). IL-1β polymorphisms can promote GC by augmenting IL-1β production, decreasing gastric acid production and increasing circulating cytokine levels (Furuta et al. 2002; El-Omar et al. 2001; Fox and Wang 2007). Individuals with high-expressing IL-1ß polymorphisms in conjunction with H. pylori cagA⁺ or vacA s1-type strains have a 25-fold or 87-fold increase in risk, respectively, for developing GC compared to uninfected individuals (Figueiredo et al. 2002). Polymorphisms that increase expression of the pro-inflammatory cytokine TNF, or that decrease the production of anti-inflammatory cytokines such as Il-10 are also associated with an increase in risk of developing GC (El-Omar et al. 2003).

2.5. Epstein-Barr Virus (EBV)

EBV is a human herpes virus that is associated with GCs. EBV-positive GCs comprise almost 10% of GCs and represent a distinct subset of GC identified by The Cancer Genome Atlas (TCGA) (Cancer Genome Atlas Research 2014). Synergistic interactions between EBV and H. pylori in the gastric epithelium may promote progression towards GC. A case–control study has shown that the combination of EBV and H. pylori induces severe inflammation and, in this way, augments the risk of developing intestinal type GC (Cardenas-Mondragon et al. 2015). In a recent study, EBV was shown to methylate the host phosphatase SHP1 and thereby prevent SHP1 from dephosphorylating CagA. This perturbation increases the oncogenic activity of CagA and may augment the synergistic effect of EBV and H. pylori (Saju et al. 2016).

2.6. Human Microbiome

The development of a new technology for analyzing microbial communities has expanded our understanding of the human gastric microbiome. The gastric microbiome is known to play a critical role in maintaining homeostasis, while perturbations in the microbiome can contribute to the development and progression of GC (Guo et al. 2020; Ai et al. 2023; Mannion et al. 2023). In the normal human stomach, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria are the major phyla identified (Bik et al. 2006; Liu et al. 2019, 2022; Delgado et al. 2013). In contrast, in H. pylori-infected individuals, H. pylori is the predominant bacterium found in the stomach and microbial diversity is decreased (Yu et al. 2017; Ndegwa et al. 2020; Mannion et al. 2023). In a study using reverse transcribed 16S rRNA as the amplification template, the metabolically active bacteria found in the upper gastrointestinal tract in individuals with and without H. pylori infection was analyzed (Schulz et al. 2018). In the stomach, consistent with other studies, Helicobacter species were found to dominate the microbiota in H. pylori -infected individuals, and the relative abundance of Actinobacteria, Bacteroidetes, Firmicutes and Fusobacteria were decreased (Schulz et al. 2018). Infection with H. pylori was also shown to influence the microbiota of the duodenum and oral cavity (Schulz et al. 2018). In a prospective population-based study, H. pylori was identified as one of the main factors in gastric microbial dysbiosis and this dysbiotic microbiota was associated with chronic atrophic gastritis, intestinal metaplasia and dysplasia. Successful H. pylori eradication resulted in restoration of the gastric microbiota to similar status of uninfected individuals (Guo et al. 2020). A population-based study in China using deep sequencing identified that the microbiota in chronic atrophic gastritis or intestinal metaplasia exhibited lower abundances of Actinobacteria, Bacteroidetes, Firmicutes and Fusobacteria compared to normal or superficial gastritis. Actinobacteria, Bacteroidetes and Firmicutes were found in greater abundance in dysplasia/GC compared to intestinal metaplasia (Kadeerhan et al. 2021). A meta-analysis of microbiota from 6 independent studies across the stages of GC development identified Veillonella, Dialister, Granulicatella, Herbaspirillum, Comamonas, Chryseobacterium, Shewanella and Helicobacter as biomarkers for discerning GC from superficial gastritis (Fischer et al. 2022). Further, gastric transplantation of stomach microbiota harvested from patients with premalignant and malignant lesions into germ-free (GF) mice induced intestinal metaplasia and dysplasia (Kwon et al. 2022). Other investigators have defined the gastric microbiota in adults residing in either a low-risk or a high-risk GC region in Colombia. Significant differences were present (Yang et al. 2016), and colonization with either of 2 such differentially abundant species modified the ability of H. pylori to induce gastric injury in GF mice (Shen et al. 2022). In a 15-year intervention study, antibiotic treatment targeting H. pylori significantly reduced the incidence of GC, even though fewer than half of treated individuals remained free of H. pylori infection (Ma et al. 2012), suggesting that antibiotics that modify the microbiota can attenuate the development of GC despite the presence of H. pylori. See chapter “Gastric Cancer: The Microbiome Beyond Helicobacter pylori” of this book for further details.

2.7. Yes-Associated Protein

Yes-Associated Protein (YAP) is a key effector of the Hippo tumor suppressor pathway. Li et al. reported that YAP is significantly upregulated in human gastric carcinogenesis and H. pylori infection induced YAP and downstream effectors in gastric epithelial cells in a cag-dependent manner (Li et al. 2018). Recently, it has been demonstrated that the interaction between CagA and PAR1b prevents nuclear translocation of the tumor suppressor, BRCA1, by inhibiting PAR1b kinase-mediated phosphorylation of BRCA1 (Imai et al. 2021). Nuclear expression of BRCA1 (BRCAness) was found to lead to replication fork instability and subsequent DNA double-strand breaks; however, cells expressing CagA were found to evade apoptosis. Simultaneously, apoptosis was shown to be suppressed through activation of Hippo signaling via CagA-mediated PAR1b inhibition, which prevented formation of the YAP/p73 pro-apoptotic complex and allowed cells the ability to repair double-strand breaks through error-prone mechanisms. In the presence of functional p53, proliferation of CagA expressing cells is inhibited by p21. In the absence of p53, CagA-expressing cells displaying BRCAness proliferate. Since loss of cellular p53 usually occurs due to aging-associated somatic TP53 mutation this may explain why H. pylori-infection in young individuals results in mostly mild disease while in elderly individuals, GC is more prevalent (Imai et al. 2021).

3. Mongolian Gerbils as a Model for Gastric Cancer Development

Mice remain the most frequently used animal model for the investigation of H. pylori-induced gastric carcinogenesis; however, the Mongolian gerbil is also used for reasons outlined below. The Mongolian gerbil is a small rodent member of the Cricetidae family. The gerbil has been increasingly used in research focused on H. pylori pathogenesis as it represents an efficient and cost-effective rodent model that recapitulates many features of H. pylori-induced gastric inflammation and carcinogenesis in humans, allowing for targeted investigation of the bacterial determinants and environmental factors that lead to H. pylori-induced disease.

3.1. Gastric Cancer Development in Mongolian Gerbils

In 1991, the first published description of the Mongolian gerbil model reported that following oral inoculation, H. pylori colonized the gastric-mucosal layer of gerbils and induced mild gastritis following a two-month infection (Yokota et al. 1991). Similar to the disease process in humans, subsequent studies demonstrated that gerbils develop gastric ulcers, duodenal ulcers and intestinal metaplasia following long-term infection with H. pylori (Hirayama et al. 1996; Matsumoto et al. 1997; Honda et al. 1998; Ikeno et al. 1999; Ohkusa et al. 2003; Franco et al. 2008). Carcinomas that developed in H. pylori-infected gerbils typically occurred in the distal stomach and the pyloric region and contained well-differentiated intestinal-type epithelium, reflecting many features of intestinal-type gastric adenocarcinoma in humans. Consistent with reports in humans, H. pylori eradication in the gerbil model significantly reduced the severity of gastritis, premalignant lesions, and incidence of gastric adenocarcinoma (Matsumoto et al. 1997; Keto et al. 2001; Nozaki et al. 2002; Nozaki et al. 2003)}.

In humans, two types of metaplasia can develop following H. pylori colonization, inflammation, and gastric atrophy: intestinal metaplasia and SPEM (see Introduction and Fig. 1). The development of intestinal-type GC is associated with intestinal metaplasia and SPEM; however, investigations into the origin of intestinal metaplasia have been somewhat limited because mice do not develop intestinal metaplasia in response to H. pylori infection (Correa 1988; Hattori 1986; Hattori and Fujita 1979; Xia et al. 2000; Schmidt et al. 1999; Yamaguchi et al. 2002; Halldorsdottir et al. 2003). Mongolian gerbils infected with H. pylori, however, do develop intestinal metaplasia, dysplasia and cancer making them fundamentally different from mouse models and more similar to humans (Hirayama et al. 1996; Honda et al. 1998; Watanabe et al. 1998; Yoshizawa et al. 2007). Recent studies using Mongolian gerbils have demonstrated that H. pylori-infected gerbils developed SPEM early and within 9 weeks of infection. SPEM initially occurred in the intermediate zone along the lesser curvature and subsequently invaded the greater curvature, which is similar to the development of metaplasia in humans. In early stages of H. pylori infection in gerbils, SPEM is organized in straight glands; however, at later stages of infections, SPEM glands became distorted and expanded. Following 6 months of infection, intestinal metaplasia developed (Yoshizawa et al. 2007).

3.2. Host Constituents

Mongolian gerbils were the first model used to identify the role of IL-1β in the development of GC. IL-1β is a Th1-type cytokine that is increased within the gastric mucosa of H. pylori-infected individuals (Noach et al. 1994; see also Sect. 2.4 Host constituents). In gerbils infected with H. pylori for 6 or 12 weeks, IL-1β levels increased, while gastric acid secretion decreased. Moreover, treatment of H. pylor- infected gerbils with an IL-1β antagonist abolished the loss of acid secretion, thus implicating IL-1β in the development of achlorhydria in the stomach of H. pylori-infected gerbils (Takashima et al. 2001).

Experiments using gerbils have also highlighted altered expression of other inflammatory mediators including inducible nitric oxide synthase (iNOS) and COX2 following H. pylori infection (Matsubara et al. 2004; Sakai et al. 2003). Cyclooxygenase 2 (Cox-2) mRNA expression was significantly increased following a 1- and 3-month infection with H. pylori, and this was not seen in gerbils infected with H. pylori lacking the T4SS component CagE (Sakai et al. 2003). Expression levels of IL-1β, TNF and iNOS mRNA were shown to be increased following a 2-week H. pylori infection, while in the fundic region, protein expression levels of IL-1β, TNF and iNOS were increased following a 4- and 8-week infection in gerbils (Matsubara et al. 2004).

The gerbil model has also been used to demonstrate the role of transcription factor NF-κB activation within the context of H. pylori-induced inflammation. In an independent study, quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) was used to compare gastric cell scrapings from H. pylori-infected and uninfected gerbils. 2764 proteins were quantified and 166 were significantly altered in abundance by H. pylori infection. Pathway mapping identified significant changes in many signaling pathways including those involved in inflammation, proliferation, differentiation, apoptosis, and regulation of the cell cycle (Noto et al. 2019a). H. pylori infection of gerbils has also been shown to increase serum levels of gastrin, which can promote gastric epithelial cell proliferation (Peek et al. 2000; Konturek et al. 2003).

The Mongolian gerbil model has also been a very useful model for investigating ‘The Colombian Enigma’. Almost 90% of the Colombian population is infected with H. pylori; however, the incidence rates of GC differ greatly in high versus low altitude regions. In the high altitude Andean region, there is a higher prevalence of precancerous lesions compared to the low risk coastal region (de Sablet et al. 2011; Correa et al. 1976). Mongolian gerbils that were infected with H. pylori strains originating from the high-risk region induced more spermine oxidase (SMOX), oxidative DNA damage, dysplasia and adenocarcinoma than H. pylori from the low-risk region suggesting that activation of polyamine-driven oxidative stress could be used as a marker of GC risk and a target for chemoprevention (Chaturvedi et al. 2015).

3.3. Crucial H. pylori Determinants in Mongolian Gerbils

Many advances have been made utilizing the gerbil model to investigate cancer-associated microbial determinants. As discussed in Sect. 2.2, cagPAI is one of the most studied H. pylori virulence factors. One limitation of using murine models of H. pylori infection is that clinical cag⁺ strains often fail to colonize (Sozzi et al. 2001; Philpott et al. 2002). In contrast, clinical cag⁺ strains of H. pylori readily colonize gerbils and maintain a functional cagT4SS secretion system (Peek et al. 2000), which together with the development of more severe disease, allows for a robust investigation of the role of cagPAI in the context of H. pylori-induced inflammation and cancer. As with human infections, gerbils infected with cag⁺ strains develop significantly more severe gastritis than those infected with cag− strains, echoing the importance of the cag island in H. pylori-mediated inflammation and pathogenesis (Ogura et al. 2000; Saito et al. 2005; Ohnita et al. 2005; Shibata et al. 2006).

In vivo adaptation of H. pylori strains has been demonstrated to increase the virulence potential of H. pylori strains in gerbils. In one study, a single gerbil was infected with a human H. pylori isolate, 3 weeks post-challenge a single colony output derivative was isolated and used to infect an independent population of gerbils. Following in vivo adaptation, gastric dysplasia and adenocarcinoma developed, phenotypes that were not observed with the input H. pylori strain (Franco et al. 2005). Serial infections of gerbils with H. pylori have provided insights into how the host modifies cagT4SS function through alterations in cagY, a structural component of the cag T4SS. Changes in the genetic composition of cagY were shown to parallel cagT4SS function, and the development of dysplasia or cancer selected for attenuated cagT4SS virulence phenotypes (Suarez et al. 2017).

In terms of genetic analysis, using whole genome sequencing techniques, sequences of H. pylori strains isolated from experimentally infected gerbils were compared to the sequence of the input strain (Beckett et al. 2018). The mean annualized SNP rate per site was similar to rates reported in H. pylori-infected humans. Many of the mutations occurred within or upstream of genes that are associated with iron-related functions (fur, tonB1, fecA2, fecA3, and frpB3) or which encoded outer membrane proteins (alpA, oipA, fecA2, fecA3, frpB3 and cagY). One of the SNPs detected in the output strains, FurR88H, conferred a survival advantage when H. pylori was co-cultured with neutrophils (Beckett et al. 2018).

In studies focused on the T4SS per se, a recent study utilized a tetracyclin repressor (tetR)/tetracycline operator (tetO) system to conditionally regulate cagT4SS activity in the gerbil model of H. pylori infection (Lin et al. 2020). In experiments, where gerbils were exposed to more than three months of continuous cagT4SS activity, higher rates of dysplasia and/or GC were observed than when cagT4SS activity was limited to early or late stages of infection. However, when the activity of cagT4SS was confined to just the initial 6 weeks of infection, gastric inflammation still developed, and GC was detected in a small fraction of gerbils (Bartpho et al. 2020). These data support a hit-and-run model of carcinogenesis whereby an infectious agent triggers carcinogenesis during the initial stages of infection and the ongoing presence of the infectious agent is not required for development of cancer.

The Mongolian gerbil model has also been used to investigate other H. pylori virulence factors including the role of the OMP OipA in H. pylori-induced gastric pathogenesis (Franco et al. 2008; Sugimoto et al. 2009a). Mongolian gerbils infected with oipA-deficient mutants developed significantly less inflammation than gerbils infected with wild-type strains and did not develop gastric dysplasia or GC (Franco et al. 2008). These findings are consistent with human population data (see Sect. 2.2 H. pylori virulence factors).

3.4. Dietary Factors in the Gerbil (Salt/Iron)

The gerbil model has been widely used to investigate potential relationships between diet and GC risk in the context of H. pylori infection. Epidemiologic studies of diet in humans are limited by reliance on accurate patient reporting and difficulty in ascertaining diets that were consumed decades prior to the development of GC. Increased salt consumption has been shown to increase the risk for GC in humans (Sect. 2.3 Dietary factors), and the effects of high-salt diets on H. pylori infection and GC have also been investigated using the gerbil model. One study showed that gerbils maintained on a high-salt diet that were H. pylori-infected had a significantly higher incidence of gastric adenocarcinoma than H. pylori-infected gerbils maintained on a normal-salt diet (Gaddy et al. 2013). In a follow up study, H. pylori strains were analyzed from experimentally infected gerbils, and compared to input strains; the output strains from gerbils maintained on a high-salt diet produced higher levels of proteins involved in iron acquisition, including a mutation in fur (encoding the ferric uptake regulator variant Fur-R88H) and resistance to oxidative stress (Loh et al. 2015). In a recently published work, it was reported that the fur-R88H mutation augments H. pylori fitness in vitro under high-salt conditions and exerts the opposite effect under normal-salt conditions. FecA is a known ferric citrate transporter and analysis of the transcription profiles revealed that fecA2 plays a role in H. pylori fitness under both high-salt environments and normal salt environments (Loh et al. 2023).

The role of iron deficiency in influencing disease outcome in the context of H. pylori infection has also been studied in Mongolian gerbils. In a study where gerbils were maintained on iron-replete or iron-depleted diets and then challenged with H. pylori, more severe gastritis and increased frequency of gastric dysplasia and gastric adenocarcinoma were reported among gerbils maintained on iron-depleted diets compared to gerbils maintained on iron-replete diets (Noto et al. 2013b). These phenotypes were only present in animals infected with a cagA⁺ strain and infection with a cagA− isogenic mutant strain abrogated the response (Noto et al. 2013b). H. pylori output strains isolated from gerbils maintained on an iron-depleted diet exhibited an enhanced ability to translocate CagA and induced higher levels of IL-8 compared to output strains isolated from gerbils maintained on an iron-replete diet (Noto et al. 2013b). It has also been demonstrated that H. pylori infection causes iron deficiency anemia in the Mongolian gerbil model. In the presence of H. pylori infection, gerbils maintained on a high-salt/low-iron diet for 16 weeks exhibited a higher incidence and an increased severity of iron deficiency anemia compared to H. pylori-infected gerbils maintained on a regular diet (Beckett et al. 2016).

3.5. Gerbil Microbiome

In comparison to the human and mouse microbiome, very little is known about the gerbil microbiome. There have been a limited number of studies to determine if H. pylori induces dysbiosis of the gastric mucosal microbiota similar to what occurs in humans. Using qualitative and quantitative DNA- and RNA-based taxonomic microbiota analyses, human, mouse, and gerbil stomach samples were demonstrated to exhibit similarities at higher taxonomic levels but differences at lower taxonomic levels (Wurm et al. 2018). Microbiota changes in H. pylori infected Mongolian gerbils have also been studied and—like in humans—the microbiota of Mongolian gerbils is modified by long-term infection with H. pylori (Yin et al. 2011; Osaki et al. 2012; Heimesaat et al. 2014). Lactobacillus, Bifidobacterium, Clostridia, and Enterococcus were abundantly expressed among both H. pylori-infected and uninfected gerbils; however, the abundance of Bifidobacterium and Clostridia were significantly lower among H. pylori-negative gerbils (Osaki et al. 2012). Recently, the gerbil gastric mucosal microbiota, within the context of H. pylori infection and low iron has been more definitively defined using 16S rRNA sequencing. Infection with H. pylori was found to significantly decrease α-diversity and alter microbial community structure in a cagA-dependent manner. Concordant with earlier reports, Lactobacillus was found in abundance, but other abundant operational taxonomic units were different and included Enterobacteriaceae and Porphyromonadaceae. When gerbils were infected with H. pylori and maintained on an iron-deplete diet there were no significant differences in α- or β-diversity, phyla, or operational taxonomic unit abundance compared to infected gerbils maintained on iron-replete diets, despite increased H. pylori-induced injury in the gerbils maintained on an iron-deplete diet. Interestingly, when microbial composition was stratified based only on the severity of gastric injury, significant differences in α- and β-diversity were present among gerbils harboring premalignant or malignant lesions compared to gerbils with gastritis alone (Noto et al. 2019b).

4. Concluding Remarks

Globally, GC leads to a high number of cancer-related deaths each year. Infection rates of H. pylori vary across the globe, with some areas approaching 100%, however, 97–99% of colonized persons will never develop GC. The risk of developing GC is dependent on numerous factors including H. pylori strain-specific virulence factors, the host genotype, environmental factors such as diet as well as the microbiome. The gerbil model has provided and will continue to provide critical information on the interactions among these factors and will aid in understanding the dynamic of host genetics, dietary factors, and the microbiome in the context of chronic H. pylori infection, with the goal of identify individuals who are at the highest risk of developing GC.