This is an author version of the contribution published on:
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[Minerva Biotecnologica, 29(4), 2017, DOI:10.23736/S1120-4826.17.02354-0]
ovvero [Gian Paolo Caviglia, Caterina Bosco, 29, ed. Minerva medica, 2017,
pagg.180-187]
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Helicobacter pylori, gastric cancer and gastric cancer stem cells
Gian Paolo CAVIGLIA, Caterina BOSCO
Department of Medical Sciences, University of Turin, Turin, Italy
*Corresponding author: Gian Paolo Caviglia, Department of Medical Sciences, University of Turin, Turin 10100, Italy. E-mail: caviglia.giampi@libero.it
Abstract
Chronic Helicobacter pylori (H. pylori) infection is considered the main cause of gastric cancer (GC). Gastric carcinogenesis is the result of a series of precancerous changes from atrophic gastritis to intestinal metaplasia and dysplasia in which H. pylori plays a key role both through direct and indirect mechanisms. Chronic inflammation caused by H. pylori infection leads to gastric mucosal damage and consequently increases turnover in gastric epithelium. During this process, resident stem cells may, over time, accumulate genetic and epigenetic changes inducing an aberrant phenotype and thus leading to the emergence of GC stem cells. Furthermore, H. pylori has direct oncogenic properties regarded to the cytotoxin-associated gene A (CagA). This bacterial oncoprotein is delivered into host cells and promotes neoplastic transformation by interacting with several intracellular signaling pathways. In addition, chronic inflammation and tissue injury followed by recruitment and engraftment of bone marrow derived stem cells into the gastric epithelium, may represent another source of GC stem cells. In this scenario, GC stem cells have been identified as the cell compartment able of self-renewal and differentiation responsible of tumor continuous growth. The possibility to isolate and identify these cells via specific surface markers may provide, in the future, novel targets to develop specific molecules able to prevent GC development or to counteract tumor evolution.
Key words: Gastric cancer – Helicobacter pylori – cancer stem cells – bone marrow-derived stem cells
Gastric cancer (GC) is a major global health issue representing the third leading cause of cancer death worldwide.1 In 2012, an incidence of 951,600 GC cases and more than 723,000 GC-related deaths has been estimated.2 Despite an overall decline of GC incidence and mortality rates observed in many Western Countries, in Eastern Asia GC remains the most frequent malignancy in both sexes.3 In particular, the incidence rate is very high in Japan, China and Republic of Korea reaching 66.7/100 000 among males and 25.4/100 000 among females.1, 3
Chronic infection with Helicobacter pylori (H. pylori) is associated with around 90% of non-cardia GC cases in the world;4 in 1994, the International Agency for Research on Cancer classified H.
pylori as group I carcinogen, i.e. a definite cause of GC.5 As a matter of fact, improved sanitation together with implementation of national endoscopic screening programs led to a significant reduction of GC incidence and tumor related death, respectively.6, 7
The most accepted model of gastric carcinogenesis provides a multifactorial and multistep pathogenesis, involving a number of initiators and other continuator agents, in which H. pylori
infection plays a key role inducing a chronic inflamed environment leading to the emergence of gastric cancer stem cells (CSCs), a small fraction of cells that initiate and drive the tumor’s continuous
growth.8
Here, we briefly review the role of H. pylori in gastric carcinogenesis based on the concept of GC as model of stem-cell disease.
The characteristics of gastric cancer
GC is extremely heterogeneous in terms of location, histopathological and molecular features.9 The majority of GCs are adenocarcinomas that can arise from the gastric proximal stomach (cardia), usually not linked to H. pylori infection, or from the distal stomach. According to Lauren classification,10 tumors from the distal stomach can be differentiated on the basis of histology: diffuse and so-called
intestinal-type carcinoma.The intestinal type is the most frequent, and compared to the diffuse type, appears late in life, showing a long-standing, stepwise evolution with mucosal changes from superficial gastritis, multifocal atrophic gastritis, intestinal metaplasia (IM), dysplasia and finally in situ GC and metastatic carcinoma.11 This progressive phenotypic alteration is supported by genetic conditions (including activation of oncogenes, deletion and/or mutations of tumor suppressor genes) caused by repeated DNA damage.12
In consideration of the wide spectrum of genetic alterations and molecular pathways involved in gastric carcinogenesis, a molecular characterization of GCs has been recently proposed by The Cancer Genome Atlas Research Network.13 Following an integrative genomic analysis of 295 GCs, 4 tumor subtypes have been identified: microsatellite-instable, showing elevated mutation rates of genes encoding for oncogenic signaling proteins; Epstein-Barr virus-positive, showing extreme DNA hypermethylation and amplification of JAK2, PD-L1 and PD-L2; chromosomal-instable, showing marked aneuploidy and focal amplification of receptor tyrosine kinases; and genomically stable GC, enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins (Table I).13
The characteristics of Helicobacter pylori
H. pylori is a microaerophilic, spiral-shaped, gram-negative bacterium acquired mainly during
childhood.14 In order to live and grow in gastric acid environment, H. pylori produces high levels of urease; this enzyme degrades urea to cell-toxic ammonia neutralizing gastric acids, thus raising the pH of the stomach and thereby allowing H. pylori to safely penetrate beneath the mucous layer to the epithelium surface.15 Interestingly, as the pH approaches neutrality, in order to avoid excessive
ammoniaproduction, a negative feedback-based mechanism of regulation denies substrate access to the enzyme.16
Motility and adhesion are other characteristics essential for colonization of the stomach favoring the onset of a persistent infection (Table II).17, 18 The presence of flagella (each flagellum ~3 µm long) allows the bacterium to spread into the viscous mucous layer covering gastric epithelium where it can specifically bind via adhesion pedestals. A large amount of outer membrane proteins (OMPs) are encoded by H. pylori genome;19 the blood group antigen binding adhesin (BabA) is the most known and well-studied.20 Through this adhesin, the bacterium binds to the host Lewis b (Leb) antigen usually located at the upper epithelium.21 However, several other OMPs enable H. pylori adherence to the host epithelium including the sialic acid-binding adhesin (SabA) that recognizes the Lex antigen which is particularly expressed during chronic gastric inflammation,22 adherence-associated lipoprotein A and B (AlpA/B) that bind host laminin,23 and the cytotoxin-associated gene L (CagL) protein containing an RGD (Arg-Gly-Asp) motif that binds to the host α5β1 integrin and allows the translocation of CagA into the cytosol of gastric epithelium.24, 25
Once colonization is established, the infection can last for decades and even be lifelong.26 However, virulence and pathogenicity of H. pylori are complex phenomena involving the interaction of host, bacteria and environmental factors.27 Among bacterial determinants, the vacuolating cytotoxin protein A (VacA) and the translocated effector protein CagA are the two main pathogenic factors that can influence the outcome of chronic H. pylori infection.28
A classical feature of VacA is the capability to induce small translucent cytoplasmic vacuole in the gastric cells of the host through a mechanism based on channel formation within cellular
membranes.29 However, the most important target sites of VacA is the mitochondrial inner membrane; the bacterial toxin can depolarize the inner membrane of the organelle inducing mitochondrial
disruption and, thus, an alteration in cellular energy homeostasis. Consequently, the drop in cellular ATP levels together with the release of pro-apoptotic protein effectors from the inner membrane space into the cytosol leads to cellular apoptosis.30
The CagA protein is a widely accepted pathogenicity marker that is associated with a severe outcome of infection.31 This protein is encoded by cagA gene, localized at the end of the cag
pathogenicity island (cag PAI), and is inoculated into the host cells through the type IV secretion system (T4SS). Delivered CagA can interact with multiple host signaling molecules affecting their expression in a phosphorylation-dependent and independent manner.32 Currently, CagA is considered the most important bacterial oncoprotein.33, 34
Role of Helicobacter pylori in gastric carcinogenesis
H. pylori plays an important role in causing peptic ulcer disease, gastric adenocarcinoma and gastric
mucosa-associated lymphoid tissue.35 Since H. pylori infection is easily cured, 36, 37 understanding its role in gastric carcinogenesis is crucial, especially because ≈1-2% of H. pylori-positive patients develop GC.38
Chronic inflammation is known to be a factor of progression towards cancer and inflammatory cells, and mediators are essential components of the tumor microenvironment.39 In effect, H. pylori infection induces a long-term inflammation causing apoptosis of gastric epithelial cells and subsequent compensative proliferation for the cell loss.40 In particular, the cell wall component lipopolysaccharide (LPS) is a driving factor for chronic gastric inflammation and GC.41 The interaction of LPS with the gastric mucosal Toll-like receptor 4 (TLR4) triggers the initiation of downstream signal transduction events that involve mitogen-activated protein kinase (MAPK) cascade activation, in turn leading to the activation of cyclooxygenase (COX) enzymes, thus resulting in an increased production of
pro-inflammatory mediators such as prostaglandin E2 and nitric oxide (NO).42 The production of free radicals (superoxides [O+] and NO+) induces oxidative DNA damage leading to errors during mitosis and accumulation of mutations.43
Beside LPS, urease activity, resulting in the production of high levels of ammonia, seems to be involved in gastric carcinogenesis, too. Indeed, ammonia is a toxic substance with ulcerative action that favours the development of atrophy and cell proliferation. However, it has been showed that urease itself displays biological effects independent of enzymatic activity, enhancing neutrophil degranulation, up-regulating IL-8 expression by activated neutrophils, and inducing T-cell migration, thus
contributing to the establishment of an inflammatory environment.44
Apart from these indirect mechanisms, more recent data have shown that H. pylori may also have a direct oncogenic effect exerted by the CagA protein that is directly delivered into epithelial cells through the bacterial T4SS. Approximately, 30–40% of H. pylori strains isolated in Western countries do not carry the cag PAI, and thus are CagA-negative, whereas almost all of the H. pylori isolates from East Asian countries contain the cag PAI and are thus CagA-positive.33, 34 This feature, together with individual genetic susceptibility and dietary factors, could explain the different rates of progression towards malignancy observed among Asians, Caucasians and Latin-America populations.45 From a pathophysiological point of view, phosphorylated CagA is able to interact with the pro-oncogenic tyrosine phosphatase 2 (SHP2) which in turn leads to the activation of the Ras-ERK mitogen-activated protein kinase (RAS-MAPK) pathway which provokes pro-mitogenic cellular response while
morphologically inducing the so-called “hummingbird” phenotype, characterized by an elongated cell shape with elevated cell motility and scattering.46 It has been shown that this phenotypic change corresponds to an epithelial-to-mesenchymal (EMT) transition, in which epithelial cells acquire the motile, migratory properties of mesenchymal cells, unveiling cancer stem cell (CSC)-like properties.47
Gastric cancer stem cells
H. pylori’s capability to induce EMT of gastric epithelial cells may represent a source of CSC
differentiation into various cell types.48 Despite being a very small fraction of the entire GC cellular population, CSCs can initiate and drive the tumour’s continuous growth.49 Although it may be possible for non–stem cells to contribute to gastric regeneration and cancer, chronic inflammation associated with H. pylori infection typically induces a regenerative and reparative response orchestrated by gastric resident stem cells;50 such cells, in a state of relative immaturity and greater vulnerability are thus exposed to carcinogens present in gastric lumen.51 Under physiological conditions, the main function of gastric stem cells is to maintain the epithelial integrity and replenish all the mature cell lineages, especially after injury.52 Through the accumulation of genetic alterations in genes encoding for key proteins of pathways that control normal cell development, those cells escape all checkpoints and acquire growth advantages over their counterparts.53, 54 Furthermore, resident stem cells are those cells that live long enough to serve as reservoirs of mutated stem cells being the ideal cellular targets for the accumulation of genetic alterations.55, 56
Several approaches have been developed to identify and/or isolate CSCs from GC both in vitro and in vivo by using cell surface markers.57-59 In particular, Villin and leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) are the first biomarkers reported to label gastric stem cells, being able to identify gastric progenitors with the characteristics of multilineage potential differentiation required to generate the different cell types of the whole gastric gland.60 Villin is an actin-binding-protein that plays a key role in the morphogenesis of microvillar actin filaments.61 Villin-marked stem cells are localized in the antral glands and are quiescent in the unstimulated stomach; however,
following pro-inflammatory insults, they undergo both symmetric and asymmetric divisions replacing multiple entire pyloric glands. In the mouse model, Li et al. observed that transformation of Villin-marked stem cells can initiate GC following selective deletion of Klf4, a tumor suppressor gene that encodes for zinc-finger transcription factor that mediates the pro-inflammatory responses.62
Lgr5-marked stem cells reside in the gland base and show immature morphology with a large nuclear-to-cytoplasmic ratio and limited organelles.61 These cells are able to drive the repopulation of gastric glands giving rise to highly proliferative progenitor cells located in the midglandular
compartment that rapidly divide and differentiate into epithelial lineages.63 It has been shown that inappropriate Wnt signalling pathway occurs in a subset of GC arising predominantly in the pylous.64 Interestingly, in mice model knockout for APC, an essential Wnt signalling gene, Lgr5 stem cells may give rise to highly proliferative, β-catenin-positive adenocarcinomas.63
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is involved in cell signalling, migration, proliferation and differentiation.64 Despite the fact that EpCAM is a
commonly surface marker used for CSCs identification in various types of cancers like colon, breast, liver and pancreas cancers,65 the clinical and prognostic significance of EpCAM in GC remains controversial. Interestingly, a recent meta-analysis including a total of 11 studies (1960 GC patients), revealed that there were significant differences in EpCAM overexpression and tumour size (OR=2.97, 95%CI 2.13-4.13, p<0.001), the nature of the tissue (OR=80.30, 95%CI 29.21-220.81, p<0.001), lymph node metastasis (OR=2.78, 95%CI 1.23-6.27, p=0.010) and the cumulative 5-year overall survival rate (OR=0.54, 95% CI 0.29-0.99, p=0.050),66 suggesting that EpCAM may be useful as a novel prognostic factor in GC patients.
Another surface marker identified in CSCs from several types of cancers is cluster of differentiation (CD)44 antigen,67 in particular the splicing variants with variable exons (CD44v).68 Early studies on human GC showed that overexpression of CD44 was significantly associated with metastasis and invasive phenotype, which may indicate poor prognosis.69, 70 Furthermore, recent reports highlighted that the isoforms CD44v8-10 may be reliable CSC marker in GC.71 In particular, Choi et
al. confirmed that CD44v8-10 is the major isoform of the CD44 variants in GC and, more importantly,
anti-CD44v9 antibody.72 This clinically-relevant finding may provide a new therapeutic strategy to increase the disease-free survival rates of GC patients.
More work still needs to be done to identify biomarkers useful to follow the evolution to cancer overtime, from healthy to inflamed, metaplastic and tumoral epithelium. At present, the molecular characterization of gastric stem cells and gastric CSCs still suffers from the lack of specific markers. Furthermore, there are no universal and specific markers applying to all GC cell lines and/or human primary GC.59
In the last decades, studies from mouse models of H. pylori-induced GC suggested that cancer may develop from bone marrow-derived cells (BMDC) which are frequently recruited to sites of tissue injury and inflammation, representing a potential source of malignancy (Figure 1).73 Among BMDCs, mesenchymal stem cells represent a heterogeneous population of cells that can give rise to adipocytes, chondrocytes, osteocytes and the marrow mesenchyme.74 The pioneer work of Houghton et al. showed that acute injury, acute inflammation, or transient parietal cell loss within the stomach do not lead to BMDC recruitment, while chronic infection of C57BL/6 mice with Helicobacter induces repopulation of the stomach with BMDCs, contributing over time to metaplasia, dysplasia, and cancer.75 More recently, Varon et al. investigated the bacteria's potential to induce premalignant lesions in mice and studied the kinetics of BMDC settlement in the gastric epithelium. By using a mouse model of C57Bl/6 mice irradiated and transplanted with bone marrow from transgenic mice expressing green fluorescent protein, the authors found that H. pylori infection leads to development of chronic inflammation, hyperplasia, metaplasia, and dysplasia, as well as the recruitment and accumulation of BMDCs in the gastric epithelial mucosa. Furthermore, nearly 25% of dysplastic lesions showed green fluorescent protein-positivity indicating the presence of cells originating from bone marrow.76 Likely, the
recruitment of mesenchymal BMDCs within a chronically infected gastric mucosa, leads to an incorrect differentiation of these cells which are unable to repair tissue injury and may convert into CSC.
Importantly, the role of microRNAs (miRNAs) have also been studied in the field of GC . MiRNAs which are small (~20-22 nucleotides) non-coding RNAs, are the biggest class of genetic regulators that act post-transcriptionally with a mechanism based on sequence complementarities between miRNA and target messenger RNA (mRNA).77 MiRNAs of exosomes harbored within cancer cells have been shown to induce inflammatory conditions that accelerate tumor growth and metastasis. Interestingly, the oncogenic role of bone marrow mesenchymal stem cells (BM-MSCs) in the
modulation of immunosuppression, tumor invasion, and metastasis was found to be partly mediated through the secretion of exosomes. Ma et al, in a recent study, have shown that high expression of miRNA-221 (miR-221) in exosomes originating from BM-MSCs was positively correlated with the poor clinical prognosis of GC.78 Notably, the use of a miR-221 inhibitor with an excellent restraining effect in exosomes provides therapeutic potential for GC in future clinical medicine.
The possibility to counteract carcinogenesis via gastric CSCs is intriguing. In addition to its anti-diabetic properties,79 metformin is known to inhibit the proliferation of tumour
cells in vitro and in vivo .80 Recently, Courtois et al. evaluated the anti-tumoural effect of metformin on gastric cancer in vitro and in vivo and investigated whether this molecule could target the gastric CSCs. Metformin effects were evaluated on the proliferation and tumorigenic properties of the gastric CSCs from patient-derived primary tumourxenografts (PDXs) and cancer cell lines (MKN45, AGS and MKN74) in vitro in conventional 2 dimensional (2D) and in 3 dimensional (3D) culture systems, in which only CSCs are able to form tumorspheres and in mouse xenograft models in vivo. Metformin induced a cell cycle arrest, which decreased cell proliferation in the 2D cultures. In a 3D culture system, metformin decreased the number of tumorspheres, revealing its capacity to target the CSCs. This effect was confirmed by analysis of the expression of CSC markers (CD44 and Sox2) and differentiation markers (Kruppel-like factor 4 and MUC5AC), which were decreased or increased in response to metformin, respectively. Finally, in vivo treatment of PDXs with metformin led to a tumour
growth delay and decreased the self-renewal ability of the CSCs. These results suggested that the use of metformin could represent an efficient strategy to inhibit tumour growth by targeting gastric CSCs.81
Conclusions
Chronic H. pylori infection is presently considered the principal cause of GC, promoting carcinogenesis via multiple mechanisms. Current available treatment options allow bacterial
eradication with high efficacy,82-85 representing to date the best strategy to prevent GC. Nevertheless, the origin of gastric CSCs is still under investigation; the identification and the study of surface markers of stemness may be useful to better understand the evolution of GC overtime and to provide the basis for the development of novel therapeutic strategies. 86-88
Conflicts of interest. The authors certify that there is no conflict of interest with any financial
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Table I. Main features of GC subtypes according to The Cancer Genome Atlas Research Network. GC subtype Salient features
MSI Hypermethilation
Gastric-CIMP
MLH1 silencing
Mitotic pathways
EBV PIKCA mutation
PD-L1/2 overexpression
EBV-CIMP
CDKN2A silencing
Immune cell signaling
CIN Intestinal histology
TP53 mutations RTK-RAS activation GS Diffuse histology CDH1, RHOA mutations CLDN18-ARHGAP fusion Cell adhesion
Abbreviations: CIMP, CpG island methylator phenotype; CIN, chromosomally unstable; EBV, Epstein–Barr virus–positive; GS, genomically stable; MSI, microsatellite-instable.
Table II. Principal proteins of H. pylori and related functions.
Protein Function
Survival in acid environment ureA/B
ureI/E/F/G/H
AmiE/F NixA
ABC-transporter
Urease subunits (apoenzyme) for generation of ammonia
Urease regulatory proteins (UreI determines urease survival in acidic conditions, UreE facilitates Ni2+ incorporation into the apoenzyme, UreF facilitates carbamylation of the Ni2+-bridging lysine residue and blocks premature Ni2+ binding to the active site; UreG provides energy during urease assembly and UreH stabilizes the apoenzyme)
Accessory enzymes for generation of ammonia High-affinity nickel transport protein
ABC-transport system involved in nickel uptake Motility and adhesion
FlaA/B HAP-2 BabA SabA AlpA/B OipA HopQ
Subunits of the flagella
Protein required for the assembly of flagellar filaments
Adhesin that mediates Leb binding at an initial stage of infection Adhesin that mediates sialyl-Lex and sialyl-Lea binding
Adhesins that mediate laminin binding and inflammatory response enhancement Mediates adhesion and induces inflammatory cytokine production
Mediates adhesion to host cell and translocation of CagA via the T4SS Virulence and pathogenicity factors
VacA CagA
T4SS complex HtrA
Induces vacuoles in host cells, increased epithelial permeability and apoptosis Promotes cell growth and dysplastic changes
Translocation of CagA into host cell cytoplasm
Serine protease that cleaves the cell adhesion protein and tumor suppressor E-cadherin of gastric epithelial cells
Abbreviations: ABC-transporter, ATP-binding protein cassette-transporter; AlpA/B, adherence-associated lipoprotein A/B; AmiE/F, aliphatic amidase E/F; BabA, blood-group-antigen-binding adhesion; CagA, cytotoxin-associated gene A; HAP-2, hook-associated protein 2; HopQ, outer membrane protein Q; HtrA, high temperature requirement A; NixA, nickel-binding protein A; OipA, outer inflammatory protein A; SabA, sialic acid binding adhesion; T4SS, type IV secretion system; VacA, vacuolating cytotoxin A.
Figure 1. Model of differentiation of bone marrow derived stem cells.
Abbreviations: BMDSCs, bone marrow derived stem cells; H. pylori, Helicobacter pylori; HSCs, hematopoietic stem cells; MSCs, mesenchymal stem cells.
