Helicobacter pylori virulence factor CagA promotes tumorigenesis of gastric cancer via multiple signaling pathways

Helicobacter pylori (H. pylori) is the most common human pathogen worldwide, infecting an estimated 50 % of the
global population 1]. During H. pylori infection, sustained inflammation and abnormal epithelial proliferation may be major
factors that lead to H. pylori-associated gastric diseases, such as gastritis, peptic ulcers, mucosa-associated
lymphoid tissue lymphoma and gastric cancer. Further studies have indicated that H. pylori is responsible for several extragastric diseases, such as iron deficiency anemia
and idiopathic thrombocytopenic purpura 1], 2]. Interesting associations have also been noted between H. pylori and other extragastric
diseases, such as cardiovascular, neurological, hepatobiliary, colonic and pancreatic
diseases 2]. However, gastric cancer is one of the most malignant types of tumor and represents
a major health problem worldwide. The association of H. pylori with gastric cancer has received a great deal of attention and has been thoroughly
studied. The World Health Organization and the International Agency for Research declared
H. pylori to be a Group I human carcinogen for gastric cancer in 1994, and a prospective cohort
study further implied that H. pylori is a necessary cause of gastric cancer 3]. H. pylori also affects the prognosis of gastric cancer. Fukase et al. performed a multi-center,
open-label, randomized controlled trial to clarify that eradication of H. pylori should be performed after endoscopic resection of early gastric cancer to prevent
the development of metachronous gastric cancer 4].

The majority of the H. pylori-infected population remains asymptomatic, and few individuals may develop gastric
cancer. As the clinical outcomes caused by H. pylori infection are considered to be associated with a complex combination of host susceptibility,
environmental factors and bacterial isolates 5]. The H. pylori genome shows genetic diversity among distinct isolates, and H. pylori pathogenicity is different in distinct isolates. Bacterial virulence factors exert
an important influence in determining the clinical outcomes, and clinically isolated
H. pylori strains are therefore classified according to bacterial virulence factors. The strongest
candidates include the cag pathogenicity island (cag PAI) and vacuolating cytotoxin
A (VacA). Clinically isolated H. pylori strains are often subdivided into two types according to the cag PAI-encoded cytotoxin-associated
gene A (CagA) protein. Infections involving H. pylori strains that possess a functional cag PAI confer a higher risk for gastric cancer
than those involving cag-negative strains 6]. The cag PAI is a 40 kb DNA fragment that encodes the CagA protein and functional
components of a type IV secretion system (T4SS). The CagA protein, which is injected
into gastric epithelial cells through the T4SS, behaves as a bacterial oncoprotein
7]. Ohnishi N et al. generated CagA transgenic mice that showed a significant increase
in the incidence of gastric cancer. These results provide first direct evidence of
the role of CagA as a bacterial oncoprotein that acts in mammals 8]. Meta-analyses further indicates that individuals infected with CagA-positive strains
of H. pylori show an increased risk for gastric cancer over the risk associated with H. pylori infection alone 9], 10].

The molecular mechanism underlying CagA-positive H. pylori-induced gastric cancer has been widely studied. Translocation of CagA into gastric
epithelial cells is the first step in the processes of CagA-induced tumorigenesis.
Several different Cag proteins are involved in the translocation of CagA. One of these
proteins, CagL, functions as a component of the T4SS that binds to and activates ?5?1
integrin receptors, triggering the delivery of the bacterial effector protein CagA
to the cytoplasm of host cells 11]. Additional Cag proteins (CagY, CagI, CagA) have also been shown to bind ?1 integrin
and permit translocation of the bacterial effector protein CagA 12], 13]. Another structural component, CagE, was reported to be essential for CagA translocation.
Furthermore, infection with H. pylori can induce phosphatidylserine (PS) externalization in epithelial cells, and CagA
then interacts with the externalized PS to initiate its entry into cells 14]. Collectively, these findings indicate that H. pylori exploits host cell surface molecules such as integrins and PS to deliver CagA to
the host cells. Additionally, CagA translocation requires energy-dependent host cell
processes distinct from endocytic pathways. Cytomembrane cholesterol and actin polymerization
are also necessary for CagA translocation 14].

Once the protein has entered these target cells, parts of CagA molecules are tyrosine-phosphorylated
by Src and Abl family kinases within several repeat Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs,
while other CagA molecules remain unphosphorylated 15]–17]. CagA then binds to various signaling proteins and causes dysregulation of multiple
signaling pathways in either a phosphorylation-dependent or phosphorylation-independent
manner 18]. Phosphorylated CagA causes epithelial cells elongation and scattering, a morphology
was originally referred to as the “hummingbird phenotype”, due to its effect on host
cell signaling pathways, such as the extracellular signal-regulated kinase (ERK)/mitogen-activated
protein kinase (MAPK) pathway, by interacting with SHP2, Grb2 and Crk/Crk-L 19], 20]. On the other hand, unphosphorylated CagA interacts with various signaling proteins,
such as Met, E-cadherin, Grb2 and Par1b, and then activates corresponding signaling
pathways, such as the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway,
nuclear factor-?B (NF-?B) signaling pathway, Wnt/?-catenin signaling pathway and Ras
signaling pathway, among others 21]–23]. These interactions and the activation of these signaling pathways contribute to
the epithelial proliferation and pro-inflammatory processes as well as the disruption
of cell-to-cell junctions, or loss of cell polarity.

This review focuses on recent findings regarding CagA, related to various kinds of
vital signaling proteins and several classic signaling pathways. In combination with
previous studies on corresponding proteins or signaling pathways, we hope to discuss
the possible molecular mechanism underlying the CagA-induced abnormal expression of
vital signaling proteins and the dysregulation of signaling pathways, and to further
recognize the relationship of CagA and the clinical outcomes of H. pylori infection, especially gastric cancer, which is the most severe outcome.

CagA mediates dysregulation of the Wnt/?-catenin signaling pathway

The Wnt/?-catenin signaling pathway is a key pathway regulating embryonic development
and adult tissue homeostasis. Aberrant Wnt/?-catenin signaling plays an essential
role in disease pathogenesis, especially in tumorigenesis and progression 24], 25]. The Wnt/?-catenin signaling pathway is highly conserved among species, and the core
of this pathway is the versatile protein ?-catenin, encoded by CTNNB1. The canonical
Wnt/?-catenin signaling pathway is activated by a secreted extracellular Wnt ligand
that binds to frizzled (Fzd) receptors and their co-receptors the low-density lipoprotein
receptor related protein (LRP) families. The activated Fzd receptor switches on the
intracellular signaling cascade. Then the phosphorylation of LRP activates the disheveled
protein (Dvl), which in turn inactivates a complex of proteins collectively termed
the “destruction complex”, including the core proteins glycogen synthase kinase-3?
(GSK-3?), Axin, adenomatosis polyposis coli (APC) and casein kinase-1 (CK-1). In the
absence of Wnt ligands or the presence of abundant competitive ligands, such as Wnt
inhibitory factor-1(WIF-1), secreted Frizzled related proteins (sFRPs), or Dickkopf
family proteins (DKKs), the destruction complex phosphorylates the N-terminus of cytosolic
?-catenin, and targets it for proteasomal degradation, thereby maintaining ?-catenin
at low baseline cytosolic levels. However, in the presence of Wnt ligands, cytosolic
?-catenin can not be phosphorylated due to inactivation of the destruction complex.
As a result, ?-catenin is stabilized and accumulates in the cytoplasm and subsequently
translocates to the nucleus. In the nucleus, ?-catenin interacts with the T cell factor/lymphoid
enhancer factor (TCF/LEF) family of transcription factors to induce target gene transcription
26], 27].

Dysregulation of the Wnt/?-catenin signaling pathway is widely implicated in gastrointestinal
cancers, including colorectal cancer and gastric cancer 27], 28]. Mutation of pathways components (eg. Axin and ?-catenin), inhibition of antagonists
(eg. sFRPs) or crosstalk with other signaling pathways leads to continuous dysregulation
of the Wnt/?-catenin signaling pathway. Infection by Pathogenic microorganisms, such
as H. pylori, Hepatitis virus, is another important factor influencing the Wnt/?-catenin signaling
pathway 29]–33]. Franco et al. reported that CagA-positive H. pylori altered ?-catenin localization and increased ?-catenin nuclear accumulation in gastric
epithelial cells AGS. Along with ?-catenin nuclear translocation, the Wnt/?-catenin
signaling pathway is activated. These authors also observed this phenomenon in gerbil
gastric mucosae and human gastric mucosae colonized by CagA-positive H. pylori29]. Another study further confirmed that the nuclear translocation of ?-catenin and
subsequent activation of the Wnt/?-catenin signaling by CagA required the EPIYA repeat
region and was independent of CagA tyrosine phosphorylation 30]. Neal et al. generated transgenic zebrafish expressing CagA and found the Wnt/?-catenin
target genes significant upregulation in the transgenic zebrafish. The functional
consequences of the CagA-activated Wnt/?-catenin signaling pathway included increased
intestinal proliferation. These results provided in vivo evidence of CagA-induced
Wnt/?-catenin signaling pathway activation 31].

Under physiological conditions, ?-catenin interacts with the cytoplasmic tail of E-cadherin
to form adherens junctions between epithelial cells. However, upon infection with
CagA-positive H. pylori strains, CagA will become competitive in combination with E-cadherin and disrupt
complex formation between E-cadherin and ?-catenin, causing cytoplasmic and nuclear
accumulation of ?-catenin. In addition, the interaction between CagA and E-cadherin
is independent of CagA tyrosine phosphorylation, but the specific binding sites involved
are not clear (Fig. 1a) 22]. Therefore, they may be directly bound or indirectly bound via another component
of the adherens junction complex. Although the details of the interaction between
CagA and E-cadherin remain to be elucidated, this research provided the molecular
mechanism of CagA deregulation of ?-catenin and subsequent activation of the canonical
Wnt/?-catenin signaling pathway. AGS gastric epithelial cells are commonly used in
the study of H. pylori because these cells are highly susceptive to H. pylori infection. Interestingly, AGS do not form E-cadherin/?-catenin complexes due to a
lack of E-cadherin expression, and CagA is therefore not able to release of ?-catenin
from the E-cadherin/?-catenin compelx. Nevertheless, CagA still increases the cytoplasmic
and nuclear accumulation of ?-catenin in AGS cells and activates the Wnt/?-catenin
signaling pathway 21], 29]. Thus, there may be other mechanisms through which CagA regulates ?-catenin and the
Wnt/?-catenin signaling pathway.

Fig. 1. CagA mediates dysregulation of the Wnt/?-catenin signaling pathway. a. CagA competitively
combines with E-cadherin and disrupts the E-cadherin/?-catenin complex formation,
causing cytoplasmic and nuclear accumulation of ?-catenin. b. H. pylori induces rapid phosphorylation and activation of LRP6. c. CagA induces GSK-3? inactivation
via the PI3K/Akt signaling pathway. d. CagA binds GSK-3? directly and depletes GSK-3?
activity, inhibiting the phosphorylation and proteasomal degradation of cytosolic
?-catenin

Wnt ligands/receptors or the components of the destruction complex may be involved
in the CagA-positive H. pylori-induced activation of the Wnt/?-catenin signaling pathway. It has been reported H. pylori induces rapid activation of the Wnt/?-catenin signaling pathway co-receptor LRP6,
which is dependent on proteins of Dvl family (Fig. 1b) 34]. Although this process was found to be independent of CagA, H. pylori lacking a functional T4SS failed to induce LRP6 phosphorylation and activation. It
is still unclear how H. pylori activates LRP6 and whether there is a direct relationship between H. pylori and Dvl. Sokolova et al. reported that H. pylori suppressed GSK-3? activity to promote ?-catenin activity in a CagA-independent manner
35]. Subsequently, Nakayama et al. reported that H. pylori VacA induced the phosphorylation and inactivation of GSK-3? through the PI3K/Akt
signaling pathway, and subsequent nuclear translocation of ?-catenin regulated transcriptional
activity 36]. Tabassam et al. came to a similar conclusion, that H. pylori induced GSK-3? inactivation via the PI3K/Akt signaling pathway, but their results
showed that CagA was responsible for GSK-3? inactivation and downstream ?-catenin
activation (Fig. 1c) 37]. These opposing results may be due to the differences in the cell lines and H. pylori strains used in the research. Sokolova et al. mainly employed MDCK cells and an H. pylori strain P1 expressing CagA. They confirmed that CagA could be translocated and phosphorylated
at tyrosine residues in both AGS and MDCK cells, but they did not verify the following
results in AGS cells. Similarly, Nakayama et al. only used the AZ-521 cell line in
their research, and this cell line has been used relatively less frequently in research
on H. pylori. Recently, Korean researchers explained H. pylori CagA-induced GSK-3? inactivation from another perspective. They indicated that CagA
could directly bind GSK-3? and deplete GSK-3? activity. Furthermore, they found that
the C-terminal region of CagA harbored an EPIYA motif and a multimerization domain
that played an important role in the binding and depletion of GSK-3? (Fig. 1d) 38]. This result is consistent with previous findings showing that the EPIYA repeat region
is responsible for ?-catenin activation 30]. Taken together, these results show that CagA mediates dysregulation of the Wnt/?-catenin
signaling pathway via effecting the release and degradation of ?-catenin.

CagA activates PI3K/Akt and the downstream signaling pathways

PI3K, which is involved in tumorigenesis, is a heterodimer consisting of a p85 regulatory
subunit and a p110 catalytic subunit. The main function of PI3K is to phosphorylate
phosphatidylinositol in the cytomembrane and convert it into phosphatidylinositol
3, 4, 5-triphosphate (PIP3). PIP3 interacts with the pleckstrin homology (PH) domain
of a ser/thr kinase Akt and allows Akt phosphorylation at Ser473 or Thr308. This phosphorylation
activates Akt, which in turn, activates or inactivates downstream tyrosine kinase-
and G-protein-coupled receptors in turn. GSK-3?, NF-?B and mammalian target of rapamycin
(mTOR) are key representative of PI3K/Akt downstream target proteins 37], 39]–44], and the MEK/ERK signaling pathway is a representative of PI3K/Akt downstream signaling
pathway 45].

The PI3K/Akt signaling pathway is hyperactive in some cancers, including gastric cancer
46], 47]. H. pylori infection is a major factor in the activation of PI3K/Akt and downstream signaling
pathways 21], 37], 44]. The activation of PI3K/Akt signaling pathways is a response to growth factors (eg.
epidermal growth factor, hepatocyte growth factor). As the specific receptors of epidermal
growth factor and hepatocyte growth factor, the epidermal growth factor receptor (EGFR)
and c-met, respectively, mediate the activation of the PI3K/Akt signaling pathway.
H. pylori infection induces the phosphorylation of EGFR Tyr 992 and the transactivation of
EGFR, which is dependent on CagA and another virulence factor, OipA. As a result,
the PI3K p85 subunit is activated, and two specific phosphorylation sites (Thr 308
and Ser 473 of Akt) are phosphorylated. Importantly, the CagA mutation reduces the
activation of Akt Thr 308, whereas the oipA mutation reduces the levels of Akt Ser
473 in response to H. pylori infection. Double CagA/OipA mutation results in complete inhibition of the phosphorylation
of Akt Ser 473, confirming that CagA mainly affects the phosphorylation of Akt Ser
473 37]. Interestingly, EGFR is activated by H. pylori during early infection, but H. pylori CagA inactivates EGFR during prolonged infection via reducing the phosphorylation
of EGFR tyrosine residues 48]. This research revealed that CagA-positive H. pylori regulates EGFR activation and inactivation to support persistent infection. But it
is regrettable that the EGFR/PI3K/Akt signaling pathway is not mentioned in this paper.
Thus, the role of EGFR in H. pylori-induced signaling pathways is not completely clear and needs to be investigated more
thoroughly in the future. However, it has been confirmed that c-met is activated in
H. pylori-infected conditions, and CagA is able to interact with activated c-met 21], 49] Moreover, the C-terminal region of CagA, which is designated CRPIA (conserved repeat
responsible for phosphorylation-independent activity), is involved in the interaction
with c-met and the activation of the downstream PI3K/Akt signaling pathway. As a results
of CagA-induced PI3K/Akt signaling pathway activation, GSK3?, which is a downstream
target of PI3K/Akt, is inactivated, and ?-catenin transcription is subsequently activated
21], 37]. In other words, there is a crosstalk between the CagA-induced Wnt/?-catenin signaling
pathway and the PI3K/Akt signaling pathway.

Recently, Zhang et al. found CagA EPIYA repeat region was also involved in the activation
of PI3K/Akt signaling pathway 50]. The EPIYA repeat region of CagA is classified into 4 tyrosine phosphorylation motifs
(TPMs), A-, B-, C- or D-TPMs. Western CagA has A-, B-, and C-TPMs, while East Asian
CagA possesses A-, B-, and D-TPMs 51], 52]. Zhang et al. demonstrated CagA activated PI3K/Akt signaling pathway by interaction
with PI3K via a functional B-TPM. Through database searching and silico analysis,
they revealed a strong non-random distribution of the B-TPM polymorphisms (A/T polymorphism,
including EPIYA and EPIYT) in Western H. pylori isolates. And Matsunari et al. previously also reported that B-TPM polymorphisms
(EPIYT, EPIYA, ESIYA and so on) and that EPIYT of B-TPM was more predominant in Western
H. pylori isolates and EPIYA of B-TPM was more predominant in East Asian H. pylori isolates 53]. Interestingly, during co-culture with AGS cells, an H. pylori strain with EPIYT B-TPM had higher affinity to PI3K and significantly enhanced induction
of PI3K/Akt signaling pathway, compared to the isogenic strain with EPIYA B-TPM 50]. These results suggest that the A/T polymorphisms in B-TMP could regulate PI3K/Akt
signaling pathway activity through influencing the interaction between CagA and PI3K.
In addition, H. pylori strain with EPIYT B-TPM induced less hummingbird cells and IL-8 levels than the isogenic
strain with EPIYA B-TPM 50]. In this regard, the differential EPIYT and EPIYA functions might be part of the
reason that the incidence of gastric carcinoma is much higher in East Asian countries
than in Western countries. However, it is regrettable that there is no direct result
confirms the relationship between PI3K/Akt signaling and hummingbird phenotype and
IL-8 levels. Indeed, it is difficult to use a simple linear relationship to clarify
the regulation of PI3K/Akt signaling on hummingbird phenotype and IL-8 levels, because
(i) PI3K/Akt downstream target proteins or signaling pathways are pleiotropic and
have complex crosstalk with other signaling pathways, (ii) H. pylori could induce AGS hummingbird phenotype and IL-8 production through multiple CagA-mediated
mechanisms.

NF-?B, which is a downstream target of PI3K/Akt, is a crucial regulator of many cellular
processes, including inflammation, immune responses and tumorigenesis. NF-?B is a
p65/p50 heterodimer that forms a complex with cytoplasmic inhibitors (eg. I?B) in
resting condition. PI3K/Akt can activate I?B kinase (IKK), which phosphorylates I?B
and subsequently releases NF-?B from the complex. Then NF-?B translocates to the nucleus,
and phosphorylation of the p65 subunit plays a key role in the NF-?B-mediated transcriptional
response. It has been accepted that H. pylori cag PAI is required for NF-?B activation 54], 55], but the role of CagA in the regulation of NF-?B activity is still a subject of intense
discussion. Early studies found that H. pylori induced NF-?B activation in time-, multiplicity of infection-, and cag PAI-dependent
manners 54]. Recently, Sokolova further confirmed that H. pylori cag PAI encoding T4SS was required for activation of NF-?B and release of downstream
IL-8, but CagA had only a partial or minor role in NF-?B activation and IL-8 release
at early infection 55]. In contrast, Brandt et al. found that CagA could activate NF-?B and induce downstream
IL-8 release in a time-dependent manner 56]. In this study, CagA activates NF-?B and induces IL-8 induction via the MEK/ERK signaling
pathway. Subsequently, Kim et al. constructed CagA and its fragments eukaryotic expression
vectors, and further confirmed that NF-?B activation and IL-8 release induced by CagA
occurred via the MEK/ERK signaling pathway activation 57]. Recently, Kang et al. compared different CagA-positive and -negative H. pylori strains for their ability to activation of NF-?B in different gastric cancer cells.
They confirmed that CagA was required for H. pylori-induced activation of NF-?B and that CagA selectively induced Phospholipase D1 expression
via NF-?B 58]. The different H. pylori strains used by different laboratories may be responsible for different components
of H. pylori that drive NF-?B signaling pathway. However, the time point of H. pylori infection may be the main factor determing which component drive NF-?B signaling
pathway. Most of the previous studies compared different CagA-positive and -negative
H. pylori strains for their ability to activation of NF-?B at early time points of infection
(less than 3 h). They found H. pylori induced NF-?B activation and IL-8 release in a CagA-independent manner 54], 55]. Interestingly, during persistent infection (more than 12 h), NF-?B activation and
IL-8 release further increased in a CagA-dependent manner 56], 58]. It is may be meaningful to explore how T4SS initiate NF-?B signaling pathway and
how CagA is involved in NF-?B activation during persistent infection. Transforming
growth factor-?-activated kinase 1 (TAK1) is a key regulator of signal transduction
cascades that lead to the activation of IKK and NF-?B. CagA physically interacts with
TAK1 and enhances the activity of TAK1, which is required for NF-?B activation by
CagA 59]. However, a later study has shown that this is a misinterpretation because the antibody
against TAK1 immunoprecipitated to some extent the CagA protein by unknown reasons,
while in a reverse immunoprecipitation using a CagA antibody they also could not recognise
co-immunoprecipitated TAK1 60]. Although they indicate TAK1 is not a target of the H. pylori CagA, TAK1 is required for H. pylori-induced NF-?B activation in a T4SS-dependent and CagA-independent manner during early
infection. Actually, H. pylori-induced interaction between TAK1 and IKK complex may be involved in NF-?B initial
activation in a T4SS-dependent manner during early infection, while CagA may be play
a role in extend the activation of NF-?B during persistent infection because CagA
takes more time to be delivered into the host cells and regulates its target proteins
or signaling pathways. As mentioned in the previous studies, CagA need more time to
enhance NF-?B activity and increase IL-8 release during persistent infection 56], 58]. Therefore, further studies are necessary to elucidate the mechanisms of H. pylori-induced interaction between TAK1 and IKK complex during early infection and the exact
function of CagA in the regulation of NF-?B activity during persistent infection.

H. pylori CagA has the ability to activate of the PI3K/Akt signaling pathway, but the regulation
of the PI3K/Akt downstream signaling pathways by H. pylori is still not completely clear. As an upstream regulator of NF-?B, PI3K/Akt is not
mentioned in H. pylori-mediated activation of NF-?B. Similarly, it has been reported that H. pylori activates the PI3K/Akt/mTOR signaling pathway in a CagA-independent manner 44]. As we mentioned above, CagA and OipA can phosphorylate Akt and activate the PI3K/Akt
signaling pathway, but only OipA is required for H. pylori-induced inactivation of the Forkhead transcription factors of class O (FoxO) family
members FoxO1 and FoxO3a, which are the downstream of PI3K/Akt 61]. These findings indicate that H. pylori can activate PI3K/Akt and downstream signaling pathways via different molecules and
mechanisms, maintaining the dysregulation of these signaling pathways and forming
a complex network.

The role of CagA in other oncogenic signaling pathways

Similar to the Wnt/?-catenin signaling pathway, the Hedgehog (Hh) signaling pathway
plays a critical role in embryonic development, adult tissue homeostasis and tumorigenesis
62]. H. pylori infection induces upregulation of sonic hedgehog (Shh), which activates the Hh signaling
pathway in gastric cancer cells. Moreover, Shh overexpression is CagA-dependent and
is mediated through the NF-?B signaling pathway 63]. Shh is mainly expressed in parietal cells, influencing fundic gland differentiation
and function. Recently, Schumacher et al. used a mouse model that expresses Shh fused
to green fluorescent protein, in place of wildtype Shh to visualize Shh ligand expression
in response to H. pylori infection in vivo. They found that H. pylori induced Shh overexpression in parietal cells that was consistent with the expression
pattern observed in the native tissue. Furthermore, they confirmed that NF-?B signaling
mediated H. pylori-induced Shh overexpression and that CagA is involved in this process 64]. In addition, in the early stages of H pylori infection, H. pylori-induced Shh overexpression from parietal cells acts as a macrophage chemoattractant
to drive the innate immune response during the initiation of gastritis 65].

Since it was first discovered, the c-Jun NH2-terminal kinase (JNK) signaling pathway
has been demonstrated to exhibit both tumor suppressor and pro-tumorigenic functions
in different cell types and organs 66], 67]. Infection with H. pylori has been shown to activate the JNK signaling pathway, and CagA is an important mediator
of the activation of this signaling pathway during infection 68], 69]. Wandler et al. used transgenic Drosophila to express CagA, and they found that CagA
triggered JNK signaling pathway activation, which caused apoptosis in epithelial cells.
Interestingly, when these authors employed a Ras oncogene-overexpression Drosophila
metastasis model, they found that coexpression of CagA could enhance the growth and
invasive potential of tumor cells through activation of the JNK signaling pathway
69]. This finding indicates that, in addition to the presence of virulence factors (eg.
CagA), host genetics must also play an important role in determining the outcome of
H. pylori infection. Indeed, H. pylori infection can persist for many years before the occurrence of gastric cancer. Therefore,
JNK-mediated apoptosis may be an effective mechanism for limiting pathogenic effects
and protecting the gastric epithelium in early infection. Under persistent H. pylori infection and the influence of other factors, accumulation of genetic mutations is
observed. Following the acquisition of an oncogenic mutation, CagA-mediated JNK signaling
pathway activation promotes tumor progression. It will be particularly valuable to
further confirm the role of CagA-mediated JNK signaling pathway activation in clinical
tissue samples, but there has been no report of such studies to date.

Dysregulation of the Janus kinase (JAK)/signal transducers and activators of transcription
3 (STAT3) signaling pathway is observed in many cancers including gastric cancers,
and it correlates with both tumor progression and a poor prognosis. Jackson and colleagues
found that STAT3 activation was more pronounced in CagA-positive H. pylori-infected gastric tissue 70]. In this study, as the majority of bacterial pathogens mediate STAT3 activation via
autocrine IL-6, it was found that IL-6 expression was increased after H. pylori infection, and both IL-6 and IL-11 were strongly up-regulated in gastric cancer tissue.
In epithelial cells infected with H. pylori, STAT3 tyrosine phosphorylation, nuclear translocation and transcriptional activity
are dependent on unphosphorylated CagA. Although H. pylori CagA-mediated STAT3 activation requires IL-6 and the gp130 receptor, autocrine activation
of STAT3 by IL-6 and IL-11 is not involved in H. pylori CagA-mediated STAT3 activation 71]. Further studies may be required to elucidate the exact mechanism underlying the
interaction between CagA and these relevant receptors. Moreover, H. pylori-mediated STAT3 activation shows the ability to manipulate host immunity and facilitate
immune evasion 72], 73]. Recent evidence indicates that CagA increases the expression of Gram-positive specific
bactericidal lectin, regenerating islet-derived (REG) 3?, in gastric epithelial cells
via activation of the STAT3 signaling pathway 72]. While the functional basis of this response is not entirely clear, these findings
indicate that CagA-medicated REG3? overexpression may abolish the fitness of co-habiting
Gram-positive bacteria and reduce the competition for resources between H. pylori and Gram-positive bacteria in the gastric mucosal niche. Finally, H. pylori may rebuild the gastric microbiome and manipulate host immunity to favor its own
survival. The ability to evade the host immune response is another crucial factor
in the survival of H. pylori in the host gastric mucosae. As DCs are key modulators of the host adaptive immune
response, they are ideal targets for the pathogen’s immunity-manipulating efforts.
Several studies indicate that H. pylori infection promotes the development of tolerogenic dendritic cells (DCs) in a coculture
system and in murine models 74], 75]. Recently, Rizzuti and colleagues found that H. pylori activated the STAT3 signaling pathway in bone marrow-derived DCs (BMDCs). Then BMDCs
secrete IL-10, which activates STAT3 in DCs, thereby blunting DC maturation, inducing
the tolerogenic DCs 73]. This study describes another novel mechanism of H. pylori facilitation of immune evasion to maintain its persistence.

The regulation of signaling pathways may be influenced by the CagA tyrosine phosphorylation
status. It has been reported that unphosphorylated CagA showed preferentially activates
of JAK/STAT3, whereas phosphorylated CagA enhances SHP2 binding activity and ERK/MAPK
signaling pathway activation 76]. These findings indicate that the CagA tyrosine phosphorylation status affects the
signal switch, providing a novel mechanism explaining H. pylori-mediated signaling pathways. Unphosphorylated and phosphorylated CagA often exist
at the same time therefore, STAT3 and ERK/MAPK signaling activation must be abnormal
during H. pylori infection. In support of this hypothesis, a research group observed significantly
increased STAT3 and ERK/MAPK signaling activation in H. pylori-infected gastric tissue, which was further enhanced in the presence of CagA-positive
H. pylori strains 70]. Recent evidence indicates that IL-22 promotes gastric cancer development via activation
of the STAT3 and ERK signaling pathways 77]. In this study, while gastric cancer cells were co-cultured with IL-22-expressing
cancer-associated fibroblasts (CAFs) from human gastric cancer tissues, the invasive
ability of the gastric cancer cells was significantly enhanced through activation
of the STAT3 and ERK signaling pathways. In fact, H. pylori infection also stimulates peripheral mononuclear cells and CD4-positive T cells to
secrete IL-22, and IL-22 subsequently induces the expression of antimicrobial proteins
(eg. RegIII? and lipocalin-2) in gastric epithelial cells 78]. In this regard, IL-22 may play a protective role in gastric mucosae infected by
H. pylori, but more studies suggest that IL-22 may lead to pathological inflammation and thereby
promote tumorigenesis and progression via STAT3 activation 77]–80]. Th1 and Th17 are major T cell subsets that produce IL-22, and H. pylori is ability to induce Th1 and Th17 responses 81], 82]. Therefore, it is necessary to confirm that (i) the role of H. pylori-mediated upregulation of IL-22 in gastric cancer cells, (ii) the activation of STAT3
dependent on H. pylori-mediated IL-22 and/or H. pylori CagA, (iii) the role of CagA in H. pylori-mediated upregulation of IL-22.

In addition, H. pylori-mediated signaling (including p21-activated kinase1 and ERK1/2)
to actin-binding protein cortactin could regulate cell scattering and elongation in
a T4SS dependent manner 83], 84]. Injected CagA further involves in the interaction of cortactin with downstream focal
adhesion kinase (FAK), and this interaction increases FAK activity, which is important
for cell scattering and elongation phenotype 83]. Cortactin plays a central role in host signaling and involves in a variety of cellular
processes, including tumorigenesis, invasion and metastasis. Thus, further studies
may be required to elucidate the role of H. pylori-hijacked cortactin in tumorigenesis in vivo. Furthermore, CagA-positive H. pylori-induced activation of the Src/MEK/ERK signaling pathway is involved in the upregulation
of ?-enolase and ornithine decarboxylase, implying the progression of gastric diseases
85], 86]. What’s more, H. pylori is involved in protein kinase C (PKC) signaling pathway activation through PI3K,
phospholipase C? and Ca
²⁺
. As a result, PKC contributes to c-Fos upregulation and activator protein-1 activation,
leading to overexpression of matrix metalloproteinase-1 87]. Due to the large number of the related studies, it is difficult to list all of H. pylori-mediated oncogenic signaling pathways. In other words, there are several known mechanisms
underlying H. pylori-mediated gastric cancers.

The role of CagA in tumor suppressor pathways

Tumorigenesis is considered to occur as a multifactorial events. The activation of
oncogenes and the inactivation of tumor suppressor genes are two key events in most
cancers. Aberrant activation of oncogenes can be counteracted by tumor suppressor
genes. It is more significant to enrich and explain the tumorigenic mechanism of H. pylori. In addition to activating several oncogenic signaling pathways, H. pylori also plays a key role in the inactivation of tumor suppressor pathways. p53 is a
key tumor suppressor, and inactivation of p53 is a critical step in tumorigenesis
and progression. Previous reports showed that H. pylori infection increased p53 levels in the gastric mucosae 88]–90]. Subsequently, Wei et al. observed that p53 levels were dynamically altered in H. pylori-infected mongolian gerbil gastric tissues and cell lines. Consistent with previous
reports, these authors found that p53 was increased following H. pylori infection for 4–6 h in mongolian gerbils, however, p53 decreased rapidly following
the initial increase. Interestingly, p53 was shown to be increased again under continuous
H. pylori infection for 12 weeks 91]. In this study, the authors confirmed that CagA-positive H. pylori phosphorylated Human Double Minute 2 (HDM2, a main E3 ubiquitin ligase), which induced
p53 degradation. In addition, H. pylori-induced phosphorylation and activation of HDM2 could be mediated by Akt or ERK activation
(Fig. 2a) 91], 92]. Cellular stresses, such as H. pylori-induced activation of oncogenic signaling pathways, may be responsible for the initial
increase of p53. The second increase of p53 may be driven by DNA damage known to be
associated with inflammatory processes. In another report, Wei et al. indicated that
H. pylori could induce upregulation of truncated p53 isoforms that inhibit p53 function and
increase the transcriptional activity of NF-?B in gastric epithelial cells (Fig. 2b) 93]. Taken together, these findings indicate that DNA damage-mediate upregulation of
p53 may be shifted to inhibitory p53 isoforms by CagA-positive H. pylori. Inhibition of p53 may allow H. pylori to alter the cellular homeostasis, without apoptosis or triggering cell cycle arrest.

Fig. 2. The role of CagA in p53 regulation. a. CagA phosphorylation and activation of HDM2
is mediated by Akt or ERK activation. b. CagA plays a crucial role in p53 shifting
to inhibitory p53 isoforms. c. CagA-induced hypermethylation of the p14ARF promoter
results in a decrease in p14ARF protein levels that is not sufficient to sequester
HDM2 in the nucleus. d. CagA interacts with ASPP2 to recruit and bind p53, which is
then degraded by the proteasome. e. CagA induces aberrant expression of AID via NF-?B
and thereby elicits a high mutation frequency in p53

The p14ARF is a tumor suppressor that inhibits the proteasomal degradation of p53
by sequestering HDM2 and inhibiting its E3 ligase activity 94]. H. pylori CagA-induced hypermethylation of the p14ARF promoter results in a decrease of p14ARF
protein levels that is not sufficient to inhibit HDM2 and ARF-BP1 (another E3 ubiquitin
ligase) activity, and then HDM2 and ARF-BP1 then facilitates the degradation of p53
(Fig. 2c) 95]. This study provides a novel mechanism in which the CagA-mediated degradation of
p53 is controlled by two E3 ubiquitin ligases that are activated due to p14ARF promoter
hypermethylation and downregulation of p14ARF protein levels. Apoptosis-stimulating
protein of p53 (ASPP2) is best known for its role as a p53-binding protein and has
also shown to act as a tumor suppressor. Following H. pylori infection, CagA shows the ability to interact with ASPP2 to form a complex. After
this interaction, ASPP2 recruits and binds p53, which is then degraded by the proteasome.
Although the formation of a ternary complex between CagA, ASPP2, and p53 has not been
detected, it has been confirmed that the degradation of p53 is a consequence of the
recruitment and misregulation of ASPP2 by CagA. As a result, CagA-mediated degradation
of p53 leads to resistance to apoptosis (Fig. 2d) 96]. Recently, CagA- and ASPP2-interacting domains have been identified. The obtained
co-crystal structure revealed that N-terminal subdomain of CagA forms a highly specialized
three-helix bundle and that ASPP2 forms an extended helix in this groove of CagA.
Consistent with previous reports, this study provides evidence that the direct interaction
of CagA and ASPP2 also has an antiapoptotic effect during H. pylori infection 97].

P53 is inactivated by mutations in 40 %-50 % of gastric cancers. Individuals infected
with CagA-positive H. pylori show a higher likelihood of harboring p53 mutations 98], 99]. Activation-induced cytidine deaminase (AID) is a DNA and RNA mutator enzyme. In
vitro studies have revealed that CagA-positive H. pylori induces aberrant expression of AID in gastric epithelial cells via NF-?B activation
and thereby elicits a high mutation frequency in p53 (Fig. 2e) 100]. In addition, AID expression is elevated in H. pylori-positive human gastric mucosae and is reduced following H. pylori eradication 101]. A recent study in which whole-exome sequencing was performed indicated that p53
mutations accumulate in patients with H. pylori infection. In this study, the authors also used AID-transgenic mice to confirm that
AID expression plays a critical role in the accumulation of p53 mutations 102]. These studies provide a mutation-dependent mechanism of H. pylori-mediated p53 inactivation.

Similar to p53, (i) Runt-related transcription factor 3 (RUNX3) is also a tumor suppressor,
(ii) infection with CagA-positive H. pylori is associated with inactivation of RUNX3 in premalignant gastric lesions 103], (iii) CagA inhibits the expression of RUNX3 via the ERK/MAPK signaling pathway 104], (iv) CagA may increase the risk of RUNX3 promoter methylation 105], (v) CagA targets RUNX3 for ubiquitination and proteasome-mediated degradation 106]. In addition, the methylation of tumor suppressor genes is widespread in H. pylori-infected models. Cheng et al. used integrative genome-wide scans to identify genes
that were concomitantly hypermethylated in mouse and human gastric cancer samples
infected with H. pylori. They observed that the promoter hypermethylation of the Foxd3 tumor suppressor initiated
by H. pylori infection affected the prognosis of gastric cancer patients 107]. H. pylori infection causes gastric mucosal inflammatory responses, resulting in upregulation
of IL-1? and overproduction of nitric oxide (NO). IL-1? and NO play an important role
in H. pylori-induced methylation 108], 109]. Here, H. pylori-induced promoter methylation is observed not only in tumor suppressor genes but also
in microRNAs (miRNAs). Silencing of these miRNAs promotes tumorigenesis through activation
of their target oncogenes 110]. In general, CagA can decrease the levels of tumor suppressor proteins or inhibit
their activity to inactivate of tumor suppressor pathways in a variety of ways.