toxins

Review
Tyrosine Kinases in Helicobacter pylori Infections and
Gastric Cancer
Bianca E. Chichirau 1 , Sebastian Diechler 1 , Gernot Posselt 1 and Silja Wessler 2, *
1 Department of Biosciences, Paris-Lodron University of Salzburg, 5020 Salzburg, Austria;
[email protected] (B.E.C.); [email protected] (S.D.); [email protected] (G.P.)
2 Cancer Cluster Salzburg, Department of Biosciences, Paris-Lodron University of Salzburg,
5020 Salzburg, Austria
* Correspondence: [email protected]; Tel.: +43-662-8044-7210




Received: 30 August 2019; Accepted: 9 October 2019; Published: 11 October 2019


Abstract: Helicobacter pylori (H. pylori) has been identified as a leading cause of gastric cancer, which
is one of the most frequent and malignant types of tumor. It is characterized by its rapid progression,
distant metastases, and resistance to conventional chemotherapy. A number of receptor tyrosine
kinases and non-receptor tyrosine kinases have been implicated in H. pylori-mediated pathogenesis
and tumorigenesis. In this review, recent findings of deregulated EGFR, c-Met, JAK, FAK, Src, and
c-Abl and their functions in H. pylori pathogenesis are summarized.

Keywords: Helicobacter pylori; tyrosine kinases; c-Abl; CagA; Src family kinases; EGFR; c-Met

Key Contribution: In this review we provide a summary of the recent advances in the field of
Helicobacter pylori regulated tyrosine kinase signaling.

1. Introduction: Tyrosine Kinases in Health and Disease

The human ‘kinome’ consists of 535 protein kinases and represents the largest protein family
in humans [1]. Approximately 90 of these protein kinases belong to the family of tyrosine kinases,
which can be sub-classified into receptor tyrosine kinases (RTK) and non-receptor tyrosine kinases
(nRTK). In normal tissues, tyrosine kinases control a wide range of cellular functions including
differentiation, proliferation, survival, apoptosis, motility, and cell adhesion. However, deregulated
tyrosine kinase activities in response to epigenetic modifications, gene amplification or mutations
have been implicated in the development of a number of human diseases, in particular inflammation,
diabetes, and cancer [1,2].
Although the incidence in Europe has declined in the last 30 years, gastric cancer is still a leading
cause of cancer-related deaths [3]. Endoscopic resection represents a curative strategy for early gastric
neoplastic lesions, but is associated with unfavorable prognosis at advanced stages of gastric cancer [4].
The upregulation of several RTKs and nRTKs in gastric cancer has been reported, suggesting that
pharmacological targeting of tyrosine kinases might be beneficial and might increase the survival
time of patients [5]. High-throughput analyses have revealed that epidermal growth factor receptor
(EGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR),
the hepatocyte growth factor (HGF) receptor c-Met, and vascular endothelial growth factor receptor
(VEGFR) are expressed at high levels in gastric cancer compared to normal tissues [6], suggesting that
RTKs play a primary role in carcinogenesis. Additionally, epigenetic changes in the egfr locus have
been observed in gastric cancer and it has been suggested that egfr promoter hypermethylation could
serve as a potential biomarker for gastric cancer status and progression [7]. Similar observations have
been made for a number of nRTKs. Elevated mRNA and protein expression levels of Src and Lyn have

Toxins 2019, 11, 591; doi:10.3390/toxins11100591 www.mdpi.com/journal/toxins

Toxins 2019, 11, 591 2 of 15

been detected in gastric tumors that may have a role in invasiveness and metastasis [8]. Accordingly,
targeted therapy against deregulated tyrosine kinases in gastric cancer is being intensively discussed.
The paradigm of targeted therapy directed against kinases in cancer is the tyrosine kinase inhibitor
(TKI) Gleevec (STI-571, Imatinib, Novartis). Gleevec mainly blocks kinases of the Abl family, but
also PDGFR and c-Kit, all of which are implicated in chronic myelogenous leukemia (CML), acute
lymphocytic leukemia (ALL), and gastrointestinal stromal tumors (GIST) [9]. The effectiveness of
Gleevec in the treatment of CML is of particular importance, since CML is characterized by a gene
fusion of breakpoint cluster region and c-Abl (BCR-Abl) that results in a constitutively active kinase
and uncontrolled proliferation of hematopoietic stem cells. The fusion protein BCR-Abl is produced by
the genomic translocation [t(9;22)(q34;q11)], known as the Philadelphia chromosome. Gleevec binds to
BCR-Abl, blocks its kinase activity, and thus halts proliferation and induces apoptosis in CML cells [9].
Motivated by this breakthrough, a number of TKIs have been developed, e.g., gefitinib and erlotinib
(both block EGFR) for the treatment of lung cancer. However, subsequent TKIs have been unable to
replicate the great success of Gleevec, although a significant increase in the survival time of patients
has been observed in tests of small cohorts of patients [10–12].
Gastric cancer is strongly associated with the human pathogen Helicobacter pylori (H. pylori) which
triggers a cascade of inflammation-driven carcinogenesis. Chronic infection with H. pylori provokes
conditions such as acute and chronic gastritis, ulcer diseases, mucosa-associated lymphoid tissue
(MALT) lymphoma, and gastric cancer. The bacterium has therefore been classified as a group-I
carcinogen by the WHO [13]. The current model of H. pylori-dependent gastric cancer development
follows a sequence of chronic gastritis leading to atrophy and intestinal metaplasia and finally dysplastic
changes and gastric cancer [14].
RTK expression has been found to be highly deregulated in transformed gastric cancer tissues.
However, H. pylori directly affects RTK gene expression to a limited extent. Expression analyses of
egfr in H. pylori-infected patients have not revealed any clear pattern and show only minor alterations.
In the literature, EGFR levels are reported to be regulated in both directions or remain unchanged in
biopsies obtained from H. pylori-positive individuals [15–18], whereas cell culture models suggest an
upregulation of EGFR [19–22]. c-Met expression has also been shown to increase in gastric epithelial
cell lines after H. pylori exposure, but no significant difference was found in c-met expression in tumor
tissues from H. pylori-positive and -negative patients [23]. Expression of VEGFR decreased slightly
in human umbilical vein endothelial cells (HUVECs) upon infection with H. pylori [24], whereas in
another study, H. pylori urease stimulated VEGFR expression in human microvascular endothelial cells
(HMEC-1) [25]. In summary, these data suggest that overexpression of RTKs in gastric cancer occurs
late in the transformation process and is largely independent of H. pylori. Nevertheless, neoplastic
cancer cells display deregulated expression and accumulation of mutations in RTKs. According
to the Cancer Genome Atlas (TCGA) project, tumor protein p53 (TP53) and myeloid/lymphoid or
mixed-lineage leukemia 4 (MLL4) mutations occur with the highest frequencies in gastric cancer, but
gene alterations of members of the human epidermal growth factor receptor family (HER3, ERBB4 and
EGFR) are also prevalent in gastric cancer and are considered to be important driver mutations [26].

1.1. H. pylori Utilizes Specific Virulence Factors to Control a Complex Network of Tyrosine Kinases
Even though the expression of RTKs and nRTKs is only marginally regulated by H. pylori, drastic
effects on kinase activities can be seen. A number of H. pylori virulence factors highjack signal
transduction pathways and thus interfere with host cell functions. Many significantly correlate with the
risk of developing H. pylori-dependent gastric cancer. A central disease-promoting factor of H. pylori
is a specific type-IV secretion system (T4SS) which is encoded by the cytotoxin-associated gene (cag)
pathogenicity island (cagPAI). The T4SS critically interacts with surface receptors on host cells and
also delivers several bacterial products directly into the cytosol of infected cells (Figure 1A). The T4SS
forms a pilus with the pilus-associated factors CagL, CagI, and CagY, which bind to integrin-β1
and integrin-β6 and facilitate the injection of bacterial factors into gastric epithelial cells [27–29].
Toxins 2019, 11, 591 3 of 15

These factors include the oncoprotein cytotoxin-associated gene A (CagA) [30,31], peptidoglycan [32],
chromosomal DNA, and ADP-glycero-β-D-manno-heptose (ADP-heptose) [33–35]. ADP-heptose is a
metabolite derived from lipopolysaccharide (LPS)-biosynthesis and has only recently been described
as a novel T4SS effector. Importantly, ADP-heptose mediates NF-κB activation via the alpha-kinase
1 (ALPK1)-TRAF-interacting protein with FHA domain (TIFA) axis and this is a central step in
the regulation of innate immune pathways [34,35]. The T4SS-mediated translocation of CagA has
been well investigated and plays a major role in pathogenesis. Injection of CagA causes intense
cytoskeletal rearrangements, leading to cell elongation and increased cell migration [36,37]. Recently,
CagA translocation has also been found to be associated with carcinoembryonic antigen-related cell
adhesion molecule (CEACAM) surface molecules, which serve as receptors for the H. pylori adhesin
HopQ [38,39]. However, the interplay between integrins and CEACAMs is not well understood [38,39].
Integrin receptors have been suggested to induce H. pylori-induced kinase signaling as H. pylori binding
triggers the activation of β1-integrin and, subsequently, of focal adhesion kinase (FAK), Src, EGFR,
and HER3 (heregulin receptor 3)/ErbB3 [28] (Table 1). The nRTK FAK is an important regulator of cell
adhesion, spreading, motility, differentiation, and death [40]. H. pylori-mediated FAK activation is
characterized by phosphorylation on Y397, Y407, Y576, Y577, Y861 and Y925. The drastic morphological
changes seen in H. pylori-infected gastric epithelial cells have been attributed to β1-integrin-mediated
FAK activation [28,41,42]. Apparently, the actin cytoskeleton regulator cortactin is also involved in
integrin-induced FAK activity. Cortactin tyrosine phosphorylation is lost early in infection (cf. 1.2 Src
family kinases (SFK) signaling), but serine 405 and serine 418 are phosphorylated at later time points,
which leads to an interaction with and activation of FAK [42]. On the other hand, phosphorylated CagA
diminishes FAK phosphorylation via recruitment of the Src homology region 2 domain-containing
phosphatase-2 (SHP2) [43], suggesting that CagA counterbalances β1-integrin-FAK signaling. A similar
effect has been described for the H. pylori-secreted virulence factor vacuolating cytotoxin A (VacA),
which also reduces FAK activity [44]. The RTK c-Met also takes center stage in signaling events
hijacked in H. pylori infections. HGF is the only known ligand for c-Met and activates signaling
pathways involved in proliferation, motility, migration, and invasion [45]. The HGF-c-Met interaction
induces autophosphorylation of the intracellular domain, which forms a binding platform for signaling
molecules, such as growth factor receptor-bound protein 2 (Grb2) and Grb2-associated-binding protein
1 (Gab1) [46] (Figure 1B). CagA has been shown to interact with the intracellular domain of c-Met,
leading to the activation of ligand-independent signaling. It has been suggested that CagA acts as an
adaptor protein mimicking Gab1 and recruits additional signaling proteins [47,48], such as E-cadherin,
p120 catenin, or zonula occludens-1 (ZO-1) [49,50]. The resulting signaling triggers cell migration [51],
CagA-mediated autophagy [52], and proliferation of primary organoid-derived cells [53,54] (Table 1).
Importantly, H. pylori activates the matrix metalloproteases MMP-2 and MMP-9 in a c-Met-dependent
fashion [55]. Both MMPs are considered important factors in the motogenic response to H. pylori,
as well as in metastasis of gastric cancer and tumor-associated angiogenesis [56]. Additionally,
c-Met ectodomain shedding has been observed as a consequence of H. pylori-induced activation of A
Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) [57]. This might explain
why soluble c-Met can be considered an important biomarker for gastric cancer [58].
Toxins 2019, 11, 591 4 of 15
Toxins 2019, 11, x; doi: FOR PEER REVIEW 4 of 15

Figure1.1.RTK
Figure RTKand andnRTK
nRTK networks
networks in H.inpylori
H. pylori pathogenesis.
pathogenesis. (A) H.(A) H. pylori
pylori expresses
expresses adhesinsadhesins (e.g.,
(e.g., HopQ)
HopQ) and secretes virulence factors (e.g., VacA) and T4SS-delivered effectors
and secretes virulence factors (e.g., VacA) and T4SS-delivered effectors such as CagA or ADP-heptose such as CagA or ADP-
heptose
for for persistent
persistent colonization
colonization of the humanof the human
gastric gastric epithelium.
epithelium. Integrin stimulation
Integrin stimulation leads to theleads to the
activation
activation
of FAK, SFK,ofandFAK, c-Abl,SFK,
whichand c-Abl,CagA
stimulate which stimulate CagA
phosphorylation, phosphorylation,
cytoskeleton rearrangements,cytoskeleton
and cell
rearrangements, and
motility/migration. cell motility/migration.
Injection of ADP-heptose Injection
leads to aofstrong
ADP-heptose leads to
NF-κB-driven a strong NF-κB-driven
inflammation. (B) c-Met
inflammation.
receptors (B) c-Met
are targeted receptors
via differentare targeted via CagA
mechanisms: differentcanmechanisms:
substitute Gab1 CagA can substitute
adapter proteinsGab1and
adapter proteins and stimulate proliferation and pro-survival signals.
stimulate proliferation and pro-survival signals. Furthermore, MMP-2 and MMP-9 activities, Furthermore, MMP-2
whichand aid
MMP-9
the activities,
motogenic whichareaid
response, the motogenic
induced response, are
via c-Met-dependent inducedMoreover,
signaling. via c-Met-dependent
the sheddase signaling.
ADAM10
Moreover,
is activatedthe
andsheddase ADAM10
targets the is activated
extracellular domain andoftargets
c-Met. the (C)extracellular domainEGFR
H. pylori mediates of c-Met. (C) H.
activation
pylori
and mediates
stabilizes EGFR activation
its surface expression and by thestabilizes
inhibitionitsof surface expression
its endocytosis by the inhibition
and proteasomal of its
degradation.
endocytosis and proteasomal degradation. (D) H. pylori induces JAK/STAT3
(D) H. pylori induces JAK/STAT3 signaling during early infections, whereas the pathway is shut down signaling during early
infections,
during whereas
persistent the pathway
colonization. CagAis shut down
mediates theduring persistent
recruitment of thecolonization. CagA and
SHP2 phosphatase mediates the
prevents
recruitment
STAT bindingofviathe SHP2
IL-R phosphatase and
dephosphorylation. prevents STAT
Additionally, H. pylori binding via IL-Rcholesterol
CGT facilitates dephosphorylation.
extraction
Additionally,
from the outerH. pylori
cell CGT facilitates
membrane and, thus,cholesterol
disruptsextraction
lipid raftsfrom whichtheprovide
outer cell membrane and,
membranous thus,
signaling
disrupts lipid
platforms for therafts which pathway.
JAK/STAT provide membranous signaling
Abbr.: RTK, receptor platforms
tyrosine kinase;for the non-receptor
nRTK, JAK/STAT pathway.
tyrosine
kinase, HopQ,
Abbr.: RTK, H. pylori
receptor outer membrane
tyrosine kinase; nRTK, protein Q; VacA, vacuolating
non-receptor cytotoxin
tyrosine kinase, HopQ, A; H.
T4SS, Type
pylori IV
outer
secretion
membrane system
proteinA; CagA,
Q; VacA,cytotoxin-associated
vacuolating cytotoxin gene A; FAK,T4SS,focal
Typeadhesion kinase;
IV secretion SFK, Src
system A; family
CagA,
kinase; NF-kB, nuclear
cytotoxin-associated factor
gene A; kappa B; MMP,
FAK, focal matrixkinase;
adhesion metalloprotease; ADAM10,
SFK, Src family kinase;A Disintegrin
NF-kB, nuclearand
metalloproteinase
factor kappa B; MMP, domain-containing protein 10; EGFR,
matrix metalloprotease; epidermal
ADAM10, growth factor
A Disintegrin andreceptor; JAK/STAT3,
metalloproteinase
Janus kinase/Signal Transducers
domain-containing protein 10; and EGFR, Activators
epidermalof Transcription
growth factor 3; SHP2, Src homology
receptor; JAK/STAT3, region
Janus2
domain-containing phosphatase-2,
kinase/Signal Transducers IL-R, interleukin-receptor;
and Activators of Transcription 3; CGT, SHP2,cholesterol-glucosyltransferase.
Src homology region 2 domain-
containing phosphatase-2, IL-R, interleukin-receptor; CGT, cholesterol-glucosyltransferase.

Table 1. The role of receptor tyrosine kinases and non-receptor tyrosine kinases in H. pylori
pathogenesis.

RTKs Function Role in Hp Pathogenesis1 References

Differentiation,  Activated by Hp-integrin-β1 [28]
EGFR
growth, interaction
migration,  Activates MAPK→ JAK→ hBD3 [59]
Toxins 2019, 11, 591 5 of 15

Table 1. The role of receptor tyrosine kinases and non-receptor tyrosine kinases in H. pylori pathogenesis.

RTKs Function Role in Hp Pathogenesis 1 References

Differentiation, growth, migration, proliferation,  Activated by Hp-integrin-β1 interaction [28]
EGFR
matrix adhesion  Activates MAPK→ JAK→ hBD3 [59]
 Interacts with CagA [51]
 Induces cell migration and autophagy [51,52]
Differentiation, migration, mitogenic activity,
HGFR/c-Met  Induces proliferation of primary cells [53,54]
invasive growth, wound healing
 Promotes invasive growth via MMP activation [55]
 Leads to c-Met ectodomain shedding by Hp-induced ADAM10 [57]
Angiogenesis, chemotaxis, differentiation,
FGFR  n.d.
migration, embryonic development
Angiogenesis, chemotaxis, differentiation,
PDGFR  n.d.
migration, survival, wound healing
Angiogenesis, chemotaxis, differentiation,
VEGFR  n.d.
migration, survival, wound healing
c-Kit Hematopoiesis, survival, proliferation  n.d.
nRTKs Function Role in Hp pathogenesis References
 CagA kinase [60–62]
Implicated in cell elongation and migration
 [60–62]
Abl family Apoptosis, differentiation, migration, proliferation  Promotes cell death in B cells [63]
 Acts as switch between apoptosis and cell survival in epithelial cells [64]
 Inhibits EGFR endocytosis and limits EGFR turn-over [65,66]
 Leads to c-myc transactivation and enhanced cell migration [67]
Immunity, cell division, cell death,
JAK  Involved in the synthesis of hBD3 [59,68]
tumor formation
 Involved in NF-κB-mediated IL-8 expression [69]
 Activated by Hp/integrin-β1 interaction [28]
FAK Adhesion, cytoskeleton rearrangements, migration
 Promotes cytoskeleton rearrangement, cell motility, scattering [28,41,42]
 Activated by Hp/integrin-β1 interaction [28]
Adhesion, cytoskeleton rearrangements, growth,  CagA kinase [60–62]
Src family
RTK signaling  Implicated in cell elongation and migration [60–62]
 Promotes cell death in B cells [63]
1 Abbr.: Hp, H. pylori; n.d., not determined.
Toxins 2019, 11, 591 6 of 15

1.2. The nRTKs c-Abl and Src Phosphorylate CagA

CagA delivery into the host cell cytoplasm is a key process in H. pylori pathogenesis and development
of gastric cancer [70,71]. After translocation, CagA rapidly becomes tyrosine-phosphorylated, which is a
prerequisite for the strongly elongated cell morphology [72,73]. The C-terminal part of CagA contains a
variable number of EPIYA motifs (Glu-Pro-Ile-Tyr-Ala), which have been identified via mass spectrometry
and site-directed mutagenesis as unique CagA phosphorylation sites [72–74]. Four distinct EPIYA motifs
have been described based on their surrounding sequence, namely EPIYA -A, -B, -C and -D, and these can
be assigned to the geographic regions from where the bacterial strains were isolated [75]. EPIYA-A and -B
motifs are present in all strains, whereas EPIYA-C is mainly conserved in Western isolates and replaced
with EPIYA-D in strains from East Asian countries [75,76]. The number of EPIYA motifs varies from one to
seven, with an average of three EPIYA segments present in the majority of strains [77].
Upon infection of gastric epithelial cells with H. pylori, CagA is phosphorylated by two families
of nRTKs, SFKs and c-Abl, in a highly time-coordinated manner (Table 1). Both SFKs and c-Abl are
implicated in processes ranging from cytoskeleton rearrangements, cell motility, and proliferation to DNA
damage response and apoptotic pathways [78,79]. During earlier stages of infection (0.5–2 h), the SFK
members c-Src and Lyn are rapidly activated by an unknown mechanism and directly phosphorylate CagA.
Specific c-Src kinase inhibitors cause a decrease in CagA phosphorylation levels and efficiently prevent
the elongation phenotype induced by H. pylori, whereas overexpression of c-Src in infected cells leads
to an increase in CagA phosphorylation [73,80]. Interestingly, during prolonged infection (2–8 h), c-Src
activity is downregulated by a negative feedback-loop involving phosphorylated CagA and its interaction
with the C-terminal Src kinase (Csk), which inactivates c-Src [81]. Discontinued c-Src activity leads to the
dephosphorylation of the actin-binding proteins vinculin, cortactin, and ezrin [82–84]; however, CagA
tyrosine phosphorylation is maintained by activated c-Abl [60–62]. At this stage, a complex composed
of phosphorylated CagA, activated c-Abl, and its substrates, Crk adaptor proteins, is formed, which is
essential for the H. pylori-induced elongation phenotype [60,61,85]. The model of CagA phosphorylation
includes a hierarchically ordered action of SFKs and c-Abl. Early c-Src activity exclusively favors the
phosphorylation of EPIYA-C or -D motifs, whereas c-Abl phosphorylates EPIYA-C or -D motifs followed
by EPIYA-A and -B motifs. Two-dimensional electrophoresis has revealed that only one or two motifs
are phosphorylated at the same time and mutational tyrosine residue replacement in the EPIYA motifs
has shown that none of the phosphorylated EPIYA motifs alone are able to initiate the characteristic cell
elongation phenotype. Moreover, phosphorylation of EPIYA-A and EPIYA-C induces a similar phenotype
to that induced by the presence of three intact EPIYA motifs [62]. Depending on its phosphorylation
status, CagA binds different sets of signaling molecules. Tyrosine-phosphorylated CagA interacts with
Crk proteins, Csk, or SHP2. Binding partners for non-phosphorylated CagA include E-cadherin, Grb2,
c-Met, and ZO-1 [50], underlining the importance of the CagA phosphorylation status for the control of
cellular responses to H. pylori. The influence of H. pylori on kinase networks also extends to the Janus kinase
(JAK)/ Signal Transducers and Activators of Transcription (STAT) signaling axis (Figure 1 D). CagA tyrosine
phosphorylation also functions as a switch between the SHP2 and JAK/STAT pathways mediated by
glycoprotein gp130 receptor chains. In the presence of phosphorylated CagA, SHP2 is recruited to gp130,
whereas non-phosphorylated CagA preferentially causes JAK2-mediated STAT3 activation, which leads to
c-myc transactivation and enhanced cell migration [67]. JAK2 phosphorylation has also been observed in
response to H. pylori lipopolysaccharide (LPS) stimulation [86,87]. However, another study has indicated
that H. pylori activates EGFR→mitogen-activated protein (MAP) kinase→JAK/STAT signaling, leading to
the synthesis of the antimicrobial human beta-defensin 3 (hBD3) [59]. Furthermore, JAK/STAT signaling has
been associated with NF-κB-mediated interleukin-8 (IL-8) expression in gastric epithelial cells [69] (Table 1).
H. pylori has been shown to interfere with JAK/STAT signaling by disrupting lipid rafts via cholesterol
depletion in the outer membrane using a cholesterol-α-glucosyltransferase. This results in abrogated hBD3
production and reduced expression of interferon response genes even in the presence of high interferon
gamma (IFN-γ) levels, thereby creating a protective niche for H. pylori [68].
synthesis of the antimicrobial human beta-defensin 3 (hBD3) [59]. Furthermore, JAK/STAT signaling
has been associated with NF-κB-mediated interleukin-8 (IL-8) expression in gastric epithelial cells
[69] (Table 1). H. pylori has been shown to interfere with JAK/STAT signaling by disrupting lipid rafts
via cholesterol depletion in the outer membrane using a cholesterol-α-glucosyltransferase. This
results in abrogated hBD3 production and reduced expression of interferon response genes even in
Toxins 2019, 11, 591 7 of 15
the presence of high interferon gamma (IFN-γ) levels, thereby creating a protective niche for H. pylori
[68].
Taking
Taking into intoconsideration
consideration thatthat chronic
chronic infection
infection with H.with pyloriH. pylori recruitment
initiates initiates recruitment
of neutrophils of
neutrophils
and lymphocytes and lymphocytes
into the gastric into the gastric
mucosa, a direct mucosa, a direct
interaction interaction
between between
infiltrating cells infiltrating cells
and the bacteria
and the bacteria
is highly plausibleis[14,88].
highly Therefore,
plausible [14,88].
differentTherefore,
immune cell different immuneTHP-1,
lines (MEC-1, cell lines
U937,(MEC-1,
J774A.1)THP-1,
have
U937, J774A.1) have
been employed been employed
to demonstrate to demonstrate
the successful the successful
translocation translocation of
and phosphorylation and phosphorylation
CagA in these cells
of CagA
upon in these
infection cells upon
[82,88]. infection
However, [82,88].ofHowever,
the model successivethe model of successive
phosphorylation of CagAphosphorylation
by SFK and Abl of
CagA
family by SFK derived
kinases and Ablfrom family
the kinases derived from
gastric epithelial the gastric
cells does not apply epithelial
to immune cellscells
does[63].
notUsing
applythe to
immune
MEC-1 cell cells
line[63]. UsingBthe
(chronic MEC-1 cell
lymphocyte line (chronic
leukemia cells) asB lymphocyte
an infection leukemia
model, it hascells) as an
been infection
shown that
model,
expression it has
and been
activityshown
of c-Srcthat expression two
is up-regulated andhours
activity of c-Src isand
post-infection up-regulated
remains stable,twowhereas
hours c-Abl
post-
infection
activity is and remains
rapidly stable,However,
increased. whereasthe c-Abl activity
impact is rapidly
on the increased.pattern
phosphorylation However, the impact
of EPIYA motifs on
by the
phosphorylation
simultaneous activation patternof of EPIYA
both tyrosinemotifs by the
kinases simultaneous
in immune activation
cells is not yet known.of both tyrosineinhibition
Individual kinases in of
immune cells is not yet known. Individual inhibition of c-Src and Abl kinases
c-Src and Abl kinases moderately decreases CagA phosphorylation levels, while simultaneous inhibition moderately decreases
CagA
of bothphosphorylation
kinases causes a levels,complete whilelosssimultaneous inhibition of both
of CagA phosphorylation. kinasescell
Moreover, causes a complete
viability loss
assays have
of CagA
shown phosphorylation.
a significant reduction Moreover, cell viability assays
in H. pylori-mediated cell death havein shown a significant
the presence of c-Src reduction in H.
and Abl kinase
pylori-mediated cell deathSFKs
inhibitors [63]. Therefore, in the andpresence of c-Src
Abl family andcontribute
kinases Abl kinasenot inhibitors [63]. Therefore,
only to CagA SFKs and
phosphorylation but
Abl family
also to kinases contribute
the regulation of associated not only to CagA
downstream phosphorylation
signaling pathways (Figure but 2).
also to the regulation of
associated downstream signaling pathways (Figure 2).

H. pylori-induced
Figure 2. H. pylori-induced CagA phosphorylation by Src and Abl family kinases. (A) H. pylori injects
CagA via its T4SS into human gastric epithelial cells and sequentially activates SFK and c-Abl to
induce CagA phosphorylation at the EPIYA motifs. Early CagA CagA phosphorylation
phosphorylation triggers recruitment
activation of
and activation ofCsk,
Csk,which
whichfeeds
feedsinto
intoa a negative
negative feedback
feedback loop,
loop, inactivates
inactivates SFK,
SFK, andandthusthus causes
causes the
the dephosphorylation
dephosphorylation of targets
of Src Src targets cortactin,
cortactin, ezrin,ezrin, and vinculin.
and vinculin. At thisAtpoint,
this point, c-Abl maintains
c-Abl maintains CagA
CagA phosphorylation.
phosphorylation. (B) The(B) The H.metabolite
H. pylori pylori metabolite ADP-heptose
ADP-heptose is transferred
is transferred into hostinto
cellshost
and cells and
activates
activates PKC through a yet undescribed mechanism. PKC then phosphorylates
PKC through a yet undescribed mechanism. PKC then phosphorylates threonine 735 of c-Abl. (C) threonine 735 of
c-Abl. (C) Phosphorylated T735
c-Ablas a docking
serves as a for
docking site for the interaction
Phosphorylated c-AblT735 serves site the interaction with proteins with
of theproteins of the
14-3-3 family,
14-3-3
which family, which
prevent prevent
nuclear nuclear shuttling
shuttling of c-Abl.of c-Abl. Cytoplasmic
Cytoplasmic tethering
tethering of c-Ablforces
of c-Abl forces CagA
CagA
phosphorylation and increases cell survival, cell motility, and proliferation, whereas c-Abl activities in
the nucleus are attenuated. Abbr.: PKC, protein kinase C; Csk, C-terminal Src kinase.

1.3. c-Abl Is more than a CagA Kinase: Control of Cell Migration and Apoptosis
The regulation of c-Abl in H. pylori-infected cells has attracted much attention, because the c-Abl
proto-oncoprotein has been strongly implicated in tumorigenesis and cancer [89]. Abl proteins share
Toxins 2019, 11, 591 8 of 15

structural similarities with SFKs and are characterized by an N-terminal Src homology 2 (SH2) domain
binding to phosphorylated tyrosine residues and a Src homology 3 (SH3) domain which preferentially
binds to proline-rich protein sequences. The C-terminal tail of c-Abl consists of a DNA-binding domain,
F- and G-actin-binding domains, nuclear localization signals (NLS), and nuclear export signals (NES),
and is of high importance for the regulation of kinase activity and localization [90]. Abl kinases are
activated by various factors, such as PDGFR and EGFR, substrate binding, oxidative stress, or bacterial
pathogens [89–91]. H. pylori is included in the list since many studies have provided strong evidence
showing that it utilizes c-Abl-CagA signaling for efficient colonization and pathogenesis [92,93].
The presence of H. pylori leads to a continuous activation of c-Abl and phosphorylation of the kinase
on tyrosine residues 245 and 412 [60,61,64]. Moreover, kinase inhibition of c-Abl, silencing by RNA
interference, or expression of a kinase-dead version cause a significant reduction in cell elongation and
scattering and a decrease in late CagA phosphorylation upon H. pylori infection [60,61]. Furthermore,
cross-talk between c-Abl and EGFR signaling has been observed. c-Abl-mediated phosphorylation of
EGFR on tyrosine residue 1173 inhibits receptor endocytosis leading to an increase in EGFR surface
expression in H. pylori infections [65,66]. This process has been shown to be dependent on CagA, but
independent of CagA phosphorylation [65]. Additionally, EGFR protein turnover is slowed down
by activated c-Abl via suppression of the ubiquitin ligase casitas B-lineage lymphoma (Cbl), which is
involved in EGFR degradation [66] (Figure 1 C).
In H. pylori-infected gastric epithelial cells, c-Abl kinase activation is triggered in a CagL-dependent
fashion; however, the pathways upstream of c-Abl activation are not fully understood [60,61,64].
Activation results in major conformational changes and is accompanied by phosphorylation at tyrosine
245 and tyrosine 412 [94,95]. Abl kinases have functions in manifold cellular responses, like cell
migration, survival, proliferation, and cell death. These opposing effects are mainly regulated via the
subcellular localization of the kinase. Depending on the cellular context and environmental signals,
NLS and NES sequences regulate shuttling of c-Abl between the cytoplasm and the nucleus. In the
cytoplasm, c-Abl is involved in the regulation of actin dynamics and proliferation. Accordingly, many
of the identified kinase substrates (Crk proteins, cortactin, Wave, etc.) are closely associated with cell
morphology, migration, and proliferation [90,96,97] (Figure 2A). In contrast, nuclear c-Abl contributes
to the DNA damage response [78] and apoptosis [98,99]. The importance of the subcellular localization
of c-Abl is accentuated by the action of the BCR-Abl fusion protein. In untreated cells, BCR-Abl
is constitutively active solely in the cytoplasm, which leads to uncontrolled proliferation, whereas
treatment with kinase inhibitors also allows shuttling of c-Abl to the nuclear compartment and cell
death [100]. Therefore, a balanced nucleo-cytoplasmic transport of c-Abl is a tightly regulated process
in normal cells.
The pivotal role of the subcellular localization of c-Abl in cell fate decisions implies the existence
of several levels of regulation. Shuttling mediated via NLS and NES motifs has been associated with
the interaction of members of the 14-3-3 protein family. The 14-3-3 proteins have been shown to
interact preferentially with phosphorylated threonine 735 in the c-Abl protein (pAblT735 ) and thereby
mask the NLS motifs [101,102] (Figure 2 C). The phosphorylation of pAblT735 is mediated via the dual
specific kinase monopolar spindle 1 (Mps1, TTK) [102] and protein kinase C (PKC) kinases [64]. Under
genotoxic or oxidative stress, c-Abl is released from 14-3-3 interaction and preferentially shuttles to the
nucleus. This release is mediated via JNK-dependent 14-3-3 phosphorylation on serine 84 (as shown
for 14-3-3ζ), which abrogates 14-3-3 interaction with c-Abl irrespective of AblT735 phosphorylation
status. Nuclear c-Abl is central to the DNA damage response and contributes to cell fate decisions
between cell cycle arrest and DNA repair on the one hand, and cell death and apoptosis on the other.
Nuclear c-Abl is activated via ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase
(DNA-PK) and has been shown to directly phosphorylate p73 [79]. Interaction with p53 is thought
to trigger cell cycle arrest, whereas p73 and MAP-kinase signaling feed into an apoptotic signaling
cascade. Interestingly, apoptotic caspases have been shown to cleave c-Abl, which results in a loss of
the N-terminal NES signals and thus amplifies nuclear c-Abl accumulation and apoptosis in a positive
Toxins 2019, 11, 591 9 of 15

feedback loop [103]. Further, nuclear c-Abl leads to elevated JNK activity levels in a c-Jun-dependent
signaling circuit [104]. Again, JNK-dependent 14-3-3 phosphorylation might lead to a release of c-Abl
from cytoplasmic tethering, resulting in nuclear accumulation. In combination, all these mechanisms
reinforce nuclear c-Abl localization and activity, which sequentially could reach a point of no return
and thus inevitably induce cell death.
The cytoplasmic functions of c-Abl in the course of H. pylori infections are well established and its
roles in CagA phosphorylation and CagA-independent mechanisms have been well described [105].
However, the signaling events upstream of c-Abl activation are still unknown. Recently, we found that
H. pylori additionally triggers the phosphorylation of threonine 735 and binding to 14-3-3, leading to
cytoplasmic retention of pAblT735 [64]. The phosphorylation at the critical tyrosine residues 245 and
412 during c-Abl activation is independent of threonine phosphorylation and is elicited via a distinct
mechanism. Both threonine as well as tyrosine phosphorylation strictly depend on the presence of an
intact T4SS. We could show that while c-Abl activation requires the T4SS structural component CagL,
the threonine phosphorylation is enabled by the recently described H. pylori metabolite ADP-heptose,
which has been shown to be injected into host cells [34,35]. In uninfected gastric epithelial cells,
c-Abl activation levels are low and the subcellular distribution between cytoplasm and nucleus is in
equilibrium. This picture drastically changes in response to H. pylori infection, when phosphorylation
of c-Abl threonine 735 forces 14-3-3 binding and hence cytoplasmic localization [64]. This activation and
localization pattern favors the cytoplasmic actions of c-Abl and contributes to CagA phosphorylation
and cytoskeleton rearrangements leading to cell elongation and enhanced cell motility. Concomitantly,
nuclear exclusion prevents apoptotic signaling and thus limits infection-induced cell death (Table 1).
In contrast to the situation under conditions of genotoxic or oxidative stress, in which TTK has been
identified as the kinase which phosphorylates AblT735 [102], we identified PKC kinases upstream
of pAblT735 (Figure 2B). Syk kinase activation has been suggested to contribute to PKC activity in
H. pylori infections in an LPS/TLR-4-mediated manner [106]. Mutational disruption of the c-Abl/14-3-3
interaction renders the anti-apoptotic action of this signaling cascade ineffective and, consequently, leads
to a strong increase in apoptosis after H. pylori infection. In vivo, we found constant phosphorylation
of the 14-3-3 interaction site AblT735 in patients suffering from chronic H. pylori gastritis, whereas
pAblT735 levels were low in chemically induced gastritis and healthy individuals. In a mouse model
of chronic H. pylori infection, the cytoplasmic c-Abl activity contributes to the aggravation of disease
progression, as animals that received the c-Abl inhibitor Gleevec showed an overall milder gastric
phenotype despite equal colonization levels [64].

2. Concluding Remarks
Tyrosine kinase inhibition has proved to be a promising strategy to support cancer therapy against
non-small cell lung carcinoma (EGFR inhibitors) and leukemic malignancies (CML: ABL inhibitors;
AML: FLT3 inhibitors; ALL: ABL inhibitors). TKIs of the latest generation have high target specificity
and cause fewer side effects than conventional chemotherapy. Unfortunately, many TKI targets are
subject to mutation and can acquire resistance to TKI inhibition. Therefore, many TKIs are used in
combinatory regimens to circumvent drug resistance. In gastric cancer, changed expression levels
and mutation of RTKs are implicated in tumor progression and metastasis [103]. This highlights the
importance of a personalized precision medicine to specifically target deregulated signaling pathways
when using TKIs. Furthermore, TKIs are not only used to directly target driver kinases: promising
results have also been reported for anti-angiogenic drugs targeting VEGF signaling to prevent tumor
vascularization and metastasis [5,107]. The c-Abl inhibitor Gleevec is also used in the treatment of the
rare GIST cancer and some studies have suggested a role for Gleevec in combination therapy regimens
targeting gastric cancer. Although kinase inhibition might be inappropriate for the treatment of
non-malignant H. pylori-associated disease, it would be highly interesting to study disease progression
in a cohort of H. pylori-positive CML patients being treated with TKIs. This would give important
insights into the in vivo relevance of H. pylori-regulated kinase networks.
Toxins 2019, 11, 591 10 of 15

Author Contributions: Conceptualization: S.W. and G.P.; Writing-Original Draft: S.W., B.E.C., S.D. and G.P.;
Visualization: S.D. Writing-Review & Editing, S.W. and G.P.
Funding: The work was supported by the grant W_1213 from the Austrian Science Fund (FWF).
Acknowledgments: The work was supported by the open Access Funding by the Austrian Science Fund (FWF).
Conflicts of Interest: The authors declare no conflict of interest.

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