Mechanisms of the Epithelial-Mesenchymal Transition and Tumor Microenvironment in Helicobacter pylori-Induced Gastric Cancer.

Helicobacter pylori (H. pylori) is one of the most common human pathogens, affecting half of the world's population. Approximately 20% of the infected patients develop gastric ulcers or neoplastic changes in the gastric stroma. An infection also leads to the progression of epithelial-mesenchymal transition within gastric tissue, increasing the probability of gastric cancer development. This paper aims to review the role of H. pylori and its virulence factors in epithelial-mesenchymal transition associated with malignant transformation within the gastric stroma. The reviewed factors included: CagA (cytotoxin-associated gene A) along with induction of cancer stem-cell properties and interaction with YAP (Yes-associated protein pathway), tumor necrosis factor α-inducing protein, Lpp20 lipoprotein, Afadin protein, penicillin-binding protein 1A, microRNA-29a-3p, programmed cell death protein 4, lysosomal-associated protein transmembrane 4β, cancer-associated fibroblasts, heparin-binding epidermal growth factor (HB-EGF), matrix metalloproteinase-7 (MMP-7), and cancer stem cells (CSCs). The review summarizes the most recent findings, providing insight into potential molecular targets and new treatment strategies for gastric cancer.

During this process, epithelial cells undergo a series of biochemical changes, which lead to the loss of polarity and migratory capacity of cells, resulting in cell shape changes (cell elongation). EMT promotes the transformation of immobile epithelial cells into motile mesenchymal cells, enhancing the metastatic properties [69,70]. Further, adherens and tight junctions become impaired, resulting in a mesenchymal phenotype [12,[71][72][73]. Altered E-and N-cadherin levels and the following β-catenin activation promote the expression of many tumor-associated proteins, including cyclin D1, CD44, or c-MYC [54,[74][75][76][77][78][79]. A transformation of cell phenotype enhances the migratory properties, Figure 1.
Schematic of the epithelial-mesenchymal transition and chosen epithelial and mesenchymal markers.
During this process, epithelial cells undergo a series of biochemical changes, which lead to the loss of polarity and migratory capacity of cells, resulting in cell shape changes (cell elongation). EMT promotes the transformation of immobile epithelial cells into motile mesenchymal cells, enhancing the metastatic properties [69,70]. Further, adherens and tight junctions become impaired, resulting in a mesenchymal phenotype [12,[71][72][73]. Altered E-and N-cadherin levels and the following β-catenin activation promote the expression of many tumor-associated proteins, including cyclin D1, CD44, or c-MYC [54, [74][75][76][77][78][79]. A transformation of cell phenotype enhances the migratory properties, invasiveness, and apoptosis resistance of cells [80]. Moreover, EMT is involved in the induction of cancer stem cell properties, which leads to chemoresistance and tumor dormancy [81][82][83].
H. pylori infection significantly affects the gastric microenvironment by induction of several inflammatory responses via infiltrating macrophages, neutrophils, regulatory T-cells, and natural killer cells [84,85]. Inflammatory mediators such as cytokines, chemokines, and metalloproteinases Cells 2020, 9, 1055 3 of 28 that are released by gastric and infiltrating cells promote the EMT process within gastric cells; transforming growth factor β (TGF-β) is probably one of the most relevant EMT inducers [86][87][88]. Thus, chronic inflammation might significantly contribute to EMT progression and carcinogenesis [89][90][91]. A significant number of H. pylori virulence factors are considered being associated with the promotion of EMT in gastric cells, which consequently causes neoplasia and malignant transformation. This review summarizes several mechanisms associated with epithelial-mesenchymal transition, gastric tumor microenvironment, and the influence of H. pylori infection, although some described mechanisms are not only H. pylori-specific. Even though H. pylori-induced carcinogenesis is not fully understood, several mechanisms have already been deciphered.

CagA and EMT
Among many virulence factors of H. pylori, cag pathogenicity island (cagPAI) probably plays a key role in carcinogenesis [92][93][94]. It encodes a type 4 secretion system (T4SS) and the CagA oncoprotein [95,96]. The T4SS forms a pilus that allows the injection of CagA into a cell, transforming its shape into the so-called "hummingbird phenotype" characterized by an elongated cell shape commonly observed in EMT [97,98]. Injection of the CagA into the cell via the T4SS induces signal transduction, with one of the most relevant mecahnisms being the nuclear factor κB (NF-κB) signaling pathway involving extracellular regulated kinases 1/2 (ERK-1/2) [99][100][101]. These kinases are involved in the conformational changes of the cytoskeleton, which might enhance the EMT process [102]. The inhibition of ERK and c-Jun N-terminal kinase (JNK) results in the lower expression of the 'hummingbird' phenotype induced by H. pylori [103]. CagA in host cells is tyrosine phosphorylated and interacts with protein tyrosine phosphatase 2 (SHP-2), also inducing the progression of the "hummingbird phenotype" [104,105]. CagA enhances EMT via the stabilization of Snail protein, which is essential in carcinogenesis, mainly by the reduction of glycogen synthase kinase-3 (GSK-3) activity [106]. CagA-positive H. pylori strains with CagA containing phosphorylation-functional EPIYA motifs present significantly higher expression of mesenchymal markers such as vimentin, Snail, and ZEB-1 and the stem cell marker CD44 [96, [107][108][109][110]. Many studies have shown that CagA-positive H. pylori strains induce a higher probability of gastric carcinogenesis and induction of EMT process [111][112][113]. Incidents of CagA-positive H. pylori infection present poor clinical outcome, and higher invasion and metastatic characteristics [114]. Besides, there is an increasing interest in microRNAs (miRNAs), since these are reported to play a role in gastric carcinogenesis and progression [115,116].

CagA and Cancer Stem Cell Properties
Recent research suggests that cells that undergo EMT obtain the ability to acquire cancer stem cell (CSC) properties [117][118][119][120][121][122][123]. The main source of gastric CSCs includes stem cells and progenitor cells; other studies suggest that CSCs originate from bone marrow-derived cells [124,125]. Due to the ability of self-renewal and differentiation into a vast number of cells, CSCs have the property of tumorigenesis induction [126,127]. Gastric CSCs are primarily generated by CagA-positive H. pylori strain infection [128,129]. High expression of CD44, a compelling marker of CSCs, predisposes cells to the induction of mesenchymal phenotype and EMT [130][131][132][133][134][135][136].  studied the role of H. pylori in the generation of cells with CSC properties, including several gastric epithelial cell lines (AGS, MKN-45, MKN-74) [103]. The role of CagA in the induction of CSC properties was studied on H. pylori 7.13 wild-type (WT) CagA-positive strain and its knock-out mutants-7.13CagA-negative and 7.13DCagE-negative H. pylori strains. Only the wild-type (WT) (7.13CagA-positive) strain-induced mesenchymal changes and EMT in cells; cells infected by CagA-positive strains, presented higher expression of mesenchymal markers-CD44, Snail1, vimentin, or zinc finger E-box binding homeobox 1 (ZEB1), while an expression of epithelial markers-cytokeratin 7 (CK7) or osteopontin (SSP-1) was lowered [103]. The migration and invasion The Wnt/β-catenin signaling pathway is involved in CagA-positive H. pylori EMT and the induction of CSC properties [137]. Likewise, chronic CagA-positive H. pylori infection with N-nitrosoguanidine (MNNG) stimulation causes CSC features with dysplastic lesions and mesenchymal phenotype. Amieva and Peek (2016) observed that CD44+ cells with CSC features displayed increased CagA half-life [26]. Accumulation of intracellular CagA was confirmed in CD44+ gastric CSCs [138]. H. pylori-infected (CagA-positive) gastric cancer cells exhibit CSC properties via increased expression of surface markers-CD44 and Lgr5 with Nanog, Oct4, and c-myc upregulation [139,140]. CagA impairs transcription factor CDX1 expression, promoting EMT, and CSC features in gastric epithelial cells [141].

CagA and Yes-Associated Protein Pathway
Yes-Associated-Protein (YAP) is an element of the Hippo tumor suppressor signaling pathway, which plays a crucial role in the maintenance of the proper size of organs, tissue homeostasis, cell proliferation, and stem cell maintenance [142][143][144]. Since YAP induces the progression of carcinogenesis, it is considered to be an oncogenic protein [145][146][147]. Increased expression of YAP activates several oncoproteins including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), or MYC oncogene [148,149]. Besides, the overexpression of YAP lowers the expression of epithelial markers including E-cadherin, which induce mesenchymal changes and EMT [150].  showed a significantly higher expression of YAP and TAZ (YAP paralog) in cancerous gastric tissues with a positive correlation between YAP, TAZ levels, and tumor size [151]. During early tumor stages, YAP is expressed mainly in the cell cytoplasm, whereas in advanced stages it is expressed in the nucleus. CagA-positive H. pylori strain induces higher YAP expression and decreases E-cadherin, N-cadherin, and Slug levels, promoting EMT.
Other studies have shown a potential role of GSK3/β-catenin or AMP-activated protein kinase (AMPK) pathways, which are regulated by CagA, in the enhancement of the YAP pathway [152,153]. Molina-Castro et al. (2020) showed that infection by CagA-positive H. pylori upregulates YAP1 and large tumor suppressor 2 (LATS2) expression in gastric epithelial cells [154]. Overexpression of the oncogenic YAP1 is associated with aggressiveness and poor prognosis [155]. Gastric cancer development and progression are further promoted by the dysregulated YAP1/SLC35B4 axis; another mechanism involves altered IL-1β levels [156,157]. These findings show that YAP might become a potential target in gastric cancer treatment.
Researchers have suggested the role of Tipα and nucleolin receptor (88 kDa) on the migration properties of gastric cancer cells [161,181]. Fujiki et al. showed that EMT phenotype induced by Tipα could be inhibited via small interfering RNAs targeted for the nucleolin receptor, showing its role in EMT progression [182]. Thus, potential ligands of nucleolin receptors (e.g., lactoferrin) might inhibit H. pylori infection by inhibiting Tipα and nucleolin interaction [183,184].
Tipα induces the formation of filopodia and a reduction of cell stiffness, predisposing cells to higher motility [185]. It has been reported that Tipα induces higher levels of IL-1β, TNF-α, and IL-8 in SGC7901 cells [164]. Furthermore, H. pylori infection promotes the Il-6/STAT3 signaling pathway in AGS cells, which is one factor promoting EMT [186]. Inoue et al. observed that vaccinations with Tipα did successfully eradicate H. pylori infection [187].

Antigenic Lpp20 Protein
The Lpp20 protein plays an important role as an H. pylori virulence factor, promoting its colonization and survival properties [188]. Lpp20 is a lipoprotein embedded in the external membrane of H. pylori, which enhances the stimulation of cell proliferation. Lpp20 expression is increased in the acidic pH of stomach juice and during iron depletion [189,190].
Lpp20 (a structural homologue of Tipα), is one of the carcinogenic factors released by H. pylori via NF-κB pathway activation [183]. Tipα and Lpp20 induce EMT mainly by stimulating cell proliferation, migratory properties of cells, and filopodia formation [191].
Even though Lpp20 and Tipα have different locations within the bacterium (external and inner membranes respectively), these proteins show similar effects on gastric cancer cells by stimulating EMT and gastric cancer progression.

Afadin Protein Downregulation
Afadin is a cytoplasmic actin-filament-binding protein responsible for the formation and stabilization of tight and adherens junctions [192][193][194][195]. Marques et al. (2018) observed that infection by H. pylori lowered the expression of Afadin protein independently of CagA and VacA in MKN74 and NCI-N87 cell lines [196]. Downregulation of Afadin resulted in dysregulation of tight junctions and adherens junctions, which caused inappropriate cell permeability, stiffness, and impaired transepithelial electrical resistance. The decreased level of Afadin induced higher expression of Snail (EMT marker) and increased actin stress fiber formation; no changes were observed in Slug, ZEB1, vimentin, and N-cadherin levels. So far, this is the first study suggesting a role of Afadin in the EMT induction in gastric cancer cells infected by H. pylori.

Penicillin-Binding Protein 1A
Penicillin-binding protein (PBP) is a specialized acyl serine transferase released by H. pylori with an affinity for penicillin binding; any modifications within PBP might induce resistance to β-lactam antibiotics (amoxicillin, penicillin G, ampicillin) [197]. These mainly include either mutations or mosaics (or both) of PBP2X, PBP2B, and PBP1A [198]. It was shown that H. pylori PBPs might present unique characteristics because of several putative genes encoding PBPs.
Huang et al. (2019) showed significant differences between PBP1A mutation-positive H. pylori (H. pylori CagA+/P+) strain and CagA+/P− in terms of clinicopathological characteristics of gastric cancer and EMT induction [199]. H. pylori CagA+/P+ infection resulted in progressive and clinically significant EMT and gastric cancer severity. Researchers have observed decreased levels of E-cadherin and increased α-smooth muscle actin (α-SMA) levels; likewise, it was observed that PBP1A mutation is associated with decreased miR-134 levels. MiR-134 suppresses gastric carcinogenesis by targeting the KRAS gene and Golgi phosphoprotein 3 (GOLPH3) downregulation; another essential target gene of miR-134 is FoxM1 [200,201]. The inhibition of PBP via paepalantine is suggested to be a promising Cells 2020, 9, 1055 6 of 28 method of H. pylori activity decline [202]. Marcus et al. showed that at pH 3.0, the expression of PBPs genes is significantly decreased [203].

Upregulation of MicroRNA-29a-3p
Micro-RNAs constitute a family of small noncoding RNAs, which are regulators of posttranscriptional gene expression by binding to the 3 -untranslated region of mRNA, promoting an inappropriate translation of targeted mRNA. There is an increasing interest in micro-RNA involvement in many diseases and malignancies [204][205][206][207]. A vast number of micro-RNAs are directly associated with the progression of gastric cancer, including microRNA-4513, microRNA-95, and microRNA-4268 [208][209][210].
Sun et al. presented the relevance of upregulation of microRNA-29a-3p and its role in the decreased expression of A20 in patients infected by H. pylori [211]. A20 is the ubiquitin-editing enzyme, which negatively regulates the NF-kB pathway, controlling proper cellular activation, development, and differentiation [212]. H. pylori infection induced the overexpression of microRNA-29a-3p in gastric cancer tissue samples, which resulted in an enhanced ability of the migration of gastric epithelial cells. Furthermore, the silencing of A20 increased the EMT markers Snail, vimentin, and N-cadherin, and decreased E-cadherin levels. The authors have suggested that the overexpression of micro-RNA-29a-3p might be associated with a higher probability of EMT induction in gastric cells.

Downregulation of Programmed Cell Death Protein 4
Programmed cell death protein 4 (PDCD4) is a tumor suppressor responsible for the inhibition of cell growth, tumor invasion, metastasis, or induction of apoptosis [213]. It is localized in the nucleus of proliferating cells and binds to the eukaryotic initiation factor-4A (eIF4A) or eukaryotic translation initiation factor 4 G (eIF4G), inhibiting translation [214,215]. PDCD4 controls the inhibition of cancer invasion by regulating the expression of mitogen-activated protein 4 kinases 1 or urokinase plasminogen activator receptor [216]. It is also responsible for the inhibition of the Y-box binding protein expression and connecting with the DNA-binding domain of the TWIST1 gene [217].
PDCD4 is one factor responsible for the induction of carcinogenesis [218]. Yu et al. showed that the downregulation of PDCD4 expression resulted in a decreased level of E-cadherin, increased TWIST1, and vimentin levels in gastric cancerous tissues [219]. PDCD4 downregulation and subsequent alterations in epithelial-mesenchymal markers were induced by CagA-positive H. pylori strain infection. PDCD4 overexpression reduced EMT. MicroRNA-21 overexpression in gastric cancer modulates the expression of the tumor suppressors phosphatase and tensin homolog (PTEN) and PDCD4, altering molecular pathways associated with cell growth, invasion, migration, and apoptosis [220].
It has been reported that H. pylori infection leads to the aberrant expression of LAPTM4B in gastric epithelial cells, which eventually induces the EMT process and further malignant transformation [225]. Researchers have observed that LAPTM4B upregulation by H. pylori infection increases the levels of EMT markers (N-cadherin, vimentin) while lowering E-cadherin levels and dysregulating cell-cell junctions. Additionally, there was a noticeable increase of ZEB1, β-catenin, Snail, and Slug markers, whereas tight junction protein-1 (zonula occludens-1, ZO-1) level was decreased. LAPTM4B seems to be a factor strictly associated with EMT induction since it is responsible for regulating cells' filopodia, increasing motility of gastric cancer cells, and a higher invasion potential [226]. Overexpression of LAPTM4B results in poorer prognosis in various cancers [227].
Krzysiek-Maczka et al. observed the overexpression of mesenchymal markers (α-SMA, N-cadherin, vimentin, Snail, Twist) and lowered levels of E-cadherin, antiapoptotic B-cell lymphoma 2 (Bcl-2), and proliferative marker Ki-67 protein within fibroblasts [246]. CagA+VacA+ H. pylori infection resulted in the aberrant apoptosis process, an increased level of collagen production, and impaired fibroblast and myofibroblast differentiation into CAFs. This induced desmoplastic reactions and EMT within gastric cells, promoting carcinogenesis. The increased levels of β1-integrin and COX-2, which are crucial in invasion, metastasis, and angiogenesis, enhanced the migration properties of the infected cells. CagA+Vac+ H. pylori infection results in the upregulation of HIF-1α and tenascin-C (TNC), enhancing EMT and tumor progression, the release of proangiogenic factors, and further CAFs activation [250][251][252]. CagA+Vac+ H. pylori infection activates gastric fibroblasts and induces the release of TGF-β [253].
Matrix metalloproteinase-7 (MMP-7) is an enzyme responsible for the degradation of adherence between cells in the extracellular matrix, and overexpression of MMP-7 induces a more aggressive course of gastric cancer [270][271][272][273]. MMP-7 and HB-EGF expression is upregulated by H. pylori infection and is associated with EMT [274,275]. Likewise, MMP-7 overexpression is because of increased levels of gastrin, activator protein 1 (AP-1), and NF-kB induced by H. pylori infection [276][277][278][279]. Levels of MMP-7 might correlate with the degree of rehabilitation after treatment and poor prognosis in gastric cancer survival [270,280].
Yin et al. showed that excessive gastrin secretion due to H. pylori infection results in the increased release of MMP-7 and soluble HB-EGF, promoting EMT [281]. HB-EGF might promote tumor growth and EMT by IL-4-conditioned media from macrophages [282]. Besides HB-EGF, other ErbB ligands (transforming growth factor-α (TGF-α) and amphiregulin (AREG)) have a prognostic impact on gastric cancer and might make up potential targets in cancer therapy [283][284][285]. Furthermore, trastuzumab sensitivity in gastric cancer can be predicted by HB-EGF expression [286]. Gastric cancer invasion can be increased by nuclear translocation of the cytoplasmic domain of HB-EGF [287,288]. However, Krakowiak et al. showed that MMP-7 might limit H. pylori-induced inflammation and damage via M1 macrophage polarization suppression [289].

Stem Cells
Mesenchymal stem cells (MSCs) present a multipotent potential to differentiate into various cell types, while keeping a capacity for self-renewal [290,291]. MSCs are recruited to the areas of H. pylori infection, neoplasia, or cancer, being involved in the migration properties of gastric cancer cells [292][293][294]. MSCs promote EMT, tumor growth, and metastasis [295,296]. Nanog Homeobox Retrogene P8 (NANOGP8) makes up the main regulator of gastric CSCs [297].

Conclusions
The association between H. pylori infection and gastric carcinogenesis remains controversial. In fact, some mechanisms are yet not fully understood. H. pylori infection seems to play an important role in terms of EMT induction within gastric mucosa (Figure 2).

Conclusions
The association between H. pylori infection and gastric carcinogenesis remains controversial. In fact, some mechanisms are yet not fully understood. H. pylori infection seems to play an important role in terms of EMT induction within gastric mucosa (Figure 2). The severity and progression of gastric cancer depends on the presence of specific H. pylori virulence factors. Likewise, cellular components that are associated with EMT progression can be influenced by H. pylori infection (Table 1). EMT and its progression depend on the particular strain of H. pylori along with its properties and virulence factors (Table 1). Specific strains may promote EMT with various intensity, thus influencing the eventual outcome of gastric cancer differently. Even though most known virulence factors, along with cellular components and their mechanisms of action, have been studied, more research should be performed to investigate the relationships between those factors, as well as to search for unknown processes and aspects. Notwithstanding, there is potential for further pharmacological research since all virulence factors constitute potential molecular targets for new medications. This may eventually provide new treatment strategies for gastric cancer, along with the possible eradication of H. pylori during the early stages of infection, which could significantly decrease the number of gastric cancer incidents. The severity and progression of gastric cancer depends on the presence of specific H. pylori virulence factors. Likewise, cellular components that are associated with EMT progression can be influenced by H. pylori infection (Table 1). EMT and its progression depend on the particular strain of H. pylori along with its properties and virulence factors (Table 1). Specific strains may promote EMT with various intensity, thus influencing the eventual outcome of gastric cancer differently. Even though most known virulence factors, along with cellular components and their mechanisms of action, have been studied, more research should be performed to investigate the relationships between those factors, as well as to search for unknown processes and aspects. Notwithstanding, there is potential for further pharmacological research since all virulence factors constitute potential molecular targets for new medications. This may eventually provide new treatment strategies for gastric cancer, along with the possible eradication of H. pylori during the early stages of infection, which could significantly decrease the number of gastric cancer incidents.  Funding: This research received no external funding.

Conflicts of Interest:
The author declares no conflict of interest.