Helicobacter pylori‐induced DNA double‐stranded break in the development of gastric cancer

Abstract Infection with cagA‐positive Helicobacter pylori strains plays an etiological role in the development of gastric cancer. The CagA protein is injected into gastric epithelial cells through a bacterial Type IV secretion system. Inside the host cells, CagA promiscuously associates with multiple host cell proteins including the prooncogenic phosphatase SHP2 that is required for full activation of the Ras–ERK pathway. CagA–SHP2 interaction aberrantly activates SHP2 and thereby deregulates Ras–ERK signaling. Cancer is regarded as a disease of the genome, indicating that H. pylori‐mediated gastric carcinogenesis is also associated with genomic alterations in the host cell. Indeed, accumulating evidence has indicated that H. pylori infection provokes DNA double‐stranded breaks (DSBs) by both CagA‐dependent and CagA‐independent mechanisms. DSBs are repaired by either error‐free homologous recombination (HR) or error‐prone non‐homologous end joining (NHEJ) or microhomology‐mediated end joining (MMEJ). Infection with cagA‐positive H. pylori inhibits RAD51 expression while dampening cytoplasmic‐to‐nuclear translocalization of BRCA1, causing replication fork instability and HR defects (known as “BRCAness”), which collectively provoke genomic hypermutation via non‐HR‐mediated DSB repair. H. pylori also subverts multiple DNA damage responses including DNA repair systems. Infection with H. pylori additionally inhibits the function of the p53 tumor suppressor, thereby dampening DNA damage‐induced apoptosis, while promoting proliferation of CagA‐delivered cells. Therefore, H. pylori cagA‐positive strains promote abnormal expansion of cells with BRCAness, which dramatically increases the chance of generating driver gene mutations in the host cells. Once such driver mutations are acquired, H. pylori CagA is no longer required for subsequent gastric carcinogenesis (Hit‐and‐Run carcinogenesis).


| INTRODUC TI ON
Cancer, a major cause of human death worldwide, has been considered to be primarily a disease evoked by malfunctioning of the host cell genome. Many cancers including gastric cancer are recognized as inflammation-related cancers, which are generally associated with chronic viral/bacterial/parasitic infection, where inflammation and direct genotoxicity by oncogenic pathogens may be critical events that promote carcinogenesis. A causal link between viruses and particular cancers has been well accepted because several viruses possess viral oncogenes. For example, chronic infection with an RNA virus HCV plays an etiologic role in the development of HCC. At least four HCV gene products (core, NS3, NS5A, and NS5B) have been shown to deregulate oncogenic signaling pathways in the infected host cells. 1 The human retrovirus human T-cell leukemia virus type 1 (HTLV-1), which caused ATL, possesses two viral oncogenes, HBZ and tax. 2 Similarly, oncogenic DNA viruses encode oncoproteins that neutralize the function of tumor suppressors, such as p53 and pRB, in infected host cells. For instance, HBV is an etiological agent of HCC.
HBV not only uses viral DNA integration into the host genome but also inhibits p53 function by binding with viral oncoprotein HBx. 1 It is well understood that high-risk types of HPV are causative agents of cervical cancers. The HPV oncoproteins E6 and E7, which are viral oncoprotein that are responsible for initiation and progression of cervical cancer, inactivate p53 and pRB, respectively, by physical complex formation. 3 In contrast, bacteria have long been considered not to be etiologically involved in the development of human cancer. However, the results of recent epidemiological studies as well as those of molecular cell biological analyses have suggested that there is a causal link between several bacterial infections and carcinogenesis. Specifically, Helicobacter pylori has been recognized as a bacterium that is etiologically associated with the development of gastric cancer. 4,5 Genotoxic colibactin-producing pks-positive Escherichia coli has also been recognized as a causative agent of colorectal cancer. 6,7 Salmonella typhi has been associated with gallbladder cancer. 8 and Chlamydia trachomatis has been involved in the development of cervical cancer or ovarian cancer. 9,10 Although the mechanisms by which these bacteria promote neoplastic transformation of the host cells have not been fully understood, a common feature is that infection with these bacteria induces DNA DSBs in the host genome.
DSBs are the most dangerous type of DNA damage, in which the phosphate backbones of the two complementary DNA strands are broken simultaneously. Unrepaired DSBs induce apoptosis or cellular senescence, whereas misprocessing of DSBs leads to various types of mutation including insertion, deletion, and translocation, thereby causing genome instability that promotes carcinogenesis. 11 Helicobacter pylori is a micro-aerophilic Gram-negative bacterium, which was discovered by Barry Marshall and Robin Warren in 1984. 12 H. pylori colonizes the human stomach and is estimated to infect at least half of the world's human population. 5 Gastric adenocarcinoma is histopathologically divided into two major types: intestinal type and diffuse type. Long-term infection with H. pylori is intimately associated with intestinal-type gastric cancer, which proceeds through Correa's cascade with the following sequential stages: chronic gastritis, atrophy, intestinal metaplasia, and dysplasia culminating in gastric cancer. 13 The causative role of H. pylori in gastric carcinogenesis has been shown by clinical evidence that eradication of H. pylori significantly decreased the rate of gastric cancer in H. pylori-infected patients without precancerous lesions. 14 H. pylori is subdivided into cagA-positive and cagA-negative strains based on the presence or absence of the cag pathogenicity island (cagPAI). The cagPAI, an ~40-kb DNA segment, contains ~30 genes (open reading frames), which include cagA and several genes encoding components of a bacterial microsyringe, termed the Type IV secretion system (T4SS), that delivers CagA into the attached gastric epithelial cell ( Figure 1). 15 Whereas almost all the H. pylori isolates circulating in East Asian countries, including Japan, China, and Korea, are cagApositive strains, ~40% of the H. pylori isolates in other countries are cagA-negative. 16 Chronic infection with cagA-positive strains plays a critical role in the development of gastric cancer. 4,17 Consistently, the oncogenic potential of CagA has been directly demonstrated by its transgenic expression in Drosophila, zebrafish, and mouse. [18][19][20] Inside the host cells, CagA undergoes tyrosine phosphorylation by Src family kinases and c-Abl kinase. 21,22 Tyrosine-phosphorylated CagA acquires the ability to bind to SHP2 and thereby stimulates the pro-oncogenic phosphatase activity, which potentiates RAS-ERK signaling. [23][24][25] CagA also interacts with the polarity-regulating serine/threonine kinase PAR1b (also known as MARK2) in a tyrosine phosphorylation-independent manner through the CM motif, resulting in junctional and polarity defects of gastric epithelial cells. 26,27 CagA also interacts with the c-MET HGF receptor through the CM motif, thereby activating the PI3K-AKT pathway. 28 These CagA activities have been thought to play a central role in gastric carcinogenesis ( Figure 2). 5,29 In addition to CagA, H. pylori produces several virulence factors that contribute to its pathogenesis ( Figure 2). Vacuolating cytotoxin A (VacA), which is secreted from H. pylori and internalized into gastric epithelial cells by endocytosis, inducing vacuole formation and apoptosis. 30,31 Although almost all H. pylori strains possess the vacA gene, ~50% of H. pylori strains produce the toxigenic and pathogenic VacA due to gene sequence variations. 32 All H. pylori strains produce urease that catalyzes the hydrolysis of urea to yield ammonia, thereby neutralizing the acidic environment of the stomach. In vitro studies have shown that urease-mediated ammonia production disrupts tight junctions of the gastric epithelial cells. 33,34 Therefore, urease functions as both a colonization factor and a virulence factor. All H. pylori strains express γ-glutamyl transpeptidase (GGT) that catalyzes the consumption of glutamine and glutathione, thereby producing ammonia and reactive oxygen species (ROS), which play a role in the colonization of gastric mucosa. 35 Neutrophil-activating protein (NapA), produced by all the H. pylori strains, has also been identified as a virulence factor, which attracts and activates neutrophiles, thereby provoking gastric inflammation ( Figure 2). 36,37 In addition to CagA, metabolic precursors of H. pylori lipopolysac- and ADPβd-manno-heptose (ADP-heptose), are delivered into host cells through the T4SS. 38,39 In gastric epithelial cells, HBP and ADPheptose are recognized by alpha-kinase 1 (ALPK1) to activate the tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with the forkhead-associated domain (TIFA)-mediated innate immune response to elicit NF-κB activation, which triggers the production of proinflammatory cytokines such as IL-8 ( Figure 2). 39 Helicobacter pylori has been thought to promote neoplastic transformation of gastric epithelial cells via CagA-dependent deregulation of cell proliferation, as well as cell motility in conjunction with CagA-independent chronic inflammation. 40 Intriguingly, while H. pylori infection appears to be mostly essential for gastric cancer initiation, the presence of this bacterium is no longer required for the later phase of gastric carcinogenesis. Therefore, previous studies that investigated pro-mitogenic activity of delivered CagA, as well as chronic inflammation induced by H. pylori-infected gastric mucosa, do not fully explain the mechanism of the later phase of gastric carcinogenesis. In this regard, recent studies have revealed that H. pylori infection induces DSBs in the host genome, which may elicit genome instability in the infected host cells. In this review, we focus on the mechanisms of DSBs induction by H. pylori, and discuss the possible mechanisms leading to genome instability that underscores gastric carcinogenesis.

| INDUC TI ON OF DS BS BY H . PYLORI I N A C AG A-DEPENDENT MANNER
The main repair pathways used to resolve DSBs are HR, nonhomologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ). HR is an error-free pathway as it uses an homologous template for repair, whereas NHEJ and MMEJ are error-prone DSB repair pathways. 41 The tumor suppressor BRCA1 is indispensable for HR-mediated DSB repair. "BRCAness" is defined by a defect in HR-mediated DSB repair by mutational loss of BRCA1, BRCA2, or genes constituting components of the BRCA1/BRCA2 pathway. 42,43 Infection with cagA-positive H. pylori induces DSBs in gastric epithelial cells, which is concomitantly associated with reduced expression of RAD51 that protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation, while mediating HR as a recombination factor. 44 Consistently, cagA-positive H. pylori induces long noncoding RNA SNHG17 that functions as a decoy for miR-3909, thereby reducing the expression of RAD51 (Figure 3). 45 More recently, we found that CagA-mediated PAR1b kinase inhibition also gives rise to DSBs in H. pylori-infected human gastric epithelial cells. 46 Plasmid-mediated expression of CagA in gastric epithelial cells leads to DSB induction in a CM motif-dependent manner, indicating that CagA delivery on its own gives rise to DSBs. 46  In normal cells, PAR1b phosphorylates BRCA1 on S616, which is responsible for the cytoplasmic-to-nuclear translocation of BRCA1. CagA prevents S616 phosphorylation of BRCA1 through PAR1b inhibition. CagA reduces RAD51 expression via the induction of long noncoding RNA SNHG17. These CagA activities cause replication fork instability leading to DSBs and HR defects two NLS, proximal NLS (residues 503-508) and distal NLS (residues 606-615). PAR1b phosphorylates BRCA1 on S616 that is located immediately downstream of the distal NLS, which is responsible for the cytoplasmic-to-nuclear translocation of BRCA1 (Figure 3). 46 CagA prevents phosphorylation of BRCA1 on S616 by inhibiting PAR1b kinase activity, resulting in a shortage of nuclear BRCA1. Consequently, CagA elicits replication fork instability that gives rise to DSBs and, at the same time, impairs HR-mediated DSB repair, which cooperatively provoke genome instability through error-prone end joining. 46 In vivo experiments have also shown that CagA induces dysfunction of BRCA1.
Floxed cagA-transgenic mice that conditionally express the cagA transgene by tamoxifen treatment show reduced nuclear BRCA1 in the stomach mucosa upon CagA induction. Also, CagA induction in mice increases DSBs in the stomach mucosa. 46 Furthermore, loss of nuclear BRCA1 is observed in human stomach mucosa infected with chronic cagA-positive H. pylori but not in those without H. pylori infection. 46

| INDUC TI ON OF DS BS BY H . PYLORI I N A C AG A-INDEPENDENT MANNER
Helicobacter pylori induces DSBs in infected gastric epithelial cells, and the DSB induction requires bacterial T4SS and host cell β1integrin. 48

| IMPAIRMENT OF THE DNA DAMAG E RE S P ON S E BY H . PYLORI
As described above, H. pylori inhibits HR that is essential for errorfree DSB repair by depleting nuclear BRCA1 in a CagA-dependent manner. DSBs that are associated with impairment of HR factors are also induced by cagA-positive H. pylori. 44,63,64 In addition, H. pylori has been reported to impair multiple DDRs including the DNA repair system. DNA mismatch repair (MMR) corrects DNA mismatches generated during DNA replication, thereby preventing mutations from being inherited in daughter cells. 65 In vitro studies showed that H. pylori infection is significantly lower than that in H. pylori-negative control subjects. The reduced level of MGMT is due to hypermethylation of the MGMT promoter, which is rescued after H. pylori eradication. 69 These findings indicate that DNA repair is impaired during H. pylori colonization, further increasing genome instability in H. pylori-infected gastric epithelial cells ( Figure 6).
Under physiological conditions, the expression level of p53 is kept low through its ubiquitination by MDM2, an E3 ubiquitin ligase for p53, which is followed by proteasomal degradation. In response to DNA damage, however, p53 is rapidly activated via elevated protein levels and increased DNA-binding activity, causing transactivation of target genes that mediate cell cycle arrest, DNA repair, apoptosis, and cell senescence. 70 The expression of AID is highly restricted to B cells in germinal centers, in which it plays an important role for somatic hypermutation and class-switch recombination in immunoglobulin genes.
However, aberrant expression of AID in non-B cells can induce mutations that contribute to tumorigenesis. 71,72 Strikingly, the infection F I G U R E 5 Induction of DSBs through oxidative stress induced by Helicobacter pylori. VacA, γ-glutamyl-transferase (GGT), urease, and NapA contribute to ROS production that causes DSBs of gastric epithelial cells with H. pylori induces TP53 mutations through ectopic expression of AID in an NF-κB-dependent manner. 73,74 Recent studies have further shown that H. pylori can induce p53 inactivation without its mutation. MDM2 activity is regulated by serine-threonine kinase ERK and AKT that phosphorylate MDM2 at Ser166. 75,76 H. pylori activates MDM2 through ERK/AKT activation in a CagA-dependent manner, which negatively regulates p53 by increasing ubiquitination and proteasomal degradation. 77,78 It was further reported that another virulence factor, VacA, also activates MDM2 through AKT activation. 79 In addition, CagA interacts with the human tumor suppressor apoptosis-stimulating protein of p53 (ASPP2) that functions as a proapoptotic protein by associating with and activating p53. 80 CagA/ASPP2 interaction leads to cytoplasmic retention of p53 and thereby promotes proteasomal degradation of p53. 81 Moreover, H. pylori activates the transcription factor AP1 and thereby inducing N-terminally truncated p53 isoforms, Δ133p53 and Δ160p53, that inhibit p53 and p73 activities and increase the survival of infected cells (Figure 6). 82 The TP53 gene is the most frequently mutated gene in human gastric cancer and, indeed, mutations are found in intestinal metaplasia (38%), dysplasia (58%), and intestinal-type gastric cancer (71%), indicating that the TP53 mutation is intimately associated with this type of gastric cancer that typically arises after long-term H. pylori infection. Recently, we showed that sustained expression of CagA in gastric epithelial cells gives rise to SBS3 and ID6 corroborating that CagA provokes transient and reversible BRCAness in the delivered host cells, thereby causing genome instability. 46 While H. pylori infection can evoke genome instability in a CagA-independent manner, CagAdependent BRCAness greatly increases the chance of driver gene mutations.
Although cagA-positive H. pylori plays a critical role in the early phase of gastric carcinogenesis, the bacterial pathogen is no longer necessary in the carcinogenic process once key driver mutations have been introduced into cancer precursor cells. It can be speculated that additional triggers and stimulants, both cell intrinsic and cell extrinsic, may also exist in the later phase of gastric carcinogenesis. F I G U R E 6 Impairment of the DNA damage response by Helicobacter pylori. The expression levels of MLH1, MSH2, and MGMT are downregulated in H. pylori-infected gastric epithelial cells. CagA induces TP53 mutations through the aberrant expression of AID. CagA and VacA activate MDM2 through an AKT/ ERK-dependent S166 phosphorylation. CagA/ASPP2 interaction promotes the proteasomal degradation of p53. H. pylori T4SS activates AP1, and thereby induces Δ133p53 and Δ160p53 that inhibit p53 and p73 As a result, H. pylori CagA directs the neoplastic transformation of gastric epithelial cells through the Hit-and-Run mechanism. 87 In the CagA-mediated Hit-and-Run oncogenesis, cagA-positive H. pylori eradication is neither capable of inhibiting gastric cancer development, nor poly(ADP-ribose) polymerase (PARP) inhibitors, which are utilized for the treatment of cancer cells with BRCAness. 42,88 This does not work as the activity of BRCA1 is fully restored in the established gastric cancers in the absence of CagA delivery.

ACK N OWLED G M ENTS
This study was supported by Japan Society for the Promotion of

CO N FLI C T S O F I NTE R E S T
The authors have no conflict interest. Masanori Hatakeyama is the Editor-in-Chief of Cancer Science.