Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Coevolution of the ileum with Brk/Ptk6 family kinases confers robustness to ileal homeostasis.

Brk/Ptk6, Srms, and Frk constitute a Src-related but distinct family of tyrosine kinases called Brk family kinases (BFKs) in higher vertebrates. To date, however, their biological roles have remained largely unknown. In this study, we generated BFK triple-knockout (BFK/TKO) mice lacking all BFK members using CRISPR/Cas9-mediated genome editing. BFK/TKO mice exhibited impaired intestinal homeostasis, represented by a reduced stem/progenitor cell population and defective recovery from radiation-induced severe mucosal damage, specifically in the ileum, which is the most distal segment of the small intestine. RNA-seq analysis revealed that BFK/TKO ileal epithelium showed markedly elevated IL-22/STAT3 signaling, resulting in the aberrant activation of mucosal immune response and altered composition of the ileal microbiota. Since single- or double-knockout of BFK genes did not elicit such abnormalities, BFKs may redundantly confer robust homeostasis to the ileum, the most recently added intestinal segment that plays crucial roles in nutrient absorption and mucosal immunity. Given that BFK diversification preceded the appearance of the ileum in vertebrate phylogeny, the present study highlights the coevolution of genes and organs, the former of which shapes up the latter in higher vertebrates.

The Helicobacter pylori CagA oncoprotein disrupts Wnt/PCP signaling and promotes hyperproliferation of pyloric gland base cells.

Helicobacter pylori strains that deliver the oncoprotein CagA into gastric epithelial cells are the major etiologic agents of upper gastric diseases including gastric cancer. CagA promotes gastric carcinogenesis through interactions with multiple host proteins. Here, we show that CagA also disrupts Wnt-dependent planar cell polarity (Wnt/PCP), which orients cells within the plane of an epithelium and coordinates collective cell behaviors such as convergent extension to enable epithelial elongation during development. Ectopic expression of CagA in Xenopus laevis embryos impaired gastrulation, neural tube formation, and axis elongation, processes driven by convergent extension movements that depend on the Wnt/PCP pathway. Mice specifically expressing CagA in the stomach epithelium had longer pyloric glands and mislocalization of the tetraspanin proteins VANGL1 and VANGL2 (VANGL1/2), which are critical components of Wnt/PCP signaling. The increased pyloric gland length was due to hyperproliferation of cells at the gland base, where Lgr5+ stem and progenitor cells reside, and was associated with fewer differentiated enteroendocrine cells. In cultured human gastric epithelial cells, the N terminus of CagA interacted with the C-terminal cytoplasmic tails of VANGL1/2, which impaired Wnt/PCP signaling by inducing the mislocalization of VANGL1/2 from the plasma membrane to the cytoplasm. Thus, CagA may contribute to the development of gastric cancer by subverting a Wnt/PCP-dependent mechanism that restrains pyloric gland stem cell proliferation and promotes enteroendocrine differentiation.

Exploring Allosteric Inhibitors of Protein Tyrosine Phosphatases Through High-Throughput Screening.

High-throughput screening (HTS) using a natural or synthetic chemical or natural product library is a powerful technique for discovering novel small-molecular-weight compounds in order to develop drugs that specifically inhibit or activate molecular targets, malfunctioning of which underlies the development of diseases, especially malignant neoplasms. In contrast to a large number of successful cases in obtaining inhibitors against protein tyrosine kinases (PTKs) using HTS, however, the development of selective inhibitors for protein tyrosine phosphatases (PTPs) has lagged since PTP family members share highly conserved catalytic domain structures. Here, in this chapter we describe a novel method for exploring seed compounds of allosteric PTP inhibitors from a chemical/natural product library through HTS.

Diet-mediated constitutive induction of novel IL-4+ ILC2 cells maintains intestinal homeostasis in mice.

Group 2 innate lymphoid cells (ILC2s) expressing IL-5 and IL-13 are localized at various mucosal tissues and play critical roles in the induction of type 2 inflammation, response to helminth infection, and tissue repair. Here, we reveal a unique ILC2 subset in the mouse intestine that constitutively expresses IL-4 together with GATA3, ST2, KLRG1, IL-17RB, and IL-5. In this subset, IL-4 expression is regulated by mechanisms similar to but distinct from those observed in T cells and is partly affected by IL-25 signaling. Although the absence of the microbiota had marginal effects, feeding mice with a vitamin B1-deficient diet compromised the number of intestinal IL-4+ ILC2s. The decrease in the number of IL-4+ ILC2s caused by the vitamin B1 deficiency was accompanied by a reduction in IL-25-producing tuft cells. Our findings reveal that dietary vitamin B1 plays a critical role in maintaining interaction between tuft cells and IL-4+ ILC2s, a previously uncharacterized immune cell population that may contribute to maintaining intestinal homeostasis.

Helicobacter pylori, Homologous-Recombination Genes, and Gastric Cancer.

BACKGROUND: Helicobacter pylori infection is a well-known risk factor for gastric cancer. However, the contribution of germline pathogenic variants in cancer-predisposing genes and their effect, when combined with H. pylori infection, on the risk of gastric cancer has not been widely evaluated. METHODS: We evaluated the association between germline pathogenic variants in 27 cancer-predisposing genes and the risk of gastric cancer in a sample of 10,426 patients with gastric cancer and 38,153 controls from BioBank Japan. We also assessed the combined effect of pathogenic variants and H. pylori infection status on the risk of gastric cancer and calculated the cumulative risk in 1433 patients with gastric cancer and 5997 controls from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). RESULTS: Germline pathogenic variants in nine genes (APC, ATM, BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, and PALB2) were associated with the risk of gastric cancer. We found an interaction between H. pylori infection and pathogenic variants in homologous-recombination genes with respect to the risk of gastric cancer in the sample from HERPACC (relative excess risk due to the interaction, 16.01; 95% confidence interval [CI], 2.22 to 29.81; P = 0.02). At 85 years of age, persons with H. pylori infection and a pathogenic variant had a higher cumulative risk of gastric cancer than noncarriers infected with H. pylori (45.5% [95% CI, 20.7 to 62.6] vs. 14.4% [95% CI, 12.2 to 16.6]). CONCLUSIONS: H. pylori infection modified the risk of gastric cancer associated with germline pathogenic variants in homologous-recombination genes. (Funded by the Japan Agency for Medical Research and Development and others.).

Impact of the Helicobacter pylori Oncoprotein CagA in Gastric Carcinogenesis.

Helicobacter pylori CagA is the first and only bacterial oncoprotein etiologically associated with human cancer. Upon delivery into gastric epithelial cells via bacterial type IV secretion, CagA acts as a pathogenic/pro-oncogenic scaffold that interacts with and functionally perturbs multiple host proteins such as pro-oncogenic SHP2 phosphatase and polarity-regulating kinase PAR1b/MARK2. Although H. pylori infection is established during early childhood, gastric cancer generally develops in elderly individuals, indicating that oncogenic CagA activity is effectively counteracted at a younger age. Moreover, the eradication of cagA-positive H. pylori cannot cure established gastric cancer, indicating that H. pylori CagA-triggered gastric carcinogenesis proceeds via a hit-and-run mechanism. In addition to its direct oncogenic action, CagA induces BRCAness, a cellular status characterized by replication fork destabilization and loss of error-free homologous recombination-mediated DNA double-strand breaks (DSBs) by inhibiting cytoplasmic-to-nuclear localization of the BRCA1 tumor suppressor. This causes genomic instability that leads to the accumulation of excess mutations in the host cell genome, which may underlie hit-and-run gastric carcinogenesis. The close connection between CagA and BRCAness was corroborated by a recent large-scale case-control study that revealed that the risk of gastric cancer in individuals carrying pathogenic variants of genes that induce BRCAness (such as BRCA1 and BRCA2) dramatically increases upon infection with cagA-positive H. pylori. Accordingly, CagA-mediated BRCAness plays a crucial role in the development of gastric cancer in conjunction with the direct oncogenic action of CagA.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Transgenically expressed Helicobacter pylori CagA in vascular endothelial cells accelerates arteriosclerosis in mice.

Arteriosclerosis is intimately associated with cardiovascular diseases. Recently, evidence accumulated that infection with Helicobacter pylori cagA-positive strains, which causes gastritis, peptic ulceration, and gastric cancer, is also involved in the development of arteriosclerosis. The cagA-encoded CagA protein is injected into the attached gastric epithelial cells via the type IV secretion system. We previously showed that CagA-containing exosomes are secreted from CagA-injected gastric epithelial cells and enter the systemic blood circulation, delivering CagA into endothelial cells. In the present study, transgenic mice were established in which CagA was selectively expressed in endothelial cells by Cre-loxP system. Treatment of the mice with a high-fat diet revealed that atherogenic lesions were induced in mice expressing CagA in vascular endothelial cells but not in CagA-nonexpressing mice. To investigate the effects of CagA on endothelial cells, we also established conditional CagA-expressing human vascular endothelial cells using the Tet-on system. Upon induction of CagA, a dramatic change in cell morphology was observed that was concomitantly associated with the loss of the endothelial cells to form tube-like structures. Induction of CagA also activated the pro-inflammatory transcription factor STAT3. Thus, exosome-delivered CagA deregulates signals that activates STAT3 in endothelial cells, which accelerates inflammation that promotes arteriosclerosis/atherosclerosis.

Mouse Gastric Epithelial Cells Resist CagA Delivery by the Helicobacter pylori Type IV Secretion System.

The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ-Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.

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

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).

The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis.

Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.

Protocol for visualizing conditional interaction between transmembrane and cytoplasmic proteins.

This protocol visualizes dynamic interaction between a transmembrane protein and an intracellular protein induced by clusterization/oligomerization of the transmembrane protein. Association-dissociation of the intracellular region of the transmembrane protein with cytoplasmic protein(s) is detected by proximity ligation assay. Since a transmembrane protein often resists extraction, biochemical analysis of its dynamic interaction with cytoplasmic effectors is cumbersome. This protocol quantitatively visualizes protein-protein interaction occurring in the membrane periphery, providing a powerful tool to elucidate signal transduction across the membrane. For complete details on the use and execution of this protocol, please refer to Ooki et al. (2019).

Alternative splicing reverses the cell-intrinsic and cell-extrinsic pro-oncogenic potentials of YAP1.

In addition to acting as a transcriptional co-activator, YAP1 directly mediates translocalization of the pro-oncogenic phosphatase SHP2 from the cytoplasm to nucleus. In the cytoplasm, SHP2 potentiates RAS-ERK signaling, which promotes cell proliferation and cell motility, whereas in the nucleus, it mediates gene regulation. As a result, elucidating the details of SHP2 trafficking is important for understanding its biological roles, including in cancer. YAP1 comprises multiple splicing isoforms defined in part by the presence (as in YAP1-2γ) or absence (as in YAP1-2α) of a γ-segment encoded by exon 6 that disrupts a critical leucine zipper. Although the disruptive segment is known to reduce co-activator function, it is unclear how this element impacts the physical and functional relationships between YAP1 and SHP2. To explore this question, we first demonstrated that YAP1-2γ cannot bind SHP2. Nevertheless, YAP1-2γ exhibits stronger mitogenic and motogenic activities than does YAP1-2α because the YAP1-2α-mediated delivery of SHP2 to the nucleus weakens cytoplasmic RAS-ERK signaling. However, YAP1-2γ confers less in vivo tumorigenicity than does YA1-2α by recruiting tumor-inhibitory macrophages. Mechanistically, YAP1-2γ transactivates and the YAP1-2α-SHP2 complex transrepresses the monocyte/macrophage chemoattractant CCL2 Thus, cell-intrinsic and cell-extrinsic pro-oncogenic YAP1 activities are inversely regulated by alternative splicing of exon 6. Notably, oncogenic KRAS down-regulates the SRSF3 splicing factor that prevents exon 6 skipping, thereby creating a YAP1-2α-dominant situation that supports a "cold" immune microenvironment.

Hyaluronan Degradation Promotes Cancer via Hippo-YAP Signaling: An Intervention Point for Cancer Therapy.

High-molecular-weight hyaluronan acts as a ligand of the tumor-suppressive Hippo signal, whereas degradation of hyaluronan from a high-molecular-weight form to a low-molecular-weight forms by hyaluronidase 2 inhibits Hippo signal activation and thereby activates the pro-oncogenic transcriptional coactivator yes-associated protein (YAP), which creates a cancer-predisposing microenvironment and drives neoplastic transformation of cells through both cell-autonomous and non-cell-autonomous mechanisms. In fact, accumulation of low-molecular-weight hyaluronan in tissue stroma is observed in many types of cancers. Since inhibition of YAP activity suppresses tumor growth in vivo, pharmacological intervention of the Hippo-YAP signal is an attractive approach for future drug development. In this review, pharmacological intervention of excessive hyaluronan degradation as a novel approach for inhibition of the Hippo-YAP signal is also discussed. Development of hyaluronidase inhibitors may provide novel therapeutic strategies for human malignant tumors.

Helicobacter pylori CagA oncoprotein interacts with SHIP2 to increase its delivery into gastric epithelial cells.

Chronic infection with Helicobacter pylori cagA-positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA-encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) segments (EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D), which are present in various numbers and combinations in its C-terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain-containing host cell proteins, including the prooncogenic SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain-containing phosphatidylinositol 5'-phosphatase, is a hitherto undiscovered CagA-binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA-specific EPIYA-C segment or East Asian CagA-specific EPIYA-D segment through the SH2 domain in a tyrosine phosphorylation-dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA-C than to EPIYA-D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4-diphosphate [PI(3,4)P2 ]. The CagA-SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA-deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori-host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.

Molecular anatomy and pathogenic actions of Helicobacter pylori CagA that underpin gastric carcinogenesis.

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer. The cagA gene product, CagA, is delivered into gastric epithelial cells via the bacterial type IV secretion system. Delivered CagA then undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in its C-terminal region and acts as an oncogenic scaffold protein that physically interacts with multiple host signaling proteins in both tyrosine phosphorylation-dependent and -independent manners. Analysis of CagA using in vitro cultured gastric epithelial cells has indicated that the nonphysiological scaffolding actions of CagA cell-autonomously promote the malignant transformation of the cells by endowing the cells with multiple phenotypic cancer hallmarks: sustained proliferation, evasion of growth suppressors, invasiveness, resistance to cell death, and genomic instability. Transgenic expression of CagA in mice leads to in vivo oncogenic action of CagA without any overt inflammation. The in vivo oncogenic activity of CagA is further potentiated in the presence of chronic inflammation. Since Helicobacter pylori infection triggers a proinflammatory response in host cells, a feedforward stimulation loop that augments the oncogenic actions of CagA and inflammation is created in CagA-injected gastric mucosa. Given that Helicobacter pylori is no longer colonized in established gastric cancer lesions, the multistep nature of gastric cancer development should include a "hit-and-run" process of CagA action. Thus, acquisition of genetic and epigenetic alterations that compensate for CagA-directed cancer hallmarks may be required for completion of the "hit-and-run" process of gastric carcinogenesis.

Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer.

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly (P < 0.005) elevated continuously from intramucosal carcinoma to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus, which co-occurred in intramucosal carcinomas, were significantly (P < 0.005) increased only in multiple polypoid adenomas and/or intramucosal carcinomas. Metabolome analyses showed that branched-chain amino acids and phenylalanine were significantly (P < 0.005) increased in intramucosal carcinomas and bile acids, including deoxycholate, were significantly (P < 0.005) elevated in multiple polypoid adenomas and/or intramucosal carcinomas. We identified metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls. Our large-cohort multi-omics data indicate that shifts in the microbiome and metabolome occur from the very early stages of the development of colorectal cancer, which is of possible etiological and diagnostic importance.

Classification of Helicobacter pylori Virulence Factors: Is CagA a Toxin or Not?

Since its discovery, Helicobacter pylori has been identified as the causative agent of various gastric diseases. H. pylori produces myriads of disease-associated virulence factors. These bacterial determinants can be distinguished as cell-binding factors, immunoregulatory components, survival factors, toxins, and effector proteins. For most of these factors there is consensus about their classification. However, there is a strong dispute in the literature as to whether one of the best-studied factors, CagA, represents a toxin or not. CagA displays unique functions that are clearly different from conventional toxins, and CagA counteracts the activities of an established H. pylori toxin, VacA. Canonical toxins commonly have specific (and narrow) targets, can act even in the absence of the bacterial cell, and elicit acute damage to host cells. However, there is still no agreement on the classification of CagA. Here we discuss whether CagA acts as a toxin, and propose a classification consensus for CagA.