The stomach is an important digestive organ with various biological functions. However, because of the complexity of its cellular and glandular composition, its precise cellular biology has yet to be elucidated. In this study, we conducted single-cell RNA sequencing (scRNA-seq) and subcellular-level spatial transcriptomics analysis of the human stomach and constructed the largest dataset to date: a stomach encyclopedia. This dataset consists of approximately 380,000 cells from scRNA-seq and the spatial transcriptome, enabling integrated analyses of transcriptional and spatial information of gastric and metaplastic cells. This analysis identified LEFTY1 as an uncharacterized stem cell marker, which was confirmed through lineage tracing analysis. A wide variety of cell-cell interactions between epithelial and stromal cells, including PDGFRA+BMP4+WNT5A+ fibroblasts, was highlighted in the developmental switch of intestinal metaplasia. Our extensive dataset will function as a fundamental resource in investigations of the stomach, including studies of development, aging, and carcinogenesis.

Solanine is the main component of the plant Solanum, which has been shown to provide growth-limiting activities in a variety of human cancers. However, little is known about its function in gastric cancer (GC).

We investigated the effect of solanine on GC in vivo and in vitro. The inhibition rate of solanine on the tumor was observed by constructing a subcutaneous tumor in nude mice. Morphological changes were analyzed with H&E staining. The expression of ATF4 was detected by IF analysis. MTT assays, EdU staining, and colony formation assays were used to detect the inhibition rate of solanine on GC cells. Matrigel transwells were used to detect the invasion of GC cells. Cell migration was measured using the wound healing assay. The flow cytometric analysis was used to monitor changes in the cell cycle and cell apoptosis. Western blotting was used to detect major proteins in cells and tumors.

Solanine suppressed gastric tumorigenesis. Solanine also inhibited the proliferation, invasion and mitigation of GC cells, and induced cell cycle arrest and apoptosis in vitro. Moreover, the growth-limiting activities of solanine in gastric cancer were related to the suppression of the AAMDC/MYC/ATF4/Sesn2 pathway-mediated autophagy. Overexpression of AAMDC reversed the inhibitory effect of solanine on autophagy and gastric cancer.

In summary, our findings indicate that solanine confers growth-limiting activities by deactivating the AAMDC-regulated autophagy in gastric cancer.

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression.NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.

In women, serum levels of CTSB, GKN2, LIPF, LIPFG, AZGP1, TOP2A and PGA4 are proposed as predictive markers of gastric cancer. It is unknown whether GKN1 expression varies with the sex of patients with chronic gastritis or gastric cancer. We studied 36 patients with histopathological diagnosis of chronic gastritis from the state of Guerrero, Mexico. PCR was performed for H. pylori detection and GKN1 expression was determined by RT-qPCR and western blot. GKN1 mRNA expression was significantly lower in patients with chronic follicular gastritis than in those with chronic chemical gastritis (p = 0.00071). The mRNA and protein level of expression of GKN1 were significantly lower in women with chronic follicular gastritis than in men with the same condition (p = 0.0279 and p = 0.0014, respectively); the lowest levels of GKN1 were detected in women with H. pylori-positive follicular gastritis (p = 0.0175 and p = 0.0111, respectively). Through a bioinformatic analysis, estrogen response elements were identified in the GKN1 promoter.

Gastrokine 1 (GKN1) is a protein expressed on the surface mucosa cells of the gastric antrum and fundus, which contributes to maintaining gastric homeostasis, inhibits inflammation and is a tumor suppressor. The expression of GKN1 decreases in mucosa that are either inflamed or infected by Helicobacter pylori, and is absent in gastric cancer. The measurement of circulating GKN1 concentration, the protein itself, or the mRNA in gastric tissue may be of use for the early diagnosis of cancer. The mechanisms that modulate the deregulation or silencing of GKN1 expression have not been completely described. The modification of histones, methylation of the GKN1 promoter, or proteasomal degradation of the protein have been detected in some patients; however, these mechanisms do not completely explain the absence of GKN1 or the reduction in GKN1 levels. Only NKX6.3 transcription factor has been shown to be a positive modulator of GKN1 transcription, although others also have an affinity with sequences in the promoter of this gene. While microRNAs (miRNAs) are able to directly or indirectly regulate the expression of genes at the post‑transcriptional level, the involvement of miRNAs in the regulation of GKN1 has not been reported. The present review analyzes the information reported on the determination of GKN1 expression and the regulation of its expression at the transcriptional, post‑transcriptional and post‑translational levels; it proposes an integrated model that incorporates the regulation of GKN1 expression via transcription factors and miRNAs in H. pylori infection.

The accepted paradigm for intestinal-type gastric cancer pathogenesis is a multistep progression from chronic gastritis induced by Helicobacter pylori (H. pylori) to gastric atrophy, intestinal metaplasia, dysplasia and ultimately gastric cancer. The genetic and molecular mechanisms underlying disease progression are still not completely understood as only a fraction of colonized individuals ever develop neoplasia suggesting that bacterial, host and environmental factors are involved. MicroRNAs are noncoding RNAs that may influence H. pylori-related pathology through the regulation of the transcription and expression of various genes, playing an important role in inflammation, cell proliferation, apoptosis and differentiation. Indeed, H. pylori have been shown to modify microRNA expression in the gastric mucosa and microRNAs are involved in the immune host response to the bacteria and in the regulation of the inflammatory response. MicroRNAs have a key role in the regulation of inflammatory pathways and H. pylori may influence inflammation-mediated gastric carcinogenesis possibly through DNA methylation and epigenetic silencing of tumor suppressor microRNAs. Furthermore, microRNAs influenced by H. pylori also have been found to be involved in cell cycle regulation, apoptosis and epithelial-mesenchymal transition. Altogether, microRNAs seem to have an important role in the progression from gastritis to preneoplastic conditions and neoplastic lesions and since each microRNA can control the expression of hundreds to thousands of genes, knowledge of microRNAs target genes and their functions are of paramount importance. In this article we present a comprehensive review about the role of microRNAs in H. pylori gastric carcinogenesis, identifying the microRNAs downregulated and upregulated in the infection and clarifying their biological role in the link between immune host response, inflammation, DNA methylation and gastric carcinogenesis.

Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.

Gastrokine 1 (GKN1) is a stomach-specific protein important in the replenishment of the surface lumen epithelial cell layer and in maintaining mucosal integrity. A role in cell proliferation and differentiation has also been hypothesized. Despite these findings, the function(s) as well as the cellular localization of GKN1 in the cellular machinery are currently not clarified. The investigation of subcellular localization of GKN1 in gastric cancer cells can provide insights into its potential cellular roles. Subcellular fractions of gastric cancer cells (AGS) transfected with full-length GKN1 (flGKN1) or incubated with recombinant GKN1 (rGKN1) lacking the first 20 amino acids at N-terminal were analyzed by Western blot and confocal microscopy and compared with those from normal gastric tissue. Wild type GKN1 (wtGKN1) and flGKN1 were revealed in the cytoplasm and in the membrane fractions of gastric cells, whereas rGKN1 was revealed in the cytoplasmic fractions, but a high amount was detected in the membrane pellet of the AGS lysate. The cellular distribution of GKN1 was also confirmed by confocal microscopy. The purified protein was also used to highlight its possible association with actin through confocal microscopy, pelleting assay, and size-exclusion chromatography. GKN1 co-localizes with actin in normal gastric tissue, but no direct interaction was observed between the two proteins in vitro. Most likely, GKN1 indirectly participates in actin stabilization since its overexpression in gastric cancer cells strongly increases the expression of tight and adherens junction proteins.

The present study aims to investigate whether gastrokine 1 (GKN1) induces senescence and apoptosis in gastric cancer cells by regulating telomere length and telomerase activity. Telomere length, telomerase activity, and hTERT expression decreased significantly in AGSGKN1 and MKN1GKN1 cells. Both stable cell lines showed increased expression of TRF1 and reduced expression of the hTERT and c-myc proteins. In addition, TRF1 induced a considerable decrease in cell growth, telomerase activity, and expression of hTERT mRNA and protein. GKN1 completely counteracted the effects of c-myc on cell growth, telomere length, and telomerase activity. Interestingly, GKN1 directly bound to c-myc and down-regulated its expression as well as inhibited its binding to the TRF1 protein and a hTERT promoter. Furthermore, GKN1 triggered senescence, followed by apoptosis via up-regulating the p53, p21, p27, and p16 proteins and down-regulating Skp2. Telomere length in 35 gastric cancers was shortened significantly compared with the corresponding gastric mucosae, whereas GKN1 expression was inversely correlated with telomere length and c-myc and hTERT mRNA expression. Taken together, these results suggest that GKN1 may shorten telomeres by acting as a potential c-myc inhibitor that eventually leads to senescence and apoptosis in gastric cancer cells.

Homeostatic imbalance between cell proliferation and death in gastric mucosal epithelia may lead to gastritis and gastric cancer. Despite abundant gastrokine 1 (GKN1) expression in the normal stomach, the loss of GKN1 expression is frequently detected in gastric mucosa infected with Helicobacter pylori, as well as in intestinal metaplasia and gastric cancer tissues, suggesting that GKN1 plays an important role in gastric mucosal defense, and the gene functions as a gastric tumor suppressor. In the stomach, GKN1 is involved in gastric mucosal inflammation by regulating cytokine production, the nuclear factor-κB signaling pathway, and cyclooxygenase-2 expression. GKN1 also inhibits the carcinogenic potential of H. pylori protein CagA by binding to it, and up-regulates antioxidant enzymes. In addition, GKN1 reduces cell viability, proliferation, and colony formation by inhibiting cell cycle progression and epigenetic modification by down-regulating the expression levels of DNMT1 and EZH2, and DNMT1 activity, and inducing apoptosis through the death receptor-dependent pathway. Furthermore, GKN1 also inhibits gastric cancer cell invasion and metastasis via coordinated regulation of epithelial mesenchymal transition-related protein expression, reactive oxygen species production, and PI3K/Akt signaling pathway activation. Although the modes of action of GKN1 have not been clearly described, recent limited evidence suggests that GKN1 acts as a gastric-specific tumor suppressor. This review aims to discuss, comment, and summarize the recent progress in the understanding of the role of GKN1 in gastric cancer development and progression.

Epstein-Barr Virus (EBV) latently infects ~10% of gastric carcinomas (GC). Epstein-Barr Nuclear Antigen 1 (EBNA1) is expressed in EBV-associated GC, and can bind host DNA, where it may impact cellular gene regulation. Here, we show that EBNA1 binds directly to DNA upstream of the divergently transcribed GC-specific tumor suppressor genes gastrokine 1 (GKN1) and gastrokine 2 (GKN2).

We use ChIP-Seq, ChIP-qPCR, and EMSA to demonstrate that EBNA1 binds directly to the GKN1 and GKN2 promoter locus. We generate AGS-EBV, and AGS-EBNA1 cell lines to study the effects of EBNA1 on GKN1 and GKN2 mRNA expression with or without 5' azacytidine treatment.

We show that gastrokine genes are transcriptionally silenced by DNA methylation. We also show that latent EBV infection further reduces GKN1 and GKN2 expression in AGS gastric carcinoma cells, and that siRNA depletion of EBNA1 partially alleviates this repression. However, ectopic expression of EBNA1 slightly increased GKN1 and GKN2 basal mRNA levels, but reduced their responsiveness to demethylating agent.

These findings demonstrate that EBNA1 binds to the divergent promoter of the GKN1 and GKN2 genes in GC cells, and suggest that EBNA1 contributes to the complex transcriptional and epigenetic deregulation of the GKN1 and GKN2 tumor suppressor genes in EBV positive GC.

Interleukin-4 (IL-4) -524C > T polymorphism has been implicated to alter the risk of colorectal cancer (CRC), but the results are controversial. The objective of this study was to quantitatively evaluate the association between IL-4 -524C > T polymorphism and CRC risk. A comprehensive search was conducted to identify all eligible studies of IL-4 -524C > T polymorphism and CRC risk. Statistical analysis was performed with Review Manager 5.0 and Stata 11. A total of 5 case-control studies, including 1,224 cases and 1,551 controls, were included. The combined results based on all eligible studies suggested that IL-4 -524C > T polymorphism was not associated with CRC susceptibility. When stratifying for race, the data showed that the IL-4 -524C > T polymorphism was also not associated with an increased CRC susceptibility in Caucasians. Our study suggests that IL-4 -524C > T polymorphism may be not associated with an increased CRC susceptibility.

Accessing localized proteomic profiles has emerged as a fundamental strategy to understand the biology of diseases, as recently demonstrated, for example, in the context of determining cancer resection margins with improved precision. Here, we analyze a gastric cancer biopsy sectioned into 10 parts, each one subjected to MudPIT analysis. We introduce a software tool, named Shotgun Imaging Analyzer and inspired in MALDI imaging, to enable the overlaying of a protein's expression heat map on a tissue picture. The software is tightly integrated with the NeXtProt database, so it enables the browsing of identified proteins according to chromosomes, quickly listing human proteins never identified by mass spectrometry (i.e., the so-called missing proteins), and the automatic search for proteins that are more expressed over a specific region of interest on the biopsy, all of which constitute goals that are clearly well-aligned with those of the C-HPP. Our software has been able to highlight an intense expression of proteins previously known to be correlated with cancers (e.g., glutathione S-transferase Mu 3), and in particular, we draw attention to Gastrokine-2, a "missing protein" identified in this work of which we were able to clearly delineate the tumoral region from the "healthy" with our approach. Data are available via ProteomeXchange with identifier PXD000584.

Published studies on the association between interleukin-4 (IL-4) -590C>T polymorphism and gastric cancer risk have yielded conflicting results. Thus, a meta-analysis of published studies was performed to assess the possible association. All eligible studies of -590C>T polymorphism and gastric cancer risk were collected from the PubMed, the Cochrane Library, and the Embase electronic databases. Statistical analyses were performed by Review Manager 5.0 and Stata 11.0. When all groups were pooled, we did not detect a significant association of -590C>T polymorphism with gastric cancer risk. When stratifying for race, there was a significant association between -590C>T polymorphism and decreased gastric cancer risk under dominant model and allelic model in the subgroup of Caucasians. However, significant association was absent in Asians. Based on our meta-analysis, -590C>T polymorphism was associated with a lower gastric cancer risk under dominant model and allelic model in Caucasians. Nevertheless, we suggest that further studies should be made to confirm these findings.

In a screen for genes expressed specifically in gastric mucous neck cells, we identified GKN3, the recently discovered third member of the gastrokine family. We present confirmatory mouse data and novel porcine data showing that mouse GKN3 expression is confined to mucous cells of the corpus neck and antrum base and is prominently expressed in metaplastic lesions. GKN3 was proposed originally to be expressed in some human populations and a pseudogene in others. To investigate that hypothesis, we studied human GKN3 evolution in the context of its paralogous genomic neighbors, GKN1 and GKN2. Haplotype analysis revealed that GKN3 mimics GKN2 in patterns of exonic SNP allocation, whereas GKN1 appeared to be more stringently selected. GKN3 showed signatures of both directional selection and population based selective sweeps in humans. One such selective sweep includes SNP rs10187256, originally identified as an ancestral tryptophan to premature STOP codon mutation. The derived (nonancestral) allele went to fixation in Asia. We show that another SNP, rs75578132, identified 5 bp downstream of rs10187256, exhibits a second selective sweep in almost all Europeans, some Latinos, and some Africans, possibly resulting from a reintroduction of European genes during African colonization. Finally, we identify a mutation that would destroy the splice donor site in the putative exon3-intron3 boundary, which occurs in all human genomes examined to date. Our results highlight a stomach-specific human genetic locus, which has undergone various selective sweeps across European, Asian, and African populations and thus reflects geographic and ethnic patterns in genome evolution.

During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.