Advertisement

Hedgehog Signaling Links Chronic Inflammation to Gastric Cancer Precursor Lesions

  • Juanita L. Merchant
    Correspondence
    Correspondence Address correspondence to: Juanita L. Merchant, MD, PhD, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109-2200. fax: (734) 763-4686.
    Affiliations
    Department of Internal Medicine-Gastroenterology, University of Michigan, Ann Arbor, Michigan

    Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
    Search for articles by this author
  • Lin Ding
    Affiliations
    Department of Internal Medicine-Gastroenterology, University of Michigan, Ann Arbor, Michigan
    Search for articles by this author
Open AccessPublished:January 14, 2017DOI:https://doi.org/10.1016/j.jcmgh.2017.01.004
      Since its initial discovery in Drosophila, Hedgehog (HH) signaling has long been associated with foregut development. The mammalian genome expresses 3 HH ligands, with sonic hedgehog (SHH) levels highest in the mucosa of the embryonic foregut. More recently, interest in the pathway has shifted to improving our understanding of its role in gastrointestinal cancers. The use of reporter mice proved instrumental in our ability to probe the expression pattern of SHH ligand and the cell types responding to canonical HH signaling during homeostasis, inflammation, and neoplastic transformation. SHH is highly expressed in parietal cells and is required for these cells to produce gastric acid. Furthermore, myofibroblasts are the predominant cell type responding to HH ligand in the uninfected stomach. Chronic infection caused by Helicobacter pylori and associated inflammation induces parietal cell atrophy and the expansion of metaplastic cell types, a precursor to gastric cancer in human subjects. During Helicobacter infection in mice, canonical HH signaling is required for inflammatory cells to be recruited from the bone marrow to the stomach and for metaplastic development. Specifically, polarization of the invading myeloid cells to myeloid-derived suppressor cells requires the HH-regulated transcription factor GLI1, thereby creating a microenvironment favoring wound healing and neoplastic transformation. In mice, GLI1 mediates the phenotypic shift to gastric myeloid-derived suppressor cells by directly inducing Schlafen 4 (slfn4). However, the human homologs of SLFN4, designated SLFN5 and SLFN12L, also correlate with intestinal metaplasia and could be used as biomarkers to predict the subset of individuals who might progress to gastric cancer and benefit from treatment with HH antagonists.

      Keywords

      Abbreviations used in this paper:

      ATPase (adenosine triphosphatase), DAMP (damage-associated molecular pattern), GLI (glioma-associated protein), Gr-MDSC (granulocytic myeloid-derived suppressor cell), HH (hedgehog), HHIP (hedgehog-interacting protein), IFN (interferon), IL (interleukin), MDSC (myeloid-derived suppressor cell), Mo-MDSC (monocytic myeloid-derived suppressor cell), mRNA (messenger RNA), PTCH (Patched), SHH (sonic hedgehog), SLFN4 (Schlafen 4), SMO (Smoothened), SP (spasmolytic polypeptide), SPEM (spasmolytic polypeptide–expressing mucosa), SST (somatostatin), TLR (Toll-like receptor)
      Hedgehog signaling plays an essential role in gastric development, homeostasis, and neoplastic transformation. This article reviews the evidence for its role in the initiation of gastric inflammation due to Helicobacter infection but then chronically polarizes myeloid cells into myeloid-derived suppressor cells creating a microenvironment favoring cancer development.
      Hedgehog (HH) signaling initiates cancer in several organ systems,
      • Yun J.I.
      • Kim H.R.
      • Park H.
      • et al.
      Small molecule inhibitors of the hedgehog signaling pathway for the treatment of cancer.
      • Sahebjam S.
      • Siu L.L.
      • Razak A.A.
      The utility of hedgehog signaling pathway inhibition for cancer.
      but a clear etiologic role has not been shown for this pathway in gastric cancer. Because HH inhibitors currently are undergoing clinical trials for different types of cancer, understanding the role of HH signaling in regulating the tumor microenvironment becomes an important target to consider.
      • Merchant A.A.
      • Matsui W.
      Targeting Hedgehog–a cancer stem cell pathway.
      Based on prior mouse studies of increased HH signaling in preneoplastic lesions,
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      • Saqui-Salces M.
      • Coves-Datson E.
      • Veniaminova N.A.
      • et al.
      Inflammation and Gli2 suppress gastrin gene expression in a murine model of antral hyperplasia.
      • Merchant J.L.
      Hedgehog signalling in gut development, physiology and cancer.
      we have suggested that the use of HH inhibitors in human subjects chronically infected with Helicobacter might prevent progression of chronic atrophic gastritis to mucous gland metaplasias, a sentinel lesion that increases the likelihood of gastric cancer.
      • El-Zimaity H.M.
      • Ota H.
      • Graham D.Y.
      • et al.
      Patterns of gastric atrophy in intestinal type gastric carcinoma.
      • Fox J.G.
      • Wang T.C.
      Inflammation, atrophy, and gastric cancer.
      • Wu C.
      • Cheng J.
      • Hu S.
      • et al.
      Reduced proliferation and increased apoptosis of the SGC7901 gastric cancer cell line on exposure to GDC0449.
      • Abdel-Rahman O.
      Hedgehog pathway aberrations and gastric cancer; evaluation of prognostic impact and exploration of therapeutic potentials.
      Thus, the focus of the current review is to understand the basis for HH signaling in normal adult stomach and how this developmental pathway might play a role in neoplastic transformation. Because our current understanding of HH signaling in the stomach arises from transgenic mouse models, the information presented refers to the mouse except when information from human studies exists.

      Role of Hedgehog Signaling in Gastric Homeostasis

      To date, there are 3 known mammalian genes encoding the hedgehog ligands: Sonic hedgehog (SHH), Indian hedgehog, and Desert hedgehog.
      • Ruiz i Altaba A.
      • Sanchez P.
      • Dahmane N.
      Gli and hedgehog in cancer: tumours, embryos and stem cells.
      • Marigo V.
      • Roberts D.J.
      • Lee S.M.
      • et al.
      Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog.
      • Katoh Y.
      • Katoh M.
      Comparative genomics on Sonic hedgehog orthologs.
      During embryonic development, SHH is expressed throughout the gut and in other foregut-derived organs (eg, lung, pancreas).
      • Litingtung Y.
      • Lei L.
      • Westphal H.
      • et al.
      Sonic hedgehog is essential to foregut development.
      • Shannon J.M.
      • Hyatt B.A.
      Epithelial-mesenchymal interactions in the developing lung.
      • Kim S.K.
      • Melton D.A.
      Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor.
      Although its function in mature gastric epithelium was not initially studied in adult mammals, it became apparent that SHH remains highly expressed in the stomach once expression in the intestine diminishes.
      • Willet S.G.
      • Mills J.C.
      Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis.
      • Ramalho-Santos M.
      • Melton D.A.
      • McMahon A.P.
      Hedgehog signals regulate multiple aspects of gastrointestinal development.
      Subsequently, it was reported that SHH regulates epithelial cell maturation and differentiation in the adult stomach.
      • Van Den Brink G.R.
      • Hardwick J.C.
      • Tytgat G.N.
      • et al.
      Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.
      • van den Brink G.R.
      • Hardwick J.C.
      • Nielsen C.
      • et al.
      Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.
      Normally, SHH is expressed in mature acid-secreting glands of the adult mouse and human stomachs, primarily within parietal cells
      • Van Den Brink G.R.
      • Hardwick J.C.
      • Tytgat G.N.
      • et al.
      Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.
      • Fukaya M.
      • Isohata N.
      • Ohta H.
      • et al.
      Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer.
      • Stepan V.
      • Ramamoorthy S.
      • Nitsche H.
      • et al.
      Regulation and function of the sonic hedgehog signal transduction pathway in isolated gastric parietal cells.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis progressing to gastric cancer.
      (Figure 1). During progression from the inflamed stomach to gastric cancer, the acid-producing parietal cells fail to produce acid and eventually are replaced by mucous-secreting cells that express spasmolytic polypeptide (SP) or trefoil factor 2.
      • El-Zimaity H.M.
      • Ota H.
      • Graham D.Y.
      • et al.
      Patterns of gastric atrophy in intestinal type gastric carcinoma.
      • Petersen C.P.
      • Mills J.D.
      • Goldenring J.R.
      Murine models of gastric corpus preneoplasia.
      Mostly in mice, but also in human subjects, SP-expressing mucosa (SPEM) is a type of oxyntic gland atrophy.
      • Yamaguchi H.
      • Goldenring J.R.
      • Kaminishi M.
      • et al.
      Identification of spasmolytic polypeptide expressing metaplasia (SPEM) in remnant gastric cancer and surveillance postgastrectomy biopsies.
      • Engevik A.C.
      • Feng R.
      • Choi E.
      • et al.
      The development of spasmolytic polypeptide/TFF2-expressing metaplasia (SPEM) during gastric repair is absent in the aged stomach.
      In concert with parietal cell atrophy, SHH expression in these acid-producing cells also is lost.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis progressing to gastric cancer.
      • Suzuki H.
      • Minegishi Y.
      • Nomoto Y.
      • et al.
      Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pylori-infected Mongolian gerbils.
      Although SHH expression diminishes along with loss of parietal cells, the expanding mucous cell compartment or SPEM continues to produce SHH in both human subjects
      • van den Brink G.R.
      • Hardwick J.C.
      • Nielsen C.
      • et al.
      Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis progressing to gastric cancer.
      and rodents,
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      • Suzuki H.
      • Minegishi Y.
      • Nomoto Y.
      • et al.
      Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pylori-infected Mongolian gerbils.
      but remains unprocessed, maintaining the full-length 45-kilodalton form
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      (Figure 1). Surprisingly, even unprocessed Hedgehog protein (Drosophila) shows activity where it traffics to the cell membrane to participate in cell–cell signaling.
      • Tokhunts R.
      • Singh S.
      • Chu T.
      • et al.
      The full-length unprocessed hedgehog protein is an active signaling molecule.
      This result suggests that aberrant HH signaling in cancer might function as an autocrine or paracrine regulator, especially in the stem cell niche.
      • Singh S.
      • Wang Z.
      • Liang Fei D.
      • et al.
      Hedgehog-producing cancer cells respond to and require autocrine Hedgehog activity.
      • Konstantinou D.
      • Bertaux-Skeirik N.
      • Zavros Y.
      Hedgehog signaling in the stomach.
      • Saqui-Salces M.
      • Merchant J.L.
      Hedgehog signaling and gastrointestinal cancer.
      Figure thumbnail gr1
      Figure 1SHH expression in the stomach corpus of wild-type (WT) and Gli1-/- mice. Shown is the co-localization of SHH with E-cadherin, F4/80 (macrophage/myeloid marker), or α-smooth muscle actin (SMA) (myofibroblasts) protein markers in the absence or presence of Helicobacter felis infection. 4′,6-diamidino-2-phenylindole (DAPI) indicates cell nuclei. Arrows indicate the presence of SPEM.
      Reprinted with permission from El-Zaatari et al.
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      Scale bars = 100 μm.
      Processing of SHH to its active form (19 kilodaltons) in parietal cells becomes compromised in the absence of gastric acid.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      Atrophy of parietal and zymogenic (chief cell) lineages result in hypochlorhydria and reduced serum pepsinogen I (A) levels compared with pepsinogen II (C).
      • Sierra R.
      • Une C.
      • Ramirez V.
      • et al.
      Association of serum pepsinogen with atrophic body gastritis in Costa Rica.
      • Iijima K.
      • Sekine H.
      • Koike T.
      • et al.
      Serum pepsinogen concentrations as a measure of gastric acid secretion in Helicobacter pylori-negative and -positive Japanese subjects.
      • Sipponen P.
      • Ranta P.
      • Helske T.
      • et al.
      Serum levels of amidated gastrin-17 and pepsinogen I in atrophic gastritis: an observational case-control study.
      • Nomura A.M.
      • Kolonel L.N.
      • Miki K.
      • et al.
      Helicobacter pylori, pepsinogen, and gastric adenocarcinoma in Hawaii.
      • Kokkola A.
      • Louhimo J.
      • Puolakkainen P.
      • et al.
      Helicobacter pylori infection and low serum pepsinogen I level as risk factors for gastric carcinoma.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Histologic and serum risk markers for noncardia early gastric cancer.
      • Li P.
      • He C.
      • Sun L.
      • et al.
      Pepsinogen I and II expressions in situ and their correlations with serum pesignogen levels in gastric cancer and its precancerous disease.
      These zymogens are proteins encoded by different gene loci that are used clinically to indicate preneoplastic changes in the stomach.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Histologic and serum risk markers for noncardia early gastric cancer.
      • Li P.
      • He C.
      • Sun L.
      • et al.
      Pepsinogen I and II expressions in situ and their correlations with serum pesignogen levels in gastric cancer and its precancerous disease.
      Pepsinogens A and C are converted to the enzymatically active aspartic proteinases, pepsin A and pepsin C, through intramolecular self-cleavage.
      • Li P.
      • He C.
      • Sun L.
      • et al.
      Pepsinogen I and II expressions in situ and their correlations with serum pesignogen levels in gastric cancer and its precancerous disease.
      • Roberts N.B.
      Review article: human pepsins - their multiplicity, function and role in reflux disease.
      We showed previously that pepsinogen A is produced primarily in the mouse corpus by parietal cells, whereas pepsinogen C is produced primarily by both mucous neck and chief cells throughout the stomach.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      This result is consistent with the exclusive expression of pepsinogen A in the human corpus and not the antrum, whereas pepsinogen C marks mucous cells of both the antrum and corpus (www.proteinatlas.org). Pepsin A prefers to cleave proteins at hydrophobic and aromatic residues, particularly at phenylalanine (F) when the pH is less than 2. By contrast, pepsin C recognizes a broader consensus site and uses a wider pH spectrum than pepsin A.
      • Roberts N.B.
      Review article: human pepsins - their multiplicity, function and role in reflux disease.
      • Fujinaga M.
      • Chernaia M.M.
      • Tarasova N.I.
      • et al.
      Crystal structure of human pepsin and its complex with pepstatin.
      Specifically, we showed using site-directed mutagenesis that pepsin A cleaves the nascent 45-kilodalton SHH polypeptide at residue 200 (SGGCF200|P) to generate the active 19-kilodalton form, whereas pepsin C does not cleave SHH peptide
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      (Figure 2).
      Figure thumbnail gr2
      Figure 2SHH processing methods compared. Two mechanisms for processing SHH ligand have been reported. The best known is the autocatalytic mechanism of SHH in which the C-terminus functions as a cholesterol esterase by adding the sterol to cysteine residue 199 followed by adding the fatty acid palmitate to residue 25. The fatty acid permits SHH to be tethered to the plasma membrane until it is cleaved by A Disintegrin and Metalloproteinase Domain Containing Protein protease. The cleaved SHH molecules form miscelles in the presence of a transport protein called Dispatched. This mechanism has been described for Drosophila cells and mammalian cells derived from the mesenchyme. By contrast, parietal cells produce both gastric acid and pepsinogen A, a zymogen that undergoes autocatalytic cleavage at a low pH (pH < 2). Thus, in the stomach SHH is cleaved by the acid-dependent aspartic proteinase at the C-terminal side of the phenylalanine (at residue 200), suggesting that the addition of lipid is not required, perhaps facilitating its solubility in a more polar microenvironment. Nevertheless, whether SHH produced from the parietal cell is modified post-translationally with cholesterol or a fatty acid is not known.
      Reprinted with permission from Merchant.
      • Merchant J.L.
      Hedgehog signalling in gut development, physiology and cancer.

      Processing of Sonic Hedgehog

      Perhaps because of difficulties in measuring SHH protein during development, most studies primarily have relied on messenger RNA (mRNA) levels and not protein to study SHH expression. Nevertheless, prior studies examining generation of SHH protein showed that the protein undergoes a complex series of processing steps that includes the initial generation of a 45-kilodalton precursor polypeptide, subsequent removal of the N-terminal 24 amino acid residue signal peptide, and then cleavage of the amino terminus to generate a 19-kilodalton protein that can be modified post-translationally by palmitate and cholesterol
      • Bumcrot D.A.
      • Takada R.
      • McMahon A.P.
      Proteolytic processing yields two secreted forms of sonic hedgehog.
      • Wendler F.
      • Franch-Marro X.
      • Vincent J.P.
      How does cholesterol affect the way Hedgehog works?.
      • Goetz J.A.
      • Suber L.M.
      • Zeng X.
      • et al.
      Sonic Hedgehog as a mediator of long-range signaling.
      • Lee J.J.
      • Ekker S.C.
      • von Kessler D.P.
      • et al.
      Autoproteolysis in hedgehog protein biogenesis.
      (Figure 2). These studies performed initially in Drosophila showed that cholesterol transferase activity resides in the C-terminal portion of the Hedgehog molecule such that esterification of cysteine 198 by the transferase results in intramolecular autocatalytic cleavage of the 45-kilodalton precursor
      • Roelink H.
      • Porter J.A.
      • Chiang C.
      • et al.
      Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis.
      (Figure 2). Additional studies have shown that the extent of lipid modification modulates SHH diffusion away from the cell of origin. Apparently, a shorter range of diffusion correlates with a higher degree of lipid (palmitate) modification and membrane association.
      • Gritli-Linde A.
      • Lewis P.
      • McMahon A.P.
      • et al.
      The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides.
      • Goetz J.A.
      • Singh S.
      • Suber L.M.
      • et al.
      A highly conserved amino-terminal region of sonic hedgehog is required for the formation of its freely diffusible multimeric form.
      By contrast, we showed in the adult mouse and human stomach that both SHH processing and gene expression are linked to acid secretion.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      Specifically, infusion of the hormone gastrin over 2 weeks using osmotic pumps stimulates SHH gene expression in a hypochlorhydric gastrin-deficient mouse in concert with re-establishing acid secretion.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      Moreover, post-translational processing of SHH precursor to its secreted form depends on cleavage by the acid-activated protease pepsin A generated from pepsinogen A. Thus, we concluded that generation of the biologically active form of SHH in the stomach is regulated. If gastric acidity is reduced, as a result of inhibition of acid secretion (omeprazole therapy) or loss of the parietal cell (atrophy), then pepsin A is not activated and most of the precursor SHH protein produced is not cleaved into its functionally active 19-kilodalton form.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      These observations are consistent with the finding that oxyntic gland atrophy (specifically loss of the parietal cell) correlates with reduced pepsinogen A to C ratios.
      • Shiotani A.
      • Iishi H.
      • Uedo N.
      • et al.
      Histologic and serum risk markers for noncardia early gastric cancer.
      More importantly, these observations suggest that SHH is processed within parietal cells. SHH co-localizes to the tubulovesicle fraction with the H+, K+-adenosine triphosphatase (ATPase) enzyme. With the addition of a secretogogue, movement of the SHH precursor to the canalicular membrane coincides with insertion of the proton pump into the apical membrane.
      • Zavros Y.
      • Orr M.A.
      • Xiao C.
      • et al.
      Sonic hedgehog is associated with H+,K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation.
      This thesis would account for the ability of SHH processing and secretion to coincide with the production of gastric acid. Moreover, this mechanism would predict that a significant amount of SHH would be less lipid-modified and capable of diffusing both basolaterally and apically throughout the gastric gland and into the circulation.
      • Zavros Y.
      • Orr M.A.
      • Xiao C.
      • et al.
      Sonic hedgehog is associated with H+,K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation.
      Indeed, we and others have found that blood levels of SHH peptide originate in part from the parietal cell
      • Schumacher M.A.
      • Donnelly J.M.
      • Engevik A.C.
      • et al.
      Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      and can be detected in the circulation of human volunteers.
      • El-Zaatari M.
      • Daignault S.
      • Tessier A.
      • et al.
      Plasma sHH levels reduced in pancreatic cancer patients.

      Hedgehog Signaling in the Adult Stomach

      Canonical HH signaling involves epithelial expression of ligand (typically SHH in the stomach), which subsequently binds to its receptor Patched (PTCH) and relieves its inhibitory influence on an adjacent transmembrane HH activator called Smoothened (SMO). Once SMO inhibition is relieved, glioma-associated protein 2 (GLI2) is processed to an activator form, translocates to the nucleus, and then binds to the promoters of HH effectors including PTCH, hedgehog-interacting protein (HHIP), and GLI1.
      • Bai C.B.
      • Auerbach W.
      • Lee J.S.
      • et al.
      Gli2, but not Gli1, is required for initial SHH signaling and ectopic activation of the SHH pathway.
      • Pan Y.
      • Bai C.B.
      • Joyner A.L.
      • et al.
      Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation.
      Thus, GLI1, PTCH, and HHIP are transcriptional read-outs of canonical HH signaling activity.
      • van den Brink G.R.
      Hedgehog signaling in development and homeostasis of the gastrointestinal tract.
      The extracellular signals regulating shh gene expression in the stomach are not well defined but might correlate with those reported in other tissues. For example, during pancreatic development, shh expression appears to be regulated by activin A.
      • van Eyll J.M.
      • Pierreux C.E.
      • Lemaigre F.P.
      • et al.
      SHH-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.
      During limb bud development, fibroblast growth factors and bone morphogenetic proteins regulate shh expression.
      • Zuniga A.
      • Haramis A.P.
      • McMahon A.P.
      • et al.
      Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds.
      • Khokha M.K.
      • Hsu D.
      • Brunet L.J.
      • et al.
      Gremlin is the BMP antagonist required for maintenance of SHH and Fgf signals during limb patterning.
      Shh null (shh-/-) mice do not survive past postnatal day 1.
      • Ramalho-Santos M.
      • Melton D.A.
      • McMahon A.P.
      Hedgehog signals regulate multiple aspects of gastrointestinal development.
      However, the stomachs of these mice were hyperplastic and further underscored that loss of HH signaling shows important functional consequences.
      • Ramalho-Santos M.
      • Melton D.A.
      • McMahon A.P.
      Hedgehog signals regulate multiple aspects of gastrointestinal development.
      Subsequent studies have been performed in adult mice using a H+,K+-ATPase-Cre transgene to delete the shh gene locus only in parietal cells. Conditional deletion of the shh gene resulted in parietal cell atrophy and foveolar hyperplasia.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      • Xiao C.
      • Feng R.
      • Engevik A.C.
      • et al.
      Sonic Hedgehog contributes to gastric mucosal restitution after injury.
      • Xiao C.
      • Ogle S.A.
      • Schumacher M.A.
      • et al.
      Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.
      Indeed, SHH signaling is required for optimal H+,K+-ATPase expression.
      • Stepan V.
      • Ramamoorthy S.
      • Nitsche H.
      • et al.
      Regulation and function of the sonic hedgehog signal transduction pathway in isolated gastric parietal cells.
      In addition, a prior study in a gastric cancer cell line showed that increased gastric acidity stimulates shh expression.
      • Dimmler A.
      • Brabletz T.
      • Hlubek F.
      • et al.
      Transcription of sonic hedgehog, a potential factor for gastric morphogenesis and gastric mucosa maintenance, is up-regulated in acidic conditions.
      Accordingly, modulators of gastric acid such as gastrin and somatostatin (SST) have been shown to modulate SHH levels and HH signaling.
      • Zavros Y.
      • Waghray M.
      • Tessier A.
      • et al.
      Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
      • Zavros Y.
      • Orr M.A.
      • Xiao C.
      • et al.
      Sonic hedgehog is associated with H+,K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation.
      • El-Zaatari M.
      • Zavros Y.
      • Tessier A.
      • et al.
      Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.
      • Waghray M.
      • Zavros Y.
      • Saqui-Salces M.
      • et al.
      Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.

      Inflammation Regulates Gastric Acid Secretion and SHH

      We previously examined modulation of gastric acid secretion by proinflammatory cytokines and reported that both gastrin and SST are regulated, albeit in a reciprocal manner, by cytokines in vivo and in primary cell cultures.
      • Zavros Y.
      • Rathinavelu S.
      • Kao J.Y.
      • et al.
      Treatment of Helicobacter gastritis with interleukin-4 requires somatostatin.
      • Zavros Y.
      • Merchant J.L.
      Modulating the cytokine response to treat Helicobacter gastritis.
      Interferon γ (IFNγ), a T1-helper cytokine, stimulated gastrin and inhibited SST, and interleukin-4 (IL4), a T2-helper cytokine, stimulated SST and inhibited gastrin.
      • Li P.
      • He C.
      • Sun L.
      • et al.
      Pepsinogen I and II expressions in situ and their correlations with serum pesignogen levels in gastric cancer and its precancerous disease.
      Thus, similar to the negative feedback regulation known to exist for gastrin and SST, immune modulators impart parallel control of these peptides and therefore acid secretion. Teleologically, it makes sense that the innate immune system regulates gastric acid because acid is one of the first defense mechanisms that is activated by the gastrointestinal tract to combat invading organisms. However, prior studies by Beales
      • Beales I.L.
      Effect of cytokines on acid secretion and gastrin secretion in Helicobacter pylori infection and aspirin-induced gastritis.
      and other investigators
      • Okumura T.
      • Uehara A.
      • Okamura K.
      • et al.
      Inhibition of gastric pepsin secretion by peripherally or centrally injected interleukin-1 in rats.
      • Uehara A.
      • Okumura T.
      • Sekiya C.
      • et al.
      Interleukin-1 inhibits the secretion of gastric acid in rats: possible involvement of prostaglandin.
      • Wallace J.L.
      • Keenan C.M.
      • Cucala M.
      • et al.
      Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy.
      have shown by using a rabbit primary culture system that either IL1β or tumor necrosis factor-α infusion into rodents suppresses acid secretion. Subsequently, more ominous implications became attributed to cytokine suppression of acid secretion when El-Omar et al
      • El-Omar E.M.
      • Carrington M.
      • Chow W.H.
      • et al.
      Interleukin-1 polymorphisms associated with increased risk of gastric cancer.
      and other investigators
      • Furuta T.
      • El-Omar E.M.
      • Xiao F.
      • et al.
      Interleukin 1beta polymorphisms increase risk of hypochlorhydria and atrophic gastritis and reduce risk of duodenal ulcer recurrence in Japan.
      • Garza-Gonzalez E.
      • Bosques-Padilla F.J.
      • El-Omar E.
      • et al.
      Role of the polymorphic IL-1B, IL-1RN and TNF-A genes in distal gastric cancer in Mexico.
      • Atsuta Y.
      • Ito L.S.
      • Oba-Shinjo S.M.
      • et al.
      Associations of TNF-A-1031TT and -857TT genotypes with Helicobacter pylori seropositivity and gastric atrophy among Japanese Brazilians.
      showed that IL1β, but not tumor necrosis factor-α polymorphisms, predispose human subjects to gastric atrophy and gastric cancer. Testing the significance of the polymorphism, Tu et al
      • Tu S.
      • Bhagat G.
      • Cui G.
      • et al.
      Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice.
      reported that transgenic overexpression of IL1β in mouse parietal cells induced gastric inflammation and dysplasia. By contrast, IFNγ polymorphisms do not appear to correlate with gastric atrophy.
      • Rad R.
      • Dossumbekova A.
      • Neu B.
      • et al.
      Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during Helicobacter pylori infection.
      Indeed, we reported that proinflammatory cytokines show differential effects on SHH expression with IFNγ stimulating SHH expression and IL1β inhibiting expression when added acutely (6 h) to parietal cell cultures.
      • Waghray M.
      • Zavros Y.
      • Saqui-Salces M.
      • et al.
      Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.
      It generally has been assumed that all proinflammatory cytokines generated during bacterial colonization exert the same effect on gastric cells. However, our results in primary parietal cell cultures coupled with differences in the association of cytokine polymorphisms support the likelihood that the effects of these proinflammatory cytokines on parietal cells are distinct.

      SHH Regulates Gastrin and Gastric Acidity

      To examine the impact of HH signaling in vivo, we generated a transgenic mouse that secreted the natural -inhibitor of HH ligands called HHIP expressed from the cell-specific H+,K+-ATPase β subunit promoter.
      • El-Zaatari M.
      • Zavros Y.
      • Tessier A.
      • et al.
      Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.
      Our results showed that loss of HH signaling in parietal cells, caused by the production of secreted HHIP, reduced H+,K+-ATPase gene expression and gastric acid.
      • El-Zaatari M.
      • Zavros Y.
      • Tessier A.
      • et al.
      Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.
      Normally, hypochlorhydria stimulates gastrin gene expression through a decrease in SST.
      • Brand S.J.
      • Stone D.
      Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria.
      Accordingly, we found coincident with increased plasma gastrin occurring in the hhip transgenic mice that sst gene expression also decreased. This result showed that modulation of HH signaling in parietal cells is sufficient to activate the normal feedback mechanisms typically attributed to gastrin and SST. Indeed, we reported that both antral G and D cells possess primary cilia, organelles protruding from the plasma membrane, which transduce HH signaling.
      • Saqui-Salces M.
      • Keeley T.M.
      • Grosse A.S.
      • et al.
      Gastric tuft cells express DCLK1 and are expanded in hyperplasia.
      • Saqui-Salces M.
      • Dowdle W.E.
      • Reiter J.F.
      • et al.
      A high-fat diet regulates gastrin and acid secretion through primary cilia.
      Therefore, gastric endocrine cells are capable of responding directly to the SHH ligand. We showed that transgenic overexpression of GLI2 suppresses gastrin gene expression.
      • Saqui-Salces M.
      • Coves-Datson E.
      • Veniaminova N.A.
      • et al.
      Inflammation and Gli2 suppress gastrin gene expression in a murine model of antral hyperplasia.
      Thus, gastrin stimulates gastric acid and SHH expression whereas HH signaling suppresses gastrin expression. Taken together, the production of SHH by parietal cells and the ability of gastric endocrine cells to sense the ligand through primary cilia are consistent with a central role for HH signaling in the feedback regulation of gastric acidity.

      Cross-Talk Between Gastric Epithelium and Mesenchyme

      Canonical HH signaling typically involves cross-talk between epithelial cells that produce the ligands, for example, SHH, Indian hedgehog, and cells that express the receptor-signaling complexes, such as PTCH, SMO, HHIP, and transcription factors GLI1, GLI2, and GLI3. Therefore, to identify gastric cells that respond to HH ligands in the absence and presence of Helicobacter, we used the Gli-LacZ reporter mouse. The β-galactosidase complementary DNA was recombined into the gli1 gene locus to create heterozygous or homozygous genotypes.
      • Bai C.B.
      • Auerbach W.
      • Lee J.S.
      • et al.
      Gli2, but not Gli1, is required for initial SHH signaling and ectopic activation of the SHH pathway.
      In the absence of a Helicobacter infection, we found that α-smooth muscle actin–positive myofibroblasts were the major population expressing the LacZ reporter.
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      However, during a Helicobacter infection, the LacZ+ cells infiltrating the gastric mesenchyme were myeloid cells and correlated with parietal cell atrophy and the emergence of SPEM.
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      Strikingly, when either the Gli1LacZ/+ or Gli1LacZ/LacZ mice were infected, they did not develop SPEM. This result showed that canonical HH signaling was required for SPEM.
      • Goldenring J.R.
      • Nam K.T.
      Oxyntic atrophy, metaplasia, and gastric cancer.
      Use of microarray analysis to identify GLI1 target genes showed that induction of a myeloid differentiation factor called Schlafen 4 (SLFN4) contributed to the GLI1-dependent development of SPEM. However, the induction of slfn4 mRNA coincident with SPEM was time-dependent, and required 6 months to be expressed in wild-type mice or 4 months in the presence of constitutively elevated levels of SHH ligand (pCMVShh).
      • El-Zaatari M.
      • Kao J.Y.
      • Tessier A.
      • et al.
      Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
      Overexpression of SHH accelerated the development of SPEM, but only in the presence of a Helicobacter infection.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      Therefore, both SPEM and the appearance of SLFN4+ myeloid cells required GLI1. We concluded that HH signaling is required to polarize a subset of myeloid cells and transition gastric mucosa from a proinflammatory to a preneoplasia state, but only in the presence of the bacterial infection (Figure 3).
      Figure thumbnail gr3
      Figure 3Schematic of SLFN4-positive cells migrating from the bone marrow during a Helicobacter infection. SHH released by parietal cells into the circulation is sensed by SLFN4-positive myeloid cells (yellow cells). Presumably, the concentration of SHH is highest in the acid-secreting stomach (blue triangles), which encourages the SLFN4-positive cells to home to the infected stomach. Eventually, the SLFN4-positive myeloid cells become polarized to Gr-MDSCs (red cells) by tissue IFNα induced by DAMP signals that accumulate as a result of cellular atrophy during chronic Helicobacter infection. The genes expressed by SLFN4-positive-Gr-MDSCs are indicated (expanded red cell), as well as how slfn4 gene expression is regulated by both HH signaling (Gli1) and the inducible inflammatory signal (IFNα). Therefore, polarization to Gr-MDSCs can be achieved only in the infected stomach where the SLFN4-positive myeloid cells encounter increased IFNα inducing maximal SLFN4 levels. ISRE, interferon-stimulated response element.
      Modified with permission from Ding et al.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      The heterogeneous populations of myeloid cells that emerge during chronic atrophic gastritis are phenotypically myeloid-derived suppressor cells (MDSCs). MDSCs show both monocytic (Mo-MDSC) and granulocytic (Gr-MDSC) features, suggesting that they might represent the reprogramming of monocytes and neutrophils recruited to the tissue.
      • Millrud C.R.
      • Bergenfelz C.
      • Leandersson K.
      On the origin of myeloid-derived suppressor cells.
      MDSCs suppress T-cell function by consuming L-arginine through arginase 1 and inducible nitric oxide synthase activated to generate reactive oxygen species. L-arginine is required for T-cell proliferation and its ability to block cancer growth.
      • Gabrilovich D.I.
      • Ostrand-Rosenberg S.
      • Bronte V.
      Coordinated regulation of myeloid cells by tumours.
      Therefore, the presence of MDSCs has been strongly linked to tumor promotion, owing to their immunosuppressive role once cancer has emerged.
      • Bronte V.
      • Brandau S.
      • Chen S.H.
      • et al.
      Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.
      By contrast, our recent studies strongly have suggested that a subset of MDSCs appear during the preneoplastic phase of cancer development and require HH signaling.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      Schlafens are a family of molecules strongly induced by type 1 IFNs (IFNα), and their expression typically correlates with immune cell quiescence.
      • Schwarz D.A.
      • Katayama C.D.
      • Hedrick S.M.
      Schlafen, a new family of growth regulatory genes that affect thymocyte development.
      • Puck A.
      • Aigner R.
      • Modak M.
      • et al.
      Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells.
      Specifically, SLFN4 modulates myelopoiesis.
      • van Zuylen W.J.
      • Garceau V.
      • Idris A.
      • et al.
      Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis.
      Coincident with the apoptosis of parietal cells, tissue levels of damage-associated molecular patterns (DAMPs) accumulate, culminating in increasing IFNα secretion from plasmacytoid-derived dendritic cells.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      • Panda S.K.
      • Kolbeck R.
      • Sanjuan M.A.
      Plasmacytoid dendritic cells in autoimmunity.
      Toll-like receptors (TLRs) 3, 7, 8, and 9 are the intracellular TLRs that recognize DAMPs by initiating a complex cascade of signaling molecules (eg, myeloid differentiation primary response gene 88, interferon factor regulatory transcriptions, and signal transducer and activator of transcriptions) that ultimately induce expression of type 1 interferons (IFNα and IFNβ)
      • Yamamoto M.
      • Takeda K.
      Current views of toll-like receptor signaling pathways.
      (Figure 3). Recent studies have indicated that chronic Helicobacter infection in both mice and human beings induces TLR9 expression.
      • Otani K.
      • Tanigawa T.
      • Watanabe T.
      • et al.
      Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis.
      Apparently, DAMPs and their subsequent activation of TLR9 are associated with immune suppression.
      • Varga M.G.
      • Piazuelo M.B.
      • Romero-Gallo J.
      • et al.
      TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.
      • Hernandez C.
      • Huebener P.
      • Schwabe R.F.
      Damage-associated molecular patterns in cancer: a double-edged sword.
      Moreover, there is an increased incidence of gastric neoplasia in subjects with TLR9 polymorphisms and H pylori infection.
      • Varga M.G.
      • Shaffer C.L.
      • Sierra J.C.
      • et al.
      Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system.
      • Wang X.
      • Xue L.
      • Yang Y.
      • et al.
      TLR9 promoter polymorphism is associated with both an increased susceptibility to gastric carcinoma and poor prognosis.
      Increased tissue levels of type I interferons can suppress inflammation.
      • Varga M.G.
      • Piazuelo M.B.
      • Romero-Gallo J.
      • et al.
      TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.
      In addition, DAMPs have been implicated in the reprogramming of monocytic cells to become Mo-MDSCs.
      • Millrud C.R.
      • Bergenfelz C.
      • Leandersson K.
      On the origin of myeloid-derived suppressor cells.
      In addition, our results show that DAMP signals also polarize Gr-MDSCs as observed for Mo-MDSCs (Figure 3). In particular, the novelty of this observation is that Gr-MDSCs appear during the metaplastic phase of the transforming gastric mucosa before frank cancer emerges.
      Collectively, the emergence of SLFN4+ MDSCs might be cogent biomarkers because the slfn4 promoter remains quiescent until both transcriptional regulators—one constitutive (GLI1) and one inducible (IRFs/STAT)—engage the promoter (Figure 3). In this way, immune suppressor function only becomes active under the appropriate conditions (ie, to dampen the chronic gastritis initiated by Helicobacter). The T-cell suppressor activity exerted by immature myeloid cells occurs because they restrict T-cell access to L-arginine, a substrate for the MDSC enzymes arginase 1 and inducible nitric oxide synthase.
      • Gabrilovich D.I.
      • Ostrand-Rosenberg S.
      • Bronte V.
      Coordinated regulation of myeloid cells by tumours.
      We showed previously that small interfering RNA knockdown of slfn4 significantly reduces arg1 and inos mRNA in SLFN4+ MDSCs,
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      suggesting that SLFN4 is required for myeloid cells to acquire their immune-suppressor function.
      Because GLI1 gene expression blocks maturation of an immature myeloid cell subpopulation, creating a gastric microenvironment favorable for metaplasia and transformation, we examined the pattern for SLFN4 homologs in human subjects.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      • Companioni Napoles O.
      • Tsao A.C.
      • Sanz-Anquela J.M.
      • et al.
      SCHLAFEN 5 expression correlates with intestinal metaplasia that progresses to gastric cancer.
      Human SLFN 5, SLFN12, and SLFN12L show the closest homology to mouse SLFN4 protein (the slfn4 gene does not exist in the human genome). Consistent with the mouse model, we recently reported in a 13-year follow-up study that SLFN5 is increased most significantly in those subjects with intestinal metaplasia whose lesions progressed to gastric cancer.
      • Companioni Napoles O.
      • Tsao A.C.
      • Sanz-Anquela J.M.
      • et al.
      SCHLAFEN 5 expression correlates with intestinal metaplasia that progresses to gastric cancer.
      Although SLFN5 is expressed in myeloid cells, we found that its expression also occurred primarily in T cells.
      • Companioni Napoles O.
      • Tsao A.C.
      • Sanz-Anquela J.M.
      • et al.
      SCHLAFEN 5 expression correlates with intestinal metaplasia that progresses to gastric cancer.
      When we examined the expression pattern of SLFN12L, we found its expression correlated with the human surface markers for Gr-MDSCs.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      Therefore, human myeloid cells express SLFN12L as observed for SLFN4 in mice.
      • Ding L.
      • Hayes M.M.
      • Photenhauer A.
      • et al.
      Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
      Furthermore, we would predict that increased SLFN12L levels, like SLFN5, might predict those individuals with metaplasia who are more likely to develop gastric cancer.

      Summary

      HH signaling in the stomach plays a significant role in gastric development, homeostasis, and neoplastic transformation.
      • Merchant J.L.
      Hedgehog signalling in gut development, physiology and cancer.
      Although extensive developmental literature on SHH protein and downstream targets exists, essentially none of the information was applied to the stomach, despite the evidence that SHH is highly expressed in gastric cancer cell lines.
      • Fukaya M.
      • Isohata N.
      • Ohta H.
      • et al.
      Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer.
      Although increased levels of SHH have been reported in gastric cancers, its specific role in gastric transformation remains elusive but carries significance because of the availability of HH antagonists. Here, we reviewed the role of HH signaling in normal gastric homeostasis, inflammation, and transformation. In particular, we highlighted our studies showing that the phenotype of infiltrating myeloid cells changes over time to become MDSCs and that the polarization requires HH signaling. More importantly, expression of GLI1, which targets SLFN4 (mice) and SLFN12L and SLFN5 (human beings), is an early indicator that the myeloid cells recruited during chronic inflammation have become polarized toward Gr-MDSCs, a cell type that appears to favor neoplastic development. In addition to MDSCs, other bone marrow–derived cells are recruited to the stomach and have been implicated in facilitating gastric transformation.
      • Petersen C.P.
      • Mills J.D.
      • Goldenring J.R.
      Murine models of gastric corpus preneoplasia.
      • Donnelly J.M.
      • Engevik A.
      • Feng R.
      • et al.
      Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach.
      • Petersen C.P.
      • Weis V.G.
      • Nam K.T.
      • et al.
      Macrophages promote progression of spasmolytic polypeptide-expressing metaplasia after acute loss of parietal cells.
      • Buzzelli J.N.
      • Chalinor H.V.
      • Pavlic D.I.
      • et al.
      Il33 is a stomach alarmin that initiates a skewed Th2 response to injury and infection.
      The ability to track these cell types in the preneoplastic state broadens options for more effective screening of subjects predisposed to eventually develop gastric cancer as well as to expand options for prophylactic therapy once atrophic gastritis develops, including antagonists of mTOR (mechanistic antagonist of rapamycin).
      • Goldenring J.R.
      Gastric intestinal metaplasia and tamoxifen: can we reverse the inevitable?.
      • Choi E.
      • Hendley A.M.
      • Bailey J.M.
      • et al.
      Expression of activated Ras in gastric chief cells of mice leads to the full spectrum of metaplastic lineage transitions.
      • Syu L.-Y.
      • Zhao X.
      • Zhang Y.
      • et al.
      Invasive mouse gastric adenocarcinomas arising from Lgr5+ stem cells are dependent on crosstalk between the Hedgehog/GLI2 and mTOR pathways.
      Although Hedgehog antagonists have been used for other cancer types, their use in clinical trials for gastric cancer is still in its infancy.
      • Yun J.I.
      • Kim H.R.
      • Park H.
      • et al.
      Small molecule inhibitors of the hedgehog signaling pathway for the treatment of cancer.
      • Konstantinou D.
      • Bertaux-Skeirik N.
      • Zavros Y.
      Hedgehog signaling in the stomach.
      Where initiated, those trials have focused on targeting CD44-positive gastric stem cells to treat metastatic disease.
      • Yoon C.
      • Park D.J.
      • Schmidt B.
      • et al.
      CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance.

      References

        • Yun J.I.
        • Kim H.R.
        • Park H.
        • et al.
        Small molecule inhibitors of the hedgehog signaling pathway for the treatment of cancer.
        Arch Pharm Res. 2012; 35: 1317-1333
        • Sahebjam S.
        • Siu L.L.
        • Razak A.A.
        The utility of hedgehog signaling pathway inhibition for cancer.
        Oncologist. 2012; 17: 1090-1099
        • Merchant A.A.
        • Matsui W.
        Targeting Hedgehog–a cancer stem cell pathway.
        Clin Cancer Res. 2010; 16: 3130-3140
        • El-Zaatari M.
        • Kao J.Y.
        • Tessier A.
        • et al.
        Gli1 deletion prevents helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.
        PLoS One. 2013; 8: e58935
        • Saqui-Salces M.
        • Coves-Datson E.
        • Veniaminova N.A.
        • et al.
        Inflammation and Gli2 suppress gastrin gene expression in a murine model of antral hyperplasia.
        PLoS One. 2012; 7: e48039
        • Merchant J.L.
        Hedgehog signalling in gut development, physiology and cancer.
        J Physiol. 2012; 590: 421-432
        • El-Zimaity H.M.
        • Ota H.
        • Graham D.Y.
        • et al.
        Patterns of gastric atrophy in intestinal type gastric carcinoma.
        Cancer. 2002; 94: 1428-1436
        • Fox J.G.
        • Wang T.C.
        Inflammation, atrophy, and gastric cancer.
        J Clin Invest. 2007; 117: 60-69
        • Wu C.
        • Cheng J.
        • Hu S.
        • et al.
        Reduced proliferation and increased apoptosis of the SGC7901 gastric cancer cell line on exposure to GDC0449.
        Mol Med Rep. 2016; 13: 1434-1440
        • Abdel-Rahman O.
        Hedgehog pathway aberrations and gastric cancer; evaluation of prognostic impact and exploration of therapeutic potentials.
        Tumour Biol. 2015; 36: 1367-1374
        • Ruiz i Altaba A.
        • Sanchez P.
        • Dahmane N.
        Gli and hedgehog in cancer: tumours, embryos and stem cells.
        Nat Rev Cancer. 2002; 2: 361-372
        • Marigo V.
        • Roberts D.J.
        • Lee S.M.
        • et al.
        Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog.
        Genomics. 1995; 28: 44-51
        • Katoh Y.
        • Katoh M.
        Comparative genomics on Sonic hedgehog orthologs.
        Oncol Rep. 2005; 14: 1087-1090
        • Litingtung Y.
        • Lei L.
        • Westphal H.
        • et al.
        Sonic hedgehog is essential to foregut development.
        Nat Genet. 1998; 20: 58-61
        • Shannon J.M.
        • Hyatt B.A.
        Epithelial-mesenchymal interactions in the developing lung.
        Annu Rev Physiol. 2004; 66: 625-645
        • Kim S.K.
        • Melton D.A.
        Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor.
        Proc Natl Acad Sci U S A. 1998; 95: 13036-13041
        • Willet S.G.
        • Mills J.C.
        Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis.
        Cell Mol Gastroenterol Hepatol. 2016; 2: 546-559
        • Ramalho-Santos M.
        • Melton D.A.
        • McMahon A.P.
        Hedgehog signals regulate multiple aspects of gastrointestinal development.
        Development. 2000; 127: 2763-2772
        • Van Den Brink G.R.
        • Hardwick J.C.
        • Tytgat G.N.
        • et al.
        Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.
        Gastroenterology. 2001; 121: 317-328
        • van den Brink G.R.
        • Hardwick J.C.
        • Nielsen C.
        • et al.
        Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.
        Gut. 2002; 51: 628-633
        • Fukaya M.
        • Isohata N.
        • Ohta H.
        • et al.
        Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer.
        Gastroenterology. 2006; 131: 14-29
        • Stepan V.
        • Ramamoorthy S.
        • Nitsche H.
        • et al.
        Regulation and function of the sonic hedgehog signal transduction pathway in isolated gastric parietal cells.
        J Biol Chem. 2005; 280: 15700-15708
        • Shiotani A.
        • Iishi H.
        • Uedo N.
        • et al.
        Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis progressing to gastric cancer.
        Am J Gastroenterol. 2005; 100: 581-587
        • Petersen C.P.
        • Mills J.D.
        • Goldenring J.R.
        Murine models of gastric corpus preneoplasia.
        Cell Mol Gastroenterol Hepatol. 2017; 3: 11-26
        • Yamaguchi H.
        • Goldenring J.R.
        • Kaminishi M.
        • et al.
        Identification of spasmolytic polypeptide expressing metaplasia (SPEM) in remnant gastric cancer and surveillance postgastrectomy biopsies.
        Dig Dis Sci. 2002; 47: 573-578
        • Engevik A.C.
        • Feng R.
        • Choi E.
        • et al.
        The development of spasmolytic polypeptide/TFF2-expressing metaplasia (SPEM) during gastric repair is absent in the aged stomach.
        Cell Mol Gastroenterol Hepatol. 2016; 2: 605-624
        • Suzuki H.
        • Minegishi Y.
        • Nomoto Y.
        • et al.
        Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pylori-infected Mongolian gerbils.
        J Pathol. 2005; 206: 186-197
        • Zavros Y.
        • Waghray M.
        • Tessier A.
        • et al.
        Reduced pepsin a processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.
        J Biol Chem. 2007; 282: 33265-33274
        • Tokhunts R.
        • Singh S.
        • Chu T.
        • et al.
        The full-length unprocessed hedgehog protein is an active signaling molecule.
        J Biol Chem. 2010; 285: 2562-2568
        • Singh S.
        • Wang Z.
        • Liang Fei D.
        • et al.
        Hedgehog-producing cancer cells respond to and require autocrine Hedgehog activity.
        Cancer Res. 2011; 71: 4454-4463
        • Konstantinou D.
        • Bertaux-Skeirik N.
        • Zavros Y.
        Hedgehog signaling in the stomach.
        Curr Opin Pharmacol. 2016; 31: 76-82
        • Saqui-Salces M.
        • Merchant J.L.
        Hedgehog signaling and gastrointestinal cancer.
        Biochim Biophys Acta. 2010; 1803: 786-795
        • Sierra R.
        • Une C.
        • Ramirez V.
        • et al.
        Association of serum pepsinogen with atrophic body gastritis in Costa Rica.
        Clin Exp Med. 2006; 6: 72-78
        • Iijima K.
        • Sekine H.
        • Koike T.
        • et al.
        Serum pepsinogen concentrations as a measure of gastric acid secretion in Helicobacter pylori-negative and -positive Japanese subjects.
        J Gastroenterol. 2005; 40: 938-944
        • Sipponen P.
        • Ranta P.
        • Helske T.
        • et al.
        Serum levels of amidated gastrin-17 and pepsinogen I in atrophic gastritis: an observational case-control study.
        Scand J Gastroenterol. 2002; 37: 785-791
        • Nomura A.M.
        • Kolonel L.N.
        • Miki K.
        • et al.
        Helicobacter pylori, pepsinogen, and gastric adenocarcinoma in Hawaii.
        J Infect Dis. 2005; 191: 2075-2081
        • Kokkola A.
        • Louhimo J.
        • Puolakkainen P.
        • et al.
        Helicobacter pylori infection and low serum pepsinogen I level as risk factors for gastric carcinoma.
        World J Gastroenterol. 2005; 11: 1032-1036
        • Shiotani A.
        • Iishi H.
        • Uedo N.
        • et al.
        Histologic and serum risk markers for noncardia early gastric cancer.
        Int J Cancer. 2005; 115: 463-469
        • Li P.
        • He C.
        • Sun L.
        • et al.
        Pepsinogen I and II expressions in situ and their correlations with serum pesignogen levels in gastric cancer and its precancerous disease.
        BMC Clin Pathol. 2013; 13: 22
        • Roberts N.B.
        Review article: human pepsins - their multiplicity, function and role in reflux disease.
        Aliment Pharmacol Ther. 2006; 24: 2-9
        • Fujinaga M.
        • Chernaia M.M.
        • Tarasova N.I.
        • et al.
        Crystal structure of human pepsin and its complex with pepstatin.
        Protein Sci. 1995; 4: 960-972
        • Bumcrot D.A.
        • Takada R.
        • McMahon A.P.
        Proteolytic processing yields two secreted forms of sonic hedgehog.
        Mol Cell Biol. 1995; 15: 2294-2303
        • Wendler F.
        • Franch-Marro X.
        • Vincent J.P.
        How does cholesterol affect the way Hedgehog works?.
        Development. 2006; 133: 3055-3061
        • Goetz J.A.
        • Suber L.M.
        • Zeng X.
        • et al.
        Sonic Hedgehog as a mediator of long-range signaling.
        Bioessays. 2002; 24: 157-165
        • Lee J.J.
        • Ekker S.C.
        • von Kessler D.P.
        • et al.
        Autoproteolysis in hedgehog protein biogenesis.
        Science. 1994; 266: 1528-1537
        • Roelink H.
        • Porter J.A.
        • Chiang C.
        • et al.
        Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis.
        Cell. 1995; 81: 445-455
        • Gritli-Linde A.
        • Lewis P.
        • McMahon A.P.
        • et al.
        The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides.
        Dev Biol. 2001; 236: 364-386
        • Goetz J.A.
        • Singh S.
        • Suber L.M.
        • et al.
        A highly conserved amino-terminal region of sonic hedgehog is required for the formation of its freely diffusible multimeric form.
        J Biol Chem. 2006; 281: 4087-4093
        • Zavros Y.
        • Orr M.A.
        • Xiao C.
        • et al.
        Sonic hedgehog is associated with H+,K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation.
        Am J Physiol Gastrointest Liver Physiol. 2008; 295: G99-G111
        • Schumacher M.A.
        • Donnelly J.M.
        • Engevik A.C.
        • et al.
        Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori.
        Gastroenterology. 2012; 142: 1150-1159 e1156
        • Ding L.
        • Hayes M.M.
        • Photenhauer A.
        • et al.
        Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.
        J Clin Invest. 2016; 126: 2867-2880
        • El-Zaatari M.
        • Daignault S.
        • Tessier A.
        • et al.
        Plasma sHH levels reduced in pancreatic cancer patients.
        Pancreas. 2012; 41: 1019-1028
        • Bai C.B.
        • Auerbach W.
        • Lee J.S.
        • et al.
        Gli2, but not Gli1, is required for initial SHH signaling and ectopic activation of the SHH pathway.
        Development. 2002; 129: 4753-4761
        • Pan Y.
        • Bai C.B.
        • Joyner A.L.
        • et al.
        Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation.
        Mol Cell Biol. 2006; 26: 3365-3377
        • van den Brink G.R.
        Hedgehog signaling in development and homeostasis of the gastrointestinal tract.
        Physiol Rev. 2007; 87: 1343-1375
        • van Eyll J.M.
        • Pierreux C.E.
        • Lemaigre F.P.
        • et al.
        SHH-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.
        J Cell Sci. 2004; 117: 2077-2086
        • Zuniga A.
        • Haramis A.P.
        • McMahon A.P.
        • et al.
        Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds.
        Nature. 1999; 401: 598-602
        • Khokha M.K.
        • Hsu D.
        • Brunet L.J.
        • et al.
        Gremlin is the BMP antagonist required for maintenance of SHH and Fgf signals during limb patterning.
        Nat Genet. 2003; 34: 303-307
        • Xiao C.
        • Feng R.
        • Engevik A.C.
        • et al.
        Sonic Hedgehog contributes to gastric mucosal restitution after injury.
        Lab Invest. 2013; 93: 96-111
        • Xiao C.
        • Ogle S.A.
        • Schumacher M.A.
        • et al.
        Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.
        Gastroenterology. 2010; 138: 550-561
        • Dimmler A.
        • Brabletz T.
        • Hlubek F.
        • et al.
        Transcription of sonic hedgehog, a potential factor for gastric morphogenesis and gastric mucosa maintenance, is up-regulated in acidic conditions.
        Lab Invest. 2003; 83: 1829-1837
        • El-Zaatari M.
        • Zavros Y.
        • Tessier A.
        • et al.
        Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.
        Gastroenterology. 2010; 139: 2061-2071 e2062
        • Waghray M.
        • Zavros Y.
        • Saqui-Salces M.
        • et al.
        Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.
        Gastroenterology. 2010; 138 (572 e561–562): 562-572
        • Zavros Y.
        • Rathinavelu S.
        • Kao J.Y.
        • et al.
        Treatment of Helicobacter gastritis with interleukin-4 requires somatostatin.
        Proc Natl Acad Sci U S A. 2003; 100: 12944-12949
        • Zavros Y.
        • Merchant J.L.
        Modulating the cytokine response to treat Helicobacter gastritis.
        Biochem Pharmacol. 2005; 69: 365-371
        • Beales I.L.
        Effect of cytokines on acid secretion and gastrin secretion in Helicobacter pylori infection and aspirin-induced gastritis.
        Scand J Gastroenterol. 1998; 33: 1230-1232
        • Okumura T.
        • Uehara A.
        • Okamura K.
        • et al.
        Inhibition of gastric pepsin secretion by peripherally or centrally injected interleukin-1 in rats.
        Biochem Biophys Res Commun. 1990; 167: 956-961
        • Uehara A.
        • Okumura T.
        • Sekiya C.
        • et al.
        Interleukin-1 inhibits the secretion of gastric acid in rats: possible involvement of prostaglandin.
        Biochem Biophys Res Commun. 1989; 162: 1578-1584
        • Wallace J.L.
        • Keenan C.M.
        • Cucala M.
        • et al.
        Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy.
        Gastroenterology. 1992; 102: 1176-1185
        • El-Omar E.M.
        • Carrington M.
        • Chow W.H.
        • et al.
        Interleukin-1 polymorphisms associated with increased risk of gastric cancer.
        Nature. 2000; 404: 398-402
        • Furuta T.
        • El-Omar E.M.
        • Xiao F.
        • et al.
        Interleukin 1beta polymorphisms increase risk of hypochlorhydria and atrophic gastritis and reduce risk of duodenal ulcer recurrence in Japan.
        Gastroenterology. 2002; 123: 92-105
        • Garza-Gonzalez E.
        • Bosques-Padilla F.J.
        • El-Omar E.
        • et al.
        Role of the polymorphic IL-1B, IL-1RN and TNF-A genes in distal gastric cancer in Mexico.
        Int J Cancer. 2005; 114: 237-241
        • Atsuta Y.
        • Ito L.S.
        • Oba-Shinjo S.M.
        • et al.
        Associations of TNF-A-1031TT and -857TT genotypes with Helicobacter pylori seropositivity and gastric atrophy among Japanese Brazilians.
        Int J Clin Oncol. 2006; 11: 140-145
        • Tu S.
        • Bhagat G.
        • Cui G.
        • et al.
        Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice.
        Cancer Cell. 2008; 14: 408-419
        • Rad R.
        • Dossumbekova A.
        • Neu B.
        • et al.
        Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during Helicobacter pylori infection.
        Gut. 2004; 53: 1082-1089
        • Brand S.J.
        • Stone D.
        Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria.
        J Clin Invest. 1988; 82: 1059-1066
        • Saqui-Salces M.
        • Keeley T.M.
        • Grosse A.S.
        • et al.
        Gastric tuft cells express DCLK1 and are expanded in hyperplasia.
        Histochem Cell Biol. 2011; 136: 191-204
        • Saqui-Salces M.
        • Dowdle W.E.
        • Reiter J.F.
        • et al.
        A high-fat diet regulates gastrin and acid secretion through primary cilia.
        FASEB J. 2012; 26: 3127-3139
        • Goldenring J.R.
        • Nam K.T.
        Oxyntic atrophy, metaplasia, and gastric cancer.
        Prog Mol Biol Transl Sci. 2010; 96: 117-131
        • Millrud C.R.
        • Bergenfelz C.
        • Leandersson K.
        On the origin of myeloid-derived suppressor cells.
        Oncotarget. 2017; 8: 3649-3665
        • Gabrilovich D.I.
        • Ostrand-Rosenberg S.
        • Bronte V.
        Coordinated regulation of myeloid cells by tumours.
        Nat Rev Immunol. 2012; 12: 253-268
        • Bronte V.
        • Brandau S.
        • Chen S.H.
        • et al.
        Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.
        Nat Commun. 2016; 7: 12150
        • Schwarz D.A.
        • Katayama C.D.
        • Hedrick S.M.
        Schlafen, a new family of growth regulatory genes that affect thymocyte development.
        Immunity. 1998; 9: 657-668
        • Puck A.
        • Aigner R.
        • Modak M.
        • et al.
        Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells.
        Results Immunol. 2015; 5: 23-32
        • van Zuylen W.J.
        • Garceau V.
        • Idris A.
        • et al.
        Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis.
        PLoS One. 2011; 6 (e15723)
        • Panda S.K.
        • Kolbeck R.
        • Sanjuan M.A.
        Plasmacytoid dendritic cells in autoimmunity.
        Curr Opin Immunol. 2016; 44: 20-25
        • Yamamoto M.
        • Takeda K.
        Current views of toll-like receptor signaling pathways.
        Gastroenterol Res Pract. 2010; 2010: 240365
        • Otani K.
        • Tanigawa T.
        • Watanabe T.
        • et al.
        Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis.
        Biochem Biophys Res Commun. 2012; 426: 342-349
        • Varga M.G.
        • Piazuelo M.B.
        • Romero-Gallo J.
        • et al.
        TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.
        Am J Physiol Gastrointest Liver Physiol. 2016; 311: G852-G858
        • Hernandez C.
        • Huebener P.
        • Schwabe R.F.
        Damage-associated molecular patterns in cancer: a double-edged sword.
        Oncogene. 2016; 35: 5931-5941
        • Varga M.G.
        • Shaffer C.L.
        • Sierra J.C.
        • et al.
        Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system.
        Oncogene. 2016; 35: 6262-6269
        • Wang X.
        • Xue L.
        • Yang Y.
        • et al.
        TLR9 promoter polymorphism is associated with both an increased susceptibility to gastric carcinoma and poor prognosis.
        PLoS One. 2013; 8: e65731
        • Companioni Napoles O.
        • Tsao A.C.
        • Sanz-Anquela J.M.
        • et al.
        SCHLAFEN 5 expression correlates with intestinal metaplasia that progresses to gastric cancer.
        J Gastroenterol. 2017; 52: 39-49
        • Donnelly J.M.
        • Engevik A.
        • Feng R.
        • et al.
        Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach.
        Am J Physiol Gastrointest Liver Physiol. 2014; 306: G1075-G1088
        • Petersen C.P.
        • Weis V.G.
        • Nam K.T.
        • et al.
        Macrophages promote progression of spasmolytic polypeptide-expressing metaplasia after acute loss of parietal cells.
        Gastroenterology. 2014; 146: 1727-1738 e1728
        • Buzzelli J.N.
        • Chalinor H.V.
        • Pavlic D.I.
        • et al.
        Il33 is a stomach alarmin that initiates a skewed Th2 response to injury and infection.
        Cell Mol Gastroenterol Hepatol. 2015; 1: 203-221
        • Goldenring J.R.
        Gastric intestinal metaplasia and tamoxifen: can we reverse the inevitable?.
        Dig Dis Sci. 2014; 59: 1078-1079
        • Choi E.
        • Hendley A.M.
        • Bailey J.M.
        • et al.
        Expression of activated Ras in gastric chief cells of mice leads to the full spectrum of metaplastic lineage transitions.
        Gastroenterology. 2016; 150: 918-930 e913
        • Syu L.-Y.
        • Zhao X.
        • Zhang Y.
        • et al.
        Invasive mouse gastric adenocarcinomas arising from Lgr5+ stem cells are dependent on crosstalk between the Hedgehog/GLI2 and mTOR pathways.
        Oncotarget. 2016; 7: 10255-10270
        • Yoon C.
        • Park D.J.
        • Schmidt B.
        • et al.
        CD44 expression denotes a subpopulation of gastric cancer cells in which Hedgehog signaling promotes chemotherapy resistance.
        Clin Cancer Res. 2014; 20: 3974-3988