Sonic hedgehog and CDX2 expression in the stomach

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1 doi: /j x GASTROENTEROLOGY Sonic hedgehog and CDX2 expression in the stomach Akiko Shiotani, Tomoari Kamada, Yoshiyuki Yamanaka, Noriaki Manabe, Hiroaki Kusunoki, Jiro Hata and Ken Haruma Department of Internal Medicine, Kawasaki Medical School, Okayama, Japan Key words CDX2, gastric cancer, Helicobacter pylori, intestinal metaplasia, sonic hedgehog (Shh). Correspondence Dr Akiko Shiotani, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima Kurashiki City, Okayama Prefecture , Japan. Conflict of Interest No conflict of interest have been declared by the authors. Abstract Sonic hedgehog (Shh) is an essential regulator of patterning processes throughout development, and CDX proteins act as the master regulators for intestinal development and differentiation. Shh and CDX2 seem to be interdependently linked with cellular differentiation through different signal cascades. We have recently shown that the loss of Shh and aberrant expression of CDX2 in Helicobacter pylori (H. pylori) associated atrophic gastritis can be modified by H. pylori eradication prior to incomplete intestinal metaplasia. On the other hand, abnormal signaling of the hedgehog pathway has been reported in gastric cancer, especially diffuse-type cancer and advanced gastric cancer, and Shh acts as a proliferation factor in both the normal mucosa and malignant lesions. CDX2 expressed in the early stage of gastric carcinogenesis is associated with the intestinal phenotypic region and thus with a better outcome. However, it remains unclear how Shh and CDX2 are involved with intestinal transformation and further carcinogenesis. Introduction The risk of developing gastric cancer is closely related to Helicobacter pylori (H. pylori) associated progressive gastric inflammation, especially in the corpus. 1 4 Previous studies have suggested that the type and location of intestinal metaplasia (IM) provides important additional information regarding the risk of developing gastric cancer, 2,5,6 and IM is thought to be an important precancerous lesion in the multistep progression of gastric carcinoma as proposed by Correa. 7 However, there are data suggesting that cancer and IM arise from different cell lineages, such that IM may not be a precursor lesion but rather is likely a marker of increased risk. 8,9 Sonic hedgehog (Shh) is an essential regulator of patterning processes throughout development and CDX proteins act as master regulators of intestinal development and differentiation We have recently shown that the loss of Shh and aberrant expression of CDX2 in H. pylori-associated atrophic gastritis are early changes correlating with the presence of IM and can be modified by H. pylori eradication. 6,16,17 In contrast, abnormal activation of the Shh pathway has recently been reported in malignant lesions, including in the gastrointestinal (GI) tract, 18,19 and CDX2 has been shown to be strongly associated with intestinal phenotypic expression in gastric carcinomas However, the role of Shh and CDX2 in gastric carcinoma is not clearly understood. Shh act as a proliferation factor and CDX2 may suppress expansion of malignant cells. Here, we review the current and emerging concepts of Shh and CDX2 expression in the human stomach. Shh Embryonal differentiation processes are regulated through a number of signal transduction cascades and one of these begins with the secretion of the peptide morphogen called hedgehog (Hh). 23 Hh was initially identified in a genetic screen of the fruit fly Drosophila, 24 and subsequently, several Hh homologs (Shh, Indian hedgehog [Ihh], and desert hedgehog [Dhh]) were identified in vertebrates. 24 The Hh family are critical regulators in many patterning processes throughout both vertebrate and invertebrate development during embryogenesis, such as the left right asymmetry decision, anterior posterior axis decision in limb pattern determination, and patterning of various organs including brain, spinal cord, eye, craniofacial structures, lung, teeth, eye, and hair. 10 Moreover, Shh plays a key role in maintenance of adult tissue homeostasis, tissue repair during chronic persistent inflammation, and carcinogenesis. 25 Shh null mice display GI malformations, such as failure of the trachea and esophagus to separate normally, gut malrotation, small intestinal, and anal atresias, and an overgrowth of stomach epithelium that, upon histological examination, appears to be glandular epithelium reminiscent of an intestinal phenotype. 26,27 Shh and Ihh are coexpressed in the gut endoderm in partially overlapping patterns from the early somite stages. 11,12 Shh and/or Dhh may promote differentiation of parietal cells, whereas Ihh may induce that of pit cells. In a study of humans by Van den Brink et al. Shh mrna and Shh protein were shown to be abundantly expressed in the normal gastric fundus and in fundic gland metaplasia, whereas no Shh protein was observed in the esophagus or intestines. 28,29 Pit cells migrate up from the precursor cell and S161

2 Shh and CDX2 expression in the stomach A Shiotani et al. Shh is exclusively expressed by the downward migrating parietal cells of the gastric gland. Shh expression in humans is restricted to the large triangular parietal cells and high expression in the parietal cells near the pit-gland transition gradually diminishes toward the gland base. 29 Shh binds to its receptor Patched (PTC-PTCH1, PTCH2), which reduces the inhibitory effect of PTCH on another transmembrane protein, smoothened (SMO). Binding results in de-repression of SMO, activating a cascade that leads to the translocation of the active form of the transcription factor gliomaassociated oncogene homolog (GLI) to the nucleus Hh signaling activation leads to cell proliferation through cell cycle regulation. Nuclear GLI activates expression of a variety of target genes such as BMP4, FOXAA2, ISL1, and FOXM1 which play important roles in gastric organogenesis and maintenance of organ structure. 18,29,33 CDX2 The CDX proteins are intestine-specific transcription factors encoded by human cdx1 and cdx2 genes, and constitute the mammalian homolog of the Drosophilia homeobox gene, caudal. 13,14 In adult mammals, the expression of these genes is restricted to the epithelium of the gut from the duodenum to the large intestine, where they act as master regulators of intestinal development and differentiation. 15 CDX1 is predominantly expressed in the undifferentiated cells of the intestinal crypts and play a role in the transdifferentiation of the mucosa to an intestinal type, whereas CDX2 is mostly present in the villi or differentiated cell compartment of the small intestine. 15,34 In particular, the maintenance of intestinal differentiation appears to depend on the presence of CDX2, and loss of CDX2 expression leads to focal gastric differentiation in the colon, 35 whereas aberrant expression of CDX2 in the upper GI tract is a key event in the pathogenesis of Barrett s esophagus and of IM in the stomach. 36,37 Cdx2 plays a role in the extracellular matrix mediated intestinal cell differentiation. Runx3, a runt domain transcription factor, is a major growth regulator of gastric epithelial cells, and some gastric epithelial cells in the Runx3 / mouse differentiate into intestinal-type cells expressing CDX2. 38 Sucrase-isomaltase, glucagon, carbonic anhydrase I, lactase, and MUC2, etc. have been suggested as candidates for CDX2-regulated target genes. 22,38 Moreover, CDX2 appears to directly regulate the LI-cadherin (cadherin 17) gene and hephaestin. LI-cadherin is an intestine-specific cell adhesion molecule and expressed in IM and intestinal-type gastric cancer, and hephaestin plays a critical role in the transport of iron into the intestinal epithelium. 39 Gastric atrophy and intestinal metaplasia The degree of damage by H. pylori infection is initially most severe in the antrum, but over time the damage progresses into the gastric corpus and can be visualized as an advancing atrophic border that involves the lesser curvature more rapidly than the greater curvature. 1,40 42 Gastric IM has been classified as complete (small intestine) or incomplete (colonic) using immunohistochemical staining techniques and into three types based on the staining pattern of its mucins: I (complete), and II and III (incomplete). Type I is the most common type: Paneth cells are present and goblet cells secrete sialomucins. Types II and III are characterized by the presence of columnar cells and goblet cells secreting sialomucins and/or sulfomucins; the columnar cells secrete sialomucins in type II and sulfomucins in type III. 43 IM has also been classified according to the cell classification status using both gastric and intestinal phenotypic markers, and IM is divided into two major types: gastric and intestinal mixed (GI) type, and a solely intestinal (I) type. Mucins are heavily glycosylated proteins that constitute the major component of the mucous protective layer above mucosal surfaces. MUC5AC and MUC6 and galactose oxidase-schiff, etc. are markers of the gastric phenotype, and MUC2, CD10, Villin, sucrase, and etc. are intestinal phenotypic markers The normal gastric mucosa shows cell-type specific expression of MUC1, 5AC, and 6, and does not express MUC2. Underexpression of MUC1, MUC5AC, and MUC6 and de novo expression of MUC2 have been described in IM. The most incomplete IM (types II and III) preserving gastric mucin is considered to be the GI type, whereas the complete type is considered to be type I in the corpus. 5 In retrospective studies, type III IM has been found in 75 90% of cases of intestinal-type gastric cancer that underwent surgical resection. 49,50 The presence of type III IM has also been associated with an increased risk of developing early gastric cancer, but not with diffuse-type gastric carcinoma. 54 However, the detection of IM in routinely obtained endoscopic biopsy material is common and this alone makes its presence an unsuitable marker for identifying patients in whom surveillance is indicated. In addition, the detection of IM is subject to sampling error. Using a systematic endoscopic protocol with fixed-point biopsy samples, we previously confirmed that IM in the corpus, especially at the lesser curvature, was the most significantly increased risk marker of gastric cancer. 2 In our recent study examining the role of the type of IM and pattern of mucin expression in IM as histological risk markers of gastric cancer, patients with incomplete IM in the lesser curvature were likely to have severe atrophic pan-gastritis and such patients are at increased risk of developing gastric cancer. 5 However, most of the studies on IM as a risk marker have been done in patients who already had gastric cancers, and further long-term prospective studies are needed to confirm the usefulness of the classification of IM by histochemical staining in detecting the high-risk group for gastric cancer. Shh and CDX2 expression in intestinal metaplasia The loss of fundic glands in gastric IM in humans is accompanied by loss of Shh expression; 28 Shh-expressing cells are adjacent to metaplastic glands containing goblet cells expressing MUC 2, and the expression of Shh and MUC 2 is mutually exclusive. 28 On the other hand, aberrant expression of CDX2 correlates with development of IM. The expression of CDX2, as well as of MUC2, in isolated gastric glands is progressively up-regulated with intestinalization from the gastric type to the gastric/intestinal-mixed type to the intestinal type. 44 In our previous study, Shh expression correlated with the atrophy score and type of IM, and its expression decreased in H. pylori-negative and -positive controls, and in the cancer group, S162

3 A Shiotani et al. Shh and CDX2 expression in the stomach (a) (b) Figure 1 Expression of sonic hedgehog (Shh) in a section from the corpus greater curve of a control subject before and after eradication of Helicobacter pylori. (a) Before eradication, some Shh-stained cells can be seen in the fundic glands. (b) In the same subject after eradication, the number of Shhstained cells dramatically increases. (a) (b) Figure 2 (a) Alcian blue high-iron diamine (AB-HID) staining and (b) expression of CDX2 in serial sections taken after Helicobacter pylori eradication from the corpus lesser curve of a patient with a history of endoscopic mucosal resection for early gastric cancer. AB-HID staining shows some goblet cells and columnar cells stained brown (sulfomucin), indicating incomplete intestinal metaplasia (IM). In the patients with incomplete IM before eradication, there was persistent inflammation and IM and CDX2 staining cells were not significantly decreased after eradication. in that order (Fig. 1). 6 CDX2 staining in the lesser curvature was higher in the cancer group than in the controls, and CDX2 indices increased according to the increase of the atrophy score and increased in the ascending order of those without IM, those with complete IM, and those with incomplete IM (P < 0.001; Fig. 2). 16 Ramalho-Santos et al. reported that Shh-null mice exhibit an overgrowth of stomach epithelium that, upon histological examination, appears to be glandular epithelium reminiscent of an intestinal phenotype. Moreover, the transformation of the stomach epithelium does not depend on alterations in the expression of the Cdx2 genes. 26 Thus, Shh appears to be an essential signal of fundic gland differentiation and might prevent the modulation of differentiated gastric mucosa to an IM phenotype. In contrast, CDX2 seems to be a signal essential for modulation to an IM phenotype, and might function by preventing the execution of a fundic gland differentiation pattern. Atrophic change, specifically the loss of parietal cells, leads to the loss of Shh expression, which might in turn lead to aberrant expression of CDX2. However, CDX2 and Shh seem to be involved in intestinal transformation interdependently through different signal cascades. In H. pylori infection, parietal cells may be present, but their function, specifically of acid secretion, is suppressed. It is not known yet whether reduced acid secretion also reduces Shh expression. 6,28,29 Recent studies have shown that H +,K + -ATPase gene expression is regulated by Shh. 55 Further, Shh gene expression in a gastric cancer cell line was stimulated by acid conditions. 56 In contrast, CDX2 expression was down-regulated under acidic conditions in an intestinal cell line. 56,57 CDX2 expression is lost in duodenal gastric metaplasia, a condition in which acid can be secreted in the duodenum. 57 Overall, these findings suggest that a change in gastric acidity plays a role in the development or improvement of atrophy via changes in Shh expression and signaling, which might create a permissive environment for the expression of CDX2. In Foxa 3/Cdx2 transgenic mice, the majority of intestinal-type goblet cells produce sulfated mucin, which indicates type III IM, and intestinal genes such as alkaline phosphatase, villin, Muc 2, and trefoil family factor 3 (Tff3) are also activated. 37 The molecular pathogenesis underlying the morphological alterations caused by complete and incomplete IM remains largely unknown. It is also unclear whether CDX2 and Shh are specifically and interactively involved with these intestinal transformations. Eradication of H. pylori In the study by Wong et al. in a high-risk region of China, H. pylori eradication reduced the incidence of gastric cancer development only in the subgroup without atrophy, IM or dysplasia. 58 We recently compared the improvement in corpus gastritis with Shh and CDX2 expression after H. pylori eradication between subjects at high risk for gastric cancer and controls. 17 The difference between the control and cancer groups became remarkable for corpus atrophy after eradication, and likewise in Shh and CDX2 expressions in the corpus. Eradication of H. pylori was associated with a significant increase in Shh expression at the corpus greater (P < 0.001; Fig. 1) and lesser curves (P = 0.001) in S163

4 Shh and CDX2 expression in the stomach A Shiotani et al. both groups of patients studied. CDX2 expression at the corpus lesser curve also significantly decreased (P = 0.001) after eradication. Interestingly, Shh expression and CDX2 expression in the lesser curvature significantly improved in the mucosa without incomplete IM, but not in the mucosa with incomplete IM (Fig. 2). Advanced atrophic gastritis and incomplete IM continued to show more inflammation and persistent IM after H. pylori eradication and thus were likely to continue to be a significant cancer risk. The loss of Shh and aberrant expression of CDX2 in H. pyloriassociated atrophic gastritis can be modified by H. pylori eradication probably through modulation of the inflammatory condition in the gastric mucosa, and eradication prior to incomplete IM improves corpus gastritis and may prevent intestinal-type gastric cancer. However, there is still the possibility that it might take more time to improve inflammation in the lesion with severe atrophy or IM and inflammation might be sustained by incomplete IM. Further studies with long-term follow-up after eradication are required to investigate the effect of eradication in the high-risk group. Shh in gastric cancer Gastric cancers are classified mainly into the intestinal type and diffuse type, based on the tendency of gland formation. Shh is implicated in stem/progenitor cell restitution of damaged gastric mucosa during chronic infection with H. pylori. Shh and Ihh up-regulation and Hh-interacting protein (HHIP) down-regulation lead to aberrant activation of Hh signaling through PTCH1 to GLI1 in gastric cancer. 18 Fukaya et al. recently reported that Shh was expressed not in tumor cells but in myofibroblasts in diffuse-type gastric cancer and Hh signaling molecules express in a subset of gastric cancer. Despite Shh mrna expression, intestinal-type gastric cancers show a low and infrequent expression of Shh and the primary target genes, GLI1 and GLI 2, although most of the diffuse-type cancers express GLI1 and GLI2. 18 Hh inhibitors, such as cyclopamine, cause growth inhibition and regression of the cancerous xenograph of many tissues and might be a novel way of treating Hh-responsive tumors Shh in the normal stomach and in gastric tumors acts as a proliferation factor. However, loss of Shh in the non-cancerous mucosa in patients with intestinal-type gastric cancer is recognized and H. pylori eradication induces recovery of Shh expression. Shh in the normal stomach may play an inhibitory role in carcinogenensis, whereas in gastric cancer, especially the diffuse type, it seems to have a progressive role in carcinogenensis. CDX2 in gastric cancer CDX2 is associated with intestinal-type gastric cancer and is expressed at the early stage of gastric carcinogenesis in the intestinal phenotypic region. 22,62 Recently, Mutoh et al. reported that gastric polyps developing from the intestinal metaplastic mucosa of all stomachs in Cdx2-transgenic mice consisted of intestinaltype adenocarcinoma containing p53 and APC gene mutations. 63 The phenotypic expression of gastric cancer can be classified into gastric and intestinal epithelial cell types by immunohistochemistry using the same epithelial cell marker for IM. The hypothesis that intestinal-type carcinomas arise in intestinalized mucosa, whereas the diffuse-type develops from the proper gastric mucosa, is based on morphological similarities between the cancers and IM. However, there are several contradictions, and it has been shown that the early stage of gastric cancer, independent of the histological type, mainly consists of the gastric phenotype and shifts to the intestinal phenotype during tumor progression. 64 Mizoshita et al. studied the expression CDX1/2 in relation to phenotypic gastric cancers. They divided them into gastric type (G-type), gastric intestinal mixed type (GI type), intestinal type (I type), and null type (N type). CDX2 expression was detected not only in the I type region, but also in the N type region. The GI and I types have significantly greater CDX2 expression than the N type (P < 0.001), whereas the G type has no Cdx2 expression. 65 On the other hand, previous studies suggest that cdx2 is a tumorsuppressor gene with regard to colorectal carcinogenesis. CDX2 up-regulates transcription of p21/waf1/cip1, which plays a critical role in differentiation and tumor suppression, and promotes intestinal differentiation as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest. 66,67 CDX2-positive expressing tumors show tendencies towards less invasiveness and fewer lymph node metastases and have a better outcome than CDX2-negative tumors. 62,68 The role of CDX2 in gastric cancer still remains unclear and CDX2 may have different roles depending on cancer type. Conclusions Shh and CDX2 seem to be interdependently linked with cellular differentiation through different signal cascades. The loss of Shh and aberrant expression of CDX2 in the stomach are associated with H. pylori-associated atrophic gastritis and can be modified by H. pylori eradication. Eradication prior to incomplete IM improves corpus gastritis and may prevent intestinal-type gastric cancer. 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