Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Cancer Prevention Research Research Article Ink4a/Arf-Dependent Loss of Parietal Cells Induced by Oxidative Stress Promotes CD44-Dependent Gastric Tumorigenesis Ryo Seishima1,2,Takeyuki Wada2, Kenji Tsuchihashi1, Shogo Okazaki1, Momoko Yoshikawa1, Hiroko Oshima3, Masanobu Oshima3,Toshiro Sato4, Hirotoshi Hasegawa2,Yuko Kitagawa2, James R. Goldenring5, Hideyuki Saya1, and Osamu Nagano1 Abstract Loss of parietal cells initiates the development of spasmolytic polypeptide–expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis. Cancer Prev Res; 8(6); 492–501.
2015 AACR. Introduction considered an early and critical event in the histopathologic progression of the stomach epithelium and eventual development of gastric cancer. The molecular mechanism underlying parietal cell loss has remained unclear, however. Chronic inflammation followed by carcinogenesis is associated with the production of reactive oxygen species (ROS; refs. 7, 8). ROS function as activators of MAPK signaling, including the Ras–Raf–MEK1/2–ERK1/2 and p38MAPK signaling pathways, and they play opposing roles in tumor promotion and suppression (9, 10). In general, ROS-mediated activation of Ras–Raf–MEK1/2–ERK1/2 signaling is associated with carcinogenesis as a result of its promotion of cell survival and proliferation, whereas ROS-mediated activation of p38MAPK signaling inhibits cell proliferation and induces cell senescence through induction of the tumor-suppressor proteins p16INK4a and p19ARF (11). The functional relevance of ROS-mediated signaling in the development of metaplasia and gastric cancer has not been elucidated, however. CD44 exists in numerous isoforms that are generated through alternative splicing of CD44 precursor mRNA (12– 14). CD44 variant (CD44v) isoforms, which contain additional insertions in the membrane-proximal extracellular region, are highly abundant in epithelial-type carcinomas (15). We previously showed that interaction of CD44v with xCT (SLC7A11), a subunit of the cystine/glutamate antiporter system xc(–), stabilizes the latter protein, and thereby promotes glutathione synthesis and potentiates the ability of cancer cells to defend themselves against ROS (16). In gastric carcinogenesis, de novo expression of CD44v is associated with the development of SPEM (17) and gastric tumors (18), and CD44v was recently identified as a cell surface marker of gastric cancer stem cells (19). Consistent with these observations, we recently showed that potentiation of ROS defense by the CD44v-xCT system Chronic inflammation induces histopathologic progression of the stomach epithelium leading to the development of metaplasia followed by gastric adenocarcinoma (1, 2). Oxyntic glands, the predominant type of gastric gland, comprise several types of fully differentiated epithelial cells, including acid-secreting parietal cells and pepsinogen-secreting chief cells. Inflammation of the gastric epithelium results in a gradual loss of parietal cells and their replacement with proliferative metaplastic cells derived from transdifferentiated chief cells (3, 4), suggesting that chronic inflammation disrupts homeostasis of the gastric epithelium by altering the viability or differentiation status of these differentiated cells. Spasmolytic polypeptide–expressing metaplasia (SPEM) is triggered by the parietal cell loss, and is recognized as a precancerous lesion (5, 6). Parietal cells, thus, play a key role in the homeostasis of gastric glands, with the loss of these cells being 1 Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan. 2Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan. 3Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 4Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan. 5Nashville VA Medical Center and the Epithelial Biology Center,Vanderbilt University School of Medicine, Nashville, Tennessee. Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). Corresponding Author: Osamu Nagano, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3981; Fax: 81-3-5363-3982; E-mail: osmna@sb3.so-net.ne.jp doi: 10.1158/1940-6207.CAPR-15-0025-T
2015 American Association for Cancer Research. 492 Cancer Prev Res; 8(6) June 2015 Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Ink4a/Arf Induces Loss of Parietal Cells plays a key role not only in tumor cells, but also in the expansion of SPEM cells in the K19-Wnt1/C2mE mouse, a transgenic model of gastric cancer induced by activation of Wnt and prostaglandin E2 (PGE2) signaling pathways (17). The populations of CD44v-expressing metaplastic cells and tumor cells in which ROS defense is bolstered by the CD44v-xCT system might, thus, be able to expand preferentially in inflamed gastric epithelium exposed to high levels of ROS. However, the role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown. Materials and Methods Animals K19-Wnt1/C2mE transgenic mice, Ink4a/Arf / mice and CD44 / K19-Wnt1/C2mE mice were described as previously (16, 20, 21). K19-Wnt1/C2mE transgenic mice were crossed with Ink4a/Arf / mice to generate Ink4a/Arf / K19-Wnt1/ C2mE mice. All animal experiments were performed according to protocols approved by the Ethics Committee of Keio University. All efforts were made to minimize the suffering of animals used in this study. Tamoxifen injection Tamoxifen (Sigma-Aldrich) was injected i.p. at a daily dose of 250 mg/kg in WT or Ink4a/Arf / mice for 3 days, and tissue was dissected for analysis at 3 days after the last injection. K19-Wnt1/ C2mE mice at 20 weeks of age were injected i.p. with tamoxifen (250 mg/kg) once a week for 5 weeks. Tamoxifen was dissolved in a vehicle consisting of 10% ethanol and 90% sunflower seed oil (Sigma-Aldrich), and control mice were injected with vehicle alone. Immunohistochemistry Immunohistochemistry was performed as previously reported (16, 17). TFF2 was detected with rabbit monoclonal antibodies (diluted 1:100; Proteintech).The TFF2-positive area in five randomly selected microscopic fields (magnification, 40) per section was measured with the use of analysis software (BZ-9000; Keyence), and the mean percentage positive area was calculated. Parietal cells were detected with mouse monoclonal antibodies to the b subunit of Hþ,Kþ-ATPase (MBL). The mean percentage of Hþ,Kþ-ATPase–positive cells per gland in five microscopic fields per mouse was calculated. CD44v8-10 was detected with a rat monoclonal antibody (diluted 1:100; ref. 16), and the proportion of positive cells was determined with the use of a TissueFAXS cell analysis system (Novel Science). Immunoblot analysis Immunoblot analysis was performed as described previously (16, 17). Gastric unit isolation and gastric organoid culture Gastric gland units were isolated as previously described (22). Other experimental procedures are described in Supplementary Materials and Methods. Statistical analysis Data are presented as means  SD and were analyzed with the unpaired Student t test as performed with the use of Microsoft www.aacrjournals.org Excel 2007. A P value of <0.05 was considered statistically significant. Results Depletion of ROS suppresses gastric tumorigenesis in K19Wnt1/C2mE mice To explore the relevance of ROS in gastric carcinogenesis, we orally administered ascorbic acid, a potent water-soluble antioxidant, to K19-Wnt1/C2mE mice from 12 to 30 weeks of age. Tumors were markedly smaller in the ascorbic acid–treated K19-Wnt1/C2mE mice than in untreated control animals at 30 weeks of age (Fig. 1A and B), suggesting that gastric tumorigenesis is promoted by ROS in these mice. Given that CD44v promotes ROS defense in tumor cells in this model (17), expansion of the CD44v-expressing tumor cells might be the result of selection by ROS in the tumor microenvironment. We therefore investigated CD44v expression in gastric tumors of K19-Wnt1/C2mE mice treated with ascorbic acid. The relative area occupied by CD44v-expressing cells in tumors was markedly reduced by ascorbic acid treatment (Fig. 1C), suggesting that CD44v-expressing tumor cells expand preferentially compared with CD44v-negative cells in the presence of ROS. The expression of CD44v in epithelial cells of the normal foregut was not diminished by ascorbic acid treatment, however, suggesting that ROS depletion suppresses the selective expansion of ROS-resistant CD44v-expressing tumor cells without affecting normal epithelial cells in K19Wnt1/C2mE mice. Given that SPEM is a premalignant lesion (4), we next investigated whether CD44v-expressing SPEM cells might be the cells of origin for CD44v-expressing tumor cells. We thus examined whether ROS depletion by ascorbic acid treatment was able to reduce not only the number of CD44v-expressing tumor cells, but also that of CD44v-expressing SPEM cells in K19-Wnt1/C2mE mice. Formation of trefoil factor 2 (TFF2)–expressing SPEM was significantly suppressed in ascorbic acid–treated mice (Fig. 1D and E), indicating that ROS promotes CD44v-expressing SPEM formation as well as tumor formation. Given that the parietal cell loss is a key event in SPEM development, we next examined the expression of the b subunit of Hþ, Kþ-ATPase, a marker of parietal cells. Immunohistochemical analysis revealed that the loss of parietal cells was significantly attenuated in ascorbic acid–treated mice (Fig. 1D and F), implicating ROS in the parietal cell loss apparent in untreated K19Wnt1/C2mE mice. Analysis of the expression of CD44v in epithelium adjacent to tumors of K19-Wnt1/C2mE mice revealed that the emergence of CD44v-expressing SPEM cells was markedly suppressed by ascorbic acid treatment (Fig. 1D). Thus, ROS play a key role in parietal cell loss leading to the development of SPEM comprising CD44v-expressing SPEM cells. p38MAPK is activated specifically in gastric parietal cells of K19Wnt1/C2mE mice To examine whether oxidative stress–induced signaling is operative in parietal cells of K19-Wnt1/C2mE mice, we investigated the activation status of p38MAPK, a major target of ROS. The phosphorylated (activated) form of p38MAPK (phosphop38MAPK) was found to be abundant in parietal cells of K19Wnt1/C2mE mice (Fig. 2A), suggesting that ROS accumulate in these cells. To address this possibility further, we measured ROS Cancer Prev Res; 8(6) June 2015 Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. 493 Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Seishima et al. Figure 1. Effects of ascorbic acid on development of gastric tumors and SPEM. A, hematoxylin and eosin (H&E) staining of gastric tumors of K19Wnt1/C2mE mice treated (or not) with ascorbic acid from 12 to 30 weeks of age. The black and red dashed lines indicate the tumor boundary and adjacent regions, respectively; T, tumor; AT, region adjacent to tumor; scale bars, 500 mm. B, mucosal thickness, which reflects the size of the tumor, in K19-Wnt1/C2mE mice treated (or not) with ascorbic acid from 12 to 30 weeks of age (n ¼ 3 and 5, respectively) relative to that in wildtype (WT) mice (n ¼ 5). Data, means  SD for the indicated numbers of mice;  , P < 0.05;   , P < 0.01 (Student t test). C, immunohistochemical staining for CD44v in gastric tumors of K19-Wnt1/ C2mE mice treated (or not) with ascorbic acid from 12 to 30 weeks of age. The boxed regions in the left are shown at higher magnification in the middle. Staining for CD44v in the foregut is also shown in the right as a positive control; scale bars, 200 mm (left and right) or 100 mm (middle). D, immunohistochemical analysis of þ þ TFF2, the b subunit of H ,K -ATPase and CD44v in regions adjacent to gastric tumors of K19-Wnt1/C2mE mice treated (or not) with ascorbic acid from 12 to 30 weeks of age; scale þ bars, 200 mm. E, mean area of TFF2 epithelium determined from sections similar to those in D. Data, means  SD for 3 or 5 K19-Wnt1/C2mE mice treated or not treated with ascorbic acid, respectively, as well as for the normal gastric epithelium of five WT mice;   , P < 0.01 (Student t test). F, the mean þ þ percentage of H ,K -ATPase–positive cells per gland in five microscopic fields per mouse was calculated in tumor-adjacent regions of sections similar to those in D. Data, means  SD for the numbers of animals indicated in E;   , P < 0.01 (Student t test). levels in parietal cells and nonparietal gastric cells isolated from wild-type (WT) and K19-Wnt1/C2mE mice. Flow cytometric analysis of cells stained with 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) revealed that DCFH-DA fluorescence intensity was much higher in parietal cells of K19-Wnt1/C2mE mice than in nonparietal cells of the same animals or in parietal cells or nonparietal cells of WT mice (Fig. 2B), suggesting that ROS activates p38MAPK signaling in parietal cells. To examine further whether p38MAPK is activated selectively in parietal cells of the stomach epithelium in the presence of high ROS levels, we exposed WT mice to ionizing radiation at a dose of 10 Gy to induce ROS production. The abundance of phospho- 494 Cancer Prev Res; 8(6) June 2015 p38MAPK was increased selectively in parietal cells of the irradiated mice (Fig. 2C), suggesting that parietal cells are highly sensitive to oxidative stress. We also examined the sensitivity of p38MAPK activity in parietal cells to ROS with the use of a gastric organ culture system (Fig. 2D). Exposure of normal gastric mucosa to the oxidative stressor hydrogen peroxide (H2O2) induced p38MAPK activation specifically in parietal cells, and this activation was attenuated in the additional presence of ascorbic acid or the specific p38MAPK inhibitor SB203580 (Fig. 2E). Together, these results indicated that parietal cells have a low capacity for ROS defense, and therefore might be a major target for ROS during gastric carcinogenesis. Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Ink4a/Arf Induces Loss of Parietal Cells Figure 2. MAPK ROS induce activation of p38 specifically in parietal cells. A, immunohistofluorescence staining of þ þ H ,K -ATPase (red) and phosphoMAPK (green) as well as staining of p38 nuclei with 40 ,6-diamidino-2phenylindole (DAPI, blue) in the gastric epithelium of a WT mouse as well as in a gastric tumor–adjacent region (AT) of a K19-Wnt1/C2mE mouse at 30 weeks of age. The boxed region in the middle is shown at higher magnification in the right; scale bars, 200 mm (left and middle) or 100 mm (right). B, sorted parietal cells and nonparietal cells from SPEM lesions of K19-Wnt1/C2mE mice at 30 weeks of age and those from the glandular stomach of WT mice were stained with DCFH-DA and subjected to flow cytometric analysis; RFI, relative fluorescence intensity. C, immunohistofluorescence staining of þ þ MAPK H ,K -ATPase and phospho-p38 as well as staining of nuclei with DAPI in the gastric epithelium either of a WT mouse exposed to 10 Gy of ionizing radiation or of a nonirradiated control mouse. Insets show corresponding higher magnification images; scale bars, 100 mm. D, schematic representation of gastric organ culture. E, immunohistofluorescence þ þ staining of H ,K -ATPase and MAPK as well as staining phospho-p38 of nuclei with DAPI in stomach tissue of WT mice cultured for 6 hours in the absence or presence of 500 mmol/L H2 O2, ascorbic acid (50 mg/mL) and 10 mmol/L SB203580 as indicated. Insets show corresponding higher magnification images; scale bars, 100 mm. Parietal cell loss is triggered by ROS p38MAPK p16INK4a/ p19ARF signaling Given that activated p38MAPK has been shown to induce gene expression at the Ink4a/Arf locus, which encodes p16INK4a and p19ARF, in cells exposed to oxidative stress (10), we next examined whether the expression of these proteins is increased in SPEM lesions of K19-Wnt1/C2mE mice. The abundance of p16INK4a and p19ARF as well as that of phospho-p38MAPK were markedly increased in SPEM of K19-Wnt1/C2mE mice compared with normal gastric mucosa of WT mice (Fig. 3A), suggesting that the p38MAPK p16INK4a/p19ARF pathway is activated in SPEM. www.aacrjournals.org To investigate the functional relevance of p16INK4a and p19ARF to parietal cell loss, we examined the effects of i.p. injection of tamoxifen, a potent inducer of parietal cell loss (23), in Ink4a/Arf knockout (Ink4a/Arf / ) mice. Immunohistochemical analysis revealed that the tamoxifen-induced parietal cell loss was markedly suppressed in Ink4a/Arf / mice compared with WT mice (Fig. 3B and C), suggesting that the Ink4a/Arf locus is essential for this effect of tamoxifen. The area of TFF2-positive mucosa was also substantially smaller in tamoxifen-treated Ink4a/Arf / mice than in tamoxifen-treated WT mice (Fig. 3B and D). Together, these results suggested that the Ink4a/Arf locus is required for the parietal cell loss, which plays a role in the development of SPEM in tamoxifen-treated mice. Cancer Prev Res; 8(6) June 2015 Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. 495 Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Seishima et al. Figure 3. The Ink4a/Arf locus is essential for parietal cell loss. A, immunoblot INK4a ARF , p19 , and analysis of p16 phosphorylated (p-) and total forms of MAPK in SPEM of 30-week-old K19p38 Wnt1/C2mE mice and in normal gastric mucosa of WT mice. b-Actin was analyzed as a loading control. B, immunohistochemical staining of þ þ H ,K -ATPase and TFF2 in gastric / mice at mucosa of WT or Ink4a/Arf 3 days after daily i.p. injection with tamoxifen (TAM) for 3 days; scale bars, þ 100 mm. C, the mean percentage of H , þ K -ATPase–positive cells per gland in five microscopic fields per mouse was calculated in gastric mucosa from sections similar to those in B. Data, means  SD for five tamoxifen-treated / WT or Ink4a/Arf mice;  P < 0.01 (Student t test). D, mean þ area of TFF2 gastric epithelium determined from sections similar to those in B. Data, means  SD for the numbers of animals indicated in C;  , P < 0.05 (Student t test). E, immunohistofluorescence staining of þ þ H ,K -ATPase and either phosphoMAPK ARF p38 or p19 in gastric mucosa of tamoxifen-treated WT or Ink4a/ / Arf mice. Nuclei were also stained with DAPI (blue). Insets show corresponding higher magnification images; scale bars, 100 mm. F, immunohistofluorescence staining of Ki67 and CD44v as well as staining of nuclei with DAPI in gastric mucosa of / tamoxifen-treated WT or Ink4a/Arf mice as well as of a nontreated WT mouse; scale bars, 100 mm. We next investigated the abundance of phospho-p38MAPK and p19ARF in tamoxifen-treated mice. Immunofluorescence analysis revealed that phospho-p38MAPK and p19ARF were highly expressed in the few remaining parietal cells of tamoxifen-treated WT mice (Fig. 3E). The number of phospho-p38MAPK–positive parietal cells in Ink4a/Arf / mice after tamoxifen treatment was much higher than that in WT mice (Fig. 3E), suggesting that activation of p38MAPK and downstream p19ARF expression contribute to tamoxifen-induced parietal cell loss. To examine whether the parietal cell loss induced by tamoxifen results in the generation of CD44v-expressing metaplastic cells in the gastric epithelium, we performed immunofluorescence analysis of CD44v and the proliferation marker Ki67. Injection of 496 Cancer Prev Res; 8(6) June 2015 tamoxifen effectively depleted parietal cells in WT mice (Supplementary Fig. S1). Proliferative CD44v-expressing cells were found to emerge from the base of gastric glands in tamoxifen-treated WT mice, whereas this phenomenon was greatly attenuated in tamoxifen-treated Ink4a/Arf / mice (Fig. 3F). These results suggested that the induction of Ink4a/Arf expression plays a key role in the parietal cell loss, and that such parietal cell loss triggers the generation of proliferative CD44v-expressing metaplastic cells. SPEM contains stem-like cells capable of generating gastric glands Given that CD44v is a gastric cancer stem cell marker (19) and plays a key role in SPEM development (16), we next Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Ink4a/Arf Induces Loss of Parietal Cells examined whether SPEM induced by tamoxifen treatment contains stem-like cells that possess the ability to reconstitute a gastric gland. To assess the stem cell potential of gastric epithelial cells, we isolated a 5-mm square piece of glandular mucosa from the stomach of mice treated (or not) with tamoxifen for evaluation of the ability to form gastric organoids (Fig. 4A; ref. 24). The number of gastric organoids generated by the TFF2-expressing gastric mucosa resected from WT mice treated with tamoxifen (Fig. 4B) was markedly increased compared with that for the gastric mucosa of nontreated mice (Fig. 4C), suggesting that SPEM tissue contains a much higher number of stem-like cells than does normal stomach mucosa. We next investigated CD44v expression in the gastric organoids. Immunofluorescence analysis revealed that organoids derived from both tamoxifen-treated and untreated WT mice contained CD44v-expressing stem-like cells, although the number of such cells was greatly increased for organoids formed by SPEM compared with those formed by normal gastric mucosa (Fig. 4D). These results suggested that CD44v-expressing stem-like cells are generated by the mechanically damaged epithelial tissue derived from normal gastric mucosa, and that parietal cell loss further increases the number of such stem-like cells in tamoxifen-induced SPEM. Given that tamoxifen-induced parietal cell loss and subsequent SPEM development were suppressed in the stomach of Ink4a/ Arf / mice, we next examined the role of the Ink4a/Arf locus in tamoxifen-induced gastric organoid formation. The number of organoids formed by gastric mucosa from tamoxifen-treated mice was significantly reduced by genetic ablation of the Ink4a/Arf locus as well as that of CD44 (Fig. 4E and F), suggesting that Ink4a/ Arf–dependent parietal cell loss induces the expansion of CD44vexpressing stem-like cells. Figure 4. SPEM induced by tamoxifen contains abundant stem-like cells. A, schematic representation of gastric gland isolation and organoid culture. B, quantitative RT-PCR analysis of TFF2 mRNA in glandular epithelium from tamoxifentreated or untreated WT mice. Data were normalized by the amount of GAPDH mRNA and are means  SD for 3 mice;  , P < 0.05 (Student t test). C, bright-field images of organoids derived from a single 5-mm square piece of gastric glandular epithelium from tamoxifen-treated or untreated WT mice. The representative images were acquired after culture for 0, 2, or 4 days; scale bars, 500 mm. D, immunohistofluorescence staining of CD44v (green) as well as staining of nuclei with DAPI (blue) in gastric organoids derived from tamoxifentreated or control WT mice after culture for 4 days. The medial and apical sections of organoids are shown. Arrowheads show CD44v-positive cells; scale bars, 25 mm. E, bright-field images of organoids (arrowheads) derived from tamoxifen-treated WT or / mice after culture for 2 or Ink4a/Arf 4 days; scale bars, 500 mm. F, mean number of organoids derived from individual 5-mm square pieces of gastric glandular epithelium from / tamoxifen-treated WT, Ink4a/Arf / and CD44 mice or from untreated WT mice. Data, means  SD for 4 mice;  , P < 0.01 (Student t test). www.aacrjournals.org Cancer Prev Res; 8(6) June 2015 Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. 497 Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Seishima et al. Figure 5. Enhancement of gastric tumorigenesis by tamoxifen-induced parietal cell loss. A, hematoxylin and eosin (H&E) staining of gastric tumors (T) in 25week-old K19-Wnt1/C2mE mice that had been injected with tamoxifen (or not) once a week beginning at 20 weeks of age; scale bars, 500 mm. B, mucosal thickness, which reflects the size of the tumor, in K19-Wnt1/C2mE mice treated (or not) with tamoxifen as determined from sections similar to those in A. Data, means  SD for 5 mice;  , P < 0.05 (Student t test). C, immunohistochemical staining of þ þ H ,K -ATPase and CD44v in tumoradjacent regions (AT) and in gastric tumors (T), respectively, for 25-weekold K19-Wnt1/C2mE mice treated as in A; scale bars, 200 mm. D, proportion of CD44v-positive cells in gastric tumors of K19-Wnt1/C2mE mice treated (or not) with tamoxifen as determined from sections similar to those in C. Tamoxifen injection promotes parietal cell loss and gastric tumor development Given that tamoxifen treatment promoted the development of SPEM concomitant with an increase in the number of stem-like cells in WT mice, we next administered tamoxifen to K19-Wnt1/ C2mE mice at 20 weeks of age to examine whether tamoxifeninduced parietal cell loss might also accelerate tumor development. I.p. injection of tamoxifen once a week for 5 weeks promoted the tumor growth in these mice (Fig. 5A and B). Furthermore, tamoxifen treatment enhanced the parietal cell loss as well as the production of CD44v-expressing tumor cells in K19-Wnt1/ C2mE mice (Fig. 5C and D). Together, these results suggested that loss of parietal cells might play a role in the production of CD44v- 498 Cancer Prev Res; 8(6) June 2015 expressing tumor cells and tumor growth in K19-Wnt1/C2mE mice. Parietal cell loss is associated with increasing the number of CD44v-expressing stem-like cells To examine the functional relevance of the Ink4a/Arf locus in the development of gastric tumors in K19-Wnt1/C2mE mice, we generated Ink4a/Arf / K19-Wnt1/C2mE mice. Tumors of Ink4a/ Arf / K19-Wnt1/C2mE mice were markedly smaller than those of K19-Wnt1/C2mE mice (Fig. 6A and B) and were similar in size to those of CD44 / K19-Wnt1/C2mE mice (16), suggesting that the Ink4a/Arf locus is required for CD44-dependent gastric tumor development in K19-Wnt1/C2mE mice. The number of parietal Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Ink4a/Arf Induces Loss of Parietal Cells Figure 6. The Ink4a/Arf locus is essential for parietal cell loss and the subsequent development of SPEM and gastric tumors. A, hematoxylin and eosin (H&E) staining of gastric / / tumors (T) in K19-Wnt1/C2mE mice, Ink4a/Arf K19-Wnt1/C2mE mice and CD44 K19-Wnt1/C2mE mice at 30 weeks of age; scale bars, 500 mm. B, mucosal thickness, / / which reflects the size of the tumor, in K19-Wnt1/C2mE mice, Ink4a/Arf K19-Wnt1/C2mE mice, CD44 K19-Wnt1/C2mE mice and WT mice at 30 weeks of þ þ age as determined from sections similar to those in A. Data, means  SD for 5 mice;   P < 0.01 (Student t test). C, immunohistochemical staining of H ,K -ATPase in / / K19-Wnt1/C2mE mice and CD44 K19-Wnt1/C2mE mice; scale bars, 200 mm. tumor-adjacent regions (AT) of 30-week-old K19-Wnt1/C2mE mice, Ink4a/Arf þ þ D, the mean percentage of H ,K -ATPase–positive cells per gland in five microscopic fields per mouse was calculated in gastric mucosa in sections similar to those  in C. Data, means  SD for 5 mice; , P < 0.01(Student t test). E, immunohistochemical staining of TFF2 in tumor-adjacent regions of 30-week-old K19-Wnt1/C2mE / / þ K19-Wnt1/C2mE mice and CD44 K19-Wnt1/C2mE mice; scale bars, 200 mm. F, mean area of TFF2 gastric epithelium determined from mice, Ink4a/Arf   sections similar to those in E. Data, means  SD for 5 mice; , P < 0.05; , P < 0.01 (Student t test). G, immunohistochemical staining of CD44v in gastric tumors and / / tumor-adjacent regions in 30-week-old K19-Wnt1/C2mE mice, Ink4a/Arf K19-Wnt1/C2mE mice and CD44 K19-Wnt1/C2mE mice; scale bars, 100 mm (top) or / / K19-Wnt1/C2mE mice and CD44 200 mm (bottom). H, proportion of CD44v-positive cells in gastric tumors of 30-week-old K19-Wnt1/C2mE mice, Ink4a/Arf K19-Wnt1/C2mE mice as determined from sections similar to those in G. I, model for metaplasia-carcinoma progression induced by chronic inflammation in the stomach. In inflamed gastric epithelium, ROS accumulate predominantly in parietal cells as a result of the low antioxidant capacity of these cells, and they activate oxidative MAPK stress–dependent signaling by p38 that leads to the induction of Ink4a/Arf gene expression and consequent parietal cell loss. In response to this parietal cell loss, chief cells undergo transdifferentiation into CD44v-expressing SPEM cells that include stem-like cells with a high antioxidant capacity. Oncogenic stimulation such as activation of aberrant Wnt signaling transforms the CD44v-expressing SPEM cells into CD44v-expressing stem-like tumor cells that give rise to gastric tumors. www.aacrjournals.org Cancer Prev Res; 8(6) June 2015 Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. 499 Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Seishima et al. cells remaining in the gastric epithelium adjacent to tumors was higher for Ink4a/Arf / K19-Wnt1/C2mE mice than for K19-Wnt1/ C2mE mice or CD44 / K19-Wnt1/C2mE mice (Fig. 6C and D), suggesting that the parietal cell loss in the initial stage of gastric tumorigenesis is regulated by the Ink4a/Arf locus but not by CD44 expression. On the other hand, the development of SPEM was significantly suppressed in both Ink4a/Arf / K19-Wnt1/C2mE mice and CD44 / K19-Wnt1/C2mE mice compared with K19Wnt1/C2mE mice (Fig. 6E and F), suggesting that the suppression of SPEM development in Ink4a/Arf / K19-Wnt1/C2mE mice and CD44 / K19-Wnt1/C2mE mice is mediated by different mechanisms. The Ink4a/Arf locus is essential for the parietal cell loss, whereas CD44 is required for the expansion of SPEM after parietal cell loss. Our results indicated that the stem-like cells in SPEM might be the cells of origin for CD44v-expressing tumor cells in K19Wnt1/C2mE mice; therefore, we examined whether the suppression of CD44v-expressing SPEM development might reduce the production of CD44v-expressing tumor cells in Ink4a/Arf / K19-Wnt1/C2mE mice. The area of CD44v-expressing tumor cells was found to be greatly reduced in Ink4a/Arf / K19-Wnt1/C2mE mice compared with K19-Wnt1/C2mE mice (Fig. 6G and H). Given that SPEM is a precancerous lesion of gastric cancer, these results suggest that CD44v-expressing metaplastic cells produced as a result of parietal cell loss might subsequently undergo conversion to CD44v-expressing tumor cells in K19-Wnt1/C2mE mice (Fig. 6I). Discussion Gastrointestinal malignancies are strongly linked to chronic inflammation (25–29). Gastric tumorigenesis in K19-Wnt1/ C2mE mice was also recently shown to be suppressed by knockout of either TNFa or its receptor TNFR1, suggesting that TNFa-dependent inflammation plays a key role in the development of gastric cancer (30). Furthermore, the development of SPEM in response to acute parietal cell loss is dependent on the recruitment of macrophages to the gastric epithelium (31). Together, these findings suggest that inflammation triggers the histopathologic alterations of gastric epithelium that leads to tumor development. We have now shown that the inflammation-associated ROS plays a key role in the parietal cell loss that is an early and critical step in the development of SPEM, and the subsequent formation of gastric tumors. The accumulation of ROS triggers the p38MAPK–p16INK4a/p19ARF signaling pathway selectively in parietal cells and the consequent induction of parietal cell loss. In fact, parietal cells have been reported to contain abundant mitochondria (32), a major source of intrinsic ROS production, and have been considered to be highly susceptible to oxidative stress (33). Together, oxidative stress– dependent p38MAPK–p16INK4a/p19ARF signaling is activated selectively in parietal cells of the stomach epithelium in the presence of high ROS level. The Ink4a/Arf locus encodes the cyclin-dependent kinase inhibitor p16INK4a as well as p19ARF, both of which have been shown to induce cell-cycle arrest, senescence, and apoptosis in response to oncogenic stimulation (34, 35). These proteins are thought to serve as key regulators in tumor-suppressor networks that are often disabled in human cancers (35). However, our present results indicate that ROS-induced expression of p16INK4a and p19ARF promotes gastric tumorigenesis by inducing parietal cell 500 Cancer Prev Res; 8(6) June 2015 loss and the emergence of CD44v-expressing SPEM cells. The activation of tumor-suppressor networks might, thus, promote carcinogenesis in some cases through induction of the loss of gatekeeper cells that play an important role in epithelial homeostasis. Carcinogenesis is often coupled to the generation of ROS, and cancer cells have, therefore, evolved mechanisms to protect themselves from oxidative stress through upregulation of both antioxidants and prosurvival molecules (36, 37). Expression of CD44v promotes cystine transport via system xc(–), and thereby contributes to ROS defense by increasing the synthesis of reduced glutathione in cancer cells (16). We previously showed that the de novo expression of CD44v in metaplasia cells potentiates ROS defense in these cells, and thereby promotes the development of SPEM (17). Depletion of CD44 or treatment with the xCT inhibitor sulfasalazine markedly suppressed the development of SPEM and gastric tumors in K19-Wnt1/ C2mE mice (16, 17), and the expression of SPEM-related genes was found to be greatly increased in CD44v-expressing tumor cells compared with CD44-negative tumor cells in this model (17). We have now found that, compared with normal gastric mucosa, SPEM induced as a result of Ink4a/Arf–dependent parietal cell loss contains an increased number of CD44vexpressing stem-like cells that are capable of initiating gastric organoid formation. Gastric organoids established from SPEM were found to express high level of Troy mRNA (data not shown), suggesting that Troy-expressing chief cells, which act as the reserve stem cells in response to gastric tissue damage, might be the candidate source of CD44v-expressing stem-like cells. Our results thus suggest that, in gastric tumorigenesis, it is likely that CD44v-expressing stem-like cells in SPEM undergo conversion to CD44v-expressing tumor cells, possibly as a result of the activation of oncogenic Wnt signaling or other genetic or epigenetic events (Fig. 6I). In conclusion, our present data provide evidence that ROS promote gastric tumorigenesis through the induction of p38MAPK–p16INK4a/p19ARF signaling in parietal cells—cells that serve as gatekeepers for homeostasis in oxyntic glands. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Authors' Contributions Conception and design: R. Seishima, J.R. Goldenring, H. Saya, O. Nagano Development of methodology: T. Wada, K. Tsuchihashi Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Seishima, T. Wada, M. Yoshikawa Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Seishima, T. Wada, K. Tsuchihashi, M. Yoshikawa, H. Hasegawa Writing, review, and/or revision of the manuscript: R. Seishima, J.R. Goldenring, O. Nagano Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Okazaki, H. Oshima, M. Oshima, T. Sato Study supervision: H. Hasegawa, Y. Kitagawa, O. Nagano Acknowledgments The authors thank I. Ishimatsu, Y. Suzuki, and S. Hayashi for technical assistance as well as M. Sato for help in preparation of the article. Grant Support This work was supported by grants (to H. Saya) from, as well as in part by the Project for Development of Innovative Research on Cancer Therapeutics Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on June 8, 2020. © 2015 American Association for Cancer Research. Published OnlineFirst March 26, 2015; DOI: 10.1158/1940-6207.CAPR-15-0025-T Ink4a/Arf Induces Loss of Parietal Cells (P-Direct; to O. Nagano) of, the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Department of Veterans Affairs Merit Review award and NIH RO1 DK071590 (to J.R. Goldenring). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received January 22, 2015; revised March 16, 2015; accepted March 22, 2015; published OnlineFirst March 26, 2015. References 1. Genta RM. 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