- Email: [email protected]
+ mice
Biomedicine & Pharmacotherapy 121 (2020) 109534
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Gastric precancerous lesions present in ApcMin/+ mice a,1
b,1
Sheng Wang , Jianbiao Kuang Lijing Wanga,* a b c
b
c
a
a
, Guifeng Li , Guilan Huang , Lingyun Zheng , Jiangchao Li ,
T
School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China School of Nursing, Guangdong Pharmaceutical University, Guangzhou 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Gastric precancerous lesions Apc gene mutation Mouse model Stomach
The ApcMin/+ mouse is an animal model for familial adenomatous polyposis, and aged ApcMin/+ mice also spontaneously develop multiple tumors in their stomachs. However, gastric premalignant lesions in ApcMin/+ mice have not been well characterized. The stomachs of ApcMin/+ mice were compared with those of their wild type littermates at 24 weeks with hematoxylin and eosin (H&E) staining and alcian blue staining. Ki67, CD68 and CA199 expression was analyzed by immunohistochemistry. The results revealed the presence of epithelial proliferation and inflammatory infiltration in the forestomachs, glandular atrophy and intestinal metaplasia in the gastric bodies, and dysplasia in the gastric antra. The effect of mutations in the Apc gene on chronic gastritis and gastric precancerous lesions was characterized in ApcMin/+ mice. These results suggest that ApcMin/+ mice represent a genetic model for mechanistic studies and drug discovery in gastric precancerous lesions.
1. Introduction Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death in the world [1]. The formation of gastric cancer is widely believed to be a multistep process that evolves from chronic gastritis [2]. In researching gastric cancer, Correa described a premalignant cascade beginning with normal epithelia, then transitioning to chronic active gastritis, atrophic gastritis, intestinal metaplasia (IM), dysplasia and finally gastric adenocarcinoma [3,4]. Many studies have found an association between the formation of IM, dysplasia and the development of gastric carcinoma [4–6]. Atrophic gastritis, IM and dysplasia are considered to be precancerous lesions for gastric cancer [6,7]. Thus, exploring the mechanisms underlying the pathological development of gastric precancerous lesions and their transition from benign to malignant is the crux of preventing and treating gastric cancer. It is important to develop a faithful animal model to imitate the process of gastric precancerous lesion development. Thus far, experiments on gastric carcinogenesis in animal models have been conducted using H. pylori infections and/or treatment with carcinogens such as l-methyl-3nitro-1-nitrosoguanidine [8,9]. However, there is rarely a spontaneous animal model that shows the progression of precursor lesions to gastric carcinoma [10]. Krishnamurthy et al. found that abnormal activation of the Wnt/β-catenin signaling pathway was closely related to gastric
carcinogenesis [11]. The adenomatous polyposis coli (APC) gene is an important tumor suppressor that inhibits the Wnt/β-catenin pathway [12]. Clinical studies suggest that APC mutations have a significant correlation with the development of gastric cancer [13]. The ApcMin/+ mouse is an animal model for studying familial adenomatous polyposis, and it harbors a dominant mutation in the Apc gene, resulting in a gene product that is truncated at amino acid 850 [14]. Although aged ApcMin/+ mice have been reported to spontaneously develop multiple gastric tumors [15], the pathological features of gastric precancerous lesions in ApcMin/+ mice have not yet been well characterized. To determine whether Apc mutations initiate gastric precancerous lesions, we examined gastric lesions of ApcMin/+ mice at 24 weeks of age. In the present study, we found that the ApcMin/+ mice had developed atrophic gastritis, IM and dysplasia multistep lesions. We showed with this animal model that the characteristics of gastric precancerous lesions exist because of Apc mutations. 2. Materials and methods 2.1. Animals C57BL/6 J-ApcMin/+ mice were obtained from the Jackson Laboratory and maintained by breeding ApcMin/+ males to C57BL/6 J
Corresponding author. Tel.: +86 20 39352231. E-mail address: [email protected] (L. Wang). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.biopha.2019.109534 Received 29 July 2019; Received in revised form 28 September 2019; Accepted 2 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 121 (2020) 109534
S. Wang, et al.
Fig. 1. Macroscopic dissection view of the stomach of the ApcMin/ + mice and wild-type (Wt) littermates at 24 weeks of age; the stomachs were opened along the greater curvature (A, wild-type littermate; B, ApcMin/+ mice). FS: forestomach; GS: glandular stomach; scale bar: 5 mm. C, The length proportion of GS and FS + GS. Columns, mean; bars, SD. *, P < 0.05.
Fig. 2. The mucosal thicknesses of different gastric areas in the ApcMin/+ mice and Wt littermates at 24 weeks of age (H&E staining). A, The forestomach of Wt littermate; B, The forestomach of ApcMin/+ mouse; D, The gastric body of Wt littermate; E, The gastric body of ApcMin/+ mouse; G, The antrum of Wt littermate; H, The antrum of ApcMin/+ mouse; scale: 100 μm. C, F and I show graphic extrapolation of the results. Columns, mean; bars, SD. *, P < 0.05, **, P < 0.01.
females. These mice were maintained under specific pathogen-free conditions in a temperature-controlled room with a 12-h light/dark cycle. They were bred and maintained on a basal diet until the termination of the study. The genotypes of all the mice at four weeks were determined by PCR analysis of tail DNA using allele-specific primers, as previously described [16]. The study protocols were approved by the Ethics Committee of the Laboratory Animal Center of Guangdong Pharmaceutical University.
into three strips that were processed by standard methods and embedded in paraffin. The paraffin sections (3–5 μm) were stained with hematoxylin and eosin (H&E) and alcian blue for mucus characterization, which was performed according to a standard protocol [17]. The stained sections were examined by two of the investigators and photographed using a BX51 Olympus microscope (Olympus Corporation, Shibuya, Japan). 2.3. Immunohistochemistry
2.2. Histology
The paraffin sections (3–5 μm) were stained for Ki67, CD68 and CA199 expression according to a previously described method [18]. The proliferation index was calculated as the percentage of Ki67stained cells per field at 40× magnification. Three fields in a section
After the mice were sacrificed, the stomachs were removed and opened along the gastric greater curvature. The mouse stomach samples were fixed in 10% neutral-buffered formalin overnight and then cut 2
Biomedicine & Pharmacotherapy 121 (2020) 109534
S. Wang, et al.
Fig. 3. Immunohistochemical staining for Ki67 in different areas of gastric mucosa of ApcMin/+ mice and wild-type littermates at 24 weeks of age. A, D and G show nuclear staining for Ki67 in the forestomach, body and antrum of wild-type littermates; B, E and H show representative nuclear staining for Ki67 in the forestomach, body and antrum of ApcMin/+ mice; scale bars: 50 μm.C, F and I show graphic extrapolation of the results. Columns, mean; bars, SD. * P < 0.05, *** P < 0.001.
were selected at random from a non-necrotic area of the tumor tissues. Antigen retrieval was performed with ethylene diaminetetraacetic acid solution (pH 9.0). Then, the slides were incubated in 3% H2O2 at room temperature for 12 min and were washed, which was followed by non-specific proteins being blocked. The antibody was applied to the slides, which were subsequently incubated at 4 °C overnight. The tissue sections were washed again, and the secondary antibody was applied to the slides. Finally, diaminobenzidine was used to visualize positive staining on the slides; a diaminobenzidine chromogen system was purchased from Gene Tech Co., Ltd. (Shanghai, China).
gastric mucosa inflammation in ApcMin/+ mice at 24 weeks of age, whereas no inflammation was found in the wild-type littermates at the same age (Fig. 1A). Macroscopically, the glandular stomach in ApcMin/ + mice showed hyperaemia and swelling (Fig. 1B). The length ratio between glandular stomach (GS) and total stomach in ApcMin/+ mice was significantly reduced compared to that of the wild-type littermates (Fig. 1C).
2.4. Statistical analysis
The entire stomach of ApcMin/+ mice showed similar histology. The epithelial thickness of the forestomach in ApcMin/+ mice was greater than that of the wild-type littermates (P < 0.001) (Fig. 2A–C). In contrast, glandular atrophy was observed in the gastric body of ApcMin/ + mice. The mucosal thicknesses of the gastric bodies in ApcMin/+ mice were lower than they were in wild-type littermates (P < 0.01) (Fig. 2D–F). The mucosal thicknesses of the antra were not different between the ApcMin/+ mice and wild-type littermates (P > 0.05) (Fig. 2G, H, L).
2 The mucosal thickness increased in different gastric areas of ApcMin/ + mice
All statistical analyses were performed using SPSS for Windows software (SPSS Inc.). The corresponding relative integrated optical density (IOD) of protein expression levels in the IHC slices was analyzed using IPP software. The differences between two groups were evaluated using a two-tailed Student’s t-test Statistically significant differences were accepted at the p < 0.05 level. 3. Results
3 Cell proliferation is significantly different in the forestomach and gastric body of ApcMin/+ mice
1 Macroscopic dissection view of the stomach of the ApcMin/+ mice and wild-type littermates
To evaluate the changes in mucosal thickness in the forestomach, body and antrum of the ApcMin/+ mice, we determined the subcellular localization of Ki67 by immunohistochemical staining. The Ki67 staining results indicated that cell proliferation had significantly
In the present study, we examined gastric lesions in ApcMin/+ mice at 24 weeks of age (n = 12) and then compared the findings with the data from age-matched wild-type littermates (n = 12). We found 3
Biomedicine & Pharmacotherapy 121 (2020) 109534
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Fig. 4. Histomorphological features in different areas of gastric mucosae in ApcMin/+ mice at 24 weeks of age. A, low magnification field view of a forestomach section (H&E staining). The inflammatory infiltrate can be seen in the squamous epithelium of the forestomach. B, higher magnification of the boxed section in A. C, invasive inflammatory cells expressed a macrophage marker, CD68. E, low magnification field view of a body section (H&E staining). Intestinal metaplasia mucosa can be seen in the gastric body. F, higher magnification of the boxed section in E. G, alcian blue staining was observed in the mucosal surface of the gastric body. Some goblet cells were observed in the gastric mucosa, which are indicated by arrows. I, Proliferation of atypical neoplastic glands can be observed in the antrum mucosae (H&E staining). H, higher magnification of the boxed section in G. Several irregular mitotic events were observed and are indicated by arrows. I, CA199 was expressed in cancerous cells of the antrum mucosae. Scale bars: 100 μm (A, E, I); 20 μm (B, C, F, G, J, K). The protein expression levels in sections were quantified using IPP software. Columns, mean; bars, SD. **: p < 0.01, ***: p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
increased in the forestomach of the ApcMin/+ mice (Fig. 3A–C), whereas cell proliferation had significantly decreased in the gastric body of the ApcMin/+ mice (Fig. 3D–F). There was no difference in the cell proliferation index of the antra between ApcMin/+ mice and wild-type littermates (P > 0.05) (Fig. 3G, H, L). These changes in cell proliferation in different gastric areas were consistent with the mucosal thickness data observed in ApcMin/+ mice.
atypical neoplastic glands was observed in the area of the antrum mucosae in ApcMin/+ mice. The irregular branching glands were lined by atypical columnar cells that showed plump nuclei with large nucleoli and mitotic activity (Fig. 4I–L). The results imply that the ApcMin/+ genotype is the main factor that increases the susceptibility of mice to the observed histological abnormalities in the stomach. In all wild-type littermate mice, gastric morphology was basically normal.
4 Chronic inflammation and dysplasia occur in the stomachs of ApcMin/+ mice
4. Discussion In this study, precancerous lesions were found in the stomachs of ApcMin/+ mice at 24 weeks of age. ApcMin/+ mice showed the following characteristic precancerous changes: atrophy of the glandular stomach, infiltration of macrophages, intestinal metaplasia and glandular hyperplasia microscopically in the gastric mucosa. These findings suggest that Apc plays a role in regulating the inflammatory response of the mouse’s stomach. APC gene expression has been detected in the normal human and mouse gastric and intestinal mucosa, and the APC gene product might have an effect on the proliferation and differentiation of gastrointestinal epithelial cells [19–21]. Gastric cancer and premalignant lesions have shown decreased APC gene expression [22]. Mutations in the APC gene have been frequently detected in gastric
Histological abnormalities, when present, were located in the gastric mucosa. Gastric submucosal inflammation in the forestomach was detectable in ApcMin/+ mice. The inflammatory infiltrate consisted mainly of mononuclear inflammatory cells with the morphology of lymphocytes and plasma cells (Fig. 4A–D). In some cases, chronic inflammatory infiltrate penetrated the squamous epithelium in the forestomach of the ApcMin/+ mice. Intestinal metaplasia and gastric dysplasia were observed in the glandular stomach of ApcMin/+ mice. We examined intestinal metaplastic cells by alcian blue staining. The goblet cells were observed in the gastric body mucosae of ApcMin/+ mice but not in wild-type littermates’ mucosae (Fig. 4E–H). Proliferation of 4
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carcinomas, particularly in intestinal-type gastric carcinomas [23,24]. Therefore, the APC gene could have a role in predicting cancer prognoses. The histopathological features of the gastric precancerous lesions from ApcMin/+ mice indicated that epithelial cell proliferation and inflammatory infiltration appeared in the forestomach, and glandular atrophy and intestinal metaplasia appeared in the gastric body; however, while there was no obvious hyperplasia or atrophy in the gastric antrum, glandular dysplasia and pathological cell division were observed. Due to the coexistence of atrophy of normal tissues and abnormal hyperplasia of glands, the epithelial cells in the gastric antra remained relatively stable in number but developed into malignant lesions. These results suggest that precancerous lesions from the forestomach to the antrum in ApcMin/+ mice at 24 weeks of age gradually progress. The mechanisms of this reaction are not known. Clearly, further studies are needed to evaluate the possible role of APC in the regulation of the immune response. Although several studies suggested the involvement of the Wnt pathway in the development of sporadic gastric cancer [25–27], the functional significance of the Wnt/β-catenin pathway in gastric carcinogenesis was still poorly defined in comparison to its established role in colon carcinogenesis. Gastric tumors in ApcMin/+ mice showed adenomatous lesions with a strong nuclear/cytoplasmic accumulation of β-catenin, thus suggesting that the Wnt/β-catenin signaling pathway is involved in tumorigenesis [15]. The gastric body and antrum regions were positive for alcian blue staining and CA199 immunostaining, respectively. The present results confirmed that the APC mutation causes epithelial hyperplasia in the forestomach, glandular atrophy and intestinal metaplasia in the gastric body mucosa, so it therefore generates a preneoplastic condition. Intestinal-type carcinoma arises in older patients in a part of the stomach where inflammation has been present for a long period [28]. The protruded form of human gastric well-differentiated adenocarcinoma is often found in the intestinal metaplastic mucosae of elderly people and is macroscopically and microscopically quite similar to the protruded form of gastric precancerous lesions in ApcMin/+ mice. Severe atrophic gastritis accompanying intestinal metaplasia is closely related to the development of intestinal-type gastric carcinoma. Our present results clearly indicate that Apc gene mutations might promote the progression of the intestinal metaplasia-dysplasiacarcinoma sequence. In conclusion, we have shown that premalignant lesions play a key role in causing malignant and invasive gastric adenocarcinoma in ApcMin/+ mice. In the present study, all of the ApcMin/+ mouse stomachs examined at 24 weeks of age had developed atrophic gastritis, intestinal metaplasia and glandular dysplasia, although the role of Apc in gastritis, intestinal metaplasia in Wnt-dependent intestinal-type tumorigenesis has not been clarified. The participation of the mutated Apc gene in gastric precancerous lesions was demonstrated by the results observed in ApcMin/+ mice. The ApcMin/+ mouse model is therefore valuable for investigating the relationship between precancerous lesion and gastric carcinogenesis.
Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This work was supported by grants from Key Project for cultivating scientific and technological innovation of college students of Guangdong Province of China (Grant ID pdjha0260), and Medical Scientific Research Foundation of Guangdong Province of China (Grant ID A2017345), National Natural Science Foundation of China (Grant ID 81773118, 31471290 and 31771578 to L.W), Innovative Research Team in University of Guangdong Province of China (2016KCXTD019 to L.W). References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (2018) 394–424. [2] P. Rawla, A. Barsouk, Epidemiology of gastric cancer: global trends, risk factors and prevention, Prz. Gastroenterol. 14 (1) (2019) 26–38. [3] P. Correa, Gastric cancer: overview, Gastroenterol. Clin. North Am. 42 (2) (2013) 211–217. [4] P. Correa, M.B. Piazuelo, The gastric precancerous cascade, J. Dig. Dis. 13 (1) (2012) 2–9. [5] A.D. Spence, C.R. Cardwell, ÚC. McMenamin, B.M. Hicks, B.T. Johnston, L.J. Murray, et al., Adenocarcinoma risk in gastric atrophy and intestinal metaplasia: a systematic review, BMC Gastroenterol. 17 (1) (2017) 157, https://doi.org/ 10.1186/s12876-017-0708-4. [6] W.J. den Hollander, I.L. Holster, C.M. den Hoed, L.G. Capelle, T.J. Tang, M.P. Anten, et al., Surveillance of premalignant gastric lesions: a multicentre prospective cohort study from low incidence regions, Gut 68 (4) (2019) 585–593. [7] B. Da, N. Jani, N. Gupta, P. Jayaram, R. Kankotia, C. Yao Yu, et al., High-risk symptoms do not predict gastric cancer precursors, Helicobacter 24 (1) (2019), https://doi.org/10.1111/hel.12548 e12548. [8] R. Sitarz, M. Skierucha, J. Mielko, G.J.A. Offerhaus, R. Maciejewski, W.P. Polkowski, Gastric cancer: epidemiology, prevention, classification, and treatment, Cancer Manage. Res. 10 (2018) 239–248. [9] D. Cai, J. Yu, J. Qiu, B. He, Z. Chen, M. Yan, et al., Dynamic changes of Sonic Hedgehog signaling pathway in gastric mucosa of rats with MNNG-induced gastric precancerous lesions, J. Cell. Physiol. 234 (7) (2019) 10827–10834. [10] W. Liu, H.F. Pan, Q. Wang, Z.M. Zhao, The application of transgenic and gene knockout mice in the study of gastric precancerouslesions, Pathol. Res. Pract. 214 (12) (2018) 1929–1939. [11] N. Krishnamurthy, R. Kurzrock, Targeting the Wnt/β-catenin pathway in cancer: update on effectors and inhibitors, Cancer Treat. Rev. 62 (2018) 50–60. [12] X.Z. Yang, T.T. Cheng, Q.J. He, Z.Y. Lei, J. Chi, Z. Tang, et al., LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway, Mol. Cancer 17 (1) (2018) 126, https:// doi.org/10.1186/s12943-018-0874-1. [13] D.C. Sample, N.J. Samadder, L.M. Pappas, K.M. Boucher, W.S. Samowitz, T. Berry, et al., Variables affecting penetrance of gastric and duodenal phenotype in familial adenomatous polyposis patients, BMC Gastroenterol. 18 (1) (2018) 115, https:// doi.org/10.1186/s12876-018-0841-8. [14] A.R. Moser, H.C. Pitot, W.F. Dove, A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse, Science 247 (1990) 322–324. [15] H. Tomita, Y. Yamada, T. Oyama, K. Hata, Y. Hirose, A. Hara, et al., Development of gastric tumors in Apc(Min/+) mice by the activation of the beta-catenin/Tcf signaling pathway, Cancer Res. 67 (9) (2007) 4079–4087. [16] C. Qi, B. Li, S. Guo, B. Wei, C. Shao, J. Li, et al., P-selectin-mediated adhesion between platelets and tumor cells promotes intestinal tumorigenesis in apc(Min/+) mice, Int. J. Biol. Sci. 11 (6) (2015) 679–687. [17] A. Saxena, R. Fayad, K. Kaur, S. Truman, J. Greer, J.A. Carson, et al., Dietary selenium protects adiponectin knockout mice against chronic inflammation induced colon cancer, Cancer Biol. Ther. 18 (4) (2017) 257–267. [18] L.J. Wang, Y. Zhao, B. Han, Y.G. Ma, J. Zhang, D.M. Yang, et al., Targeting SlitRoundabout signaling inhibits tumor angiogenesis in chemical-induced squamous cell carcinogenesis, Cancer Sci. 99 (3) (2008) 510–517. [19] W. Onuma, S. Tomono, S. Miyamoto, G. Fujii, T. Hamoya, K. Fujimoto, et al., Irsogladine maleate, a gastric mucosal protectant, suppresses intestinal polyp development in Apc-mutant mice, Oncotarget 7 (8) (2016) 8640–8652. [20] S. Ghatak, P. Chakraborty, S.R. Sarkar, B. Chowdhury, A. Bhaumik, N.S. Kumar,
5. Conclusion In summary, our study showed that Apc mutations were associated with the following main features: gastric precancerous lesions, glandular atrophy, intestinal metaplasia and dysplasia. These spontaneous lesions exhibit patterns that are similar to the pathogenesis of gastric adenocarcinoma in humans. These findings underscore the potential of ApcMin/+ mice as a valuable animal model for investigating different premalignant lesions and assisting in the planning of eradication therapy to prevent the development of gastric carcinoma.
5
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[21] [22] [23] [24]
Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer, BMC Med. Genet. 18 (1) (2017) 61, https://doi.org/10.1186/s12881-0170427-2. T. Senda, A. Iizuka-Kogo, T. Onouchi, A. Shimomura, Adenomatous polyposis coli (APC) plays multiple roles in the intestinal and colorectal epithelia, Med. Mol. Morphol. 40 (2) (2007) 68–81. Z.K. Wang, J. Liu, C. Liu, F.Y. Wang, C.Y. Chen, X.H. Zhang, Hypermethylation of adenomatous polyposis coli gene promoter is associated with novel Wnt signaling pathway in gastric adenomas, J. Gastroenterol. Hepatol. 27 (10) (2012) 1629–1634. H. Saluja, C.S. Karapetis, S.K. Pedersen, G.P. Young, E.L. Symonds, The use of circulating tumor DNA for prognosis of gastrointestinal cancers, Front. Oncol. 8 (2018) 275. R. Hida, H. Yamamoto, M. Hirahashi, R. Kumagai, K. Nishiyama, T. Gi, et al., Duodenal neoplasms of gastric phenotype: an immunohistochemical and genetic
[25] [26] [27] [28]
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study with a practical approach to the classification, Am. J. Surg. Pathol. 41 (3) (2017) 343–353. B.H. Min, J. Hwang, N.K. Kim, G. Park, S.Y. Kang, S. Ahn, et al., Dysregulated Wnt signalling and recurrent mutations of the tumour suppressor RNF43 in early gastric carcinogenesis, J. Pathol. 240 (3) (2016) 304–314. F. Molaei, M.M. Forghanifard, Y. Fahim, M.R. Abbaszadegan, Molecular signaling in tumorigenesis of gastric cancer, Iran. Biomed. J. 22 (4) (2018) 217–230. L. Song, Z.Y. Li, W.P. Liu, M.R. Zhao, Crosstalk between Wnt/β-catenin and Hedgehog/Gli signaling pathways in colon cancer and implications fortherapy, Cancer Biol. Ther. 16 (1) (2015) 1–7. F. Zhou, J. Shi, C. Fang, X. Zou, Q. Huang, Gastric carcinomas in young (younger than 40 years) Chinese patients: clinicopathology, family history, and postresection survival, Medicine (Baltimore) 95 (9) (2016) e2873.
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Gastric precancerous lesions present in ApcMin/+ mice a,1
b,1
Sheng Wang , Jianbiao Kuang Lijing Wanga,* a b c
b
c
a
a
, Guifeng Li , Guilan Huang , Lingyun Zheng , Jiangchao Li ,
T
School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China School of Nursing, Guangdong Pharmaceutical University, Guangzhou 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Gastric precancerous lesions Apc gene mutation Mouse model Stomach
The ApcMin/+ mouse is an animal model for familial adenomatous polyposis, and aged ApcMin/+ mice also spontaneously develop multiple tumors in their stomachs. However, gastric premalignant lesions in ApcMin/+ mice have not been well characterized. The stomachs of ApcMin/+ mice were compared with those of their wild type littermates at 24 weeks with hematoxylin and eosin (H&E) staining and alcian blue staining. Ki67, CD68 and CA199 expression was analyzed by immunohistochemistry. The results revealed the presence of epithelial proliferation and inflammatory infiltration in the forestomachs, glandular atrophy and intestinal metaplasia in the gastric bodies, and dysplasia in the gastric antra. The effect of mutations in the Apc gene on chronic gastritis and gastric precancerous lesions was characterized in ApcMin/+ mice. These results suggest that ApcMin/+ mice represent a genetic model for mechanistic studies and drug discovery in gastric precancerous lesions.
1. Introduction Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death in the world [1]. The formation of gastric cancer is widely believed to be a multistep process that evolves from chronic gastritis [2]. In researching gastric cancer, Correa described a premalignant cascade beginning with normal epithelia, then transitioning to chronic active gastritis, atrophic gastritis, intestinal metaplasia (IM), dysplasia and finally gastric adenocarcinoma [3,4]. Many studies have found an association between the formation of IM, dysplasia and the development of gastric carcinoma [4–6]. Atrophic gastritis, IM and dysplasia are considered to be precancerous lesions for gastric cancer [6,7]. Thus, exploring the mechanisms underlying the pathological development of gastric precancerous lesions and their transition from benign to malignant is the crux of preventing and treating gastric cancer. It is important to develop a faithful animal model to imitate the process of gastric precancerous lesion development. Thus far, experiments on gastric carcinogenesis in animal models have been conducted using H. pylori infections and/or treatment with carcinogens such as l-methyl-3nitro-1-nitrosoguanidine [8,9]. However, there is rarely a spontaneous animal model that shows the progression of precursor lesions to gastric carcinoma [10]. Krishnamurthy et al. found that abnormal activation of the Wnt/β-catenin signaling pathway was closely related to gastric
carcinogenesis [11]. The adenomatous polyposis coli (APC) gene is an important tumor suppressor that inhibits the Wnt/β-catenin pathway [12]. Clinical studies suggest that APC mutations have a significant correlation with the development of gastric cancer [13]. The ApcMin/+ mouse is an animal model for studying familial adenomatous polyposis, and it harbors a dominant mutation in the Apc gene, resulting in a gene product that is truncated at amino acid 850 [14]. Although aged ApcMin/+ mice have been reported to spontaneously develop multiple gastric tumors [15], the pathological features of gastric precancerous lesions in ApcMin/+ mice have not yet been well characterized. To determine whether Apc mutations initiate gastric precancerous lesions, we examined gastric lesions of ApcMin/+ mice at 24 weeks of age. In the present study, we found that the ApcMin/+ mice had developed atrophic gastritis, IM and dysplasia multistep lesions. We showed with this animal model that the characteristics of gastric precancerous lesions exist because of Apc mutations. 2. Materials and methods 2.1. Animals C57BL/6 J-ApcMin/+ mice were obtained from the Jackson Laboratory and maintained by breeding ApcMin/+ males to C57BL/6 J
Corresponding author. Tel.: +86 20 39352231. E-mail address: [email protected] (L. Wang). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.biopha.2019.109534 Received 29 July 2019; Received in revised form 28 September 2019; Accepted 2 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 121 (2020) 109534
S. Wang, et al.
Fig. 1. Macroscopic dissection view of the stomach of the ApcMin/ + mice and wild-type (Wt) littermates at 24 weeks of age; the stomachs were opened along the greater curvature (A, wild-type littermate; B, ApcMin/+ mice). FS: forestomach; GS: glandular stomach; scale bar: 5 mm. C, The length proportion of GS and FS + GS. Columns, mean; bars, SD. *, P < 0.05.
Fig. 2. The mucosal thicknesses of different gastric areas in the ApcMin/+ mice and Wt littermates at 24 weeks of age (H&E staining). A, The forestomach of Wt littermate; B, The forestomach of ApcMin/+ mouse; D, The gastric body of Wt littermate; E, The gastric body of ApcMin/+ mouse; G, The antrum of Wt littermate; H, The antrum of ApcMin/+ mouse; scale: 100 μm. C, F and I show graphic extrapolation of the results. Columns, mean; bars, SD. *, P < 0.05, **, P < 0.01.
females. These mice were maintained under specific pathogen-free conditions in a temperature-controlled room with a 12-h light/dark cycle. They were bred and maintained on a basal diet until the termination of the study. The genotypes of all the mice at four weeks were determined by PCR analysis of tail DNA using allele-specific primers, as previously described [16]. The study protocols were approved by the Ethics Committee of the Laboratory Animal Center of Guangdong Pharmaceutical University.
into three strips that were processed by standard methods and embedded in paraffin. The paraffin sections (3–5 μm) were stained with hematoxylin and eosin (H&E) and alcian blue for mucus characterization, which was performed according to a standard protocol [17]. The stained sections were examined by two of the investigators and photographed using a BX51 Olympus microscope (Olympus Corporation, Shibuya, Japan). 2.3. Immunohistochemistry
2.2. Histology
The paraffin sections (3–5 μm) were stained for Ki67, CD68 and CA199 expression according to a previously described method [18]. The proliferation index was calculated as the percentage of Ki67stained cells per field at 40× magnification. Three fields in a section
After the mice were sacrificed, the stomachs were removed and opened along the gastric greater curvature. The mouse stomach samples were fixed in 10% neutral-buffered formalin overnight and then cut 2
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Fig. 3. Immunohistochemical staining for Ki67 in different areas of gastric mucosa of ApcMin/+ mice and wild-type littermates at 24 weeks of age. A, D and G show nuclear staining for Ki67 in the forestomach, body and antrum of wild-type littermates; B, E and H show representative nuclear staining for Ki67 in the forestomach, body and antrum of ApcMin/+ mice; scale bars: 50 μm.C, F and I show graphic extrapolation of the results. Columns, mean; bars, SD. * P < 0.05, *** P < 0.001.
were selected at random from a non-necrotic area of the tumor tissues. Antigen retrieval was performed with ethylene diaminetetraacetic acid solution (pH 9.0). Then, the slides were incubated in 3% H2O2 at room temperature for 12 min and were washed, which was followed by non-specific proteins being blocked. The antibody was applied to the slides, which were subsequently incubated at 4 °C overnight. The tissue sections were washed again, and the secondary antibody was applied to the slides. Finally, diaminobenzidine was used to visualize positive staining on the slides; a diaminobenzidine chromogen system was purchased from Gene Tech Co., Ltd. (Shanghai, China).
gastric mucosa inflammation in ApcMin/+ mice at 24 weeks of age, whereas no inflammation was found in the wild-type littermates at the same age (Fig. 1A). Macroscopically, the glandular stomach in ApcMin/ + mice showed hyperaemia and swelling (Fig. 1B). The length ratio between glandular stomach (GS) and total stomach in ApcMin/+ mice was significantly reduced compared to that of the wild-type littermates (Fig. 1C).
2.4. Statistical analysis
The entire stomach of ApcMin/+ mice showed similar histology. The epithelial thickness of the forestomach in ApcMin/+ mice was greater than that of the wild-type littermates (P < 0.001) (Fig. 2A–C). In contrast, glandular atrophy was observed in the gastric body of ApcMin/ + mice. The mucosal thicknesses of the gastric bodies in ApcMin/+ mice were lower than they were in wild-type littermates (P < 0.01) (Fig. 2D–F). The mucosal thicknesses of the antra were not different between the ApcMin/+ mice and wild-type littermates (P > 0.05) (Fig. 2G, H, L).
2 The mucosal thickness increased in different gastric areas of ApcMin/ + mice
All statistical analyses were performed using SPSS for Windows software (SPSS Inc.). The corresponding relative integrated optical density (IOD) of protein expression levels in the IHC slices was analyzed using IPP software. The differences between two groups were evaluated using a two-tailed Student’s t-test Statistically significant differences were accepted at the p < 0.05 level. 3. Results
3 Cell proliferation is significantly different in the forestomach and gastric body of ApcMin/+ mice
1 Macroscopic dissection view of the stomach of the ApcMin/+ mice and wild-type littermates
To evaluate the changes in mucosal thickness in the forestomach, body and antrum of the ApcMin/+ mice, we determined the subcellular localization of Ki67 by immunohistochemical staining. The Ki67 staining results indicated that cell proliferation had significantly
In the present study, we examined gastric lesions in ApcMin/+ mice at 24 weeks of age (n = 12) and then compared the findings with the data from age-matched wild-type littermates (n = 12). We found 3
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Fig. 4. Histomorphological features in different areas of gastric mucosae in ApcMin/+ mice at 24 weeks of age. A, low magnification field view of a forestomach section (H&E staining). The inflammatory infiltrate can be seen in the squamous epithelium of the forestomach. B, higher magnification of the boxed section in A. C, invasive inflammatory cells expressed a macrophage marker, CD68. E, low magnification field view of a body section (H&E staining). Intestinal metaplasia mucosa can be seen in the gastric body. F, higher magnification of the boxed section in E. G, alcian blue staining was observed in the mucosal surface of the gastric body. Some goblet cells were observed in the gastric mucosa, which are indicated by arrows. I, Proliferation of atypical neoplastic glands can be observed in the antrum mucosae (H&E staining). H, higher magnification of the boxed section in G. Several irregular mitotic events were observed and are indicated by arrows. I, CA199 was expressed in cancerous cells of the antrum mucosae. Scale bars: 100 μm (A, E, I); 20 μm (B, C, F, G, J, K). The protein expression levels in sections were quantified using IPP software. Columns, mean; bars, SD. **: p < 0.01, ***: p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
increased in the forestomach of the ApcMin/+ mice (Fig. 3A–C), whereas cell proliferation had significantly decreased in the gastric body of the ApcMin/+ mice (Fig. 3D–F). There was no difference in the cell proliferation index of the antra between ApcMin/+ mice and wild-type littermates (P > 0.05) (Fig. 3G, H, L). These changes in cell proliferation in different gastric areas were consistent with the mucosal thickness data observed in ApcMin/+ mice.
atypical neoplastic glands was observed in the area of the antrum mucosae in ApcMin/+ mice. The irregular branching glands were lined by atypical columnar cells that showed plump nuclei with large nucleoli and mitotic activity (Fig. 4I–L). The results imply that the ApcMin/+ genotype is the main factor that increases the susceptibility of mice to the observed histological abnormalities in the stomach. In all wild-type littermate mice, gastric morphology was basically normal.
4 Chronic inflammation and dysplasia occur in the stomachs of ApcMin/+ mice
4. Discussion In this study, precancerous lesions were found in the stomachs of ApcMin/+ mice at 24 weeks of age. ApcMin/+ mice showed the following characteristic precancerous changes: atrophy of the glandular stomach, infiltration of macrophages, intestinal metaplasia and glandular hyperplasia microscopically in the gastric mucosa. These findings suggest that Apc plays a role in regulating the inflammatory response of the mouse’s stomach. APC gene expression has been detected in the normal human and mouse gastric and intestinal mucosa, and the APC gene product might have an effect on the proliferation and differentiation of gastrointestinal epithelial cells [19–21]. Gastric cancer and premalignant lesions have shown decreased APC gene expression [22]. Mutations in the APC gene have been frequently detected in gastric
Histological abnormalities, when present, were located in the gastric mucosa. Gastric submucosal inflammation in the forestomach was detectable in ApcMin/+ mice. The inflammatory infiltrate consisted mainly of mononuclear inflammatory cells with the morphology of lymphocytes and plasma cells (Fig. 4A–D). In some cases, chronic inflammatory infiltrate penetrated the squamous epithelium in the forestomach of the ApcMin/+ mice. Intestinal metaplasia and gastric dysplasia were observed in the glandular stomach of ApcMin/+ mice. We examined intestinal metaplastic cells by alcian blue staining. The goblet cells were observed in the gastric body mucosae of ApcMin/+ mice but not in wild-type littermates’ mucosae (Fig. 4E–H). Proliferation of 4
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carcinomas, particularly in intestinal-type gastric carcinomas [23,24]. Therefore, the APC gene could have a role in predicting cancer prognoses. The histopathological features of the gastric precancerous lesions from ApcMin/+ mice indicated that epithelial cell proliferation and inflammatory infiltration appeared in the forestomach, and glandular atrophy and intestinal metaplasia appeared in the gastric body; however, while there was no obvious hyperplasia or atrophy in the gastric antrum, glandular dysplasia and pathological cell division were observed. Due to the coexistence of atrophy of normal tissues and abnormal hyperplasia of glands, the epithelial cells in the gastric antra remained relatively stable in number but developed into malignant lesions. These results suggest that precancerous lesions from the forestomach to the antrum in ApcMin/+ mice at 24 weeks of age gradually progress. The mechanisms of this reaction are not known. Clearly, further studies are needed to evaluate the possible role of APC in the regulation of the immune response. Although several studies suggested the involvement of the Wnt pathway in the development of sporadic gastric cancer [25–27], the functional significance of the Wnt/β-catenin pathway in gastric carcinogenesis was still poorly defined in comparison to its established role in colon carcinogenesis. Gastric tumors in ApcMin/+ mice showed adenomatous lesions with a strong nuclear/cytoplasmic accumulation of β-catenin, thus suggesting that the Wnt/β-catenin signaling pathway is involved in tumorigenesis [15]. The gastric body and antrum regions were positive for alcian blue staining and CA199 immunostaining, respectively. The present results confirmed that the APC mutation causes epithelial hyperplasia in the forestomach, glandular atrophy and intestinal metaplasia in the gastric body mucosa, so it therefore generates a preneoplastic condition. Intestinal-type carcinoma arises in older patients in a part of the stomach where inflammation has been present for a long period [28]. The protruded form of human gastric well-differentiated adenocarcinoma is often found in the intestinal metaplastic mucosae of elderly people and is macroscopically and microscopically quite similar to the protruded form of gastric precancerous lesions in ApcMin/+ mice. Severe atrophic gastritis accompanying intestinal metaplasia is closely related to the development of intestinal-type gastric carcinoma. Our present results clearly indicate that Apc gene mutations might promote the progression of the intestinal metaplasia-dysplasiacarcinoma sequence. In conclusion, we have shown that premalignant lesions play a key role in causing malignant and invasive gastric adenocarcinoma in ApcMin/+ mice. In the present study, all of the ApcMin/+ mouse stomachs examined at 24 weeks of age had developed atrophic gastritis, intestinal metaplasia and glandular dysplasia, although the role of Apc in gastritis, intestinal metaplasia in Wnt-dependent intestinal-type tumorigenesis has not been clarified. The participation of the mutated Apc gene in gastric precancerous lesions was demonstrated by the results observed in ApcMin/+ mice. The ApcMin/+ mouse model is therefore valuable for investigating the relationship between precancerous lesion and gastric carcinogenesis.
Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This work was supported by grants from Key Project for cultivating scientific and technological innovation of college students of Guangdong Province of China (Grant ID pdjha0260), and Medical Scientific Research Foundation of Guangdong Province of China (Grant ID A2017345), National Natural Science Foundation of China (Grant ID 81773118, 31471290 and 31771578 to L.W), Innovative Research Team in University of Guangdong Province of China (2016KCXTD019 to L.W). References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (2018) 394–424. [2] P. Rawla, A. Barsouk, Epidemiology of gastric cancer: global trends, risk factors and prevention, Prz. Gastroenterol. 14 (1) (2019) 26–38. [3] P. Correa, Gastric cancer: overview, Gastroenterol. Clin. North Am. 42 (2) (2013) 211–217. [4] P. Correa, M.B. Piazuelo, The gastric precancerous cascade, J. Dig. Dis. 13 (1) (2012) 2–9. [5] A.D. Spence, C.R. Cardwell, ÚC. McMenamin, B.M. Hicks, B.T. Johnston, L.J. Murray, et al., Adenocarcinoma risk in gastric atrophy and intestinal metaplasia: a systematic review, BMC Gastroenterol. 17 (1) (2017) 157, https://doi.org/ 10.1186/s12876-017-0708-4. [6] W.J. den Hollander, I.L. Holster, C.M. den Hoed, L.G. Capelle, T.J. Tang, M.P. Anten, et al., Surveillance of premalignant gastric lesions: a multicentre prospective cohort study from low incidence regions, Gut 68 (4) (2019) 585–593. [7] B. Da, N. Jani, N. Gupta, P. Jayaram, R. Kankotia, C. Yao Yu, et al., High-risk symptoms do not predict gastric cancer precursors, Helicobacter 24 (1) (2019), https://doi.org/10.1111/hel.12548 e12548. [8] R. Sitarz, M. Skierucha, J. Mielko, G.J.A. Offerhaus, R. Maciejewski, W.P. Polkowski, Gastric cancer: epidemiology, prevention, classification, and treatment, Cancer Manage. Res. 10 (2018) 239–248. [9] D. Cai, J. Yu, J. Qiu, B. He, Z. Chen, M. Yan, et al., Dynamic changes of Sonic Hedgehog signaling pathway in gastric mucosa of rats with MNNG-induced gastric precancerous lesions, J. Cell. Physiol. 234 (7) (2019) 10827–10834. [10] W. Liu, H.F. Pan, Q. Wang, Z.M. Zhao, The application of transgenic and gene knockout mice in the study of gastric precancerouslesions, Pathol. Res. Pract. 214 (12) (2018) 1929–1939. [11] N. Krishnamurthy, R. Kurzrock, Targeting the Wnt/β-catenin pathway in cancer: update on effectors and inhibitors, Cancer Treat. Rev. 62 (2018) 50–60. [12] X.Z. Yang, T.T. Cheng, Q.J. He, Z.Y. Lei, J. Chi, Z. Tang, et al., LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway, Mol. Cancer 17 (1) (2018) 126, https:// doi.org/10.1186/s12943-018-0874-1. [13] D.C. Sample, N.J. Samadder, L.M. Pappas, K.M. Boucher, W.S. Samowitz, T. Berry, et al., Variables affecting penetrance of gastric and duodenal phenotype in familial adenomatous polyposis patients, BMC Gastroenterol. 18 (1) (2018) 115, https:// doi.org/10.1186/s12876-018-0841-8. [14] A.R. Moser, H.C. Pitot, W.F. Dove, A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse, Science 247 (1990) 322–324. [15] H. Tomita, Y. Yamada, T. Oyama, K. Hata, Y. Hirose, A. Hara, et al., Development of gastric tumors in Apc(Min/+) mice by the activation of the beta-catenin/Tcf signaling pathway, Cancer Res. 67 (9) (2007) 4079–4087. [16] C. Qi, B. Li, S. Guo, B. Wei, C. Shao, J. Li, et al., P-selectin-mediated adhesion between platelets and tumor cells promotes intestinal tumorigenesis in apc(Min/+) mice, Int. J. Biol. Sci. 11 (6) (2015) 679–687. [17] A. Saxena, R. Fayad, K. Kaur, S. Truman, J. Greer, J.A. Carson, et al., Dietary selenium protects adiponectin knockout mice against chronic inflammation induced colon cancer, Cancer Biol. Ther. 18 (4) (2017) 257–267. [18] L.J. Wang, Y. Zhao, B. Han, Y.G. Ma, J. Zhang, D.M. Yang, et al., Targeting SlitRoundabout signaling inhibits tumor angiogenesis in chemical-induced squamous cell carcinogenesis, Cancer Sci. 99 (3) (2008) 510–517. [19] W. Onuma, S. Tomono, S. Miyamoto, G. Fujii, T. Hamoya, K. Fujimoto, et al., Irsogladine maleate, a gastric mucosal protectant, suppresses intestinal polyp development in Apc-mutant mice, Oncotarget 7 (8) (2016) 8640–8652. [20] S. Ghatak, P. Chakraborty, S.R. Sarkar, B. Chowdhury, A. Bhaumik, N.S. Kumar,
5. Conclusion In summary, our study showed that Apc mutations were associated with the following main features: gastric precancerous lesions, glandular atrophy, intestinal metaplasia and dysplasia. These spontaneous lesions exhibit patterns that are similar to the pathogenesis of gastric adenocarcinoma in humans. These findings underscore the potential of ApcMin/+ mice as a valuable animal model for investigating different premalignant lesions and assisting in the planning of eradication therapy to prevent the development of gastric carcinoma.
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[21] [22] [23] [24]
Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer, BMC Med. Genet. 18 (1) (2017) 61, https://doi.org/10.1186/s12881-0170427-2. T. Senda, A. Iizuka-Kogo, T. Onouchi, A. Shimomura, Adenomatous polyposis coli (APC) plays multiple roles in the intestinal and colorectal epithelia, Med. Mol. Morphol. 40 (2) (2007) 68–81. Z.K. Wang, J. Liu, C. Liu, F.Y. Wang, C.Y. Chen, X.H. Zhang, Hypermethylation of adenomatous polyposis coli gene promoter is associated with novel Wnt signaling pathway in gastric adenomas, J. Gastroenterol. Hepatol. 27 (10) (2012) 1629–1634. H. Saluja, C.S. Karapetis, S.K. Pedersen, G.P. Young, E.L. Symonds, The use of circulating tumor DNA for prognosis of gastrointestinal cancers, Front. Oncol. 8 (2018) 275. R. Hida, H. Yamamoto, M. Hirahashi, R. Kumagai, K. Nishiyama, T. Gi, et al., Duodenal neoplasms of gastric phenotype: an immunohistochemical and genetic
[25] [26] [27] [28]
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study with a practical approach to the classification, Am. J. Surg. Pathol. 41 (3) (2017) 343–353. B.H. Min, J. Hwang, N.K. Kim, G. Park, S.Y. Kang, S. Ahn, et al., Dysregulated Wnt signalling and recurrent mutations of the tumour suppressor RNF43 in early gastric carcinogenesis, J. Pathol. 240 (3) (2016) 304–314. F. Molaei, M.M. Forghanifard, Y. Fahim, M.R. Abbaszadegan, Molecular signaling in tumorigenesis of gastric cancer, Iran. Biomed. J. 22 (4) (2018) 217–230. L. Song, Z.Y. Li, W.P. Liu, M.R. Zhao, Crosstalk between Wnt/β-catenin and Hedgehog/Gli signaling pathways in colon cancer and implications fortherapy, Cancer Biol. Ther. 16 (1) (2015) 1–7. F. Zhou, J. Shi, C. Fang, X. Zou, Q. Huang, Gastric carcinomas in young (younger than 40 years) Chinese patients: clinicopathology, family history, and postresection survival, Medicine (Baltimore) 95 (9) (2016) e2873.