Effect of myeloid differentiation primary response gene 88 on expression profiles of genes during the development and progression of Helicobacter-induced gastric cancer

Gastric cancer is one of the most common causes of cancer-related death worldwide with an estimated 738,000 deaths each year [ 1]. Recently, H. pylori was recognized as the foremost cause of gastric cancer [ 2– 7]. With an estimated half of the world’s population being infected, Helicobacter infection contributes significantly to the worldwide gastric cancer burden [ 7, 8]. Recognition of the factors leading up to the development and progression towards gastric cancer are critical in determination of cancer pathology. H. pylori-induced gastric carcinogenesis involves a multistep progression from normal gastric mucosa to superficial gastritis, chronic gastritis, atrophic gastritis, metaplasia, dysplasia, and finally gastric carcinoma [ 8, 9]. Molecular events associated with disease progression to gastric malignancy have not been elucidated. Considerable amount of confirmatory evidence shows that host immune response to H. pylori is crucial in determining gastric cancer predisposition [ 10– 12]. We have previously shown that a key signal transduction adaptor protein, myeloid differentiation primary response gene 88 (MyD88), regulates Helicobacter-induced gastric cancer progression in a mouse model of gastric cancer [ 13]. We demonstrated that H. felis-infected MyD88 deficient ( Myd88 ^−/−) mice exhibited severe gastric pathology and an accelerated progression to gastric dysplasia compared to wild type (WT) mice [ 13] However, the MyD88-dependent gene responsible for this pathology were not described.

MyD88 is a key adaptor molecule that is crucial in mediating innate immune signals from members of the toll-like receptor (TLR) and interleukin-1 (IL-1)/IL-18 families leading to downstream activation of nuclear factor (NF)-κB [ 14– 16]. Consistent with involvement in these inflammatory pathways, MyD88 signaling has been associated with cancer progression, which stems from the understanding that inflammation is linked to cancer promotion [ 17, 18]. Studies on the role of MyD88 cancer progression have been the subject of recent intense investigations. However, the data are contradictory, which indicate that the role of MyD88 in the development and progression of inflammation-associated cancers is complex [ 19]. Several studies using genetic or chemical carcinogenesis models involving Myd88 deficient mice have shown MyD88 to either promote [ 20– 27] or suppress [ 13, 28– 34] tumor development. The complex role of MyD88 in carcinogenesis is best typified by studies in colon cancer models [ 22, 24, 29, 35] showing contradictory roles in the same tissue. The mechanistic basis for these opposing observation is still not fully understood and could be due to many factors including, the type of inflammation, the extent of tissue damage, and immune response elicited [ 35]. Further, the MyD88 dependent genes in this accelerated progression to dysplasia remain unknown. Therefore, this study was performed to identify potential genes involved in the accelerated progression of gastric cancer.

Gastric cancer develops and progresses through a stepwise sequence of events from inflammation to atrophy, metaplasia, dysplasia, and finally to gastric cancer [ 50]. We previously demonstrated using a mouse model of gastric that mice deficient in MyD88 signaling exhibited dramatic pathology and an accelerated progression to gastric neoplasia in response to H. felis infection [ 13]. In the present study, we used microarray gene expression analysis to identify the genes involved in this progression to gastric neoplasia. Although previous studies have investigated differential gene expression in mice stomachs in response to Helicobacter infection, most have focused on H. pylori [ 51– 53], which does not result in neoplastic changes in mice [ 54, 55]. The few studies that have examined gene expression profiles in mouse model of gastric cancer have used the insulin-gastrin (INS-GAS) transgenic gastric cancer mouse model [ 56, 57]. These mice have been shown to spontaneously develop gastric cancer even in the absence of Helicobacter infection [ 58]. We have previously reported that Myd88 ^−/− mice do not exhibit abnormal pathology in the absence of Helicobacter infection [ 13]. The global transcriptional profiling of mouse gastric tissue identified a large number of significant differentially expressed genes in H. felis-infected Myd88 ^−/− mice compared to H. felis-infected WT mice. The most over expressed gene in Myd88 ^−/− mice during H. felis infection at 25 weeks was Chil4. Chitinase like proteins (CLPs) have been studied in relation to other cancers yet little has been investigated in relation to gastric cancer except for our present study and a couple other studies [ 57, 59]. Upregulation of CLPs has been shown in a number of human cancers including brain, bone, breast, ovaries, lung, prostate, colon, thyroid, and liver [ 41, 60]. For gastric cancer studies, chitinase protein 3 like 1 (Chil1) was upregulated in INS-GAS mice infected with H. felis, [ 57]. In our present study, Chil1 was not upregulated in response to H. felis infection. However, in addition to upregulation of Chil4, another CLP, Chil3 was also significantly over expressed in H. felis-infected mice at both 25 weeks ( p = 0.03) and 47 weeks ( p = 0.01) (gene not listed in Tables  2 and 3, only the top 50 are listed). An abundant over expression of Chil1, Chil4 as well as Chil3 has been reported in early preneoplastic stage in the epidermis [ 61]. Overall, CLPs have been implicated to play a role in chronic inflammation, tissue remodeling, and wound healing [ 40]. Up-regulation of genes involved in tissue remodeling is noteworthy because chronic inflammation and subsequent damage to the gastric epithelium has been suggested to play an important role in cancer development and progression [ 62]. During chronic inflammation, the resulting prolonged tissue damage creates a loss of control over normal tissue repair mechanisms resulting in persistent hyper-tissue repair, which is accompanied with sustained proliferation [ 63] and ultimately advancing to precancerous lesions. Lost tissue is then replaced with stem and progenitor cells that are under a continuous stimulus of proliferation, leading to the accumulation of replacement cells with dysregulated and altered signaling pathways [ 63]. Further, studies investigating associations between chronic inflammation, tissue repair and carcinogenesis highlight the potential of these cellular changes in inducing both pro-oncogenic and tumor suppressor pathways [ 62, 64– 67]. Our study, in addition to the work done by Li et al. [ 59] and Takaishi and Wang [ 57] show a need for further investigation into the role of CLPs in gastric carcinogenesis.

Other upregulated genes included Cd74, B2m, and interferon (IFN) induced genes such as GTPases (interferon gamma induced GTPase, lgtp, immune mediated GTPase family M member 2, lrgm2), Guanylate binding protein 2 (Gbp2), and transcription factor interferon regulatory factor 1 (Irf1). Cd74 or invariant chain (Ii) protein is a chaperone molecule responsible for regulating antigen presentation of MHC II molecules. It has been linked to chronic inflammation and carcinogenesis in the gastrointestinal tract [ 44]. Further, Cd74 was also shown to play a role as a receptor for migration inhibitory factor (MIF), a molecule reported to have pro-carcinogenic effects on gastric epithelial cells [ 68]. IFNs are known to activate signal transducer and activator of transcription 3 (STAT3) [ 69, 70] signaling leading to epithelial proliferation and inhibition of apoptosis [ 71, 72]. Currently not much is known about IFNs in gastric cancer. Ubiquitin D (Ubd), which is associated with progression of colon cancer [ 73], was also significantly expressed genes in Myd88 ^−/− in response to H. felis infection.

For downregulated genes, the significantly expressed ones included, ATPase H+/K+ transporting, alpha subunit (Atp4a), Atp4b, Mucin 5 AC (Muc5ac), apolipoprotein A-1 (Apoa1), and gastric intrinsic factor (Gif). The genes, Atp4a and Atp4b encode gastric H+/K + − ATPase alpha and beta subunits, respectively. Gastric H+/K + − ATPase alpha and beta subunits are expressed in parietal cells [ 74] and their loss has been associated with gastric dysplasia [ 58]. We observed downregulation of Atp4a and Atp4b in response to infection with H. felis, which may represent a loss of parietal cells that has been shown to precede gastric dysplasia. These results are in line with those observed in another fast progressing gastric cancer model involving the use of INS-GAS mice [ 58]. Muc5ac, which encodes gastric M1 mucin [ 75] has been reported to play a role in gastric carcinogenesis [ 76] was also downregulated in response to infection with H. felis in Myd88 ^−/− mice. Progression of gastric lesions has been reported to be associated with the gradual decrease in expression of Muc5ac [ 57, 77– 79] followed by the transformation of the gastric epithelium [ 80] resulting in gastric dysplasia. Another highly downregulated gene we found in Myd88 ^−/− mice infected with H. felis was Apoa1, which was also reported to be downregulated in a fast progressing gastric cancer mouse model [ 57]. Proteomics approach in human gastric cancer also showed downregulation of Apoa1 [ 81], but its role in gastric carcinogenesis is unknown. In the lung, downregulation of Apoa1 was associated with an increased risk of lung cancer [ 82]. Data from Apoa1-deficient mice suggest antitumorigenic properties of Apoa1 via modulation of the immune system [ 83]. Gastric intrinsic factor (Gif), another downregulated gene is secreted by parietal cells and is required for Vitamin B12 absorption [ 84]. Concomitantly, the downregulation of Gif results in vitamin B12 deficiency (pernicious anemia). Gastric intrinsic factor was downregulated in H. felis-infected Myd88 ^−/− mice at both 25 and 47 weeks post-infection. A previous study using INS-GAS mice infected with H. felis also reported downregulation of Gif [ 57]. In human gastric cancer, Gif was one of the genes found by SAGE analysis to be downregulated [ 85]. Further, studies have shown an increased risk of gastric cancer in pernicious anemic patients [ 86].

We found a number of new genes in our fast progressing gastric mouse model, i.e., Myd88 ^−/− mice infected with H. felis. The genes included up- and downregulated genes, which had not been previously linked to Helicobacter-related gastric carcinogenesis including BPI fold containing family B member 1 (Bpifb1) and proteasome subunit beta 8 (Psmb8). These genes have been linked to cancer-related processes including, apoptosis and in some cases other cancers as well as prognosis indicators [ 87, 88]. Psmb8 was shown to be significantly up regulated in cancers such as bladder, breast, kidney, lung, uterine, and head and neck [ 89]. A recent study by Kwon, et al. [ 90], which was published during the writing of our manuscript reported that Psmb8 may be a potential marker for prognosis in gastric cancer. Bpifb1 may be involved in the innate immune response particularly in response to bacterial exposure. The protein encoded for by Bpifb1 binds bacterial lipopolysaccharide (LPS) as well as modulates the cellular response to LPS [ 91]. Bpifb1 has been found to be overexpressed in mucous cells of salivary gland tumors of papillary cystadenocarcinoma [ 87]. Future studies using a gastric culture organoid system will validate these genes and some of the important novel genes we identified for their role in rapid progression of Helicobacter-induced gastric cancer.