Gastric Epithelial-specific Deletion Of Smad4Led To Gastric Carcinogenesis In Mouse

Gastric cancer is one of the most frequently occurring malignancies, and ranks asthe second leading cause of cancer-related deaths worldwide. In China, mortality dueto gastric cancer accounts for25%—30%of all cancer-related deaths, ranking first inall malignancies. Gastric cancer is thought to result from a combination of factors,dietary factors, infection by Helicobater pylori, accumulation of generalized andspecific genetic alterations. Understanding the underlying molecular mechanismcould provide insights leading to improved diagnosis and therapeutic approaches.SMAD4, a central mediator of transforming growth factor-β signaling, isfrequently inactivated in various tumor types including gastric cancer. Thetumor-suppressive role of SMAD4in gastrointestinal tract is first verified by the micebearing heterozygous deletion of Smad4that spontaneously develop gastrointestinalpolyps, sharing much similarity to those in JPS. The prominent pathologicalcharacteristic in human SMAD4-mutation-driven JPS and mouse gastrointestinalpolyps caused by haploid loss of Smad4is dense inflammatory cell infiltrates andstromal expansions, indicating that loss of Smad4in microenvironmental cellscontribute to gastrointestinal tumorigenesis. Supportively and unexpectedly, followingstudies demonstrated that T cell-restricted deletion of Smad4alone leads tospontaneous gastric or intestinal polyps.However, these findings do not omit the role of epithelial Smad4in suppressionof gastrointestinal cancer. In fact, clinical observation reported that in spontaneousgastric cancer, decreased expression of Smad4was restricted in epithelial cells. Evenin FJP patients with SMAD4germline mutation, Smad4loss of heterozygosity (LOH)was only found in tumor epithelia but not in stromal lymphocytes, as furtherconfirmed by the findings in heterozygous Smad4deficient mice which showed thatSmad4LOH was only occurred within gastrointestinal polyp epithelia, raising thepossibility that loss of the wild-type SMAD4in the epithelium of JPS polyps, togetherwith haploid loss of Smad4in microenviromental cells, initiates tumorigenesis.Furthermore, our previous in vitro functional experiments showed that Smad4 knockdown in various gastric cancer cell lines resulted in significant resistance toTGF-beta-induced cell proliferation inhibition. Although previous study reported thatsmad4deficiency induced by gastrointestinal epithelial-specific Cre systems fails tolead to gastrointestinal neoplasia, it is indeed possible that the gastrointestinalepithelial-specific Cre systems was insufficient for Smad4deletion in100%epithelialcells, or inefficient in tissue stem cells. Thus, the role of gastric epithelial Smad4ingastric homeostasis maintenance and gastric tumorigenesis has yet to be clearlydefined.The purpose of our study is to generate a mouse that specifically deletes Smad4gene in mouse gastric epithelial cells, and to obtain the effects of Smad4in gastricepithelial. We hope to establish a new mouse model that can be similar to thetumorigenesis and progression of human gastric cancer.Gastric epithelial-specific Smad4gene knockout mice are obtained by theCre-Loxp mediated recombination knockout method. Specifically, expressingTPD52-Cre (also known as PC1-Cre) mice are crossed with mice that werehomozygous for the “floxed” Smad4allele to get the conditional Smad4geneknockout mice (PC-1-Cre;Smad4fl/fl). The space and timing expression pattern ofspecific Cre-mediated recombination are confirmed by PCR, Southern Blot, LacZStaining, Western Blot and Immunohistochemistry experiments. The influences ofSmad4mutation on mice gastric cell development and cellular functions are then,determined through anatomical and histological methods.PC-1-Cre;Smad4fl/flmice exhibited progressive weakness and weight loss after3months of age. Upon dissection, PC-1-Cre;Smad4fl/flmice exhibited multiple fusedpolyps throughout gastric fundus and antro-pyloric regions. All PC-1-Cre;Smad4fl/flmice died by12months of age due largely to gastric outlet obstruction andconsequent malnutrition.. Histological examination revealed that in contrast todefined glandular structure from control mice at3months of age, Smad4mutant micepresented polypoid glandular masses with elongated, branched and dilated glands.After that, Smad4-deficient mice progressively developed aggressively invasivefeatures, which showed that neoplastic epithelial cells invaded subepithelial tissuesand replaced muscle fibers of destructive infiltrative growth. The results of BrdUlabeling showed the marked hyperplasia in Smad4mutant gastric epithelium was dueto enhanced epithelial proliferation. Cyclin D1and SPP1, as key downstream target ofSmad4pathway, we found that the expression of the two genes are increased in Smad4-deficient mice. CD44is an identified marker of gastric cancer stem cells, andC-myc also is a key marker in various tumor types. Besides, the upregulatedexpression of p-Akt and p-Smad2may be the key factors of aggressive progression inSmad4-deficient tumors.In this study, we first generated a novel Cre system under the control of theTumor protein D52promoter active within100%of gastric epithelial cells. Smad4deletion in all gastric epithelial cells led to early onset and rapid malignantprogression of gastric carcinoma with full penetrance. This mouse models couldpotentially be a useful tools for studying Smad4gene functions and moleculemechanisms in the gastric cancer...