BackgroundA large fraction of murine tumors induced by transgenic expression of SV40 large T antigen (SV40 TAg) exhibits a neuroendocrine phenotype. It is unclear whether SV40 TAg induces the neuroendocrine phenotype by preferential transformation of progenitor cells committed to the neuroendocrine lineage or by transcriptional activation of neuroendocrine genes.Methodology/Principal FindingsTo address this question we analyzed CEA424-SV40 TAg-transgenic mice that develop spontaneous tumors in the antral stomach region. Immunohistology revealed expression of the neuroendocrine marker chromogranin A in tumor cells. By ELISA an 18-fold higher level of serotonin could be detected in the blood of tumor-bearing mice in comparison to nontransgenic littermates. Transcriptome analyses of antral tumors combined with gene set enrichment analysis showed significant enrichment of genes considered relevant for human neuroendocrine tumor biology. This neuroendocrine gene signature was also expressed in 424GC, a cell line derived from a CEA424-SV40 TAg tumor, indicating that the tumor cells exhibit a similar neuroendocrine phenotype also in vitro. Treatment of 424GC cells with SV40 TAg-specific siRNA downregulated expression of the neuroendocrine gene signature.Conclusions/SignificanceSV40 TAg thus appears to directly induce a neuroendocrine gene signature in gastric carcinomas of CEA424-SV40 TAg-transgenic mice. This might explain the high incidence of neuroendocrine tumors in other murine SV40 TAg tumor models. Since the oncogenic effect of SV40 TAg is caused by inactivation of the tumor suppressor proteins p53 and RB1 and loss of function of these proteins is commonly observed in human neuroendocrine tumors, a similar mechanism might cause neuroendocrine phenotypes in human tumors.
In intestinal and pyloric epithelia, leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-expressing cells represent long-lived adult stem cells that give rise to all epithelial cell types, including endocrine cells. Ablation of the Apc gene in Lgr5-expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5-positive stem cells, we crossed Lgr5-EGFP-IRES-creER(T2) mice, which express EGFP and Cre recombinase driven by the Lgr5 promoter, with CEA424-SV40-TAg mice, which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19 day-old mice, single SV40 T antigen (TAg)-positive cells were identified preferentially at the the bases of pyloric glands, close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5-positive cells in gastrointestinal neoplasia, we could not detect Lgr5-EGFP-positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg, since β-catenin-mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424-SV40-TAg tumours, we performed tracing experiments using Lgr5-EGFP-IRES-creERT2:CEA424-SV40-TAg:ROSA26-tdRFP mice. Following tamoxifen induction, it was possible to efficiently trace the progeny of Lgr5-expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP-positive tumour cells were detected, even when RFP gene activation occurred in 7 day-old mice well before the appearance of TAg-positive tumour cells. Hence, we conclude that Lgr5-expressing stem cells probably do not constitute the cells of origin in CEA424-SV40-TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5-positive stem cells. Thus, additional long-lived LGR5-negative stem cells or progenitor cells with a low turnover rate might exist in the pyloric region, which could give rise to tumours.
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