Expression of a Neuroendocrine Gene Signature in Gastric Tumor Cells from CEA 424-SV40 Large T Antigen-Transgenic Mice Depends on SV40 Large T Antigen

A 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. To 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. SV40 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

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SV40 T antigen-specific siRNA downregulates expression of neuroendocrine genes in the <i>CEA424</i>-SV40 TAg gastric cancer cell line 424GC.

<p>Three SV40 TAg-targeting siRNAs were evaluated in 424GC cells. siRNA82 and siRNA2047 completely abrogated SV40 T antigen mRNA expression analyzed 72 h after transient transfection by RT-PCR (A) and Western blot analysis (siRNA82 only; B). The two PCR products represent both non-spliced and spliced SV40 TAg RNA. The identity of the 70–80 kDa band in the untreated cell sample is unclear. 72 h after addition of siRNA82 the transcriptome of 424GC cells was analyzed and compared to that of untreated cells. The expression levels of genes up- (negative ratios) or downregulated (positive ratios) by SV40 T antigen siRNA treatment were plotted against their expression ratio (C). The most strongly up- and downregulated and most highly expressed genes in the samples are identified by gene symbols and color codes (green, neuroendocrine; red, cell cycle/proliferation; purple, retroviral; blue, other genes). For comparison, the expression levels and fold chance values of all ribosomal protein genes were plotted. Note their low fluctuation of expression change (maximally 2-fold). The mean fold change (transcript level with/transcript level without siRNA treatment) for 244 probe sets was 1.07±0.25. The control experiment was performed trice (n = 3), the siRNA experiment twice (n = 2). GSEA of the SV40 TAg siRNA ranked gene list from treated and untreated 424GC cells using a neuroendocrine signature of 39 genes from human tumors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone-0029846-g001" target="_blank">Figure 1</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Hofsli1" target="_blank">[15]</a>; D) and 36 E2F target genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Cantalupo1" target="_blank">[19]</a>; E). ES, enrichment score; FDR, false discovery rate; TAg siRNA up/down, signature gene sets up- or downregulated in TAg siRNA-treated cells.</p

<i>CEA424</i>-SV40 TAg gastric tumors exhibit a neuroendocrine phenotype.

<p>Transcriptome analysis of RNA from tumor-bearing regions of the antrum from 30- (A), 60- (B) and 90-day-old <i>CEA424</i>-SV40 TAg-transgenic mice (C) and non-transgenic mice revealed predominant enrichment of genes typically expressed in neuroendocrine tissues (symbols shown in green). The means of the expression levels of genes preferentially expressed in tumor-bearing stomach tissues were plotted against their expression ratio tumor/normal stomach (genes with fold change >5-fold are shown; n = 3). The most strongly upregulated and most highly expressed genes in the samples are identified by gene symbols. These were statistically significant for day 90 (p<0.05). Note the strong increase of upregulated genes with age which reflects the exponential tumor growth between d30 and d90 in <i>CEA424</i>-SV40 TAg transgenic mice (D). (E, F) Gene set enrichment analysis (GSEA) of the d90 tumor ranked gene list using the neuroendocrine signature genes from murine <i>Atp4b</i>-SV40 TAg gastric tumors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Syder1" target="_blank">[4]</a>; E) and from human neuroendocrine tumors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Hofsli1" target="_blank">[15]</a>; F) comprising 305 identifiable genes out of 399 genes and 39 identifiable genes out of 52 genes, respectively. ES, enrichment score; FDR, false discovery rate; tumor up/down, signature gene sets up- or downregulated in tumors. (G) Tumor-bearing transgenic mice exhibit elevated serum serotonin levels. Serum from wildtype (n = 6 pooled; age: 130 d) and <i>CEA424</i>-SV40 TAg mice (n = 6; age: 95–110 d) were analyzed by ELISA. (H–K) Gastric tumor cells express both SV40 TAg (brown nuclear staining; H, I) and the neuroendocrine marker chromogranin A (CHGA; brown cytoplasmic staining; J, K). Parallel sections of formalin-fixed paraffin-embedded gastric tumors from 30- and 99-day-old mice were used for immunohistological staining. The tumors were localized in the antrum and duodenum, respectively. Latter tumors were commonly observed in mice older than 90 days and are supposed to have originated from pyloric tumors through invasive growth or metastatic spread. (M, N) Double-staining with fluorescently labeled anti-SV40 TAg and CHGA of gastric tumor-bearing tissue from 30- and 60-day-old mice. Note most of the cells with SV40 TAg-positive nuclei (red color) express CHGA (yellow color). The nuclei are visualized by DAPI (blue color). Bars: 50 µm. (L) Transmission EM of a typical tumor cell in a tumor from a 90-day-old <i>CEA424</i>-SV40 TAg mouse. Note the numerous electron-dense secretory granules (LDCV, large dense core vesicles, arrows; SLMV, small synaptic-like microvesicles arrowheads) and extended mesh of rough endoplasmic reticulum (ER; open arrowheads) typical of neuroendocrine and rapidly growing cells, respectively. Original magnification: ×15.300 in (L). Bars: 50 µm in H-K; 2 µm in L. (O) Western blot analysis of NEUROD1 expression in <i>CEA424</i>-SV40 TAg tumor-derived cell lines (424GC, GC3, GC8) and in tumors from 85-day-old mice. The endothelioma cell line served as a negative control, detection of β-actin as loading control.</p

The <i>CEA424</i>-SV40 TAg gastric tumor-derived cell line 424GC transcriptome reflects the neuroendocrine phenotype of the parental tumor.

<p>Transcriptome analysis of RNA from tumor-bearing regions of the antrum from 30- (red), 60- (blue) and 90-day-old mice (green) and d90 non-transgenic mice (pink) as well as from 424GC cells (brown) were performed and the data sets were subjected to hierarchical clustering and principal component analyses (n = 3 for all samples). The heatmap shows that tumors at day 90 and 424GC show the most closely related expression pattern (A). Note that in PCA, data points of 424GC cells and that of d90 antra exhibit coordinates which are substantially different from that of all other samples (B). The expression levels (mean of 3 samples and standard deviation) of selected genes (expression ratio d90 antra/normal antra >15-fold) were compared to that observed for 424GC cells and normal stomach. If a gene was represented by more than one probe set the one which displayed the highest fold change value (expression d90 versus normal antra) was chosen (C). Note high expression of genes characteristic for the neuroendocrine lineage in both d90 antra and 424GC cells (e.g. tryptophan hydroxylase, <i>Tph1</i>; chromogranin B, <i>Chgb</i>; secretin, <i>Sct</i>).</p

Gene set enrichment analyses (GSEA) of transcriptomes of d90 <i>CEA424</i>-SV40 TAg tumors and of 424GC cells after SV40 TAg siRNA treatment using neuroendocrine and E2F target gene signatures.

1<p>p values<0.001 in bold;</p>2<p>Syder et al. 2004, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846.s006" target="_blank">Table S5</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Syder1" target="_blank">[4]</a>;</p>3<p>Syder et al. 2004, Table S7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Syder1" target="_blank">[4]</a>;</p>4<p>Hofsli 2006, Table 3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Hofsli1" target="_blank">[15]</a>;</p>5<p>Schaffer et al. 2010, Figure 4a <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Schaffer1" target="_blank">[30]</a>;</p>6<p>Cantalupo et al. 2009, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846.s006" target="_blank">Table S5</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029846#pone.0029846-Cantalupo1" target="_blank">[19]</a>.</p