Marra et al. BMC Cancer 2010, 10:129 http://www.biomedcentral.com/1471-2407/10/129 Page 4 of 9 Figure 1 Characterization of SW780-shPSCA cells. A) FACS analysis of SW780-shPSCA cells in physiological condition (black line) or upon induction of shRNA against PSCA (red line): left panel shows GFP expression, which is a marker of shRNA expression; right panel shows PSCA expression, mean fluorescence intensity was decreased from 333 to 35 upon expression of shRNA. B) Viability assay in the presence of dox (empty) or without dox (filled) was carried out using the CellTiter-Blue Viability assay as described in material and methods. Error bars indicate standard error. tumor model leading to more than 50% reduction of tumor mass with respect to dox untreated tumors. Transcriptional data analysis To gain some hints on the biological role played by PSCA, expression profile was carried out in tumors. Unsupervised hierarchical clustering of genome-wide expression data showed that samples clustered accord- ing to dox treatment (Figure 3A), thus demonstrating a clear effect of the PSCA expression levels on transcrip- tional programs. To identify the genes differentially expressed in the two experimental conditions, a 1-way ANOVA analysis was carried out. Out of the 37,586 probes represented on the array, 284 and 353 were found to be respectively up- and down-regulated in dox+ samples, as compared to dox-samples (ANOVA p-value < 0.001). Similar microarray experiments were also conducted in vitro showing a similar separation between the two groups, according to dox treatment. Comparison between the in vitro and in vivo data sets revealed a statistical significant overlap for the overexpressed genes (Table 2), although it may repre- sent an underestimation due to the limited size of in vitro observations. The genes identified as differentially expressed were analyzed by gene set enrichment analysis, using the same method also used elsewhere [24]. In this way we could monitor the pathways showing a global transcrip- tional perturbation upon silencing of PSCA (Figure 3B). We defined as up- or down-regulated the pathways statistically enriched in the list of genes up- or down- regulated, respectively, in the presence or absence of treatment. A statistically significant enrichment was observed between the list of genes up-regulated upon PSCA silencing (dox+). The IFNa/b signaling pathway resulted to be up-regulated upon PSCA silencing in the in-vivo experiments but its up-regulation was not evi- dent in the in vitro datase. Out of the 27 genes listed in the Ingenuity database for interferon signaling 12 were up-regulated in vivo and 1 in vitro, STAT2. Similar results were obtained using the GeneGo database to run the analyses (8 out of 28 genes). In addition, the “anti- gen presentation” pathway resulted as perturbed with the highest statistical significance. Other immune related pathways were also affected although at lower statistical significance. In agreement with the reduced cell viability in vitro (Figure 1B) and reduced tumor growth (Figure 2A) the down-regulated pathways were “growth” and “negative regulation of apoptosis”. To measure by a more quantitative method the ampli- tude of gene regulation, selected genes were analyzed by qRT-PCR. As shown in Figure 4A, a subset of genes belonging to IFNa/b pathway such as IFIT1, IFI27 and IFI44L showed a statistical significant up-regulation (p < 0.001) in tumors where PSCA was down-regulated by dox treatment. To exclude that expression of shRNA per se could trigger expression of IFN pathway genes, tumors expressing control shRNA were analyzed. No statistical differences were observed with or without control shRNA expression. Interestingly, qPCR analysis of SW780-shPSCA in cell culture (Figure 4B) confirmed the lack of induction of the IFNa genes regardless of PSCA expression, suggesting a distinct role for PSCA in the context of tumor growth. Discussion In this study, we have identified a correlation between PSCA expression and tumor growth in vivo. High level of PSCA mRNA and protein expression has been observed in most primary prostate and pancreatic human tumors and in particular in aggressive metastatic forms. In bladder cancer, higher levels of PSCA expres- sion correlated with increasing tumor grade [25] and more recently genetic variants in this gene were asso- ciated with cancer occurrence, although the biological