Marra et al. BMC Cancer 2010, 10:129 http://www.biomedcentral.com/1471-2407/10/129 Identification of deregulated probes Differences in average probe expression between the dox + (PSCA silencing) and dox-samples were computed by 1-way ANOVA. Probes differentially expressed between the two classes were identified based on ANOVA p-value < 0.001. Gene set enrichment analysis Groups of genes identified by 1-way ANOVA were com- pared to a collection of annotated gene sets to identify the functional classes that were significantly over-repre- sented. The enrichment p-values were computed according to the Fisher’s exact test. The gene sets were obtained from public (Gene Ontology [18], KEGG [19], Interpro [20], Panther [21], oPOSSUM [22] and com- mercial sources (GeneGo (GeneGo Inc., St Joseph, MI, USA), Ingenuity (Ingenuity Systems Inc, Mountain View, CA, USA), TRANSFAC [23]. RT-PCR Microarray findings were confirmed by real-time reverse-transcription PCR (RT-PCR) using the average value of mRNA from dox untreated SW780-shPSCA samples as calibrator. Analysis of mRNA expression of selected IFNa genes was performed using Applied Bio- systems expression kit following manufacturers’ instruc- tions. Expression values were computed using the comparative CT method (ΔΔ CT) with GAPDH gene expression value serving as normaliser. Results Establishment of cell clones with inducible shRNA against PSCA Published studies on PSCA have reported limited expression in cell lines engineered, for the most part, to express this protein ectopically [7,10]. Searching in a microarray cell atlas comprising 20 cell lines of different origins including prostate, pancreas and bladder revealed a low frequency of PSCA expression. The few cell lines with a detectable level of PSCA mRNA scored negative when expression was analyzed at protein level by FACS analysis (data not shown), suggesting that display of PSCA on the cell surface is uncommon. We therefore utilized a bladder cell line, SW780, which was shown to express PSCA on the cell surface [8]. The cell line was engineered to express different shRNA sequences against PSCA (Table 1) or a control shRNA against luci- ferase. To achieve a pharmacological controlled expres- sion a dox inducible promoter was utilized in the context of a lentivirus system [14]. One out of six shRNA against PSCA showed a clear down regulation of target gene expression and was therefore utilized to gen- erate SW780-shPSCA, which expresses also the tet regu- lator. Dox treatment resulted in 75% reduction of PSCA Page 3 of 9 Table 1 shRNA sequences against PSCA shRNA 1 2 3 4 5 6 Sequence CATCCTAACGCAAGTCTGATT GCAAGTCTGACCATGTATGTT GGCAGATCAGCTCTAGTGATT CAAGTCTGACCATGTATGTTT GCAAGAAGAACATCACGTGTT GTGACACCGACTTGTGCAATT Coding Region No No No No Yes Yes N 497-515 506-524 585-603 507-525 256-274 277-295 expression as measured by FACS analysis. Since PSCA is a membrane protein a further step was undertaken to improve regulation of gene expression by sorting out cells with low PSCA expression in the presence of dox. This process led to the isolation of cells with a larger interval of regulation mediated by dox treatment. To control that the expression of shRNA was induced upon dox treatment we took advantage of GFP gene, which is cloned upstream of the shRNA and it was previously utilized as a marker of shRNA expression [14]. FACS analysis showed a quantitative GFP expression only in dox treated SW780-shPSCA cells and a strong reduction of PSCA expression (Figure 1A). These data suggest that expression of shRNA specific for PSCA can be induced and that it leads to almost 90% downregulation of the target gene. Biological effects of PSCA down-regulation in SW780 cells To characterize the impact of PSCA expression on cell growth, a viability assay was carried out. A statistical sig- nificant reduction in cell viability was observed in dox treated cells whereas no difference was observed in con- trol transfected cells (Figure 1B). To verify the impact of PSCA expression on SW780- shPSCA tumor growth dox treatment was either started in vitro one week in advance or at the time of cell implantation. A statistical significant reduction in s.c. tumor growth (p < 0.05) was observed in the cohort of animals treated with dox with respect to controls including also SW780-shCont treated with dox (Figure 2A). Interestingly, tumors generated with cells pretreated in vitro with dox were smaller than tumors treated with dox only in vivo, although this difference was not statistically significant. In contrast control shRNA did not show any statistical reduction of tumor growth. To confirm that shRNA against PSCA was induced in vivo tumors were sacrificed at day 28 and expression of shRNA was evaluated by looking at GFP expression. Only dox-treated mice showed GFP expres- sion in SW780-shPSCA tumors (Figure 2B). In line with the shRNA expression, PSCA was down-regulated as shown by IHC. All together these experiments indicate that PSCA plays a critical role in the SW780-shPSCA