Oncogene (2007) 26, 7143–7152 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector G Oxford1, SC Smith1, G Hampton2 and D Theodorescu1 1Department of Molecular Physiology and Biological Physics, Health Sciences Center, University of Virginia, Charlottesville, VA, USA and 2Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA Although the monomeric GTPases RalA and RalB have Introduction been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities Within the Ras family, RalA and RalB are closely in transcriptional programs regulated by RalA compared related proteins with both overlapping and distinct to RalB. Further, the association of these transcriptional functions (Feig, 2003). Initially discovered as Ras pathways to human carcinogenesis and progression homologs, they were shown to be regulated, at least in remains unclear. Here, we studied the role of RalA and/ part, by Ras through its ability to stimulate RalGEFs or RalB in transcriptional regulation by combining short such as RalGDS and Rlf (Wolthuis et al., 1998). interfering RNA depletion of Ral withgene expression Although studies on rodent tumorigenesis suggested profiling via microarray in the human bladder cancer cell that RalGEFs, and by extension RalA and RalB, are line, UMUC-3. A large number of genes were found to be not particularly tumorigenic (White et al., 1995), recent similarly modulated in cells withRalA and RalB results indicate more important roles for RalA and RalB depletion, suggesting that RalA and RalB impinge on in human cancer. For example, RalGEFs have been overlapping transcriptional signaling pathways. However, shown to be the most important effectors downstream smaller sets of genes were modulated by depletion of RalA of Ras in the transformation of human cells (Hamad or RalB, indicating that these closely related proteins et al., 2002) and RalA is a necessary component of Ras- also regulate nonoverlapping transcriptional pathways. driven tumorigenesis (Lim et al., 2005). Conversely, Computational analysis of upstream sequences of genes RalB has been shown to be pro-migratory in human modulated by Ral depletion identified Ras-responsive bladder and prostate cancer cells (Oxford et al., 2005), element-binding protein (RREB)-1, as a putative Ral and depletion of RalB via short interfering RNA transcriptional target, which we verified experimentally. (siRNA) has been shown to trigger apoptosis in many Importantly, as a group, Ral-regulated probe sets identi- human cancer cell lines (Chien and White, 2003). Both fied here were disproportionally represented among those RalA and RalB have been shown to be pro-metastatic differentially expressed as a function of human bladder (Ward et al., 2001) and they appear to share an transformation. Taken together, these data strongly overlapping function in regulating cancer cell prolifera- suggest that Ral family members mediate both common tion (Oxford et al., 2005). and specific transcriptional programs that are associated Ral proteins mediate cellular functions via a reper- withhumancancer and identify RREB-1 as a novel toire of effectors such as RalBP1, a GTPase-activating transcriptional effector of Ral. protein for Rac1 and Cdc42 (Jullien-Flores et al., 1995), Oncogene (2007) 26, 7143–7152; doi:10.1038/sj.onc.1210521; which also plays a role in the transport of glutathione published online 14 May 2007 conjugates (Awasthi et al., 2000), Sec5 and exo84, components of the exocyst involved in regulated Keywords: GTPases; bladder neoplasms; biomarker; secretion (Moskalenko et al., 2003), and the actin metastasis regulatory protein filamin (Ohta et al., 1999). Through these, RalA and/or RalB regulate endocytosis of membrane receptors, exocytosis and delivery of polar- ized membrane proteins (Shipitsin and Feig, 2004), cytoskeletal dynamics (Lebreton et al., 2004), drug resistance (Awasthi et al., 2003), motility (Gildea et al., 2002) and both anchorage-dependent and anchorage-independent proliferation (Chien and White, 2003). Correspondence: Professor D Theodorescu. Department of Molecular In addition, RalA and/or RalB have also been shown Physiology and Biological Physics, Health Sciences Center, University to regulate transcription factors such as nuclear factor k B of Virginia, Box 422, Charlottesville, VA 22908, USA. E-mail: [email protected] (NF-kB), AP-1, FOXO4, HSF-1 and ZONAB (Camonis Received 15 September 2006; revised 25 January 2007; accepted 5 March and White, 2005). However, a fundamentally impor- 2007; published online 14 May 2007 tant question remains in the study of transcriptional RalA and RalB functional expression profiling in human bladder cancer G Oxford et al 7144 regulation by Ral GTPases, namely whether RalA and marked overlap in the set of genes regulated by RalA RalB affect different transcriptional targets and whether and RalB, with many probe sets showing significant this is carried out via the selective use of different expression changes in both RalA- and RalB siRNA- transcription factors. Here, we sought to resolve this transfected cells. Furthermore, there is a larger number issue and to understand how transcriptional regulation of probe sets, which show changes in expression only contributes to RalA and RalB function in human when cells are depleted of both RalA and RalB cancer. These contributions were measured by selective compared to each GTPase alone (547 vs 162), suggesting depletion of RalA and/or RalB by siRNA knockdown that, in many cases, unique signaling pathways may be in human bladder cancer cells and profiling the changes triggered only when both RalA and RalB activity is in gene expression following protein depletion. Com- altered. This indicates that functional interactions mon transcriptional components were identified and between several signaling pathways occur downstream mechanistically validated. of these two proteins. Table 1 shows the top 10 genes with the largest statistically significant positive (upregulated) or negative (downregulated) fold changes in response to siRNA- Results mediated depletion of RalA, RalB or both RalA and RalB. Here also, the combination of both overlapping Expression profiles in response to protein knockdown and unique contributions of RalA and RalB to indicate both similar and unique transcriptional networks transcriptional regulation is evident. Of the top 10 downstream of Ral family members downregulated genes in response to RalA depletion, 6 Because RalA and RalB both play important roles in were also significantly downregulated in response to human cancer and also regulate various transcription RalB depletion. Conversely, 8 of the 10 most down- factors through mostly unknown mechanisms, we chose regulated genes with RalB depletion were also down- to investigate the contributions of RalA and RalB to regulated with RalA depletion. For upregulated genes, 8 overall transcriptional regulation. We chose to focus on out of the top 10 for both RalA and RalB depletion the UMUC-3human bladder cancer cell line because: were also significantly upregulated in the other case. (1) it is derived from an invasive tumor (Grossman et al., Uniquely regulated, in response to RalA depletion, were 1986) and maintains high in vitro migration (Oxford solute carrier family 11, asparagine synthetase (both et al., 2005) and invasion (Wu et al., 2004) activities, (2) upregulated), eukaryotic translation initiation factor 4E it has measurably active RalA and RalB (Oxford et al., binding protein 2 and interleukin 1 receptor accessory 2005), presumably, in part, due to harboring an protein 1 (both downregulated). With RalB depletion, activating K-Ras mutation (Jebar et al., 2005) and (3) both ubiquitin-specific protease 18 and promyelocytic we had previously shown in this cell line that siRNA- leukemia ubiquitin ligase were uniquely upregulated and mediated RalB depletion results in decreased cell natural killer cell transcript 4 was uniquely down- motility and that depletion of both RalA and RalB regulated. impairs cell proliferation (Oxford et al., 2005). We Given that depletion of RalA and/or RalB can trigger performed gene expression analysis using oligonucleo- a wide range of phenotypes in cancer cells, we analysed tide arrays on UMUC-3cells that had been transfected the gene sets regulated in response to RalA and/or RalB with siRNA specific for RalA, RalB or both RalA and siRNA for gene ontology classes. The results of gene RalB. Parallel transfections were processed for either ontology analysis are shown in Table 2, with only one protein or RNA. Figure 1a shows the effect of siRNA ontogeny class identified as statistically significant transfection on Ral protein levels, with both RalA- and (Po0.001) in both the RalA- and RalB-regulated genes, RalB-specific duplexes reducing the expression of their and six somewhat overlapping ontology classes asso- respective targets B90% while having little effect on the ciated with RalA/B double knockdown. expression of the other Ral protein, consistent with previous results (Oxford et al., 2005). Also consistent was the effect of a single siRNA duplex targeting both RalA and RalB signal via different repertoires of RalA and RalB, which inhibited RalB expression transcription factors B90% and RalA expression B65%. Figure 1b indicates Recently, tools have been developed to uncover