Oncogene (2007) 26, 2925–2938 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells JXu1, M Gao2, S Fan1, Q Meng1, ID Goldberg2, R Abounader3, H Ressom1, JJ Laterra3 and EM Rosen1 1Department of Oncology, Lombardi Comprehensive Cancer Center/Georgetown University, Washington, DC, USA; 2Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New York, NY, USA and 3Department of Neurooncology, The Kennedy Krieger Institute/Johns Hopkins University School of Medicine, Baltimore, MD, USA The cytokine scatter factor (SF) (hepatocyte growth Introduction factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. Scatter factor (SF) (hepatocyte growth factor) is a The latter is relevant to understanding the role of SF in pleiotrophic cytokine that regulates cell motility, inva- promoting tumor cell survival in different contexts, for sion, proliferation, morphogenesis, angiogenesis, tumori- example, detachment from basement membrane, growth genesis and transformation in different cell types and in metastatic sites and responses to chemo- and radio- contexts. These actions are transduced through c-Met, a therapy. Previously, we showed that SF protects cells receptor tyrosine kinase (Bottaro et al., 1991). SF can against apoptosis owing to DNA damage, by a mechanism protect various cell types against apoptosis owing to involving phosphoinositol-3-kinase/c-Akt signaling. Here, detachment from the substratum (anoikis) (Frisch and we used DNA microarray assays to identify c-Akt- Francis, 1994), autoimmune responses (Futamatsu regulated genes that might contribute to cell protection. et al., 2005), intracellular infection (Leiriao et al., DU-145 human prostate cancer cells were transfected7a 2005), staurosporine (Bardelli et al., 1996), the sphingo- dominant-negative mutant Akt, treated7SF and analysed lipid ceramide (Kannan et al., 2004), hypoxia/reperfu- for gene expression using Affymetrix arrays. These sion injury (Wang et al., 2004) and various other agents studies identified SF-regulated genes for which induction (Dworkin et al., 2004; Huh et al., 2004). Although SF is was c-Akt-dependent vs -independent. Selected micro- usually cytoprotective, it can also mediate proapoptotic array findings were confirmed by semiquantitative and effects through several mechanisms (Matteucci et al., quantitative reverse transcription–polymerase chain reac- 2003). tion. We tested the contribution of four SF-inducible/ We reported that SF protects epithelial, carcinoma c-Akt-dependent genes (AMPD3, EPHB2, MX1 and and glioma cells against apoptosis owing to DNA- WNT4) to protection against adriamycin (a DNA topo- damaging agents, including ionizing radiation and isomerase IIa inhibitor) using RNA interference. Knock- chemotherapy drugs, for example, adriamycin (ADR) down of each gene except EPHB2 caused a small but (a DNA topoisomerase IIa inhibitor) and cis-platinum significant reduction in the SF cell protection. The lack of (a crosslinking agent). This protection is due to effect of EPHB2 knockdown may be due to the fact that antiapoptotic signaling, inhibition of the mitochondrial DU-145 cells contain a single-mutant EPHB2 allele. A apoptosis pathway and stimulation of DNA strand combination of three small interfering RNAs blocked break repair (Fan et al., 1998, 2000, 2001; Bowers et al., most of the protection by SF in both DU-145 and T47D 2000; Gao et al., 2001). The protection of cancer cells cells. These findings identify novel c-Akt-regulated genes, against DNA damage-induced apoptosis is a clinically some of which contribute to SF-mediated cytoprotection. relevant problem because SF accumulates and c-Met is Oncogene (2007) 26, 2925–2938. doi:10.1038/sj.onc.1210088; often upregulated in various tumor types, including published online 13 November 2006 cancers of the breast, bladder, prostate and brain (gliomas) (Jin et al., 1997; Rosen et al., 1997; Beppu Keywords: scatter factor (SF); hepatocyte growth factor et al., 2000; Knudsen and Edlund, 2004). Thus, SF may (HGF); c-Akt; dominant-negative; gene expression; increase tumor cell resistance to radiation and chemo- DU-145 therapy by blocking DNA damage-induced apoptosis and stimulating DNA repair. We have studied the mechanisms of SF-mediated cell Correspondence: Dr EM Rosen, Department of Oncology, Lombardi protection in several cell types, including MDCK canine Comprehensive Cancer Center/Georgetown University, 3970 Reser- epithelial cells, human breast and prostate cancer cells, voir Road, NW, Box 571469, Washington, DC 20057-1469, USA. E-mail: [email protected] and human and rat glioma cells. We found that SF Received 15 August 2005; revised 1 August 2006; accepted 1 August stimulates antiapoptotic signaling from the c-Met 2006; published online 13 November 2006 receptor to phosphatidylinositol-30-kinase (PI3K) and Effect of Akt on scatter factor-regulated gene expression JXuet al 2926 the cell survival-promoting serine/threonine kinase Condition 4 vs 3 (4/3): (SF þ DN-Akt)/DN-Akt c-Akt (Bowers et al., 2000; Fan et al., 2000, 2001). The SF-inducible genes with Akt inhibited multisubstrate adapter Gab1 (Grb2-associated binder-1) and the tumor suppressor PTEN (phosphatase and Genes on both lists are SF-inducible genes whose tensin homolog) act as upstream inhibitors of c-Akt to induction does not require c-Akt, whereas those on the block SF-mediated cell protection, whereas Pak1 (p21- first list but not the second are candidate genes whose associated kinase-1) acts downstream of c-Akt to induction by SF requires c-Akt. mediate cell protection (Fan et al., 2001, 2005). SF- Gene expression alterations were considered to be mediated activation of nuclear factor (NF-kB) signaling significant if: (1) the log (base 2) signal ratios were requires c-Src, c-Akt and Pak1, and NF-kB signaling is X þ 0.8 (ratio ¼ 1.74) or pÀ0.8 (ratio ¼ 0.57) in at least required for cell protection (Fan et al., 2005). two out of three independent experiments and (2) the The purpose of this study was to identify downstream corresponding P-values were significant, using the transcriptional targets of SF that are regulated through Affymetrix algorithm. The results for the 6 and 48 h c-Akt and contribute to cell protection. These studies time points are summarized in Table 1. At 6 h, 49 genes were carried out using DU-145 prostate cancer cells, a exhibited SF-induced alterations in expression based on cell type that we utilized in previous studies of c-Met the above criteria. Among these, 19 genes (40%) were no antiapoptosis signaling. longer altered in expression when DN-Akt was used to block c-Akt signaling. At 48 h, 104 genes showed SF-induced changes in expression and 44 of these Results genes (42%) were no longer changed in the presence of DN-Akt. At 48 h, we also observed a group of genes Identification of SF- and c-Akt-regulated genes in (N ¼ 114) whose expression was altered by SF only in DU-145 cells the presence of DN-Akt. Most of these genes (90%) The goal of this study was to identify SF-inducible genes were decreased in expression. This group may represent whose expression is dependent upon c-Akt signaling. genes for which c-Akt normally blocks the ability of SF The approach was to compare the gene expression to inhibit expression. profiles of cells treated with SF in the absence or The complete lists of genes altered by SF and/or presence of a dominant-negative (DN)-Akt expression DN-Akt and the magnitudes of the alterations are provi- vector. Briefly, subconfluent proliferating cells were ded in the Supplementary data (Supplementary Tables transfected with an empty vector (pcDNA3) or DN- S1–S9). Genes similarly altered by SF at T ¼ 6h in Akt and incubated7SF (100 ng/ml) for T ¼ 6or48h. the absence vs presence of DN-Akt (SF-regulated/ Four treatment conditions were tested: (1) pcDNA3 c-Akt-independent genes) are listed in Supplementary alone (control); (2) (SF þ pcDNA3); (3) DN-Akt alone Table S1, whereas the SF-regulated/c-Akt-dependent and (4) (DN-Akt þ SF). DNA microarray analyses were genes are listed in Supplementary Table S2. The performed using the Affymetrix HG-U133A gene chips. corresponding gene lists for T ¼ 48 h are shown in To identify SF-regulated genes that are dependent upon Supplementary Tables S3 and S4. Supplementary Table c-Akt, we compared the following: S2 includes some genes that were induced by SF in the absence of DN-Akt, but whose expression was Condition 2 vs 1 (2/1): (SF þ pcDNA3)/pcDNA3 decreased by SF in DN-Akt-transfected cells in one SF-inducible genes or more experiments. At 48 h, 60 genes showed similar Table 1 Summary of results from DNA microarray experimentsa Table number Comparison Gene subset Number Number of genes of genes increased decreased S1 [pcDNA3+SF]/pcDNA3, Similarly regulated in the presence and absence of DN-Akt 29 1 [DN-Akt+SF]/DN-Akt (T ¼ 6h) S2 [pcDNA3+SF]/pcDNA3, Altered in the absence but not presence of DN-Akt 18 1 [DN-Akt+SF]/DN-Akt (T ¼ 6h) S3 [pcDNA3+SF]/pcDNA3, Similarly regulated in the presence and absence of DN-Akt 40 20 [DN-Akt+SF]/DN-Akt (T ¼ 48 h) S4 [pcDNA3+SF]/pcDNA3, Altered in the absence but not presence of DN-Akt 13 31 [DN-Akt+SF]/DN-Akt (T ¼ 48 h) S5 [pcDNA3+SF]/pcDNA3, Altered in the presence but not absence of DN-Akt 11 103 [DN-Akt+SF]/DN-Akt (T ¼ 48 h) S6 DN-Akt vs pcDNA3 (T ¼ 6 h) All 27 — S7 DN-Akt vs pcDNA3 (T ¼ 48 h) All 9 — S8 [DN-Akt+SF] vs [pcDNA3+SF] (T ¼ 6 h) All 8 — S9 [DN-Akt+SF] vs [pcDNA3+SF] (T ¼ 48 h) All 12 5 Abbreviations: DN-Akt, dominant-negative mutant Akt; SF, scatter factor.