Effect of Akt Inhibition on Scatter Factor-Regulated Gene Expression in DU-145 Human Prostate Cancer Cells

Home , CYR61, FAN1

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. aSupplementary Tables S1–S9 can be found within the Supplementary data.

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2927 SF-induced expression changes in the absence and an antibody that detects phosphor-Akt (serine-473). presence of DN-Akt, whereas 54 genes showed Although the pcDNA3 vector had no effect on the SF-induced alterations in the absence but not presence induction of phospho-Akt, expression of a constitutively of DN-Akt (Table 1). In contrast to the 6 h time point, active myristoylated mutant Akt (myr-Akt) caused at 48 h there were a large number of genes (N ¼ 114) phosphorylation of both Akt and the Akt target whose expression was altered by SF in the presence of proteins mTOR (mammalian target of rapamycin) and DN-Akt but not in its absence. For most of these genes GSK-3a/b (glycogen synthase kinase-3 a/b) (Figure 2a). (103 of 114), expression was decreased by SF. Expression of DN-Akt and myr-Akt was confirmed as There may be a low basal level of c-Akt signaling even an increase in total Akt levels. There were no differences in the absence of SF stimulation (Fan et al., 2000). in the total mTOR or GSK-3 levels, and the pcDNA3 Alternatively, expression of the DN-Akt protein vector did not cause Akt activation. Figure 2b shows (K179A) might have biological effects independent of SF-induced phosphorylation of Akt, mTOR and GSK-3 inhibition of endogenous c-Akt signaling (e.g., kinase- in untransfected cells, and Figure 2c shows the ability of independent actions of Akt). Therefore, we compared DN-Akt to block the SF-induced phosphorylation of gene expression in DN-Akt- vs pcDNA3-transfected these proteins. Again, the DN-Akt protein was cells. These comparisons revealed altered expression of well expressed and did not alter total mTOR or GSK-3. N ¼ 27 and nine genes at T ¼ 6 and 48 h, respectively To assess transfection efficiency, the cells were co- (Table 1). When the same comparison was made in transfected with control plasmid pRSV-b-gal to allow cells stimulated with SF (i.e., (DN-Akt þ SF) vs counting of transfected cells after X-gal staining. For (pcDNA3 þ SF)), there were N ¼ 8 and 17 genes whose both pcDNA3- and DN-Akt-transfected cells, transfec- expression was altered at T ¼ 6 and 48 h, respectively. tion efficiencies of about 80% were routinely obtained (Figure 2d). We used quantitative (q) RT–PCR to better quantify Confirmation of DNA microarray findings the effects of SF and DN-Akt on selected genes. We We used reverse transcription–polymerase chain reac- studied eight SF-inducible genes, six of which were tion (RT–PCR) analysis (Xu et al., 2005) to confirm induced only in the absence of DN-Akt (AMPD3, selected microarray results. Initially, we used semiquan- ECM1, EPHB2, MMP10, MX1 and WNT4) and two of titative RT–PCR analysis with quantification by densi- which were induced in the absence and presence of DN- tometry to test the effect of DN-Akt on the expression Akt (MMP1 and STC1). The data are shown as the ratio of selected SF-inducible genes from Supplementary of messenger RNA (mRNA) for the gene of interest to Tables S1–S4. Figure 1a shows RT–PCR analysis of b-actin (control gene), normalized to the control seven genes upregulated by SF at T ¼ 6 h, five of which conditions (no SF, empty pcDNA3 vector). The results were induced only in the absence of DN-Akt (MX1, are summarized as heat maps in color-coded form in TCF8, TEB4, CYR61 and SUPAR) and two of which Figure 3a and b. The values shown are means of three or were induced in the absence and presence of SF (UP and four independent experiments illustrated using the scales PLAU). The RT–PCR assays showed reasonable agree- shown on the right of the figures. The complete data ment with the microarray data. The former five genes plotted as means7s.e.m.s (corresponding to the heat showed induction by SF in the absence of DN-Akt and maps) are provided in the Supplementary data (Supple- either no induction (MX1, TCF8, TEB4 and CYR61) mentary Figures S1 and S2). The effect of SF and DN- or very slight induction (SUPAR) in the presence of Akt on each gene was tested at T ¼ 6 h (Figure 3a, top) DN-Akt. Consistent with the microarray data, UP and and 48 h (Figure 3b, top). For each gene, the mRNA PLAU were both induced in the absence and presence of expression was increased by SF at each time point DN-Akt. Although PHLDA2 expression was decreased (Po0.01–0.001, two-tailed t-tests). The magnitudes of by DN-Akt, the induction by SF was similar in the the increases were greater at 48 h than at 6 h. For presence vs absence of DN-Akt. Figure 1b shows AMPD3, ECM1, EPHB2, MX1 and WNT4, SF failed analysis of seven genes upregulated by SF at 48 h, five to induce gene expression or caused only a modest of which were induced only in the absence of DN-Akt increase in expression (o1.6-fold) in the presence of (AMPD3, ECM1, WNT4, TNIP1 and LTBP2) and two DN-Akt. For MMP10, SF induced three- to five-fold of which were induced in the absence and presence of SF increases in mRNA levels in the presence of DN-Akt, (STC1 and MMP1). Again, the RT–PCR results were but these levels were significantly less than in the absence consistent with microarray data. The former five genes of DN-Akt (Po0.001). The ability of SF to stimulate were induced by SF in the absence of DN-Akt, but expression of MMP1 and STC1 was similar in the showed no induction (AMPD3, TNIP1 and LTBP2) or presence or absence of DN-Akt. reduced induction (ECM1 and WNT4) in the presence We used qRT–PCR to analyse expression of five of DN-Akt. The other two genes (STC1 and MMP1) genes whose expression was induced by DN-Akt in the were significantly induced by SF in the absence and presence and/or absence of SF at T ¼ 6 and/or 48 h: presence of DN-Akt. MMP1 expression was inhibited BRCA1 splice variant, clone 24787 (PLXDC1), two by DN-Akt, but the induction by SF was preserved. interferon-inducible genes (IFI-6-16 and IFIT1) and The ability of DN-Akt protein (K179A, a kinase dead MEK5C (Supplementary Tables S6–S9). The results are mutant) to inhibit Akt signaling is shown in Figure 2. summarized as heat maps in Figure 3a (bottom)(T ¼ 6h) Akt activation was assessed by Western blotting using and Figure 3b (bottom)(T ¼ 48 h). The complete data

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2928 are shown in Supplementary Figures S3 and S4. Each Role of SF/Akt-regulated genes in protection gene showed Xtwo-fold induction by DN-Akt in the against ADR absence of SF at each time point (Po0.001). All except We selected four SF-induced genes for which induction BRCA1 splice variant at 48 h showed a similar induction is regulated by c-Akt for further study: AMPD3, by DN-Akt in the presence of SF. EPHB2, MX1 and WNT4, and we used RNA

a ++ - - pcDNA3 T = 6 hr -- ++ DN-Akt Log Signal Ratio bp Markers SF - ++- pcDNA3±SF DN-Akt±SF

pcDNA3 pcDNA3+SF DN-Akt DN-Akt+SF 300 750 250 200 1.1 - 300 150 MX1 150 100 (209-bp) 50 0

200 750 150 0.8 - 100 300 50 150 TCF8 0 (121-bp)

200 150 750 0.8 - 100 300 50 150 TEB4 0 (141-bp)

200 750 150 0.9 - 300 100 150 CYR61 50 (297-bp) 0

300 250 750 1.0 -

Ratio of mRNA/ -Actin (% Control) 200 300 150 150 sUPAR 100 50 (130-bp) 0

300 250 1.0 0.9 750 200 150 300 100 150 UP 50 0 (201-bp)

300 750 250 0.9 0.8 200 150 300 PLAU 100 150 50 (235-bp) 0

750 β-Actin 300 (661-bp) 150

Figure 1 Confirmation of selected microarray results by semiquantitative RT–PCR. (a) T ¼ 6 h. DU-145 cells were transiently transfected with DN-Akt or pcDNA3 vector, incubated7SF (100 ng/ml) for 6 h and harvested for semiquantitative RT–PCR assays (see Materials and methods section). The amplified cDNA products were quantified by densitometry and expressed relative to the control gene, b-actin. Abbreviations: MX1 (myxovirus (influenza) resistance 1), TCF8 (transcription factor 8), TEB4 (membrane-associated RING-CH, MARCH-VI), CYR61 (cysteine rich, angiogenic inducer, 61), SUPAR (soluble urokinase plasminogen activator receptor), UP (uridine phosphorylase) and PLAU (urokinase-type plasminogen activator). (b) T ¼ 48 h. Assays were performed as above, except that a 48 h incubation with SF was utilized rather than a 6 h incubation. Abbreviations: AMPD3 (adenosine monophosphate deaminase (isoform E)), ECM1 (extracellular matrix 1), WNT4 (wingless-type MMTV integration site family, member 4), TNIP1 (TNFAIP3 interacting protein 1), LTBP2 (latent transforming growth factor-b binding protein 2), STC1 (stanniocalcin 1) and MMP1 (matrix metalloproteinase 1).

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2929 b ++--pcDNA3 --++DN-Akt T = 48 hr bp - ++- Log Signal Ratio Markers SF pcDNA3±SF DN-Akt±SF

pcDNA3 pcDNA3+SF DN-Akt DN-Akt+SF 300 750 250 200 1.2 - 300 150 AMPD3 150 100 (171-bp) 50 0

200 750 150 1.3 - 300 100 150 50 ECM1 0 (122-bp) 300 250 750 200 150 1.1 - 300 WNT4 100 150 50 (298-bp) 0

200 150 750 0.7 - 100 300 150 TNIP1 50 0 (269-bp) Ratio of mRNA/ -Actin (% Control) 200 750 150 0.7 - 300 100 LTBP2 150 50 (286-bp) 0

400 750 300 2.2 2.7 300 200 150 STC1 100 (288-bp) 0

300 750 250 200 4.7 4.6 300 150 150 100 MMP1 50 (129-bp) 0

750 β-Actin 300 (661-bp) 150

Figure 1 (Continued ) interference to test the contribution of each gene to None of the gene-specific or control siRNAs altered protection by SF against DNA damage owing to ADR. the viability of cells treated without or with ADR. In the Cell viability was measured using 3-(4,5-dimethyl- absence of siRNA, SF increased cell viability of ADR- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) treated cells by þ 23–27% (from 47–49 to 71–75%) in assays. qRT–PCR assays confirmed that small inter- different experiments (Figure 4a–d). There were no fering (si) RNAs against each gene decreased transcript differences in cell viability owing to the control siRNAs. levels in the absence or presence of SF, but control However, three of the four gene-specific siRNAs siRNAs had little or no effect on mRNA levels. (AMPD3, MX1 and WNT4) reduced SF-mediated The qRT–PCR data are shown as heat maps in protection from þ 23–27 to þ 14–16% (Po0.02). Figure 3c (see Supplementary Figure S5 for the Although this is only a partial effect, it is reproducible. complete data). In contrast, EPHB2-siRNA had no effect on protection

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2930 150 nM of control siRNA. The combination of siRNAs reduced SF-mediated protection from þ 24 to þ 9%, suggesting that this combination is more effective than the individual siRNAs in blocking protection by SF. However, the effect was still not complete. We also tested the same combination of siRNAs for their effect on protection against ADR in another cell line (T47D human breast cancer cells). Although the degree of protection by SF was less in T47D cells ( þ 14–15%) than in DU-145 cells ( þ 24%), the combination of gene- specific siRNAs gave a substantial reduction in protection of T47D cells (from þ 14–15% down to þ 5%) (Figure 4f). Thus, the results for T47D cells were qualitatively similar to those for DU-145. We performed Western blotting to confirm the ability of each gene-specific siRNA to knock down the protein levels in DU-145 cells. Here, each gene-specific siRNA (AMPD3, EPHB2, MX1 and WNT4) significantly reduced the corresponding protein levels, whereas the control siRNAs had little or no effect on the protein levels of the specific genes, and the gene-specific siRNAs had little or no effects on the protein levels of the control gene (a-actin) (Figure 4g). We performed qRT–PCR corresponding to each assay condition tested in Figure 4a–d in order to evaluate the expression levels for AMPD3, EPHB2, MX1 and WNT4 in cells treated with different combinations of SF, ADR and siRNAs. The mRNA levels were expressed relative to b-actin and normalized to the control value (no SF, no ADR no siRNA). The gene-specific siRNAs reduced the levels of gene expression whether in the presence or absence of SF or in the presence or absence of ADR (Supplementary Figures S6a–d). Interestingly, exposure to ADR reduced the SF-induced increases of gene expression, and the gene expression levels were further reduced by addition of gene-specific siRNAs.

Role of c-Akt, Pak1 and NF-kB in SF-mediated cell protection Previously, we showed that c-Akt, Pak1 and NF-kB Figure 2 Ability of DN-Akt to block c-Akt activation and signaling are required for protection against ADR by SF downstream signaling. DU-145 cells were transiently transfected (Fan et al., 2000, 2001, 2005). c-Akt and Pak1 are also with the indicated vector or no vector (vehicle only) and then required for SF-induction of NF-kB transcriptional treated7SF (100 ng/ml) for 24 h. The cells were then harvested and activity (Fan et al., 2005). Here, we tested the combined Western blotted to detect phospho-Akt, total Akt, phospho- roles of c-Akt/Pak1 and NF-kB in the protection of mTOR, total mTOR, phospho-GSK-3a and b, total GSK-3a and b, and a-actin (loading control). (a) Ability of DN-Akt to block the DU-145 cells against ADR, by the use of wild-type Akt activity of a constitutively active myr-Akt. (b) Phosphorylation of (wt-Akt), wt-Pak1 and mutant non-phosphorylatable endogenous Akt and two downstream target proteins induced by IkB super-repressor (S32,36A) expression vectors (Fan SF. (c) Ability of DN-Akt to inhibit SF-induced phosphorylation et al., 2005). The data in Figure 5 represent the means of Akt and two target proteins. To assess transfection efﬁciency, the cells were co-transfected with DN-Akt or pcDNA3 vector and from three independent experiments, each of which with ( þ ) or without (À) control plasmid pRVS-b-gal, post- utilized six replicate wells per assay condition. SF gave a incubated for 24 h to allow gene expression, and stained with X-gal. þ 40–47% increase in cell viability of ADR-treated cells The % transfected (darkly stained) cells were measured for three (Po0.001) that was mimicked by exogenous wt-Akt independent transfections (d). (Figure 5a) and wt-Pak1 (Figure 5b) in the absence of SF. SF plus wt-Akt or (SF þ wt-Pak1) gave slightly higher survival rates than did either agent alone, but by SF (Figure 4b) (see Discussion). In Figure 4e, we mutant IkB abrogated the increases in survival owing to tested a combination of three gene-speciﬁc siRNAs wt-Akt or wt-Pak1 alone or in combination with SF. (50 nM each of AMPD3, MX1 and WNT4) compared to Thus, hyper-activation of Pak1 or Akt pathways does

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2931 not overcome the inhibition of protection by IkB, manner (reviewed by Rosen et al., 1994). Identification suggesting that NF-kB acts downstream of these of critical signaling molecules that determine which mediators. specific pathway predominates will help to elucidate the In Figure 5c, we tested the ability of SF to protect mechanisms of SF action. One such nodal protein may DU-145 cells against apoptosis, using an immunofluores- be Gab1, which diverts the SF signal toward morpho- cence assay to assess terminal deoxytransferase end genesis (Schaeper et al., 2000) and away from anti- labeling (TUNEL) as an indicator of apoptosis. These apoptosis signaling (Fan et al., 2001). assays are complementary to the MTT assays, which We confirmed that DN-Akt effectively blocked Akt measure the ability of intact mitochondria to convert a signaling by showing that the DN-Akt protein is well tetrazolium salt to formazan. The MTT assay is expressed and that it blocks phosphorylation of Akt sensitive to both apoptotic and non-apoptotic cell death, (a correlate of Akt activation). We further showed that whereas the TUNEL assay only detects cells undergoing DN-Akt blocked the ability of SF or an activated apoptosis. The upper panel shows photomicrographs of mutant Akt protein (myr-Akt) to stimulate signaling cells treated with different combinations of DN-Akt, SF downstream of Akt (phosphorylation of mTOR and and ADR and assayed for TUNEL immunoreactivity GSK3). Finally, we showed that DN-Akt reversed 24 h after exposure to ADR, and the lower panel shows the SF-mediated protection against ADR-induced the quantification of the TUNEL assays. At this time apoptosis. point, ADR-treated cultures exhibited 30–35% apopto- We confirmed a subset of the SF- and c-Akt-mediated tic cells, and (DN-Akt þ ADR)-treated cultures exhi- gene expression alterations using semiquantitative and bited a similar percentage of apoptotic cells. Cultures qRT–PCR analyses. Among four confirmed SF- and treated with (SF þ ADR) showed a reduction in the c-Akt-regulated genes tested, siRNAs for three of the percentage of apoptotic cells to basal levels (2–3%). In four genes (AMPD3, MX1 and WNT4) reduced but did contrast, cultures treated with (DN-Akt þ SF þ ADR) not abrogate the SF-mediated protection of DU-145 showed about 20% apoptotic cells, indicating that cells against ADR. Although the effects of these siRNAs DN-Akt blocked the ability of SF to protect against on cell protection were relatively small, none of them ADR-induced apoptosis. reduced survival in cells treated with ADR alone, and the reductions of survival in cells treated with SF þ ADR were reproducible. A combination of siRNAs for AMPD3, MX1 and WNT4 gave a greater effect Discussion than any individual siRNA, although the combination of siRNAs did not abrogate the SF-mediated protection. We identified genes regulated by SF in DU-145 prostate This combination of siRNAs also attenuated SF cancer cells through c-Akt-dependent vs -independent protection of T47D cells against ADR, suggesting the pathways. c-Akt is activated by growth factor signaling relevance of our findings to other cell types. None of through PI3K and, in turn, regulates the phosphoryla- these genes are known to be directly involved in tion of various downstream targets, including proapop- apoptosis or cell survival pathways. However, a role totic proteins (e.g., BAD and caspase-9), other kinases for Wnt4 in germ cell survival in the ovary has been (e.g., GSK-3, Pak1, apoptosis signal-regulating kinase 1 proposed (Yao et al., 2004), and high levels of adenosine (ASK1) and mTOR), Forkhead family transcription that might result from a deficiency of AMPD3 can cause factors (e.g., FKHR and AFX), NF-kB, a tumor toxicity and apoptosis (Barry and Lind, 2000). MX1 is suppressor (BRCA1), an E3 ubiquitin ligase (MDM2) an interferon-inducible protein that mediates resistance and steroid hormone receptors (e.g., estrogen receptor- to influenza and a member of the family of large alpha (ER-a)) (reviewed by Mitsiades et al., 2004). GTPases. A role for MX1 in cell survival has not, to our Signaling pathways downstream of c-Akt contribute to knowledge, been described. Thus, our study has cell proliferation and survival, but the gene expression identified three novel SF-regulated/c-Akt-dependent alterations that mediate these functions of c-Akt are not genes that contribute to SF protection against DNA well understood. damage. The finding that about 40% of the SF-inducible genes siRNA for a fourth SF- and c-Akt-regulated gene were not similarly induced in the presence of DN-Akt (EPHB2) had no effect on SF protection of DU-145 was not surprising. However, the finding of a large cells. EPHB2 encodes an ephrin receptor that mediates number of genes, for which SF-induced changes (mostly developmental processes (neurogenesis) and regulates decreases in expression) were observed only in the cell adhesion and migration (Cutforth and Harrison, presence of DN-Akt at T ¼ 48 h, was somewhat surpris- 2002). EPHB2 has been identified as a potential prostate ing. The significance of these gene expression changes is tumor suppressor that is genetically inactivated in DU- unclear. However, we raise the possibility that the 145 cells, in which one EPHB2 allele is deleted, whereas inhibition of c-Akt may divert the SF signal into other the other carries a truncating mutation (Huusko et al., directions that would not be observed in the presence of 2004). Thus, it is not surprising that EPHB2-siRNA had active c-Akt. The idea that SF signaling may be diverted no effect on cell survival in DU-145 cells. through some pathways at the expense of others is We also identified a set of genes regulated by DN-Akt suggested by the observations that SF mediates different (in the absence or presence of SF), including a BRCA1 biologic responses in a cell-type and context-specific splice variant, two interferon-regulated genes (IFIT1

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2932

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2933 and G1P3), a MAP kinase kinase (MEK5C) and a any case, we do note that we identified several known plexin-like domain containing protein (PLXDC1, also SF-inducible genes in our study, including uPA (uro- called tumor endothelial marker 7 (TEM7)). The kinase), its receptor (uPAR) and MMP-1 (interstitial induction of these genes by DN-Akt may be due to collagenase). inhibition of basal Akt signaling owing to other growth factors present in serum or produced by the cells. Alternatively, these gene expression changes might be Materials and methods due to sequestration of signaling proteins by the DN- Akt protein, which alters other signaling pathways. Cell lines and culture Finally, alterations in gene expression by DN-Akt may Human prostate (DU-145) and breast (T47D) cancer cells be due to introduction of foreign DNA into the cell, but were obtained from the American Type Culture Collection control conditions always included an empty vector. (Manassas, VA, USA). The cells were cultured in Dulbecco’s Considerably more is known about Akt phosphoryla- modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum, non-essential amino acids (100 mM), tion targets (e.g., BAD, GSK-3, Pak1, ASK1, mTOR, L-glutamine (5 mM), streptomycin (100 mg/ml) and penicillin Forkhead family, NF-kB and others, see above) than (100 units/ml) (purchased from BioWhittaker, Walkersville, about the downstream Akt-regulated genes. In some MD, USA), and were subcultured at weekly intervals using cases, Akt-regulated genes may be inferred by their trypsin. identification as target genes for transcription factors activated by Akt. Thus, the urokinase receptor (UPAR), Reagents a known target of NF-kB (Sliva, 2004), was upregulated Recombinant human two-chain SF was generously provided by SF in an Akt-dependent manner in our study. by Dr Ralph Schwall, Genentech Inc. (South San Francisco, Published studies on Akt-regulated gene expression have CA, USA). ADR and MTT dye were purchased from the mostly examined the effects of a constitutively active Sigma Chemical Co. (St Louis, MO, USA). Akt in different cell types and tissues. In a mouse transgenic model of prostate-restricted overexpression Expression vectors of Akt activity, increased expression of three genes that Vectors encoding wt-Akt, an active (myr) mutant Akt and a we found to be SF- and Akt-regulated (UPAR, EPHB2 DN kinase inactive (K179A) mutant Akt (DN-Akt) (in the and IFIT1) was observed in the prostates of transgenic pCIS2 vector) were provided by Dr Michael Quon (National mice relative to non-transgenic animals (Majumder Heart, Lung and Blood Institute, Bethesda, MD, USA) (Cong et al., 1997). An expression vector encoding wt-Pak1 (Adam et al., 2003). Expression of a constitutively active et al., 2000) was provided by Dr R Kumar (University of Texas (myr) Akt led to increased expression of PHLDA1 MD Anderson Cancer Center, Houston, TX, USA). The (TDAG51) (Kuhn et al., 2001), a gene that was induced pCMV4-IkB-a (S32,36A) vector encodes a non-phosphoryla- by SF at T ¼ 48 h; however, PHLDA1 expression was table mutant ‘super-repressor’ IkB-a (Brockman et al., 1995). Akt-independent in our study. A transcriptosomal analysis of the effects of chronic Akt activation in the Transient transfections heart did not reveal any overlap with our results, nor did Subconfluent proliferating cells were transfected overnight a study of Akt-induced gene expression in human with the vector of interest or the empty pcDNA3 vector umbilical vein endothelial cells (Cook et al., 2002; Kim (Invitrogen, Carlsbad, CA, USA) (15 mg of plasmid DNA per et al., 2005). Thus, cell or tissue-type specificity and the 100-mm dish) using Lipofectamine (Life Technologies, context of Akt activation (i.e., owing to a cytokine or an Gaithersburg, MD, USA) and washed several times. To activated Akt protein) may contribute to Akt-regulated measure the transfection efficiency, cells were co-transfected with plasmid pRSV-b-gal (Promega Corp., Madison, WI, gene expression. USA) to allow staining with X-gal and counting of transfected Interestingly, in this study of DU-145 cells, we found (blue-staining) cells. that SF caused an increase in the expression of thrombospondin 1 (THBS1) (Supplementary Table S2). Isolation of RNA In other cell types, SF caused a decrease in THBS1 The total cell RNA was extracted using TRIzol Reagent (Life expression (Zhang et al., 2003). Thus, the effect of SF on Technologies), according to the manufacturer’s instructions. THBS1 expression may be cell type-dependent or The RNA was treated with DNase and precipitated using dependent upon cell type-specific signaling effects. In 95% ethanol before cDNA synthesis. Isolated RNA was

Figure 3 qRT–PCR analysis of gene expression shown as heat maps. (a) SF- and Akt-regulated gene expression (T ¼ 6 h). DU-145 cells were transfected with DN-Akt or pcDNA3 vector, incubated7SF (100 ng/ml) for T ¼ 6 h and harvested for qRT–PCR assays. Expression of eight SF-inducible genes (MX1, WNT4, AMPD3, MMP1, ECM1, STC1, MMP10 and EPHB2) and five DN-Akt- inducible genes (BRCA1 splice variant, IFIT1, G1P3, MEK5C and PLXDC1) were quantified relative to control conditions (pcDNA3, no SF) and corrected for the control gene (b-actin). The values shown are means of 3–4 experiments shown in color-coded form. (b) SF- and Akt-regulated gene expression (T ¼ 48 h). Assays were performed as above, except that a 48 h incubation with SF was used rather than a 6 h incubation. (c) Knockdown of four SF- and c-Akt-regulated genes by RNA interference. siRNAs were prepared for four genes shown to be induced by SF in a c-Akt-dependent manner (WNT4, MX1, AMPD3 and EPHB2). DU-145 cells were pretreated with gene-specific or control siRNAs (50 nM) for 48 h, treated7SF (100 ng/ml) for 6 or 48 h and harvested for RNA isolation and qRT–PCR assays. The mRNA values were expressed relative to the control gene (b-actin), normalized to the control- treated cells ( þ pcDNA3, 0 SF) and plotted as a heat map.

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2934 a AMPD3 b 100 EPHB2 100

80 80

ntrol) 60

of control) 60

of co

lity (% 40 ity (% 40

Cell viabi 20 20

Cell viabil

0 0 ______SF + + + + + + SF + + + + + + ______siRNA con _ _ + _ _ + _ + _ _ + _ siRNA con + + + + AMPD3 siRNA _ _ _ + _ _ + _ + _ _ + EPHB2 siRNA _ _ _ + _ _ + _ + _ _ + ______ADR + + + + + + ADR _ _ _ _ + _ _ + + + + +

c 100 100 MX1 d WNT4

80 80

ntrol)

of co 60

of control)

ity (% 40 ity (% 40

Cell viabil

20 Cell viabil 20

0 ______0 SF + + + + + + SF _ + _ _ _ + + _ _ + + + siRNA con _ _ + _ _ + _ + _ _ + _ siRNA con ______+ + + + AMPD3 siRNA + + + + EPHB2 siRNA _ _ + _ _ + _ + ______+ ADR + + + + + + ADR _ + _ _ + + + + +

100 e 100 AMPD3+MX1+WNT4 f AMPD3,MX1,WNT4

80 80

ntrol)

60 60

of control)

of co

ity (% 40 lity (% 40

Cell viabi Cell viabil 20 20

0 0 _ + _ __++__+++ _+_ __++__+++ SF ______SF siRNA con + + + _ + siRNA con _ _ + _ _ + _ + __+ _ _ _ _ + _ _ + _ + __+ AMPD3,MX1,WNT4 AMPD3,MX1,WNT4 _ _ _ + _ _ + _ + __+ ADR ____+ _ _ + ++++ ADR ____+ _ _ + ++++

g AMPD3 (79 kDa) EPHB2 (120 kDa) MX1 (72 kDa) MNT4 (45 kDa)

α-Actin (43 kDa)

Vehicle Vehicle Vehicle Vehicle Con-siRNA Con-siRNA Con-siRNA Con-siRNA

Gene-specific siRNA Gene-specific siRNA Gene-specific siRNA Gene-specific siRNA

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2935 electrophoresed through 1.0% agarose-formaldehyde gels to each treatment condition and time point, three independent verify the quality of the RNA, and RNA concentrations were experiments were performed. The Affymetrix HG-U133A determined from absorbance measurements at 260 and 280 nm. gene chip, which contains about 22 000 probe sets for known human genes plus expressed sequences tagged, was used for these studies. This chip includes a set of maintenance Affymetrix microarrays genes to facilitate the normalization and scaling of array Microarray analyses were performed at the Microarray data. The normalization genes express consistent levels of Core Facility of the North Shore-Long Island Jewish transcripts over a diverse set of tissues. The expression Research Institute. After cell treatments and RNA extraction, value for each gene was calculated using Affymetrix cRNA synthesis, gene chip hybridizations and data analyses Microarray Suite software version 5.0. The ﬁltering criteria were performed as described earlier (Carter et al., 2002). For are described in the text.

a c Control ADR DN-Akt 100

ntrol)

of Co

ity (% 40

Cell Viabil

0 ADR(20 M) _ ++_ _ +_ +_ +_ + _ + _ + SF(100 g/ml) __ + ++______+ + + + + IkB mutant ______++++_ _ + +++ wt-Akt _ _ _ _ + +++_ _ + + _ _ + +

SF+ADR DN-Akt+ADR DN-Akt+SF+ADR b 100 50

ntrol) o 40

of C 60

ity (% 40 30

totic cells 20 Cell Viabil 20

0 of Apop

______% ADR(20 M) ++ + ++ + + + ______SF(100 g/ml) + + + + + + + + 10 IkB mutant ______+++ +++__ + + wt-Pak1 _ _ _ _ + +++_ _ + + _ _ + + 0 DN-Akt _ __+ + + ADR _ + _ +_ + + SF _ _ _ + _ + Figure 5 c-Akt and Pak1 act upstream of NF-kB in SF protection of DU-145 cells. We tested the ability of wt-c-Akt (wt-Akt) (a) and wt-Pak1 (b) to enhance the survival response to ADR and override the inhibition of SF-mediated protection against ADR owing to inhibition of NF-kB signaling. Cells in six-well dishes were pretreated7SF (100 ng/ml), transfected with 5 mg of each indicated vector, washed, post-incubated for 24 h to allow gene-expression, treated with ADR (20 mM Â 2 h) and post-incubated for 48 h. The SF-treated cells ( þ ) were incubated with SF both before and after exposure to ADR. The cells were then assayed for MTT dye reduction. Values are means7s.e.m.s of three experiments, with each assay condition tested in six replicate wells. (c)Ability of DN-Akt to block SF protection against ADR-induced apoptosis. The cells were treated as above and then assayed for apoptosis by TUNEL. The values are means7s.e.m.s of three experiments.

Figure 4 Effect of knockdown of gene expression on SF-mediated protection. (a–e) Effects of gene-specific siRNAs on protection of DU-145 cells. The effect of knockdown of four SF- and c-Akt-regulated genes, AMPD3 (a), EPHB2 (b), MX1 (c) and WNT4 (d) was tested in panels (a–d). (e) Effect of simultaneous knockdown of APMPD3, MX1 and WNT4. DU-145 cells were pretreated with gene- specific or control siRNAs (50 nM) without or with SF (100 ng/ml) for 72 h, exposed to ADR (10 mM Â 2 h at 371C), washed, and post- incubated in fresh medium containing the same concentrations of siRNAs and SF for another 48 h. In panel (e), a combination of three gene-specific siRNAs (50 nM of each type) was tested and compared with 150 nM of control siRNA. The cells were then assayed for viability based on MTT dye reduction. Cell viability was expressed relative to the 0 ADR control. The values plotted are means7s.e.m.s of three independent experiments, with each assay condition in each experiment tested in six replicate wells. (f) Effect of a combination of gene-specific siRNAs on the protection of T47D cells. The effect of knockdown of APMPD3, MX1 and WNT4 on SF-mediated protection of T47D cells against ADR was tested as described above. The values plotted are the means7s.e.m.s of six replicate wells. (g) Effects of gene-specific siRNAs on protein levels. DU-145 cells were treated with gene-specific siRNA (50 nM), control siRNA (50 nM), or vehicle only for 72 h and harvested to assess the levels of the specific proteins and a-actin (loading control).

Oncogene Effect of Akt on scatter factor-regulated gene expression JXuet al 2936 Semiquantitative RT–PCR WNT4 sense GGAUGCUCUGACAACAUCGtt, antisense RT–PCR assays were performed as described before (Fan CGAUGUUGUCAGAGCAUCCtg and EPHB2 sense GGU et al., 2001; Yuan et al., 2001). The reaction conditions were GUGCAACGUGUUUGAGtt, antisense: CUCAAACACG individually optimized for each gene product. Cycle numbers UUGCACACCtt. To knock down gene expression, cells were were adjusted so that each reaction fell into the linear range of treated with the appropriate siRNA (50 nM Â 48 h), using product amplification. b-Actin and b2-microglobulin were used siPORT Amine reagent (Ambion). In each assay, a control as control genes. PCR products were analysed by electropho- siRNA (50 nM Â 48 h) with no homology to human, mouse or resis in 1% agarose gels with 0.1 mg/ml of ethidium bromide. rat genes (provided by Ambion) was also tested. The gels were photographed under ultraviolet light. Western blotting Quantitative RT–PCR Whole-cell extracts were prepared using radioimmunoprecipi- qRT–PCR was carried out as described before (Xu et al., tation assay buffer (1 Â phosphate-buffered saline, 1% NP-40, 2005). As a control, the mRNA level of b-actin was determined 0.5% sodium desoxycholate and 0.1% sodium dodecyl sulfate in each PCR assay and used to correct for experimental (SDS)), and Western blotting was performed as described variations. The PCR primer sequences are shown in Supple- earlier (Fan et al., 1998, 2000, 2001). Equal aliquots of whole- mentary Table S10. cell protein (50 mg per lane) were electrophoresed in 4–20% SDS-polyacrylamide gradient gels and transferred to nitrocel- ADR treatment lulose membranes (Millipore, Bedford, MA, USA). The Subconfluent proliferating cells in 100-mm dishes or 96-well membranes were blotted using the primary antibodies of plates were incubated7SF (100 ng/ml for 48 h) in serum-free interest: anti-phospho-Akt (serine-473) (#9271S, Cell Signaling DMEM and then sham-treated (control) or treated with ADR Technology (Beverly, MA, USA), 1:500 dilution); anti- (10 mM for 2 h at 371C) in complete medium (DMEM plus 5% phospho-mTOR (#ab1093, Novus Biologicals (Littleton, CO, fetal calf serum). Cultures were washed three times to remove USA), 1:100); anti-total mTOR (#NB100-241, Novus, 1:500); the ADR and post-incubated in fresh, drug-free complete anti-phospho-GSK-3a/b (#9331, Cell Signaling Technology, medium at 371C for 48 h. The cells were then harvested for 1:500); anti-total GSK-3a/b (#9338, Cell Signaling Technol- Western blotting, RT–PCR or MTT assays. ogy, 1:100); anti-EPHB4 (#sc-7284, Santa Cruz Biotechnology (Santa Cruz, CA, USA), 1:500); anti-WNT4 (#sc-5214, Santa MTT assays Cruz, 1:500) and anti-a-actin (I-19, #sc-1616, Santa Cruz, This assay is based on the ability of viable mitochondria to 1:1000). A rabbit polyclonal antibody against MX1 was convert MTT (a soluble tetrazolium salt) to formazan (Alley generously provided by Dr Otto Haller (Institut fu¨ r Medizi- et al., 1988). Cells were seeded into 96-well dishes (2000 cells nische Mikrobiologie und Hygiene, Universita¨ t Freiburg, per well), incubated for 24–48 h to allow attachment and entry Freiburg, Germany) and was utilized at a 1:250 dilution into the cell cycle, pre-incubated7SF (100 ng/ml Â 48 h), (Engelhardt et al., 2004). A rabbit polyclonal anti-AMPD3 treated with ADR (2 h), post-incubated for 48 h and tested antiserum was generously provided by Dr Richard L Sabina for MTT dye conversion. Cell viability was calculated as the (Medical College of Wisconsin, Milwaukee, WI, USA) and amount of MTT dye conversion relative to sham-treated was used at a 1:150 dilution (Mahnke-Zizelman and Sabina, control cells. 2000). Because of difficulty in detecting DN-Akt protein, a TUNEL assays combination of three primary antibodies was used for Western Cells cultured on slides were subjected to the desired blotting of total Akt: anti-Akt (catalog # P8103S, New treatments and fixed in 4% paraformaldehyde (pH 7.4) for England Biolabs (Beverly, MA, USA), 1:200) plus anti-Akt 10 min, and the slides were processed using the ApopTag (#MAB17751, R&D Systems Inc. (Minneapolis, MN, USA), Fluorescein In Situ Apoptosis Detection Kit (Chemicon 1:200) plus anti-Akt (clone 302407, R&D Systems, 1:200). The International Inc., Temecula, CA, USA), according to the membranes were blotted with the appropriate secondary manufacturer’s instructions. This assay detects fragmented antibodies (Santa Cruz, 1:1000), and protein bands were apoptotic DNA by an indirect TUNEL method, using an anti- visualized via the enhanced chemiluminescence detection digoxigenin antibody that is conjugated to a fluorescein system (Amersham Pharmacia Biotech, Uppsala, Sweden), reporter molecule. The apoptotic cells were analysed by with colored markers (Bio-Rad Laboratories, Hercules, CA, fluorescence microscopy. At least 300 cells were counted, and USA) as size standards. the percent of apoptotic cells was determined. Three independent experiments were performed, and the results were Statistical analyses expressed as means7s.e.m.s. Statistical comparisons were made using the two-tailed Student’s t-test. Small interfering RNAs siRNAs for human AMPD3, EPHB2, MX1 and WNT4 were designed and synthesized by Ambion (Austin, TX, USA). The Acknowledgements sequences of these siRNAs were as follows: AMPD3 sense GGUGUUUGCUAAAGUGCUCtt, antisense GAGCACU This research was supported, in part, by United States Public UUAGCAAACACCtt; MX1 sense GGUCAGUUACCAG Health Service grants RO1-ES09169, RO1-NS43987, and GACUACtt, antisense GUAGUCCUGGUAACUGACCtt; RO1-CA82599.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene