Selective Inhibition of TGF-B Responsive Genes by Smad-Interacting Peptide Aptamers from Foxh1, Lef1 and CBP

Selective inhibition of TGF-b responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP

Qiqi Cui1, Sang Kyun Lim1, Bryan Zhao1 and Francis Michael Hoffmann*,1,2

1McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA; 2Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA

Transforming growth factor beta (TGF-b)stimulation several pathological conditions including cancer and results in the assembly of Smad-containing protein com- fibrosis (Derynck et al., 2001; Wakefield and Roberts, plexes that mediate activation or repression of TGF-b 2002). TGF-b alters cellular gene expression and cell responsive genes. To determine if disruption of specific behavior by binding and activating the Type II and Smad protein–protein interactions would selectively Type I serine kinase receptors on the cell membrane. inhibit responses to TGF-b or generally interfere with Activated Type I receptor phosphorylates Smad2 and Smad-dependent signaling, we developed three Smad- Smad3, which form heterodimeric or heterotrimeric binding peptide aptamers by introducing Smad interaction complexes with Smad4 that accumulate in the cell motifs from Smad-binding proteins CBP, FoxH1 and nucleus (ten Dijke and Hill, 2004). The Smad2–Smad4 Lef1 into the scaffold protein E. coli thioredoxin A (Trx). and Smad3–Smad4 protein complexes bind over 20 All three classes of aptamers bound to Smads by GST different nuclear proteins including DNA-binding proteins, pulldown assays and co-immunoprecipitation from mam- transcription activators and transcription repressors malian cells. Expression of the aptamers in HepG2 cells such as AML (Pardali et al., 2000a), ATF-2 (Sano did not generally inhibit Smad-dependent signaling as et al., 1999), CBP (Pouponnot et al., 1998); Evi- evaluated using seven TGF-b responsive luciferase reporter 1(Kurokawa et al., 1998), FoxH1 (Chen et al., 1996), genes. The Trx-xFoxH1b aptamer inhibited TGF- HIF-1a (Sanchez-Elsner et al., 2001), Jun (Liberati et al., b-induced expression from a reporter dependent on 1999), Lef1 (Labbe et al., 2000), Mixer/Milk (Germain the Smad–FoxH1 interaction, A3-lux, by 50%. Trx- et al., 2000), SIP1 (Verschueren et al., 1999), Ski/Sno xFoxH1b also partially inhibited two reporters not (Akiyoshi et al., 1999; Luo et al., 1999; Sun et al., 1999) dependent on a Smad–FoxH1 interaction, 3TP-lux and SP1(Feng et al., 2000; Pardali et al., 2000b), TFE3 (Hua Twntop, and endogenous PAI-1 expression. Trx-Lef1 et al., 1998), TGIF (Wotton et al., 1999) and Vitamin D aptamer only inhibited expression of the Smad–Lef1 receptor (Yanagisawa et al., 1999) responsive reporter gene TwnTop. The Trx-CBPaptamer Association of the Smad2–Smad4 or Smad3–Smad4 had no significant effect on reporter gene expression. The protein complexes with different protein partners in results suggest that Smad-binding peptide aptamers can be different cell types is believed to be largely responsible developed to selectively inhibit TGF-b-induced gene for the cell-type-specific effects of TGF-b (ten Dijke and expression. Hill, 2004). Analyses of TGF-b responsive promoters Oncogene (2005) 24, 3864–3874. doi:10.1038/sj.onc.1208556 also suggest that different Smad-interacting proteins are Published online 7 March 2005 important at different promoters within one cell type and are therefore a key determinant of TGF-b signaling. Keywords: TGF-beta; peptide aptamer; Smad; FoxH1; For example, during Xenopus early development, Lef1 activated Smad complexes interact with Mixer and Milk at the goosecoid promoter and with xFoxH1a and xFoxH1b at the Mix.2 promoter (Howell et al., 2002). The activation of the Xenopus Mix.2 gene in response to Introduction TGF-b or activin requires that a phosphorylated Smad2–Smad4 complex binds to the forkhead/winged- Transforming growth factor beta (TGF-b) regulates a helix family transcription factor xFoxH1 at the activin variety of processes in mammalian cells, including response element (ARE) (Chen et al., 1997). The ARE proliferation, apoptosis, cell migration and extracellular includes sequences recognized by the DNA–binding matrix production (Massague and Wotton, 2000; motifs of both xFoxH1 and Smad4, but there is also an Miyazono et al., 2000; Ten Dijke et al., 2002). Aberrant essential direct protein–protein interaction between increases in TGF-b signaling have been implicated in Smad2 and xFoxH1 mediated through the a helix2 region in the Smad MH2 domain (Chen et al., 1998) and *Correspondence: FM Hoffmann; E-mail: [email protected] a 24 amino-acid Smad interaction motif (SIM) con- Received 6 September 2004; revised 5 January 2005; accepted 16 January served in both the FoxH1 family of proteins and the 2005; published online 7 March 2005 Xenopus Mixer/Milk homeodomain-containing proteins Inhibition by Smad-binding peptide aptamers Q Cui et al 3865 (Randall et al., 2002). A second Smad-interacting motif, induced gene expression. The xFoxHlb-derived aptamer FM, was recently identified in FoxH1 proteins, which reduces TGF-b-induced expression from the FoxHl- binds to the phosphorylated Smad2–Smad4 complex dependent reporter gene A3-lux by 50% and provides (Randall et al., 2004). weaker but significant inhibition from the 3TP-lux and Biochemical assays have shown the physical interac- Twntop reporter genes and endogenous PAI-1 gene tion between Smad and another DNA-binding tran- expression. The Lef1-derived aptamer only inhibits scription factor, Lef1, a downstream transcription factor expression from the Lef1/TCF-dependent reporter in the Wnt signaling cascade (Labbe et al., 2000; Nishita TwnTop. Although the sites on the Smad complex used et al., 2000; Theil et al., 2002; Hu et al., 2003; Hussein to bind to the xFoxHlb, CBP or Lef1 aptamers were et al., 2003). The cooperation between TGF-b and Wnt expected to interact with other transcription factors, the signaling pathways has been reported from studies of aptamers had no significant effect on several other TGF-b Drosophila, Xenopus and mammalian development. For reporter genes. The results suggest that peptide aptamers example, in Xenopus, Xtwn gene expression is synergis- may be useful for disrupting subsets of TGF-b responses. tically activated by activin and Wnt (Crease et al., 1998). The Xtwn promoter contains Smad- and Lef1-binding sites and mutation of either site impairs both the Lef1- dependent activation and the Smad3–Smad4-dependent Results activation in reporter gene assays (Labbe et al., 2000). The Smad3–Smad4–Lef1 complex is mediated by two Generation of Smad-binding peptide aptamers regions in the HMG (high-mobility group) domain of Constrained peptides from CBP, FoxH1 and Lef1 were Lef1, the helix2 and the basic region, binding to the made by inserting Smad-interaction motifs at the active MH2 domain and MH1 domain of Smad3, respectively site of Escherichia coli thioredoxin A (Trx) (Figure 1). (Labbe et al., 2000). The resulting aptamers were tested for Smad binding In addition to binding transcription factors such as in GST-pulldown or co-immunoprecipitation assays FoxH1 and Lef1, Smad proteins also interact with (Figure 2). transcriptional coactivators and repressors. CREB- binding protein (CBP) and the structurally similar p300 are transcriptional coactivators that interact with dozens of proteins to potentiate transcription through alteration of chromatin structure (Goodman and Smolik, 2000). Overexpression of CBP or p300 increases TGF-b-induced expression of the 3TP-lux or the PAI-1 reporter genes (Lagna et al., 1996; Feng et al., 1998), whereas inhibition of CBP function by expression of the adenoviral E1A protein inhibited TGF-b-induced expression from the A3, PAI-1 and 3TP reporter genes (Feng et al., 1998; Nishihara et al., 1998; Pouponnot et al., 1998; Topper et al., 1998). Protein interactions between Smads and CBP/p300 are mediated through the Smad2 or Smad3 MH2 domains and CBP amino acids 1891–2175 (Feng et al., 1998; Janknecht et al., 1998; Topper et al., 1998; de Caestecker et al., 2000). Ligands that inhibit the functional binding of Smads to specific cellular transcription factors and regulators would be useful reagents to determine the roles of specific binding sites on the Smad complex in generating the diverse gene expression responses to TGF-b. We are developing a series of peptide aptamers that bind to Smads for this purpose. Peptide aptamers, ‘proteins that contain a conformationally constrained peptide region of variable sequence displayed from a scaffold’ (Geyer et al., 1999), on the thioredoxin scaffold have been used to disrupt cyclin-dependent kinase 2 (Colas et al., 1996), E2F-DP1 (Fabbrizio et al., 1999), p53-mdm2 (Bottger et al., 1997), p300-HIF-1a (Kung et al., 2000), EGF receptor–Stat3 interaction (Buerger et al., 2003), and Stat3–DNA binding or Stat3 dimerization (Nagel- Wolfrum et al., 2004). We have developed three Smad- binding peptide aptamers containing Smad-interacting Figure 1 A schematic representation of Smad-binding motifs motifs from xFoxHlb, CBP and Lef1. The aptamers all from (a) FoxH1, (b) CBP and (c) Lef1 introduced into Trx to bind to Smads but differ in their ability to inhibit TGF-b- produce aptamers

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3866 A 104 amino-acid portion of CBP (amino acids 1893– Smad2. Mutation of the central PPNK motif, conserved 1996) in the Trx scaffold (Trx-CBPA1), expressed as a in all FoxH1 SIMs (Randall et al., 2002), to PPAK (Trx- GST-fusion protein, bound to purified Smad3 protein xFoxH1b (M)) greatly reduced binding of the aptamer to (Figure 2a). A series of smaller Trx-CBP aptamers Smad2 in the co-immunoprecipitation assay (Figure 2b). identified a 30 amino-acid region that was sufficient to A series of Lef1 fragments were expressed in the Trx bind to Smad3 (Figures 1b and 2a). This region includes scaffold and assayed for Smad3 binding using co- a 25 amino-acid portion of CBP, predicted to form an immunoprecipitation (Figure 2c). Expression of the amphipathic a helix, which was recently reported to be entire 92 amino-acid HMG domain (Trx-Lef1A) in sufficient for binding to Smad as a GST fusion protein Trx yielded detectable binding, as did a smaller 72 (Wu et al., 2002). No Smad3 protein was detected in the amino-acid region (Trx-Lef1B) that did not contain the pulldowns with GST-Trx, GST-TrxCBP E4 or GST- basic region between 370 and 383 previously implicated TrxCBP E5. The 49 amino-acid CBP aptamer, Trx-CBP in Smad binding (Labbe et al., 2000). Trx-Lef1D, C3, provided reproducibly stronger binding to Smad3 in containing a 30 amino-acid region of Lef1 expressed as the GST pulldown assay than other CBP aptamers and a tandem duplication, yielded the strongest interaction. is used in all other experiments reported here. A single insertion of the 30 amino-acid motif in the Trx FoxH1 Trx aptamers were generated using 24 amino- scaffold gave no detectable binding to Smad3 in the co- acid sequences from the human FoxH1 SIM and the immunoprecipitation assay (data not shown). This Xenopus xFoxH1b SIM (Howell et al., 2002; Randall et al., region of the Lef1 HMG in Trx-Lef1D overlaps one of 2002). Trx-hFoxH1 and Trx-xFoxH1b aptamers were the motifs (amino acids 324–334) identified as necessary both detected in Smad2 immunoprecipitates although for Smad binding (Labbe et al., 2000). The amount of more Trx-xFoxH1b was reproducibly associated with this HA-tagged Trx-Lef1D protein recovered in a Smad3 immunoprecipitate was significantly greater than the amount of HA-tagged Lef1 recovered in the Smad3 immunoprecipitate, suggesting the Trx-Lef1D binds to Smad better than full-length Lef1 binds to Smad.

Similar Smad3 binding to three aptamers by GST-pulldown and co-immunoprecipitation To compare the relative abilities of the three different aptamers to bind to Smad3, Trx-xFoxH1b, Trx-CBP C3

Figure 2 Generation of peptide aptamers from CBP, FoxH1 and Lef1 that bind to Smad. (a) Fragments of CBP protein were expressed as GST-Trx aptamer proteins and tested for their ability to bind to puriﬁed Smad3 protein. Both the GST-Trx aptamer and Smad3 proteins were eluted from the washed glutathione beads and are indicated on the ﬁgure. The other Trx-CBP aptamers all bound to Smad3, but the strongest Smad3 band was always in the GST- TrxCBP C3 pulldown. The molecular basis of the multiple Smad3 bands is not known although variation in their intensity in different preparations is consistent with partial degradation. Variable amounts of GST-Trx degradation products are also present at the bottom of the gel. (b) Trx-hFoxH1, Trx-xFoxH1b, Trx- hFoxH1 (M) aptamers fused to an N-terminal HA-tag and nuclear localization sequence (NLS) were expressed in 293 cells and tested for their ability to co-immunoprecipitate with Flag-tagged Smad2. Association of aptamer and Smad 2 was detected by probing anti- Flag immunoprecipitates for the presence of HA-tagged aptamer on Western blots (upper panel). Cell lysates were analysed for aptamer and Smad2 expression by Western blot analysis of total cell lysates (lower two panels). HA-tagged aptamers Trx-hFoxH1 and Trx-xFoxH1b were both detected in the Smad2 immunoprecipitate but the mutant form of Trx-xFoxH1b and the negative control Trx-GA aptamer were not detected. (c) Fragments of the Lef1 HMG domain or full-length Lef1 were expressed in COS-1 cells and were tested for their ability to co-immunoprecipitate with Flag-tagged Smad3. Expression of the aptamers and Smad3 were detected by Western blot analysis of total cell lysates (lower two panels). Full-length Lef1 and the two aptamers containing the helix regions of the HMG domain were detected in the Smad3 immunoprecipitate, but neither the single HMG helix aptamer, Trx-Lef1C, nor the control Trx-GA aptamer was detected (left side upper panel). Higher levels of Trx-Lef1D aptamer were detected in the Smad3 immunoprecipitate compared to full length Lef1 (right side upper panel)

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3867 and Trx-Lef1D were compared side-by-side in GST- Lef1 (Janknecht et al., 1998; Germain et al., 2000; Labbe pulldown (Figure 3a) and co-immunoprecipitation et al., 2000). In addition to the previously reported assays (Figure 3b). The GST-Trx-xFoxH1b, GST-Trx- interactions, we found that Trx-Lef1D was co-immuno- CBP C3 and GST-Trx-Lef1D aptamers all bound precipitated with Smad7 (Figure 4c), as was full-length, Smad3 protein, whereas a similar amount of GST-Trx- epitope-tagged Lef1 (data not shown). The functional control aptamer did not bind to Smad3 (Figure 3a), significance of this interaction is not known. Expression indicating specific and direct binding between the of Trx-Lef1D did not interfere with the ability of a aptamers and Smad3 under these conditions. All three transfected Smad7 expression vector to inhibit TGF-b aptamers, but not the Trx-GA control aptamer, associa- reporter gene expression in HepG2 cells (data not ted with Smad3 when overexpressed in transfected cells shown). as detected by co-immunoprecipitation (Figure 3b). Inhibition of the A3-lux reporter gene by FoxH1 aptamers Specific Smad binding by the three aptamers The effect of the Trx-FoxH1 aptamers on Smad-FoxH1- The interaction of the aptamers with different Smad dependent regulation of gene expression was determined proteins was tested by co-immunoprecipitation. The using the A3-lux reporter gene, which has three tandem Trx-xFoxH1b and the Trx-CBP C3 aptamers bound copies of the ARE from the Xenopus Mix2 gene. Smad7, preferentially to Smad2 and Smad3 (Figure 4a, b). The which binds to the Type I receptor to block phosphor- Trx-Lef1D aptamer was associated with Smad1, 2, 3, 7 ylation of Smad2 or 3, was used as a positive control (Figure 4c). The binding specificity of the aptamers inhibitor of Smad-dependent signaling (Kavsak et al., closely parallels previously reported binding selectivity 2000). The Trx-xFoxH1b aptamer reproducibly inhib- of Smad-interaction domains from FoxH1, CBP and ited the TGF-b induction of the A3-luciferase gene by 50% (Po0.01, n ¼ 4) (Figure 5a). Consistent with its stronger Smad binding observed by co-immunoprecipi-

Figure 4 Aptamers bind to specific Smad proteins. Flag-tagged Figure 3 Smad binding to all three aptamers is direct and occurs Smad proteins were coexpressed in 293 (a, b) or COS-1 (c) cells in intact cells. (a) Similar amounts of purified GST-Trx aptamer with HA-tagged aptamers, Trx-xFoxH1b (a), Trx-CBP C3 (b)or proteins were mixed with purified Smad3 to compare the ability of Trx-Lef1D (c). The anti-Flag immunoprecipitate was analysed by the aptamers to bind Smad3. The Smad3 loading control represents Western blotting for the presence of the HA-tagged aptamer. Cell 25% of the total Smad3 added to the GST-Trx aptamer proteins. lysates were analysed by Western blotting to check for similar (b) HA-tagged Trx aptamers were coexpressed in COS-1 cells with expression levels of Smads and aptamers. (a, b) Trx-xFoxH1b Flag-tagged Smad3. Expression of the aptamers and Smad3 was aptamer and Trx-CBP C3 aptamer were immunoprecipitated detected by Western blot of total cell lysates (lower two panels). specifically by Smad2 and Smad3. (c) Trx-Lef1D aptamer was Anti-Flag immunoprecipitates were separated by SDS–PAGE and immunoprecipitated most effectively with Smad 2 and Smad 7 and analysed by Western blotting with anti-HA antibody at lower levels with Smad1 and 3

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3868 cells were performed with Trx-Lef1D and Trx-CBP C3 (Figure 5b). Neither aptamer produced a signiﬁcant reduction in TGF-b-induced A3-lux expression.

Inhibition of the Twntop reporter gene by Lef1 aptamers The Trx-Lef1D aptamer was tested for its ability to interfere with the interaction between TGF-b-activated Smad protein and the Lef1/TCF proteins at the Twntop reporter gene, which contains two Smad-binding elements and three Lef1/TCF consensus-binding elements (Labbe et al., 2000). Trx-Lef1D provided a stronger reduction in Twntop-lux response to TGF-b than Trx- Lef1B (Po0.01, n ¼ 3) (Figure 6a), indicating that the

Figure 5 FoxH1 aptamers inhibit A3-lux expression. (a) Apta- mers based on the FoxH1 SIMs were coexpressed in HepG2 cells with hFoxH1, the A3-lux reporter gene and a CMV-b galactosidase expression vector. Statistical significance is marked with an asterisk (*). Aptamer protein expression was confirmed by Western blot analysis of the same lysates used for luciferase activity. (b) Trx- xFoxH1b, Trx-xFoxH1b (M), Trx-Lef1D and Trx-CBP C3 were tested for their effect on A3-lux stimulation by 100 pM TGF-b. Statistical significance is marked with an asterisk(*). Aptamer protein expression was confirmed by Western blot analysis of the same lysates used for luciferase activity

tation, the Trx-xFoxH1b aptamer exhibited stronger inhibition of A3-luciferase than Trx-hFoxH1 (Po0.03, n ¼ 4). Mutation of the PPNK to PPAK reduced the ability of the aptamer to inhibit A3-luciferase; however, Figure 6 Lef1 aptamer inhibits Twntop expression. (a) Aptamers the mutant aptamer did exhibit statistically significant based on the Lef1 HMG domain were coexpressed in HepG2 cells inhibition compared to the Trx-GA control (Po0.01, with the Twntop-lux reporter gene and a CMV-b galactosidase n ¼ 4). As expected, expression of Smad7 inhibited expression vector. Statistical significance is marked with an asterisk (#) for basal level and pound sign (*) for TGF-b-induced level. Smad-dependent activation of the A3 reporter gene Aptamer protein expression was confirmed by Western blot (Figure 5a). Trx-xFoxHlb also reduced the expression analysis of the same lysates used for luciferase activity. (b) Trx- by 50% from a second FoxHl-dependent reporter gene, xFoxH1b, Trx-Lef1D and Trx-CBP C3 were tested for their effect Mix.2-lux that contains the promoter from the Xenopus on Twntop stimulation by 100 pM TGF-b. Statistical significance is marked with an asterisk (#) for basal level and pound sign (*) for Mix2 gene (data not shown). To determine whether TGF-b-stimulated level. Aptamer protein expression was con- other Smad-binding aptamers would also inhibit A3-lux firmed by Western blot analysis of the same lysates used for expression, similar cotransfection experiments in HepG2 luciferase activity

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3869 aptamer with the stronger binding to Smad in co-immuno- Effect of three aptamers on other TGF-b responsive precipitation assays was a more potent inhibitor. Both reporter genes the aptamer and Smad7 inhibited the basal activity and the activity in the presence of exogenous TGF-b. This Five additional well-characterized TGF-b responsive Smad-dependent signaling even in the absence of added reporter genes were coexpressed with each of the three TGF-b might be due to low levels of TGF-b in the Smad-binding aptamers (Figure 7). Smad7 expression serum or to endogenous TGF-b produced by HepG2 was able to significantly reduce the TGF-b-induced cells in culture. The effect of the Trx-Lef1D aptamer expression of all five reporters and, as was observed on Twntop luciferase reporter expression was compared earlier on the TwnTop reporter, also inhibited the basal to the effect of the other two aptamers, Trx-xFoxH1b level of several of the reporters. However, Trx-Lef1D and Trx-CBP C3 (Figure 6b). The cotransfection and Trx-CBP C3 did not inhibit the expression of any of of Trx-CBP C3 did not reduce the expression of the five TGF-b-induced reporter genes. Trx-xFoxH1b Twntop reporter, however, Trx-xFoxH1b impaired had a small, but reproducible, effect on the 3TP- the TGF-b-induced activation of Twntop, although luciferase reporter compared to the Trx-GA control it was less potent than Trx-Lef1D or Smad7 (Po0.01, (Po0.01, n ¼ 3), but caused no significant reduction on n ¼ 3). any of the other four reporter genes.

Figure 7 Aptamers selectively inhibit TGF-b responsive reporter gene expression. Trx-xFoxH1b, Trx-Lef1D and Trx-CBP C3 were tested for their effect on TGF-b-induced reporter gene expression in HepG2 cells on SBE12-lux (a), PAI-1-lux (b), Smad7-lux (c), p15- lux (d) or 3TP-lux (e). Statistical signiﬁcance is marked with an asterisk (*). Aptamer protein expression was conﬁrmed by Western blot analysis of the same lysates used for luciferase activity

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3870 Partial inhibition of endogenous PAI-1 induction by the Trx-xFoxH1b aptamer To examine the effect of the Trx-xFoxH1b aptamer on endogenous gene expression, retroviral expression vectors were used to generate populations of NMuMg cells marked by GFP that were constitutively expressing the Trx-GA control or Trx-xFoxH1b. The 10% of the infected cells expressing the highest level of GFP were isolated by fluorescence-activated cell sorter (FACS). In addition to similar levels of GFP expression, the populations had similar levels of HA-epitope tagged Trx aptamer as determined by Western blotting using anti-HA antibody (data not shown). Quantitative RT– PCR was used to examine the levels of Smad7 or PAI-1 induced by TGF-b. There was no significant difference on the induction of Smad7 transcript, but the level of PAI-1 transcript was significantly reduced in the cells expressing Trx-xFoxH1b compared to the cells expressing Trx-GA (Po0.03, n ¼ 2) (Figure 8).

Discussion

Responses to TGF-b are mediated in part through phosphorylation and activation of Smad2 and Smad3, formation of heterodimeric or heterotrimeric Smad2/3 complexes with Smad4 (Inman and Hill, 2002) and subsequent assembly of multiprotein complexes with over 20 identified Smad-binding transcription factors, activators and repressors (Derynck and Zhang, 2003). Interfering with the binding of a Smad to one of its cofactors without altering its binding to other cofactors, might disrupt a subset of the total gene expression responses to TGF-b and, ideally, only alter one physiological response to TGF-b while retaining others. To test this hypothesis, we are developing constrained peptide motifs displayed on a small protein scaffold, peptide aptamers, that bind specific sites on the activated Smad complex. The three peptide aptamers reported here all bind to Smad proteins, in two cases with significant specificity to Smad2 and Smad3; however, the functional effects of Figure 8 Trx-xFoxH1b aptamer selectively inhibits endogenous the three aptamers on reporter gene expression are PAI-1 induction by TGF-b. Retrovirus-infected NMuMG cell populations expressing Trx-GA or Trx-xFoxH1b were isolated by different. The most interesting difference is the ability of FACS using GFP expression. The gene expression levels of PAI-1 the Trx-xFoxH1b aptamer to inhibit the A3-lux reporter (a) and Smad7 (b) induced in the two different populations and, less potently, inhibit the Twntop-lux reporter; by TGF-b were measured by quantitative RT–PCR after 6 or 24 h whereas, the Trx-Lef1D aptamer has the opposite of TGF-b treatment. Statistical significance is marked with an action, inhibiting Twntop-lux but not A3-lux. In each asterisk (*) case, the level of expression and binding affinity of the aptamer were sufficient to have a biological effect, but Smad competes with the interaction between Smad and the effect differed depending on the aptamer sequence. hFoxH1 or Lef1, leading to reduced expression of Importantly, the inhibition by Trx-xFoxH1b was con- A3-lux or Twntop, respectively. sistent with previous characterization of FoxH1 motifs: The partial inhibition of A3-lux, TwnTop-lux and xFoxH1b aptamer was more potent than the hFoxH1 endogenous PAI-1 by the aptamers is consistent with aptamer and mutation of the PPNK largely eliminated incomplete competitive inhibition with endogenous biological activity of the aptamer (Howell et al., 2002; factors. HA-tagged Trx-xFoxH1b aptamer was ex- Randall et al., 2002). Inhibition of Twntop luciferase pressed at far greater levels than the HA-tagged hFoxH1 activity by Trx-Lef1D is consistent with the requirement introduced into the HepG2 cells and required for the for a Lef1–Smad interaction for TGF-b induction of the A3-lux response (data not shown); however, Trx- Twntop reporter. We conclude that aptamer binding to xFoxH1b was still unable to inhibit more than 50% of

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3871 the TGF-b-induced A3-lux expression. hFoxH1 binds to example, the Mixer SIM, homologous to the FoxH1 the Smad–Smad complex through two motifs, the SIM SIM, was reported to compete for an unidentified Smad- and FM motifs, that bind to distinct, but overlapping binding partner on the c-jun promoter (Inman and Hill, sites on Smad2 (Randall et al., 2004). Binding through 2002). The partial inhibition of 3TP-lux, Twntop-lux these two sites may greatly increase the avidity of and endogenous PAI-1 by the Trx-xFoxH1b aptamer hFoxH1 and Smad2 binding such that the monomeric reported here demonstrates the ability of the aptamer to Trx-xFoxH1b aptamer would provide only partial alter expression from promoters that do not use FoxH1. inhibition. This is supported by earlier work in which Reduced Twntop reporter gene expression by Trx- mutation of either the SIM or FM greatly reduced the xFoxH1b might be due to partial inhibition of Smad– ability of the protein to activate the A3-lux reporter gene Lef1 complex formation or inhibition of some other and in which full-length Mixer, which contains a SIM Smad protein interaction required for optimal activation but not an FM motif, could not inhibit xFoxH1b-Smad of the Twntop promoter. Since AP1 has been implicated activation of the A3-lux reporter gene (Randall et al., in 3TP-lux and PAI-1 expression, the Smad–AP1 2004). This predicts that the generation of higher affinity interaction is a likely candidate for inhibition by the aptamers, as was accomplished for aptamer inhibition of Trx-xFoxH1b aptamer, but additional experiments will Cdk2 (Colas et al., 2000), or multivalent aptamers may be required to confirm this mechanism of action. be required to obtain better inhibition. The observation One explanation for the differential inhibition by Trx- that a tandem duplication of the peptide in aptamer xFoxH1b and Trx-Lef1D is that the Smad complex may Lef1D was required for Smad binding may be due to a have separate binding sites that are used to bind bivalent interaction between the aptamer and Smad or hFoxH1 and Lef1, and that aptamer binding to one to a unique conformation of the constrained tandem site has no effect on the function of the second site. aptamer. There is biochemical evidence for multiple binding sites The inability of Trx-CBP-C3 to inhibit TGF-b on the Smad complex, for example, isolation of ternary reporter gene expression contrasts with previous reports complexes comprising Smad, TGIF and FoxH1a that implicate CBP/p300 in maximal TGF-b induction (Wotton et al., 1999) or Smad, p300 and PIAS3 (Long of several reporter genes; however, all of these studies et al., 2004). Structural analysis of Smad has also used much larger domains of CBP or p300. For defined multiple binding surfaces. The loop-helix region example, overexpression of either CBP 1892–2441 or and the helix-bundle region participate in Smad–Smad CBP 1891–2175 fragments inhibited TGF-b induction of interactions to form Smad trimers or dimmers and are 3TP-lux by 50% (Janknecht et al., 1998; DiChiara et al., therefore not available to interact with other proteins 2000) and expression of the similar region of p300, binding to the trimer or dimmer. However, these 1860–2206, inhibited 3TP-lux by 50–65%, whereas a surfaces are available in Smad monomers and are smaller 1860–2023 fragment yielded weaker (B30%) involved in binding to SARA and Ski (Wu et al., inhibition (Nishihara et al., 1999). Smaller motifs of 2000, 2002; Qin et al., 2002). Mutational analysis p300, 1884–1975, did bind to Smad in yeast two-hybrid indicates that the FoxH1 SIM binds to the same portion analysis but were not sufficient for co-immunoprecipita- of the hydrophobic shallow groove used to bind the tion of Smad (Ishisaki et al., 1999). In contrast, the Trx- rigid coil motif in SARA, a portion of the hydrophobic CBP C3 aptamer was effective in co-immunoprecipita- groove that is not involved in the Smad–Smad-binding tion assays. The larger fragments of CBP or p300 used interface (Randall et al., 2004). The structural basis for in the previous studies would occupy larger surface Smad binding to CBP and Lef1 is not currently areas on Smad that could contribute to higher binding understood, although binding of Flag-CBP to HA- affinity and more effective competition for binding to Smad3 was inhibited by coexpression of Ski protein, endogenous CBP. CBP may also interact with Smad at suggesting overlap in the binding sites of Ski and CBP other sites that are not occupied by Trx-CBP C3; for (Wu et al., 2002) example, a second region of p300, the C/H3 region, also A recent report suggests that the shallow hydrophobic was implicated in mediating Smad–p300 interactions groove on Smad2 might bind a diverse group of (Nishihara et al., 1999). unstructured protein motifs with little or no primary Trx-xFoxH1b also caused partial but significant sequence similarity (Chong et al., 2004). The hydro- inhibition of TGF-b-induced expression from the 3TP- phobic shallow groove has also been implicated in lux reporter and the endogenous PAI-1 gene even Smad-binding to the nuclear transport proteins involved though neither 3TP-lux nor PAI-1 require FoxH1 in shuttling Smad protein from the cytoplasm into the expression. Most TGF-b responsive reporter genes do nucleus (Inman et al., 2002; Xu et al., 2002). We expect not use either FoxHl or Lef1, but other Smad- that aptamers blocking a single site used by many Smad- interacting proteins are involved in optimal expression binding partners or precluding interaction with the from those reporters, for example, AP1 at 3TP-lux and nuclear transport process would have a general inhibi- c-jun-lux (Liberati et al., 1999), TFE3 at Smad7-lux and tory effect on all Smad-dependent signaling; however, PAI-1-lux (Hua et al., 1998, 2000) and SP1 at pl5-lux (Li none of the aptamers reported here have this property. et al., 1995). One or more of these proteins might TGF-b signaling has many different functions in interact with a binding site on the Smad complex that normal development and physiology but has also been would be similar to, or overlap with, sites on the Smad strongly implicated in human disease, especially in complex that bind to the FoxHl or Lefl proteins. For cancer and fibrotic diseases. The development of

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3872 therapeutic strategies that target TGF-b signaling eluted with SDS sample buffer, separated by SDS–PAGE and depends on selectively affecting the pathological actions visualized by Coomassie blue staining. of TGF-b with minimal effects on its normal functions. The identification of peptide aptamers with selective Co-immunoprecipitation and Western blotting effects on TGF-b gene expression suggests that selective Flag-Smad1, 2, 3, 4, or 7 expression vector DNA and HA-Trx disruption of Smad transcriptional complexes might be aptamer expression vector DNA were cotransfected into 80% one target that deserves further development of peptide confluent COS-1 or 293 cells using FuGene6 (Roche). As a and nonpeptide ligands. control, the Trx scaffold was expressed with a tandem repeat of Gly-Ala insert (GAGAGAGAGAG) in the RsrII site (Trx- GA). After 30 h, cells were treated by 100 pM TGF-b for 40 min. Cell lysates were centrifuged for 10 min at 15 000 g and Materials and methods the supernatants were incubated sequentially with anti-Flag antibody (Sigma) for 1.5 h and with protein G-Sepharose for Generation of peptide aptamers 1 h. The immune complex was analysed by Western blotting to The E. coli Trx protein was used as the scaffold to make detect co-precipitated aptamers with anti-HA-peroxidase- constrained peptide aptamers. Fragments of human FoxH1 conjugated antibody (Roche) using ECL (Amersham). Ali- (accession NP_003914), Xenopus FoxH1b (accession quots of the total lysate were analysed by anti-Flag and anti- CAD29460), mouse CBP (accession P45481) and mouse Lef1 HA antibody Western blotting. (accession P27782) were obtained by PCR using primers with terminal RsrII sites. The specific amino-acid sequences Reporter gene assays included in each aptamer are shown in Figure 1. Glutathione HepG2 cells were maintained in RPMI medium plus 10% FBS S-transferase (GST)-Trx fusion proteins were generated in and 1% penicillin–streptomycin. Approximately 50 000 pGEX-2T or pGEX-6P (Amersham). For mammalian expres- HepG2 cells were seeded in each well of 12-well plates. After sion of the aptamers, the Trx-aptamer fragments were 24 h, the transfection was performed using Fugene 6 (Roche) recovered from the pGEX6P-Trx-aptamer vectors by PCR and 0.5 mg DNA (including reporter vectors, normalization using flanking primers with homology to Trx and terminal vector and aptamer vectors) according to the manufacturer’s NotI sites. The forward primer fused a Kozak consensus instructions. After 6 h, the medium was replaced with fresh start codon, a nuclear localization sequence (NLS) 0.2% serum medium or fresh 0.2% serum medium containing (MGAPPKKKRKVA) and an HA-tag sequence (YPYDVP- 100 pM TGF-b. About 12 h later, the cells were lysed and DYA) in-frame to the N-terminus of Trx. The PCR products luciferase activity was analysed with the Bright-Glo Luciferase were ligated into the NotI site of pCI (Promega). In the Trx- Assay System (Promega). The same amount of pCMV-b- xFoxH1b aptamer, the N303 was mutated to alanine to make galactosidase DNA was transfected in each well and measured Trx-xFoxH1b (M) using the site-directed mutagenesis Quik- with the Galacto-Start kit (Applied Biosystem) and all change Kit (Stratagene). luciferase values were normalized to the level of b-galactosidase activity. Data in each experiment are presented as the Generation of Smad3 protein in baculovirus mean7standard deviation of triplicates from one representa- tive experiment. All experiments shown were repeated at least The Smad3 open reading frame and the cytoplasmic domain of three times. the constitutively active Alk5QD were amplified by PCR and ligated into pBlueBac 4.5 (Invitrogen). An N-terminal in-frame Retroviral infection and isolation of cell populations stably 6-His tag was introduced into the Smad3 plasmid and an N- expressing aptamers terminal myristylation sequence was introduced into the Alk5QD plasmid. Both viruses were generated in Sf9 HA-tagged Trx-xFoxH1b Aptamer and Trx-GA aptamer were (Spodoptera frugiperda) cells using BacNBlue DNA. Sf9 cells cloned into the NotI site of the pCMMV-IRES-GFP vector were coinfected with both viruses at an MOI of 5 and grown at (Ory et al., 1996; Kennedy and Sugden, 2003). The retroviruses 271C for 48 h in SF-900 II SFM medium (Invitrogen). Cells were generated by transfecting 293-HEK cells with 3 mg were harvested by centrifugation, resuspended in cold PBS, plasmid encoding Gag-Pol elements, 1 mg plasmid encoding centrifuged again 41C and stored at À801C. To isolate protein, the vesicular stomatitis virus G protein, 1 mg plasmid encoding the frozen Sf9 cell pellets were thawed on ice and resuspended NF-kB and 10 mg retrovirus plasmid encoding Trx-xFoxH1b 7 in 1 ml/1.0 Â 10 cells of chilled lysis buffer (50 mM NaPO4, or Trx-GA aptamer using Fugene 6 (Roche) according to the 300 mM NaCl, 0.5% Triton X-100, 5 mM imidazole and a manufacturer’s instruction. The next day, the medium was protease inhibitor cocktail tablet (Roche)). The cell slurry was supplemented with 50 mM HEPES. The retrovirus supernatant incubated on ice for 30 min and then centrifuged for 20 min. was harvested every 24 h for the next 2 days. Supernatant was The His-tagged Smad3 protein was purified on Talon Metal filtered through a 0.45 mm filter after harvest and stored at affinity resin (Clontech). À801C. Normal murine mammary gland (NMuMG) cells were maintained in DMEM medium plus 10% FBS and 10 mg/ml GST-pulldown insulin. NMuMG cells were seeded in complete medium GST-Trx-aptamers were purified from E. coli BL21 using supplemented with 50 mM HEPES before infection. After 24 h, glutathione Sepharose beads (Amersham Pharmacia Biotech). cells were incubated with retrovirus supernatants at 41C for Purified His-Smad3 (15 mg) was incubated in 500 ml with 15 mg about 1.5 h with gentle rocking. After the incubation period, GST-Trx aptamer bound to glutathione Sepharose beads for cells were placed back into the 37oC incubator. The infection 2 h. Unbound Smad3 was removed by washing the GST-beads was repeated each day for 3 days. FACS sorting was three times with NETN buffer (20 mM Tris, pH 8.0, 150 mM performed to obtain the top 10% GFP-positive cells using a NaCl, 5 mM EDTA, 1% Nonidet P-40 and protease inhibitor FACS Vantage SE equipped with FACSDiVa option (Becton cocktail (Roche)). Bound Smad3 and GST-Trx aptamer were Dickson Immunocytometry Systems) in the UW-Madison

Oncogene Inhibition by Smad-binding peptide aptamers Q Cui et al 3873 UWCCC Flow Cytometry Facility. The GFP signal was excited Statistical analysis by 100 mW laser beam and collected with 530/30 nm ﬁlter. Results were analysed with the multiple experiment permuta- tion test of the MStat 4.01 program (http://mcardle.oncology. Quantitative RT–PCR wisc.edu/mstat). Experimental data for the analysis were from The sorted NMuMG cells were cultured in DMEM medium multiple experiments (indicated by ‘n’ in the text) each plus 10% FBS and 10 mg/ml insulin. Each cell population was performed in duplicate or triplicate in reporter gene assay or seeded in quadruplicate wells in six-well plates. The next day, quadruplicate in quantitative RT–PCR. Po0.05 was consid- 100 pM TGF-b or 10 mM compound SB431542 (an inhibitor of ered statistically signiﬁcant. TGF-b type I receptor) (Laping et al., 2002) were added to the medium. After 6 and 24 h, total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using Acknowledgements Oligo(dT) 15 primer (Promega) and Omniscript RT enzyme We are grateful to Drs Joan Massague´ , Peter ten Dijke, Rik (Qiagen) according to the RNeasy Mini Kit (Qiagen) manual. Derynck and Liliana Attisano for Smad expression con- All qPCR reactions and data analysis were performed in the structs and luciferase reporter constructs. We thank Dr Opticon 2 thermal cycler (MJ Research) with the DyNAmo Allen Comer for establishing the Smad baculovirus expres- SYBR Green qPCR kit (MJ Research) according to the sion system, Angela Speer for technical assistance in the manufacturer’s instruction. The glyceraldehyde-3-phosphate early stages of this project and Dr Norman Drinkwater for dehydrogenase (GAPDH) was used as an internal control to advice on statistical analysis and use of the MStat 4.01 normalize gene expression. Data are presented as the program. We thank Kathy Schell, Joan Batchelder and Joey mean7standard deviation. The primer sequences for mouse Puchalski in UWCCC Flow Cytometry Facility for their PAI-1 were forward: 50 ttc agc cct tgc ttg cct c 30 and reverse: 50 assistance on FACS sorting. This work was supported by aca ctt tta ctc cga agt cgg t 30. The primer sequences for mouse R01 CA090875 to FMH, R21 DK064336 to Dr Bryan Smad7 were forward: 50 ttg cct cgg aca gct caa tt 30 and reverse: Becker and Cancer Center Support Grant P30 CA014520. 50 tgc tgc ggt tgt aaa ccc a 30. The primer sequences for mouse QC was supported by the Cremer Foundation and BZ was GAPDH were forward: 50 agg tcg gtg tga acg gat ttg 30 and supported by the pre-doctoral training grant in Cancer Reverse: 50 tgt aga cca tgt agt tga ggt ca 30. Biology T32-CA09135.

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