Rb/E2F4 and Smad2/3 Link Survivin to TGF-B-Induced Apoptosis and Tumor Progression

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Oncogene (2008) 27, 5326–5338 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Rb/E2F4 and Smad2/3 link survivin to TGF-b-induced apoptosis and tumor progression

J Yang1,2, K Song1, TL Krebs1, MW Jackson3 and D Danielpour1,4

1Division of General Medical Sciences—Oncology, Case School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 2Department of Biochemistry, Case School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 3Department of Pathology, Case School of Medicine, Case Western Reserve University, Cleveland, OH, USA and 4Department of Pharmacology, Case School of Medicine, Case Western Reserve University, Cleveland, OH, USA

Survivin is a prosurvival protein overexpressed in many form a TbRII/TbRI tetrameric complex in which TbRII cancers through mechanisms that remain poorly explored, activates TbRI. TbRI then activates intracellular receptor- and is implicated in control of tumor progression and Smads (R-Smads), Smads 2 and 3, promoting oligomeri- resistance to cancer chemotherapeutics. Here, we report a zation either with themselves or with Smad4 (Shi and critical role for survivin in the induction of apoptosis by Massague, 2003; ten Dijke and Hill, 2004). These Smads transforming growth factor-b (TGF-b). We show that then translocate to the nucleus, where they control TGF-b rapidly downregulates survivin expression in gene expression as transcription factors or coregulators prostate epithelial cells, through a unique mechanism of (Massague et al., 2005). transcriptional suppression involving Smads 2 and 3, Rb/ A number of studies support that TGF-b functions as E2F4, and the cell-cycle repressor elements CDE and a tumor suppressor of the prostate (Danielpour, 2005), CHR. This TGF-b response is triggered through a consistent with its ability to arrest cell growth and/or Smad2/3-dependent hypophosphorylation of Rb and the induce apoptosis in normal tissues or differentiated subsequent association of the Rb/E2F4 repressive com- tumors (Hsing et al., 1996). Our laboratory has derived plex to CDE/CHR elements in the proximal region of the three spontaneously immortalized cell lines (NRP-152, survivin promoter. Viral-mediated gene delivery experi- NRP-154, DP-153) from the preneoplastic prostate of ments, involving overexpressing or silencing survivin, the Lobund-Wistar rat (Danielpour et al., 1994), all of reveal critical roles of survivin in apoptosis induced by which are exquisitely sensitive to the cytostatic effects TGF-b alone or in cooperation with cancer therapeutic of TGF-b (Hsing et al., 1996). Retroviral transduction agents. We propose a novel TGF-b/Rb/survivin axis with of dominant-negative TbRII in the above cells ablates a putative role in the functional switch of TGF-b from the cytostatic effects of TGF-b and causes non- tumor suppressor to tumor promoter. tumorigenic variants to undergo malignant transforma- Oncogene (2008) 27, 5326–5338; doi:10.1038/onc.2008.165; tion (Tang et al., 1999; Song et al., 2003a). We published online 26 May 2008 previously reported that TGF-b downregulates Bcl-xL expression in NRP-154 cells, and that the loss of this Keywords: prostate cancer; promoter regulation; apop- survival protein may be critical to TGF-b-mediated tosis; CDE; CHR; E2F apoptosis (Chipuk et al., 2001). In the current study, we identified that survivin, a member of the mammalian inhibitor of apoptosis (IAP), is also downregulated by TGF-b. IAPs are a family of endogenous caspase Introduction inhibitors containing one or multiple baculoviral IAP repeat domains serving as the interface for caspase Transforming growth factor-b (TGF-b) is a multifunc- binding and inhibition (Salvesen and Duckett, 2002; tional cytokine regulating diverse cellular processes such Liston et al., 2003). Survivin is a unique mammalian as growth, differentiation, migration, apoptosis and IAP with respect to a role in mitotic regulation (Altieri, tumorigenesis (Roberts and Wakefield, 2003; Shi and 2003b). Survivin expression in normal cells is confined to Massague, 2003; Danielpour and Song, 2006). TGF-b the G2/M phase of the cell cycle and is indispensable for signals by first binding to the cell surface receptor TGF-b normal mitosis (Altieri, 2003a, b). Such discrete cell- receptor type II (TbRII), which then recruits TbRI to cycle-dependent expression is disrupted in tumors with unclear mechanisms, related to substantial elevation of Correspondence: Dr D Danielpour, Division of General Medical survivin expression in cancers. In prostate cancer, Sciences—Oncology, Case School of Medicine, Case Western Reserve survivin expression has been shown to positively University, Wolstein Research Building, 2103 Cornell Road, Room correlate with tumor stage or loss of TbRI and TbRII 3532, Cleveland, OH 44106, USA. expression (Kishi et al., 2004; Shariat et al., 2004). E-mail: [email protected] Received 2 January 2008; revised 24 March 2008; accepted 9 April 2008; We found that an intact TGF-b signaling pathway published online 26 May 2008 transcriptionally downregulates survivin expression A TGF-b/survivin regulatory axis J Yang et al 5327 through a mechanism that is dependent on Smads 2 and cIAP1, cIAP2, XIAP were efficiently decreased by TGF- 3, and two cell-cycle repressor elements (within the b1 in NRP-154 cells (Figure 1a). Another common survivin proximal promoter), namely a cell-cycle-depen- TGF-b superfamily member, BMP-4, which activates dent element (CDE) and a cell-cycle genes homology Smads 1, 5, 8, failed to suppress survivin expression region (CHR) (Lucibello et al., 1997). TGF-b causes (Figure 1a). Treatment of NRP-154 cells with 10 ng/ml hypophosphorylation of retinoblastoma protein (Rb) TGF-b1 for different lengths of time showed that the mainly through a Smad3-dependent mechanism, leading steady-state level of survivin was decreased by 12 h post- to the recruitment of the Rb/E2F4 repressive complex to treatment and became undetectable by 16 h (Figure 1b, the CDE/CHR elements of the survivin promoter. top), corresponding to a time before the induction of an Functional inactivation of Rb family proteins by apoptotic DNA ladder (B20 h post-TGF-b1 treatment oncoproteins selectively blocks downregulation of the in these cells (Hsing et al., 1996)) or the decrease in survivin promoter by TGF-b. Moreover, survivin G2/M phase of the cell cycle (>20 h post-treatment; silencing and overexpression experiments suggest a Supplementary Figure 1). Dose–response analysis at biological function of this TGF-b-dependent regulation, 16 h of treatment gave a significant response with as low which is disrupted during tumor progression. Our model as 0.3 ng/ml of TGF-b1 (Figure 1b, bottom). These provides new mechanistic insights behind the over- results suggest a physiological role of survivin in expression of survivin in cancer and the development of apoptosis induction by TGF-b in these cells. resistance to chemotherapeutic agents. Semi-quantitative reverse transcription (RT)–PCR revealed that TGF-b1 also downregulated survivin mRNA levels with kinetics similar to the loss of survivin Results protein (Figure 1c). To determine whether such regulation occurs at the transcriptional level, we analysed the Downregulation of survivin by TGF-b effect of TGF-b1 on the activity of an 832 bp fragment We used Affymetrix expression microarrays to screen of the survivin promoter (Suv-829) (Supplementary for TGF-b-regulated genes related to apoptosis control Figure 2) placed upstream of a luciferase reporter gene. and shown to be deregulated in prostate cancer. This We found that TGF-b repressed survivin promoter analysis identified survivin as one such factor. Western activity by approximately 80%, although it had no effect blot analysis revealed that the levels of survivin but not on the empty luciferase reporter vector (Figure 1d).

TGF-1 BMP-4 026121416243648 (h) TGF-1 0 24 48 24 48 (h) 15kD Survivin Survivin 15KD -actin 75KD cIAP1

75KD cIAP2 0 0.1 0.3 1 3 10 (ng/ml) TGF-1 XIAP Survivin 50KD 15KD 50KD P-Smad2 -actin P-Smad1,5,8 50KD P-Smad3 100 50KD Smad2 90 50KD Smad3 80 -actin 70 60 50 TGF- 1 (h) 40 (R.L.U.) 30 0 2 6 12 14 16 24 36 48 20 Luciferase Activity 10 Survivin 308bp 0 TGF-1 -+-+++ 500bp -actin EV Suv-829 Figure 1 Transforming growth factor-b (TGF-b) downregulates survivin levels through a transcriptional mechanism. (a) NRP-154 cells plated in GM3 medium were treated the next day with vehicle or 10 ng/ml of TGF-b1 or BMP-4 for the indicated time. Phosphorylated Smad (P-Smad) levels that are controls for the activation of downstream signals by these ligands were measured by western blot analyses. Note that P-Smad3 antibody recognizes both P-Smad3 and P-Smads 1, 5, 8as indicated by the arrows. ( b, c) NRP-154 cells were treated with TGF-b1 (10 ng/ml) for the indicated time or with various doses of TGF-b1 for 16 h. Samples were subjected to western blot (b) or RT–PCR (c). (d) NRP-154 cells were plated in GM2.1 medium and transfected with 0.4 mg of Suv-829 or empty luciferase vector (EV), 20 ng of CMV-driven renilla vector and 0.6 mg of pcDNA3. TGF-b1 (10 ng/ml) or vehicle was added 24 h after transfection, and cells were incubated for an additional 24 h before assay. Fireﬂy luciferase values were normalized to renilla luciferase.

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5328 These data support that TGF-b downregulates survivin reversed the TGF-b downregulation of survivin protein expression through a transcriptional mechanism. (Figure 2b) and promoter activity (Figure 2c). More- over, adenovirus-mediated expression of constitutively active ALK5 (CA-ALK5) also downregulated survivin TGF-b type I receptor kinase activity is required for expression (Figure 2d). Thus, these data support that the suppression of survivin by TGF-b TGF-b represses survivin expression through the kinase Seven type I receptors have been identiﬁed to relay activity of ALK5. signals from TGF-b superfamily members (ten Dijke and Hill, 2004). They are also called activin-like kinases (ALKs). To test whether TGF-b represses survivin R-Smads are essential for mediating TGF-b’s effect through its receptor kinase activity, we treated NRP- on survivin loss 154 cells with SB431542 (Inman et al., 2002), a kinase Smads remain the best characterized direct targets of inhibitor speciﬁc for ALK5 (used by TGF-bs) and TGF-b receptors (Massague and Wotton, 2000; Massa- ALK4,7 (used by activins). SB431542 effectively gue et al., 2005). Beside R-Smads, ALK5 likely activates blocked activation of R-Smads and growth suppres- other signaling cascades such as mitogen-activated sion/apoptosis by TGF-b1 (Figure 2a); this drug also protein kinase pathways (Derynck and Zhang, 2003).

--++TGF-1 - - + + SB431542 --++SB431542 - +- + TGF-1 (24h)

P-Smad2 50KD Survivin 15KD 50KD P-Smad3 -actin -actin

35 1.4 30 )

3 1.2 25 1.0 20 0.8 15

(R.L.U.) 0.6 10 0.4 Luciferase Activity Cell Number (X 10 5 0.2 0 0.0 SB431542 - - + + SB431542 - - + + TGF-1 --++ TGF-1 --++

1/250 1/500 1/1000

Adenovirus: -CA CA C A

Survivin 15KD

HA 50KD -actin

Figure 2 Transforming growth factor-b (TGF-b) type I receptor kinase activity is essential for the repression of survivin by TGF-b. (a) Top: NRP-154 cells were treated in GM3 medium with 0.1% dimethyl sulfoxide (DMSO) or 10 mM of SB431542 for 2 h, followed by 30 min treatment with or without 10 ng/ml of TGF-b1. Bottom: NRP-154 cells were seeded at a density of 104 cells per ml per 12-well plate in GM3 medium and treated with 0.1% DMSO or 10 mM of SB431542 24 h prior to TGF-b1 (10 ng/ml, 48h). To determine cell number, cells detached with trypsinization were enumerated using a Coulter Counter (Coulter Electronics, Hialeah, FL, USA). (b)NRP- 154 cells plated in GM3 medium were treated with 0.1% DMSO or 10 mM of SB431542 for 1 h, followed by treatment with vehicle or 10 ng/ml of TGF-b1 for 24 h, and expression of survivin was analysed by western blot. (c) NRP-154 cells transfected with 0.4 mgof Suv-829 luciferase reporter construct were treated the same way as in (b), and then cells were harvested for luciferase assay. (d) NRP-154 cells were infected in GM3 with different doses (indicated as the dilution fold above the brackets) of stock adenoviruses expressing control (C) or HA-CA-ALK5 (A) for 5 h. Whole cell lysates were collected after an additional 29 h and analysed by western blot.

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5329 To determine whether the repression of survivin by silenced. Silencing either Smad alone partially reversed the TGF-b is Smad dependent or independent, we generated downregulation of survivin by TGF-b, suggesting that doxycyclin-inducible short-hairpin RNA (shRNA) each of these R-Smads contributes to the TGF-b-driven expression lentiviruses to stably silence Smads 2 and/or loss of survivin (Figure 3c), similar to that of TGF-b- 3 in NRP-154 cells. As indicated in Figure 3a, compared induced apoptosis (Figure 3b). with the non-silencing control (sh-LacZ), sh-Smads 2 or 3 efﬁciently and speciﬁcally knocked down their TGF-b represses survivin transcription by recruiting respective proteins. Moreover, robust silencing of both CDE/CHR-binding factor(s) Smads 2 and 3 was obtained with both shRNAs used To study the region of the survivin promoter targeted by together, and Smad4 levels remained unchanged with TGF-b, we generated 50 deletion constructs of this either or both shRNAs (Figure 3a). We also examined the promoter (Figure 4a) and assessed activity of each role of Smads 2 and 3 in TGF-b-induced apoptosis. construct in NRP-154 cells treated with vehicle or TGF- Assaying for changes in total cell number (Figure 3b) and b1, as before. These truncations gave varying basal for changes in sub-G1 by Flow Cytometry (data not promoter activity, with the shortest construct (Suv-182) shown), we demonstrated that Smads 2 and 3 are involved giving the lowest activity. However, TGF-b1 suppressed in the induction of apoptosis by TGF-b, although Smad3 the activity of each construct with a similar magnitude is more important. Western blot (Figure 3c top), RT– (Figure 4a right), suggesting that the most critical TGF-b PCR (Figure 3c bottom) and promoter analyses responsive element(s) reside in Suv-182 that is the (Figure 3d) indicated that the suppression of survivin À182/À4 region of this promoter. protein, mRNA and promoter activity by TGF-b were Several cis-acting DNA elements within this region reversed when both Smads 2 and 3 were concomitantly were predicted using different software (TFSearch,

80 sh-lacZ sh-S2 sh-S3 sh-S2+3 70 50KD Smad2 60 50 50KD Smad3 40 Smad4 30 50KD % Cell Survival 20 -actin 10 0

sh-S2 sh-S3 sh-lacZ sh-S2+3

sh-lacZ sh-S2 sh-S3 sh-S2+3 -+-+ -+ -+TGF-1 (48h) 1.2 1.0 15KD 0.8 -actin 0.6

Relative Qty:1 0.01 1.2 0.1 1.25 0.2 1.1 1 (R.L.U.) 0.4

Luciferase Activity 0.2 sh-lacZ sh-S2 sh-S3 sh-S2+3 0.0 ---+-+++ TGF-1 (24h) TGF-1 -+-+ 250 bp Survivin shRNA LacZ S2+3

500 bp -actin

Relative Qty: 1 0.4 1.4 0.9 1.5 0.7 1.4 1.1 Figure 3 Smads are required for transforming growth factor-b (TGF-b)-mediated suppression of survivin. (a–c) NRP-154-tTR-sh- LacZ, sh-Smad2 (sh-S2), sh-Smad3 (sh-S3), or sh-Smads 2 and 3 (sh-S2 þ 3) cells were plated in GM2.1 medium containing 0.1 mg/ml of doxycycline to induce the expression of shRNAs. After 2 days, cells were plated in GM3 medium supplemented with doxycycline (0.1 mg/ml) and treated with 10 ng/ml of TGF-b1 or vehicle. Samples were collected before (a) or after 48h ( b) of TGF-b treatment or as indicated (c). Western blot analysis (a, c top), cell number assay (b), and RT–PCR (c bottom) were performed. Note that in (b), cell number was determined as in Figure 2a and percentage cell survival after TGF-b treatment was calculated from three experiments. The bands in (c) were quantiﬁed with Quantity One software, and expressed as the relative quantity (Qty) of survivin to b-actin. (d) NRP- 154-tTR-sh-LacZ or sh-Smad2 þ 3 cells were plated in GM2.1 medium containing doxycycline and transfected 2 days later with 0.4 mg of Suv-829 reporter plasmid. Luciferase assay was conducted after 24 h of treatment with TGF-b1 (10 ng/ml) or vehicle.

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5330 http://www.rwcp.or.jp/papia/ (Heinemeyer et al., 1998); Smad-binding elements (SBEs) (Zawel et al., 1998); a Genomatix MatInspector, http://www.genomatix.de/ AP-2 transcription factor-binding site (Hilger-Eversheim index.html). These elements include: two canonical et al., 2000; Wajapeyee et al., 2006), with a CDE

1.2 Vehicle TGF-1 -4 1.0 -829 Suv-829 0.8 -660 Suv-660 0.6

-555 Suv-555 (R.L.U.) 0.4 0.2

-370 Suv-370 Luciferase Activity 0.0 -182 Suv-182

Suv-829 Suv-660 Suv-555 Suv-370 Suv-182 %Suppression: 76 71 75 79 68

Vehicle Vehicle 2.0 TGF-1 1.4 TGF-1 1.2 1.5 1.0 0.8 1.0 0.6 (R.L.U.) (R.L.U.) 0.4 0.5

0.2 Luciferase Activity Luciferase Activity 0.0 0.0

WT WT CDE(m) CHR(m) SBE1(m) SBE2(m) AP2 only(m) %Suppression: 79 78 68 %Suppression: 70 38 56 5.7

10.0 Vehicle TGF-1 7.5

5.0 (R.L.U.) 2.5 Luciferase Activity 0.0

CDE(m) CHR(m)

CDE+CHR(m)

%Suppression: 70 38 5.7 -43

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5331 contained inside; and a contiguous CHR (Supplemen- Rb and E2F4 are recruited by TGF-b to survivin proximal tary Figure 2). Except for the SBE1 site, the rest of the promoter and are essential for TGF-b to repress this above-mentioned sites are all located within À182/À4 promoter activity region of the survivin promoter. Notably, CDE Rb family proteins (Rb, p107 and p130) are critical to and CHR are cell-cycle repressor elements present in cell-cycle control. When activated by hypophosphoryla- multiple cell-cycle-regulated genes, such as survivin, tion, Rb can bind E2F transcription factors and cyclin A and cdc25C, and have been shown to be downregulate the expression of genes involved in cell- required to maintain low expression levels of these genes cycle progression (Knudsen et al., 1999; Harbour and in G0/G1 phase of the cell cycle (Liu et al., 1997; Altieri, Dean, 2000; Jackson et al., 2005). Several earlier reports 2003a, b). suggested that the Rb/E2F4 molecular complex binds to To identify which cis-acting element(s) mediate the CDE/CHR elements within promoters of some genes, suppressive effect of TGF-b on the survivin promoter, such as cdc2, and represses their transcription (Le Cam we mutated these elements individually or in combina- et al., 1999; Taylor et al., 2001). To test whether Rb is tion (Figure 4b). Mutating the SBE2 site slightly involved in CDE/CHR-mediated repression of the diminished TGF-b suppression of survivin promoter, survivin promoter by TGF-b, we overexpressed several although mutating SBE1 had no effect (Figure 4c). As proteins known to inactivate all Rb family proteins recombinant Smad3 binds to SBE2 in vitro (Supplemen- (Jackson et al., 2005). These Rb-inactivating proteins tary Figure 3), the above data suggested that direct included D1/CDK, which is a fusion protein of cyclin Smad3 binding to SBE2 has limited effect on this TGF-b D1 and cdk2 (representing constitutively active cdk), E7 response. As CDE(m) also disrupts putative AP-2- (oncoprotein from HPV-18), E7D (mutant form of E7 binding motif, we generated a mutant that selectively lacking the binding domain for Rb), and T-ag (SV-40 affects AP-2 binding independent of CDE (called AP2 large T antigen). As shown in Figure 5a, overexpression only(m)) to examine the significance of AP2 in the of D1/CDK, E7 or T-ag resulted in higher survivin basal regulation of the survivin promoter by TGF-b.As promoter activity and completely reversed the repres- shown in Figure 4d, the basal activity and TGF-b sion of this promoter by TGF-b1. Rather interestingly, responsiveness of the AP2 only(m) construct were TGF-b1 actually enhanced survivin promoter activity in similar to the wild-type (WT) promoter, suggesting that the presence of these proteins. Importantly, E7D failed AP-2 is not involved in the repression of survivin to inhibit the repression of survivin promoter by TGF-b, promoter activity by TGF-b. Therefore, we speculate suggesting that Rb inactivation is the main mechanism that the effect observed with the CDE(m) construct by which E7 oncoprotein reverses the downregulation of mainly reflects CDE function. Both the CDE(m) and survivin promoter by TGF-b (Figure 5a). To examine CHR(m) promoter constructs had higher basal activity whether this effect of Rb inactivation occurs specially at than the WT construct. Mutating the CDE site reduced the level of the survivin promoter rather than by simply TGF-b’s inhibitory effect on this promoter construct by blocking TGF-b receptor/Smad activation, in parallel, about half, whereas CHR(m) promoter was hardly we assessed effects of the Rb-inactivating proteins on affected by TGF-b (Figure 4d). We conclude that TGF- 3TP-Luciferase (3TP-Lux, a PAI-1 promoter construct). b utilizes both CDE and CHR elements, although 3TP-Lux is a classic TGF-b-induced promoter construct mostly the latter, to repress survivin promoter. which depends on Smad-mediated transcription (Wrana That mutating either of these two elements reverses et al., 1992), thus serving as a good readout to test the TGF-b-suppression of the survivin promoter whether early TGF-b responses could be affected by suggest that TGF-b downregulates this promoter by these Rb-inactivating proteins. In the case for D1/CDK, modulating the association of some factor(s) to CDE 3TP-Lux induction by TGF-b was mostly retained and CHR. Interestingly, when both CDE and CHR (approximately 80%), indicating that D1/CDK were mutated, basal survivin promoter activity increased does not significantly affect TGF-b early signals dramatically (Figure 4e). Moreover, this caused TGF-b (Supplementary Figure 4). Although E7 and T-ag to enhance the activity of this promoter instead of repressed TGF-b-induced 3TP-Lux by about 70%, suppressing it (Figure 4e). These data suggest that the interception of TGF-b early signals by these two recruitment status of the potential TGF-b-induced proteins did not account for the total reversal of the factor(s) could result in TGF-b’s opposing effect on TGF-b response observed on the survivin promoter survivin levels. (Figure 5a; Supplementary Figure 4). Therefore, these

Figure 4 Transforming growth factor-b (TGF-b) represses survivin promoter within the region À182/À4, and the CDE/CHR elements within this region are essential for this TGF-b’s effect. (a) Left: diagram of survivin promoter 50-deletion constructs. Right: individual promoter constructs (0.4 mg) were transfected into NRP-154 cells and treated with or without TGF-b (10 ng/ml) for 25 h. (b) Schematic representation of the nucleotide changes in survivin promoter mutants, which were generated with Suv-370 reporter plasmid. SBE2 and AP-2 sites are underlined, whereas CDE and CHR sites are boxed. All mutated nucleotides are indicated in inverted display. Note that CDE(m) disrupts both AP-2 and CDE sites. Although not shown here, SBE1(m) has the same nucleotide changes as SBE2(m). (c–e) Suv-370 wild-type (WT) or mutant promoter reporter plasmid (0.4 mg) was transfected into NRP-154 cells and treated with 10 ng/ml of TGF-b1 or vehicle for 24 h. Suppression for each promoter construct was calculated as percentage suppression ¼ ((LVÀLT)/LV) Â 100% (L, average luciferase activity; V, vehicle; T, TGF-b). The luciferase activity of the ﬁrst bar was arbitrarily set as 1. Note that data in (d) and (e) are from the same experiment, and the negative value of percentage suppression in (e) reﬂects enhancement.

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5332 Vehicle 10 TGF-1 8 DNAP CDE+ 6 Input WT CDE(m) CHR(m)CHR(m) SBE 4 016016 16016016160 (h) TGF- 1 (R.L.U.) 50KD E2F4 2 Rb Luciferaase Activity 105KD 0 p107 105KD p130 LXSN pBabe LNCX2 105KD LXSN-E7 LXSN-E7 50KD Smad3 LNCX2-T-ag pBabe-D1/CDK

0 2 6 121416243648 (h) TGF-1 DNAP P-Rb (Ser 807/811) Input Suv-182 SAC oligo 105KD 105KD Rb 0172 0172 017(h) TGF-1 -actin 105KD Rb p107 105KD sh-lacZ sh-S2 sh-S3 sh-S2+3 160KD p130 -+-+ -+ - + TGF-1 (48h) 105KD 105KD P-Rb (Ser 807/811)

50KD E2F4 105KD Rb 50KD Smad3 -actin Smad4 50KD Figure 5 Transforming growth factor-b (TGF-b) recruits Rb/E2F4 to survivin promoter containing the CDE/CHR elements to mediate the suppression of this promoter. (a) Retroviral constructs expressing Rb-inactivating proteins, including D1/CDK (cyclin D1- cdk2 fusion protein), E7 (from HPV-18; E7D, a mutant form that does not bind Rb), T-ag (SV-40 large T antigen) or their vector controls were individually transfected into NRP-154 cells together with Suv-370 promoter luciferase construct. (b) One hundred and ﬁfty micrograms of nuclear extracts from NRP-154 cells treated with vehicle or 10 ng/ml of TGF-b1 were incubated with biotinylated DNA (Suv-182 or SAC oligo) conjugated to streptavidin magnetic beads. DNA-bound protein complexes were resolved by SDS–PAGE and analysed by western blot. Input of nuclear proteins (10 mg) was also shown. (c) Similar DNAP experiment as in (b), except that wild-type (WT) or mutated SAC oligos were used. Mutation site(s) are as illustrated in Figure 4b. SBE oligo was used as a control. In (b, c), p130 band is indicated by the arrow. (d) Phosphorylated and total levels of Rb after TGF-b1 treatment. (e) The samples used in Figure 3c (top) were assayed for phosphorylated Rb (P-Rb) and total Rb levels.

data suggest that Rb (and/or p107, p130) is critical for capture these proteins from the nuclear extracts (data the repression of survivin by TGF-b. not shown). Our preliminary chromatin immunopreci- As active Rb family proteins typically form transcrip- pitation data support that TGF-b enhances the interac- tional repressor complexes with E2F4 on target gene tion of p107, p130 and E2F4 to this promoter region in promoters, we tested whether TGF-b recruits these intact cells (data not shown). To test whether CDE and proteins to the survivin promoter, using a biotinylated CHR are critical for such binding, we performed a DNA pull-down assay (DNAP). The binding of nuclear DNAP experiment with WT or CDE/CHR mutant proteins to either a 187 bp survivin proximal promoter (individually or in combination) SAC oligos. Another (Suv-182, spanning À182/À4 region) or an internal oligo containing three tandem repeats of the SBE 32 bp oligo DNA (SAC oligo, spanning À50/À19 consensus motif was also used as a positive control for region) containing CDE/CHR was assessed. TGF-b- Smad binding and a negative control for the other induced binding of all Rb family proteins (Rb, p130 and binding proteins. As shown in Figure 5c, TGF-b p107) and E2F4 to both DNA fragments after 17 h of treatment consistently enhanced the binding of all three treatment (Figure 5b), demonstrating that TGF-b- Rb family proteins and E2F4 to the WT SAC oligo, but dependent recruitment of Rb, Rb-like proteins and not to the mutant oligos. Interestingly, although E2F4 to this promoter temporally coincide with down- mutations in CDE or CHR essentially abolished all regulation of survivin mRNA (Figure 1c). Interestingly, binding of p107, p130 and E2F4, each of the mutant both Smads3 and 4 bound to Suv-182, but only oligos showed enhanced interaction with Rb over the minimally to the SAC oligo, suggesting that Smads WT oligo. The molecular basis and biological signiﬁ- may also bind to sites outside the À50/À19 survivin cance of the interaction of Rb to mutant CDE and CHR promoter region to mediate TGF-b’s effect. As a remain to be deﬁned. Collectively, these data suggest negative control, streptavidin beads alone did not that the integrity of CDE/CHR is critical for the

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5333 recruitment of Rb family proteins and E2F4 to the TGF-b/survivin regulatory axis may be associated with survivin promoter by TGF-b. tumor progression. An array of normal and tumor As Rb family proteins need to be activated through prostatic epithelial cells was used to test this hypothesis. hypophosphorylation to bind to promoter DNA with Compared with the NRP-154 cell line (Figure 1b, top), E2F4, we next assessed whether TGF-b can activate which is tumorigenic, the NRP-152 non-tumorigenic rat Rb. As shown in Figure 5d, TGF-b efficiently reduced prostate epithelial cell line (Danielpour et al., 1994) Rb phosphorylation, starting as early as 6 h of manifested lower basal levels of survivin but similar treatment, at a time preceding survivin downregulation. temporal patterns of downregulation by TGF-b (Figures Silencing Smad3 largely abolished hypophosphorylation 7a and b), associated with their exquisite sensitivity to of Rb by TGF-b; Smad2 also contributes to Rb TGF-b-induced apoptosis (Hsing et al., 1996). DU-145 activation by TGF-b, as silencing it alone or in and PC-3 are highly malignant/androgen-independent combination with silencing Smad3 further reversed Rb human prostate cancer cell lines derived from brain and hypophosphorylation by TGF-b (Figure 5e), indicating bone metastases, respectively. Both of them possess high that Smad3 is the major mediator of TGF-b-dependent levels of endogenous survivin, which was not suppressed Rb activation. by TGF-b1 (Figure 7b) even up to 72 h treatment (data not shown). Conversely, TGF-b downregulated survivin levels in RWPE-1 (Figure 7c), a non-tumorigenic human Manipulating survivin levels in cells alters their sensitivity prostate epithelial cell line (Bello et al., 1997). We to apoptotic stimuli including TGF-b compared survivin levels in the context of tumor We next investigated whether survivin is involved in the progression, using LNCaP cell line and its bone regulation of apoptosis by TGF-b. Survivin was first metastatic/androgen refractory variant, C4-2B, which efficiently overexpressed in NRP-154 cells by adenoviral express more survivin than LNCaP cells (Figure 7d). To transduction, and changes in apoptotic response to test the TGF-b response, we had to ectopically express TGF-b1 were assessed by both analysis of percentage TbRII in these cells as they lack this receptor. Down- sub-G cells (by Flow Cytometry) and caspase-3 1 regulation of survivin by TGF-b1, which occurred activation (by western blot). Overexpression of survivin efficiently in LNCaP cells only by enforced expression substantially decreased the percentage of sub-G cells 1 of TbRII, was abolished in C4-2B cells also expressing following TGF-b treatment (Figure 6a), and diminished exogenous TbRII. Consistent with published results the levels of caspase-3 activated by TGF-b (Figure 6b), (Zhang et al., 2005), stimulation of LNCaP cells with demonstrating that the loss of survivin initiated by 5a-dihydrotestosterone (DHT) increased survivin levels. TGF-b is required for TGF-b to efficiently induce Interestingly, DHT treatment of these cells significantly apoptosis. We also treated the above-transduced prevented the loss of survivin by TGF-b (Figure 7d), cells with etoposide or taxol, and observed an inhibition suggesting crosstalk between TGF-b and androgen on of taxol-mediated apoptosis by survivin overexpression regulation of survivin expression. In summary, our data (Figure 6c). Separately, a dose of TGF-b (0.3 ng/ml) suggest that disregulation of TGF-b signaling in prostate below the threshold for apoptosis but sufficient for cancer may contribute to disease progression and poor survivin downregulation (Figure 1b bottom) signifi- therapeutic responses by elevating survivin levels. cantly enhanced taxol-induced caspase-3 activation, and such potentiation was lost by survivin overexpression (Figure 6d). This suggests that TGF-b may also lower Discussion the threshold for cellular responses to other apoptotic agents by reducing survivin levels. We next used an This is the first report of a functional union between shRNA-lentiviral approach to study the biological survivin and TGF-b, with survivin transcriptionally function of endogenous survivin in NRP-154 cells. downregulated by TGF-b in preneoplastic and differ- Knockdown of survivin expression enhanced the induc- entiated prostate carcinoma cells but not in advanced tion of apoptosis by either etoposide or taxol prostate carcinoma cells. Moreover, we show that such (Figure 6e). The sensitivity of survivin-silenced cells to downregulation is dependent on Smads 2 and 3, Rb apoptosis by TGF-b was not detectably altered, (and/or Rb-like proteins) and the CDE/CHR promoter consistent with TGF-b’s ability to drive efficient loss elements which bind the Rb/E2F4 repressor module of survivin. Taken together, our results suggest that the following TGF-b stimulation. This is also the first downregulation of survivin expression by TGF-b is demonstration that the ability of TGF-b to induce Rb involved in apoptotic responses of not only TGF-b but hypophosphorylation is dependent mostly on Smad3. also some chemotherapeutic agents. We propose that activation of Smads 2 and 3 promotes hypophosphorylation of Rb leading to association of Cancer relevance of survivin regulation by TGF-b Rb/E2F4 complex to the survivin promoter (Figure 7e). Considering the versatility of survivin to inhibit Our model is further supported by a recent study that apoptosis by TGF-b and other agents such as taxol showed Rb/E2F complexes bind to the human survivin (Figure 6), and earlier reports correlating survivin levels promoter, controlling the G2/M cell-cycle-specific ex- to the progression of prostate cancer (Kishi et al., 2004; pression of survivin in normal cells. Defects in this Shariat et al., 2004) or the loss of TbRI and TbRII mechanism may account for the elevated levels of (Shariat et al., 2004), we hypothesized that a defective survivin in cancers (Jiang et al., 2004).

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5334 350 CTL CTL+TGF- 40 Vehicle TGF-1 30 6.0 27.9 20 0 350 % sub-G1 10 Counts Suv Suv+TGF-

0 0.8 7.2 Ad-CTL Ad-Suv 0 0 103 0 103 DNA content Ad-CTL Ad-Suv Ad-CTL Ad-Suv -+ -+TGF-1 (40h) Casp-3 (active) 15KD

Survivin DMSO Etoposide Taxol DMSO Etoposide Taxol Casp-3 (active) -actin 15KD -actin

Ad-CTL Ad-Suv ---++--++ - + + - - + + Taxol (2 nM) --+-+-+ + TGF-1 (0.3 ng/ml)

15KD Casp-3 (active) -actin

sh-LacZsh-Suv 15KD Survivin -actin

sh-LacZsh-Suv sh-LacZ sh-Suv 1 1 Vehicle TGF- Vehicle TGF- DMSO Etoposide Taxol DMSO Etoposide Taxol

15KD Casp-3 (active) -actin

Figure 6 Cellular survivin levels control the sensitivity to transforming growth factor-b (TGF-b) and chemotherapeutic drugs. (a) NRP-154 cells plated in GM3 were infected overnight with 1/500 dilution of the stock control (CTL) or survivin (Suv) adenoviruses, and then treated with vehicle or TGF-b1 (1 ng/ml) for 48h. Cells were analysed for DNA content by propidium iodide staining and ﬂuorescence-activated cell sorting (FACS). The percentage of cells with hypodiploid DNA content (Sub-G1) is indicated and plotted. (b, c) Capase-3 activation was monitored by western blot analysis of cleaved form of caspase-3 (Casp-3, active). NRP-154 cells were infected in the same way as in (a) with control (Ad-CTL) or survivin (Ad-Suv) adenoviruses, and then treated with vehicle or 10 ng/ml TGF-b (b); etoposide (50 mM), taxol (5 nM) or 0.1% dimethyl sulfoxide (DMSO, c) for 40 h. The level of overexpressed survivin was also shown. (d) NRP-154 cells, after virus infection, were treated with taxol (2 nM) and/or TGF-b1 (0.3 ng/ml) for 40 h and assayed for caspase-3 cleavage. (e) NRP-154 cells expressing doxycycline-inducible shRNA targeting LacZ (sh-LacZ) or survivin (sh-Suv) were plated in GM2.1 medium containing 0.1 mg/ml of doxycycline. After 2 days, cells were either harvested to assay for survivin protein silencing (top) or treated with vehicle, TGF-b1 (10 ng/ml), DMSO (0.1%), etoposide (25 mM), or taxol (2 nM) for 40 h to assay active caspase-3 by western blot (bottom).

We showed that Smad3 binds to the survivin proximal is that Smad3 contributes to TGF-b-mediated promoter upon TGF-b treatment, and the SBE2 site survivin promoter suppression mainly by activating within this region is critical for the binding of Rb, and the physical contact of Smad3 within this recombinant Smad3 (Supplementary Figure 3). How- promoter may either occur at other sites or play a ever, only a modest (yet statistically signiﬁcant) loss minimal role. in TGF-b-mediated suppression of SBE2(m)-survivin To date, there are only a small number of natural promoter was observed. One potential explanation promoters whose repression by TGF-b have been

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5335 DU-145 PC-3 NRP-154 NRP-152 NRP-152 cells -+ -+ -+ -+TGF-1 (24h) 06 12 18 24 36 48 (h) TGF-1 Survivin Survivin Survivin (lower exp) -actin -actin

LNCaP C4-2B RWPE-1 cells 0116 24 48 72 (h) TGF-1 -- ++ -- ++ DHT (10nM) -+ -+-+-+ TGF-1 (24h) Survivin Survivin -actin Survivin (lower exp) -actin

TGF-

Cell surface TRII T RI Smad2

P Smad3 P Smad3 Smad2

P P P P P Rb Nucleus P Rb P P P Normal cells/early stage P P P P cancer: pathway intact Rb ? sensitive to apoptosis signals Smad3 E2F4 Survivin SBE CDE CHR Late-stage cancer: pathway defectiveresistant to apoptosis signals

Figure 7 Regulation of survivin by transforming growth factor-b (TGF-b) in prostate cells correlate with their malignancy. (a) TGF-b time course in NRP-152 cells. (b) Human prostate cancer cell lines DU-145 and PC-3 plated in Dulbecco’s modiﬁed Eagle’s medium/ Ham’s F-12 (DMEM/F12) þ 1% FBS medium, and rat prostate epithelial cell lines NRP-154 (tumorigenic) and NRP-152 (nontumorigenic) plated in GM3 medium were treated with vehicle or 10 ng/ml of TGF-b1 for 24 h, and cell lysates were subjected to western blot analysis. Lower exp, lower exposure. (c) Non-tumorigenic human prostate cell line RWPE-1 was treated with vehicle or 10 ng/ml of TGF-b1 for the indicated time. (d) LNCaP and C4-2B cells were plated in 1% DC-FBS þ 15 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) þ DMEM/F12, infected with adenovirus expressing TbRII (1/200 dilution), and treated with 20 ng/ml of epidermal growth factor. Cells were then treated with or without 10 nM of DHT for 24 h, followed by the incubation with vehicle or 10 ng/ml of TGF-b1 for an additional 24 h. (e) A schematic model of survivin regulation by TGF-b and its implication in prostate cancer. TGF-b ligand-bound receptors activate Smads 2 and 3, which activate Rb (and/or Rb-like proteins) by inducing its hypophosphorylation. Activated Rb can then bind E2F4 to form a repressor complex on the survivin promoter through CDE/CHR elements. Smad3, which binds to SBE upstream of CDE/CHR in vitro, may also contribute to TGF-b-mediated survivin promoter suppression through interaction with E2F4. In normal cells, the TGF-b/survivin regulatory axis is usually intact to maintain survivin levels. However, this regulatory axis is often defective in malignant cancer cells, resulting in heightened levels of survivin and thus resistance to chemotherapeutics. characterized. One such example is the c-myc promoter, repressive SBE (RSBE) within the TIE of the c-myc onto which TGF-b recruits a transcriptional inhibitory promoter (Frederick et al., 2004). Our electrophoretic complex consisting of Smad3/Smad4/E2F4/p107 to a mobility shift assay (EMSA) data investigating the TGF-b inhibitory element (TIE) (Chen et al., 2002). interaction of Smad3 to the proximal region of the However, the survivin promoter does not contain a TIE survivin promoter demonstrated that this interaction is sequence. Instead, we show that the survivin promoter is restricted to a consensus SBE (Supplementary Figure 3). repressed by TGF-b through the CDE and CHR Although this SBE (SBE2) is not required for the elements, which represent a set of cis-acting elements repression of the survivin promoter by TGF-b,itis not previously reported to be targeted by TGF-b. The likely that association of Smad3 to SBE2 (and/or other CDE and CHR are structurally different from TIE sites) on the survivin promoter may enhance the described previously in the context of the c-myc repression of survivin by TGF-b. In contrast to the promoter. Moreover, unlike the survivin promoter, RSBE within the TIE of the c-myc promoter, which regulation of the c-myc promoter by TGF-b requires requires the interaction with Smad4, the proximal region the direct binding of Smads 3 and 4 to a unique of the survivin promoter is not controlled by Smad4

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5336 (overexpression of WT and dominant-negative Smad4; SB431542 (Tocris, Ellisville, MO, USA); DHT, Taxol, data not shown) and does not bind Smad4 (Figure 5b) in and a-b-actin (Sigma, St Louis, MO, USA); etoposide the context of the interaction with E2F4, Rb, and Rb- (Calbiochem, Gibbstown, NJ, USA); a-survivin (sc-10811), like proteins. Thus, we propose a new model, whereby a-cIAP1 (sc-7943), a-cIAP2 (sc-7944), a-Smad3 (sc-8332), Smads 2 and 3 first activate Rb to promote the a-Smad4 (sc-7966), a-p130 (sc-317), a-p107 (sc-318), a-E2F4 (sc-866) (Santa Cruz, Santa Cruz, CA, USA); formation of the suppressive complex Rb/E2F4 on the a-XIAP (no. 610762) (BD Pharmingen, San Jose, CA, USA); CDE/CHR elements of the survivin promoter. The two a-Smad2 (Transduction Laboratories, San Jose, CA, Rb-like proteins, which also bind to the CDE/CHR USA); a-P-Smad2 (Ser 465/467) (no. 3101), a-P-Smad3 elements, may function similar to Rb. (Ser 423/425) (no. 9514), (Cell Signaling, Danvers, MA, Survivin has recently attracted wide interest as a USA); a- Rb, a-P-Rb (Ser 807/811) (NEB, Ipswich, unique IAP protein with dual roles in growth control, MA, USA). through enhancing mitosis and/or inhibiting apoptosis (Altieri, 2003a, b). Survivin expression is exceptionally high in numerous types of tumors (Altieri, 2003b), and Cell culture reported to aid in tumor maintenance and progression, NRP-152, NRP-154 cell lines (Danielpour et al., 1994) were maintained and treated with TGF-b as before (Wang mainly by conferring resistance to various apoptotic et al., 2005; Song et al., 2006). HEK293T (human embryonic stimuli. Targeting the loss or inactivation of survivin in kidney cell line 293 inserted with SV40 T antigen), DU-145 cancer cells often results in spontaneous apoptosis and/ and PC-3 were maintained in Dulbecco’s modified Eagle’s or enhanced apoptosis induced by chemotherapeutic medium/Ham’s F-12 (DMEM/F12) supplemented with 5% agents (Altieri, 2003a). Notably, antagonizing survivin fetal bovine serum (FBS). LNCaP and C4-2B cells were expression in DU-145 human prostate cancer cells by maintained and treated as described for LNCaP (Chipuk et al., ribozyme increased their sensitivity to cisplatin-induced 2002). RWPE-1 cells (Bello et al., 1997) were cultured in apoptosis (Pennati et al., 2004). We also observed complete keratinocyte serum-free growth medium (Invitrogen, similar results in NRP-154 cells treated with taxol. Carlsbad, CA, USA). (GM2.1 medium: DMEM/F-12 supple- Our data show that downregulation of survivin mented with 5% FBS, 5 mg/ml insulin and 0.1 mM dexamethasone; GM3 medium: DMEM/F12 containing 1% FBS, 15 mM expression by TGF-b is defective in malignant prostate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) cancer cells, consistent with the loss or inactivation of and 0.1 mM dexamethasone). TGF-b signaling components, including TGF-b receptors and/or Smads (Kishi et al., 2004; Shariat et al., 2004). Alternatively, defects in Rb, Rb-like proteins Western blot analysis and/or their association with the survivin promoter may Cell lysates were prepared and analysed by western blot as disable cancer cells from suppression of survivin by described previously (Song et al., 2003b) except for the TGF-b even in the presence of an intact upstream TGF- inclusion of 1 mM EDTA, 1 mM b-glycerophosphate (b-GP) b signaling system, as illustrated by viral oncoproteins and 2.5 mM Na4P2O7 to radioimmuno precipitation assay lysis that inactivate Rb family proteins. Rb is frequently lost buffer. or inactivated in human cancers, for example, 25–50% of prostatic adenocarcinomas have a defective Rb RT–PCR pathway (Jarrard et al., 2002). Rb-depleted prostate Five micrograms of total RNA (purified by RNeasy, Qiagen, cells fail to be growth arrested upon androgen depriva- Valencia, CA, USA) was reverse transcribed with Superscript tion (Sharma et al., 2007), and survivin deregulation II (Invitrogen) using an oligo(dT)20 primer. One microliter of could be one of the major contributing factors based on this cDNA product was used to amplify rat survivin (Forward our current study. Moreover, we show that mutations of primer: 50-GAGTGACATGCCACGGCTAA-30. Reverse pri- both CDE and CHR elements in the survivin promoter mer: 50-CCAGGCATGGAAACATCAAG-30). The positions actually invert the effect of TGF-b to the one that of these primers were nos. 458and 765 for forward and enhanced this promoter activity, suggesting that defects reverse primers, respectively, relative to rat survivin mRNA in such regulation may promote the elevation of survivin (AF276775). by TGF-b and contribute to the oncogenic behavior of TGF-b during tumor progression. Thus, keeping survi- Luciferase reporter assay vin levels ‘in check’ is likely to not only comprise one of Cells were transfected with plasmid DNA (1 mg per well) in 12- TGF-b’s tumor suppressive functions but also prevent well dishes, using 2.5 mg of polyethylenimine per mg DNA, as TGF-b from functioning as a tumor promoter. In specified by ExGen500 (Fermentas, Glen Burnie, MD, USA). conclusion, we have discovered a novel TGF-b/survivin Cells plated overnight at 5 Â 104 cells per well were transfected axis that is disregulated in prostate cancer and has with 0.4 mg of survivin promoter vector, 20 ng of CMV-renilla, significant therapeutic potential. and 0.6 mg plasmid (empty vector, or an expression vector). Medium was replaced with GM3 3 h later, TGF-b1 or vehicle was added the next day, and dual luciferase assays (Promega, San Luis, Obispo, CA, USA) were done 24 h later with Materials and methods an LMax II luminometer (Molecular devices, Sunnyvale, CA, USA). Reagents See Supplemental section for plasmids, adenovirus, lenti- Sources were a-caspase-3 active (AF-835), recombinant human virus-mediated gene silencing, flow cytometry, DNAP and TGF-b1, BMP-4, (R&D systems, Minneapolis, MN, USA); EMSA.

Oncogene A TGF-b/survivin regulatory axis J Yang et al 5337 Abbreviations SBE2, CDE and CHR elements; SBE, Smad-binding element; shRNA, short-hairpin RNA; TbRI, TGF-b receptor type I; 3TP-Lux, 3TP-luciferase; CA-ALK5, constitutively active TbRII, TGF-b receptor type II; TGF-b, transforming growth ALK5; Cdk, cyclin-dependent kinase; DC-FBS, dextran factor-b. charcoal-treated FBS; DMEM/F-12, Dulbecco’s modiﬁed Eagle’s medium/Ham’s F-12; DNAP, DNA pull-down assay; EMSA, electrophoretic mobility shift assay; FACS, ﬂuores- Acknowledgements cence-activated cell sorting; FBS, fetal bovine serum; HEK293T, human embryonic kidney cell line 293 inserted This research was supported by the Gene Expression and with SV40 T antigen; IAP, inhibitor of apoptosis; P-Rb, Genotyping Facility, the Flow Cytometry Core Facility of the phosphorylated Rb; P-Smad, phosphorylated Smad; Rb, Case Comprehensive Cancer Center (P30 CA43703). Grant retinoblastoma protein; R-Smad, receptor-regulated Smad; support: NCI grants R01CA092102 and R01CA102074 (to D SAC, a 32-bp survivin promoter region (À50/À19) containing Danielpour).

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

Oncogene