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 regula- tion 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).