Abstract
Survivin is a unique member of the inhibitor of apoptosis (IAP) proteins that is overexpressed in numerous cancers through poorly defined mechanisms. One such mechanism may be through constitutive activation of the insulin-like growth factor-I (IGF-I) signaling pathway, implicated in the development and progression of prostate cancer. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that IGF-I induces Survivin expression, and that silencing Survivin by lentiviral-mediated small hairpin RNA (shRNA) represses IGF-I-stimulated cell growth, implicating Survivin as a mediator of this growth response. Moreover, our data support that the induction of Survivin by IGF-I occurs through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. Use of various Survivin promoter-luciferase constructs revealed that the CDE and CHR response elements in the proximal region of the Survivin promoter are involved in this IGF-I response. Transforming growth factor (TGF-β) signaling antagonists similarly activated the Surivin promoter and rendered cells refractory to further promoter activation by IGF-I. IGF-I suppressed levels of phospho-Smads 2 and 3 with kinetics similar to that of Survivin induction. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTOR. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin by IGF-I. Taken together, these results suggest that IGF-I signaling through a PI3K/Akt/mTORC1 mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling.
Highlights
Survivin is the smallest member of the inhibitor of apoptosis (IAP) family of proteins, containing one or more conserved zinc-coordinated Cys/His baculoviral IAP repeat (BIR) motifs [1,2]
(#AF886) (R&D Systems); anti-P-Smad3 (Ser433/435, #9514), anti-P-Smad2 (Ser465/467, #3101), and P-Smad1/5/8 (Ser463/ 465/Ser426/428, #9511), anti-mTOR (#2972), anti-Raptor (#2114), anti-Rictor (#4978), anti-P-Rb (Ser807/811, #9308), Akt1 (#2967), Akt (Ser473, #927), anti-P-S6 (Ser235/236, #2211) antibodies (Cell Signaling); anti-Survivin and anti-Smad3 antibodies (Santa Cruz); anti-b-actin antibody (Sigma); anti-Smad2 (#S66220) antibody (Transduction laboratories); anti-XIAP (#610762, BD Biosciences); anti-PSmad3 (Ser423/425) was generous gift obtained from Dr Dr Ed Leof; U0126 and rapamycin (LC laboratories), perifosine, Ku0063794 (Selleck Chem); SB431542 (Tocris Bioscience), SB202190, SP600125, LY294002, HTS-466284 and ALK5 inhibitor-II (EMD Millipore), MK2206 (ChemieTek), DMEM/ F12 (Invitrogen), characterized fetal bovine serum (FBS) (HyClone)
We previously reported that TGF-b plays a key role in maintaining low levels of Survivin in normal prostate epithelial cells, and proposed that loss of the tumor suppressor function of TGF-b significantly elevates Survivin expression in prostate cancer (PCa)
Summary
Survivin ( called BIRC5) is the smallest member of the inhibitor of apoptosis (IAP) family of proteins, containing one or more conserved zinc-coordinated Cys/His baculoviral IAP repeat (BIR) motifs [1,2]. XIAP (an IAP with three BIR motifs) is well established to inhibit apoptosis through binding to caspases, the overall evidence supporting that Survivin directly inhibits the activity of caspases is not compelling. Studies support that a select pool of Survivin, released from mitochondria upon a death stimulus, inhibits apoptosis by binding to and stabilizing cytosolic XIAP [4] and/or associating to and neutralizing the pro-apoptotic protein Smac/DIABLO [5]. Consistent with its vital role in mitosis, expression of Survivin in normal cells is restricted to the G2/M phase of the cell cycle [6,9]. Survivin has recently been reported to function as a transcription factor or co-factor, binding to and inhibiting the p21WAF1/CIP1 promoter through a p53-dependent mechanism [15]. Histone deaceylase 6 (HDAC6), which can deacetylate Survivin [16], promotes Survivin’s nuclear export and subsequently represses its ability to control transcription and mitosis
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