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Transforming growth factor ß (TGFß) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFβ by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFβ1. Prior to the onset of death, TGFβ1 treatment downregulated the expression of the caspase inhibitor FLICE-like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFβ1-induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase-8 by siRNA knockdown partially rescued the apoptotic effects of TGFβ1, suggesting a role for death receptor signaling components in TGFß-mediated death of prostate epithelial cells. © 2008 Wiley-Liss, Inc.
Transforming growth factor ß (TGFß) is an essential paracrine mediator of androgen-withdrawal-induced apoptosis (AWIA) of prostate epithelium.1 Binding of TGF-ß to its receptors leads to phosphorylation of receptor-associated R-Smads (such as Smad2) which complex with Smad4, translocate to the nucleus and bind to Smad response elements, mediating gene expression. Although 3 TGFßs (1, 2 and 3) exist, and all are detected in the prostate, most studies have focused on TGFß1.2 In rodents, AWIA is accompanied by an increase in TGFß1 mRNA, and the kinetics of mRNA induction closely parallel those of AWIA.3 Additionally, there is a concomitant rise in the expression of the RI and RII subunits of the TGFß receptor4, 5 as well as phosphorylated Smad2, a key downstream mediator of TGFß.6 A dominant negative form of TGFß-RII blocks TGFß-induced differentiation and cell death.7, 8 Finally, instillation of TGFß1 into rat prostate results in regression of the gland because of apoptosis.3, 9
TGF-ß negatively regulates the proliferation of many adult epithelia, exerting its effects by arresting cell cycle progression or by inducing apoptosis. The mechanism of cell cycle arrest is Smad-dependent and well characterized,10 but the molecular mechanisms controlling apoptosis remain uncertain. There is support for a variety of molecular mechanisms11 including transcriptional regulation,12 which can be both Smad-dependent and Smad-independent.13 One potential downstream target is the signaling network regulated by the family of death receptor (DR) signaling ligands.14, 15
Consistent with the growth inhibitory activity of this pathway, TGFß signaling components function as tumor suppressors in many early stage cancers.11, 13 For example, human prostate cancer specimens frequently display reduced levels of TGFß receptors5, 16–18 and Smad4.18, 19 However, 2 of the well-characterized human prostate cancer cell lines (DU145 and PC3) have intact TGFß signaling pathways,20–22 yet have been reported to undergo only limited apoptosis when treated with TGFß.21, 23, 24 A rat prostate cancer cell line that is not normally growth inhibited by TGFß is inhibited when grown at low density in serum-free media,25 and this inhibition is reversed by growth factor treatment. Thus, autocrine growth factor signaling may account for the resistance of DU145 and PC3 to the apoptotic effects of TGFß. In our report, we identify the conditions that allow induction of TGFß1-induced apoptosis in the DU145 human prostate cancer cell line. We then test the hypothesis that TGFß1 acts by lowering the endogenous level of FLICE-like inhibitory protein (FLIP), thereby inducing caspase-8 activity and triggering apoptosis.
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- Material and methods
The commonly used human prostate cancer cell lines are growth inhibited by TGFß when grown in soft agar, but fail to respond to TGFß treatment when grown in monolayer culture.31 Although some prostate cancers resist TGFß-induced apoptosis because of defects in the TGFß receptor,1 growth factors have also been shown to counteract TGFß effects on prostate epithelial cell lines.25, 29, 32 In our report, we show that DU145 cells cultured in media containing CS, which has reduced growth factor levels relative to FCS, demonstrate reduced proliferation (Fig. 1a). This resembles the effects of low mitogen treatment on the rat prostate epithelial cell line NRP-152.29 Once DU145 cells have been shifted to low mitogen conditions, TGFß1 induces apoptosis as assessed by multiple criteria, including cell viability, Annexin V staining and PARP cleavage (Fig. 2). When we reduced the cell density to minimize the effect of secreted growth factors on adjacent cells,30 we observed a significant increase in the rate of TGFß1 apoptosis of DU145, but only modest affects on the survival of untreated cells (Fig. 3). Morton and Barrack have shown that FGF (and to a lesser extent EGF) is able to block TGFß-induced apoptosis, and that FGF-neutralizing antibody restores TGFß-induced apoptosis when the rat prostate cancer MatLyLu cells are grown at high density.25 Taken together, our data suggest that growth factors present in FCS stimulate DU145 cells to resist TGFß1-induced apoptosis and/or DU145 cells secrete one or more factors that inhibit apoptosis. Removing or diluting these factors causes DU145 cells to apoptose in response to TGFß1 akin to the response seen in normal prostate epithelial cells.
We have previously shown that an inhibitor of the DR signaling pathways, FLIP, is downregulated in the rat prostate after castration.27, 33 FLIP is an inactive homologue of caspase-8, an apical caspase activated in DR signaling, that acts as a dominant inhibitor of caspase-8 and thereby prevents the activation of distal caspases and cell death.34 In addition to the TGFß effects on rat prostate in vivo described in the Introduction, immortalized rat prostate NRP-152 cells (previously differentiated in mitogen-poor media) apoptose in vitro in response to TGFß1 treatment.28, 29 Similar to AWIA in the intact rat prostate gland, FLIP is downregulated in these cells by TGFß1 treatment, while enforced FLIP expression blocks apoptosis.29 Furthermore, silencing of FLIP expression using siRNA induces apoptosis, even in the absence of TGFß.29 NRP-152 cells maintained in mitogen-rich media are resistant to TGFß1-induced apoptosis.29, 35 This is due at least in part to activation of the Akt/mTOR pathway, which suppresses Smad3 activation32 and raises FLIP levels.29 FLIP is overexpressed in human prostate cancers,36 and loss of expression is associated with AWIA of the androgen-responsive human prostate cancer cell line LNCaP, when grown as a xenograft.37 In addition, in LNCaP cells, we have found that resistance to TRAIL-induced apoptosis depends on FLIP levels and that this effect is regulated by androgens as well as AKT acting via FOXO3a.33
Thus, we investigated FLIP as a regulator of TGFß1-induced apoptosis in DU145 cells. First, we observed that FLIP levels increase in DU145 cells grown in the low mitogen conditions (Fig. 1b) that enhance DU145 cell sensitivity to TGFß1-induced apoptosis. The mechanism that accounts for increased FLIP levels under these conditions is not known, but a similar increase in FLIP is seen when the NRP-152 cell line undergoes differentiation after a shift into low mitogen media.29 Notably, the kinetics of low mitogen-induced FLIP upregulation (a gradual increase over a period of days) is similar in both DU145 and NRP-152 cell lines.
Because FLIP levels are reduced after TGFß1 treatment of NRP-152 cells,29 we hypothesized that FLIP would be similarly reduced after TGFß1 treatment of DU145 cells. Indeed, there is a dramatic reduction in FLIP protein levels and this was mirrored by a large reduction in FLIP-encoding mRNA (Fig. 4). TGFß1-treated NRP-152 cells display a similar reduction in FLIP mRNA.29 Interestingly, in cells grown in CS but not treated with TGFß, we did not observe an increase in FLIP mRNA levels corresponding to the 4-fold increase seen in FLIP protein (compare Figs. 1b and 4b). This suggests that the increase in FLIP protein that accompanies the shift into low mitogen media is due to increased protein stability or mRNA translation efficiency, rather than a change in the rate of transcription or the half-life of the mRNA. Conversely, the TGFß1-induced decrease in FLIP protein is tied more closely to the abundance of FLIP mRNA.
When we silenced the expression of the FLIP gene using siRNAs, FLIP protein rapidly fell to undetectable levels and 40% of the DU145 cells apoptosed, despite the absence of added TGFß. It is important to note that FLIP siRNA mediated knockdown produced levels of cell death that were comparable to TGFß1 treatment alone (Fig. 5bversus 3b). Thus, although the data in Figure 4 provide correlative support for our hypothesis that TGFß acts via FLIP modulation, the knockdown results in Figure 5 represent strong functional evidence that FLIP plays a critical role in the induction of DU145 apoptosis. The extent of knockdown-mediated death in DU145 cells closely matches the 35% spontaneous apoptosis seen in lung carcinoma A549 cells after FLIP knockdown.38 In contrast, knockdown of FLIP in pancreatic and mesothelial cells, as well as in the human prostate cancer cell line PC3-TR, results in little spontaneous apoptosis, although it does sensitize these cells to death ligand-induced apoptosis.39–41 In addition, we previously found that in the NRP-152 cells, FLIP knockdown leads to spontaneous apoptosis, which is also enhanced by TGFß treatment.29 Taken together, these observations suggest that in prostate epithelium, FLIP could regulate apoptosis entirely by inhibiting a pre-existing death signal(s). However, TGFß might also induce DR activation via, for instance, an increase in the levels of a death ligand, such as TNF, which could synergize with FLIP downregulation. We are currently exploring this latter possibility in prostate cells.
Our observation that the decline in FLIP expression correlates with the onset of apoptosis, and that FLIP knockdown is sufficient to induce apoptosis suggests that the molecular mechanism of TGFß-induced death involves one or more of the death ligand/receptor signaling pathways. To test this idea, we used siRNA to silence caspases-8 and -10, 2 related proximal caspases that are activated by DRs,42–44 and inhibited by FLIP.45 We found that knockdown of caspase-8, but not caspase-10, abrogated TGFß1-induced death of DU145 cells (Fig. 6), providing additional functional support for our hypothesis that the TGFß induces apoptosis via FLIP. TGFß1-induced apoptosis of rat prostate epithelial NRP-152 cells also depends on caspase-8 activation, as crmA overexpression blocks cell death.29 The caspase-8 requirement also suggests that one or more of the DR ligands might be produced/secreted in response to TGFß. In our previous studies on TGFß1-induced apoptosis of NRP-152 cells, we were not able to identify a death ligand that mediated the effects of TGFß, although we did observe an increase in TNF mRNA.29 The latter observation remains unexplained, but might reflect a novel mechanism (Fig. 7). Two other cell lines that apoptose in response to TGFß, derived from a gastric carcinoma and a B-cell lymphoma, display a similar phenotype.14, 15 In both cases, death is caspase-8-dependent, but does not apparently require a secreted DR ligand. Along the same lines, it is notable that FLIP knockdown also induces apoptosis in a death ligand-independent, but caspase-8 and/or FADD dependent manner in colorectal cancer cells.46, 47 Thus, novel mechanisms that utilize caspase-8 but not secreted death ligands may also mediate TGFß-induced cell death.
Figure 7. Proposed regulation of FLIP transcription in prostate epithelial cells. (a) Regulation in intact (noncastrated) animals. TGFß expression is low but FLIP transcription is high (thick arrow) because of androgens (black circles), which activate the androgen receptor (AR). The activator of caspase-8 (C8) is unknown (see text), but FLIP (F) inhibits caspase-8 activation, blocking apoptosis. (b) Regulation in castrated animals. After castration, androgens are low (the androgen receptor remains in the cytoplasm) and TGFß, produced by nearby stromal cells, represses FLIP expression (thin arrow) and may also activate caspase-8, although the mechanism is unknown. It is also uncertain whether TGFß acts directly (e.g., via Smads), or indirectly, to repress FLIP transcription. Androgen response elements (rectangles, seen in (b)) are obscured in (a) by bound androgen receptor. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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Androgens regulate the growth of normal prostate and prostate cancer. Castration results in regression of the normal prostate, and androgen signaling blockade is the primary treatment for late-stage prostate cancer.48 We propose that FLIP may play a crucial role in the death and survival of prostate epithelial cells (Fig. 7).49 We and others have reported that FLIP mRNA27 and protein33, 36 levels decline in rat prostates after castration. Studies in LNCaP prostate cancer cells, which express the androgen receptor, demonstrate that the FLIP promoter contains androgen response elements and that FLIP transcription (and protein expression) is upregulated by androgens.33, 37 In addition, low-level AKT signaling, which induces FOXO3a translocation to the nucleus, is required to mediate FLIP transcription by androgens33 (not shown in Fig. 7). In this report and in our previous studies of NRP-152 rat prostate epithelial cells,29 we demonstrate that TGFß, which is produced in a paracrine manner after castration,3, 50 inhibits FLIP mRNA and protein expression. Integrating all of this data, we present a model where androgen withdrawal and paracrine TGFß provide distinct but cooperative signals to repress FLIP transcription (Fig. 7). Low levels of FLIP are permissive for the activation of caspase-8 and facilitate the induction of prostate epithelial cell apoptosis.