Epidermal growth factor and serum activate distinct pathways to inhibit the BH3 only protein BAD in prostate carcinoma LNCaP cells

Abstract

A better understanding of pathways involved in survival of prostate cancer cells is the key to develop effective and target-selective therapies. Presence of serum or epidermal growth factor in the culture medium of LNCaP cells decreases apoptosis induced by the inhibition of phosphatidylinositol 3-kinase with LY294002. However, intracellular pathway(s) involved in this survival signaling are not well defined. Here, we investigated the mechanism(s) involved in serum or epidermal growth factor-mediated inhibition of LY294002-induced death in LNCaP cells. Cell death was assessed by the percentage of cells in sub-G1 phase and caspase 3 activity. Phosphorylation status of BAD, ERK1/2 and RSKs were assessed by Western blot. Specific gene expression knock down of BAD, BAX, RSK1 and RSK2 were performed using siRNA transfections. Our results demonstrate that cell death induced by LY294002 is mediated by translocation of BAD and BAX proteins from the cytosol to the mitochondria. Whereas, epidermal growth factor activates a MAPK/ERK/RSK1 module leading to inactivation of BAD via Ser75 phosphorylation, the presence of serum, on the other hand, induces a nonconducive intracellular environment for mitochondrial translocation of dephosphorylated BAD. Taken together, these results indicate that phosphorylation of BAD or inhibition of its translocation to the mitochondria are critical phosphatidylinositol 3-kinase-independent survival pathways in LNCaP cells.

Introduction

LNCaP cell line is the most widely used in vitro model of prostate cancer (Horoszewicz et al., 1980, 1983). LNCaP cells harbor a frameshift mutation in the PTEN gene, which results in constitutive activation of the AKT kinase, even in the absence of growth factor stimulation (Carson et al., 1999). Therefore, LNCaP cells survival decreases upon inhibition of phosphatidylinositol 3-kinase (PI3K). However, the death inducing effect of PI3K inhibition in LNCaP cells could be blocked by the presence of serum or epidermal growth factor (EGF) in the culture medium (Carson et al., 1999). Notably, the mechanisms by which EGF and serum maintain survival in LNCaP cells are not well understood. Hence, in the present report, we set out to investigate the mechanism(s) underlying the inhibitory effect(s) of serum or EGF on cell death triggered by the PI3K inhibitor, LY294002 (LY), in LNCaP.

There is a growing body of literature supporting the involvement of the antiapoptotic members of the Bcl-2 family, Bcl-2 and Bcl-xL, in PI3K/AKT-independent survival pathways in prostate cancer cells (Raffo et al., 1995; Kajiwara et al., 1999; Lebedeva et al., 2000; Li et al., 2001; Shi et al., 2001; Yang et al., 2003). However, another important cell survival pathway that is distinct from AKT, the mitogen-activated protein kinase (MAPK) pathway, inhibits apoptotic cell death through ERK-mediated transcription of survival genes, or via phosphorylation of proapoptotic members of the Bcl-2 family (Chang et al., 2003; Boldt and Kolch, 2004), most notably BAD. In agreement with a critical role for BAD in the induction of cell death in LNCaP cells, our results provide evidence that the absence of BAD protects cells from LY-induced apoptosis. Moreover, we show that EGF-mediated survival upon induction of LNCaP cells death with LY involves activation of the MEK/ERK/RSK1 module leading to phosphorylation/inhibition of BAD. In contrast, addition of serum protects cells by creating a nonconducive intracellular milieu for translocation of the proapoptotic protein from the cytosol to the mitochondria.

Results

Serum and EGF activate PI3K-independent survival pathway(s) in LNCaP cells

The effect of serum or EGF on LY-induced inhibition of AKT phosphorylation (T308 and S473), and AKT kinase activity (P-GSK-3α/β) was assessed. Results (Figure 1a and b) show that neither serum nor EGF could overcome the inhibitory effect of LY on AKT phosphorylation and activity. However, the effect of LY on cell death, measured by the increase in subdiploid DNA (sub-G1) and by the enzymatic activities of caspases 3 and 9, was significantly inhibited by serum or EGF (Figure 2a–d). Of note, similar to what was observed with serum, treatment with EGF alone in serum-free media gave basal level of sub-G1 population and caspase 3 activity. Therefore, for the sake of brevity and clarity, data with EGF alone have been intentionally omitted from subsequent figures.

Figure 1
figure1

Serum and EGF do not prevent LY294002-mediated inhibition of AKT kinase phosphorylation and activity. LNCaP cells were serum-starved for 20 h before incubating with fresh growth media with 10% serum (serum), serum-free media (no serum), LY (25 μ M) in 10% serum-containing medium (LY+serum), LY in serum-free media (LY), EGF (100 ng/ml) in serum-free media (EGF), and LY and EGF in serum-free media (LY+EGF). After 1 h, cells were harvested and equal amount of protein was subjected to (a) SDS–PAGE and immunoblotting analysis to detect AKT phosphorylation at Ser473 and Thr308, and (b) AKT kinase activity assay. Endogenous AKT kinase of treated cells was pulled down using immobilized AKT 1G1 mouse monoclonal antibody and incubated with 1 μg of GSK-3 fusion protein in a kinase reaction buffer. Phosphorylation of GSK-3 was analysed by SDS–PAGE and immunoblotting. In (a) total AKT (t-AKT) was detected as control of equal loading. In (b) pull down AKT shows equal amount of the AKT kinase used for the assay. Results shown are from one representative experiment out of at least three.

Figure 2
figure2

Inhibition of PI3K in serum-starved LNCaP cells induces apoptosis that is abrogated by introduction of EGF or serum. LNCaP cells serum-starved for 20 h were treated with fresh medium containing 10% serum (serum), serum-free medium (No serum), medium containing 10% serum and 25 μ M LY (LY+serum), serum-free medium with 25 μ M LY (LY) or serum-free medium containing 25 μ M LY and 100 ng/ml EGF (LY+EGF) for 6, 12 and 20 h before (a) cell death was determined by propidium iodide staining and (c) caspase 3 and (d) caspase 9 activity as described in Materials and methods. Percentage of cells in sub-G1 was analysed using the WINMDI 2.8 software. DNA histograms depicting % cell in sub-G1 of one of the experiments after 20 h incubation in different treatments is shown in (b). Results shown in (a), (c) and (d) are a mean of three experiments performed in duplicates±s.e.

Unlike serum, EGF blocks LY-induced cell death in LNCaP cells via activation of the MEK/ERK pathway

Incubation of LNCaP cells with LY in the presence of serum or EGF did not change the expression of either Bcl-2 or Bcl-xL proteins, compared to cells incubated with LY in serum-free medium (data not shown). To the contrary, while no phosphorylation of ERK1/2 could be detected in LNCaP cells incubated in serum-free medium for 20 h (Figure 3a, no serum), ERK1/2 was strongly phosphorylated within 1 h of addition of EGF in serum-starved cells, even in the presence of LY (Figure 3a, compare: no serum, EGF and LY+EGF). Furthermore, ERK1/2 phosphorylation was significantly reduced upon 3 h preincubation of LNCaP cells with 10 μ M MEK inhibitor, U0126, before the addition of EGF in the culture medium (Figure 3a and b, compare EGF and LY+EGF and Figure 3c). Similar results were obtained with PD98059, another non-competitive inhibitor of the MEKs that prevents stimulation-mediated activation of ERK1/2 (Ballif and Blenis, 2001) (data not shown). Furthermore, preincubation with U0126, prevented EGF-mediated inhibition of LY-induced cell death and caspase 3 activity (Figure 3d and e).

Figure 3
figure3

EGF activates ERK phosphorylation in LNCaP cells. LNCaP cells were preincubated with or without MEK1/2 inhibitor, U0126 (10 μ M) for 3 h followed by treatment with LY, EGF and serum as in Figures 1 and 2. (a and b) cells were harvested at 1 h post-treatment, analyzed by SDS–PAGE and subsequently immunoblotted with antiphospho ERK1/2 and anti-ERK1/2 antibodies. (c) Densitometry analysis of results from (a and b). Results are shown as the ratio of phosphorylated ERK over total ERK converted to % of the ratio obtained in cells incubated with serum (% of relative intensity). At 6, 12 and 24 h post-treatment, cells were harvested and analysed for (d) % of cells in sub-G1 and (e) caspase 3 activity. Results shown in (a and b) are from one representative experiment of at least three. (d and e) are a mean of three experiments performed in duplicates±s.e.

In contrast to the effect of EGF, the presence of serum induced minimal phosphorylation of ERK that was not detectable in the presence of either LY (Figure 3a, compare serum and LY+serum) or U0126 (compare Figure 3a and b, serum). Furthermore, ERK1/2 activation was not an effector mechanism in serum-induced survival of LNCaP cells; inhibition of MEK with U0126 did not affect serum-mediated inhibition of LY-induced cells death and caspase 3 activity (Figure 3d and e).

EGF inhibits LY-induced BAD activation and cell death

According to the human protein nomenclature, BAD could be phosphorylated at, Ser75 (murine equivalent: Ser112), Ser99 (murine equivalent: Ser136) and Ser118 (murine equivalent: Ser155) (Eisenmann et al., 2003). Activation of the MEK/ERK pathway has been shown to phosphorylate BAD at Ser75, whereas phosphorylation at Ser99 and Ser118 were attributed to AKT and PKA kinase activities, respectively (Datta et al., 1997; Bonni et al., 1999; Lizcano et al., 2000). Surprisingly, no phosphorylation of endogenous BAD at Ser99 could be detected in LNCaP cells, even when AKT kinase activity was not inhibited (Figure 4a). In addition, while phosphorylation at Ser118 was very weak Ser75 phosphorylation was readily detectable in the presence of serum (Figure 4a). To the contrary, dephosphorylation of BAD at Ser75 could be detected within 3 h of PI3K inhibition in serum-starved cells (Figure 4b). The latter suggests that activation of BAD owing to its dephosphorylation at Ser75 could be the mechanism involved in LY-mediated cell death in LNCaP cells. Indeed, LY-induced death and caspase 3 activity were significantly inhibited upon transfection with BAD siRNA (Figure 4c and d). Interestingly, the level of inhibition of LY-induced apoptosis and caspase 3 activity obtained with BAD siRNA was similar to that observed upon incubation of control-transfected cells (control siRNA) with EGF (Figure 4c and d). These data are in line with the hypothesis that EGF-mediated inhibition of LY-induced apoptosis could be due to inhibition of BAD activation through Ser75 phosphorylation. Indeed, a strong phosphorylation at Ser 75 was induced by EGF in LNCaP cells cultured in the absence of serum (Figure 5a, compare: no serum and EGF). The presence of LY at the time of incubation with EGF did not significantly prevent EGF-mediated phosphorylation of BAD for the first 3 h of incubation (Figure 5a, compare EGF and LY+EGF). Moreover, in agreement with a role for MEK activation in EGF-mediated inhibition of LY-induced cell death in LNCaP cells, U0126 reduced EGF-induced phosphorylation of BAD at Ser75 (Figure 5a, compare LY+EGF and LY+EGF+UO). The induction of BAD phosphorylation by EGF in the presence of LY was further verified by transfecting LNCaP cells with a plasmid encoding for the wild-type mouse BAD protein (BADwt). Results show that phosphorylation of endogeneous BAD at Ser75 but not at Ser99 could be detected in LNCaP cells, whereas phosphorylation at both sites appeared on the transfected mouse wild-type BAD protein (mouse Ser112 and mouse Ser136) (Figure 5b). In agreement with the results shown previously with endogeneous BAD protein, incubation with LY in serum-free medium led to dephosphorylation of both, the human and the transfected murine BAD proteins, at Ser75 (human) and Ser112 (mouse). As expected, incubation of transfected LNCaP cells with LY in the presence of EGF maintained phosphorylation in the wild-type mouse as well as human proteins on Ser112 and Ser75, respectively. However, contrary to what should have been expected due to the inhibition of AKT kinase activity (Datta et al., 1997), Ser136 in mouse BAD was not dephosphorylated after 1 h incubation with LY (Figure 5b). It should be pointed out that in the experiments shown in Figures 4 and 5, the effect of U0126 alone on BAD phosphorylation was similar to that seen in the absence of serum (data not shown). Taken together, results from Figures 4 and 5 demonstrate that dephosphorylation of the proapoptotic protein BAD at Ser75 is responsible for the rapid induction of cell death following incubation of LNCaP cells with LY in serum-free medium. Moreover, inhibition of apoptosis by EGF is due to inhibition of BAD activation through its phosphorylation at Ser75. Our data also show that in presence of LY, BAD dephosphorylated after 6 h incubation with EGF, whereas phosphorylation was maintained in medium without LY. This suggests that, in addition to the MAPK pathway, EGF could also stimulate BAD phosphorylation in a PI3K-dependent manner.

Figure 4
figure4

BAD plays an important role in apoptosis mediated by LY in LNCaP cells. (a) LNCaP cells were left in 10% serum containing medium or serum starved for 20 h (0 h: serum and no serum, respectively) before being incubated with medium with 10% serum (Serum) for 1 h and analysed by SDS–PAGE and immunoblotting for phosphorylation of BAD at Ser118, Ser99 and Ser75. (b) LNCaP cells were serum starved for 20 h before being incubated with medium with 10% serum (Serum), serum-free medium (No serum) or serum-free medium with LY (LY). Cells were harvested at 30 min, 1, 3 and 6 h after treatment and analysed by SDS–PAGE and immunoblotting for phosphorylation of BAD at Ser75. (a and b) detection of total BAD (t-BAD) was used to ensure equal loading. (c and d) LNCaP cells were transfected with either BAD siRNA or control siRNA for 48 h followed by 20 h of serum withdrawal before treatment with 10% serum-containing medium (serum), serum-free medium and LY (LY) or serum-free medium and LY and EGF (LY+EGF). After 6, 12 and 24 h, cells were harvested for assessment of (c) percentage of cells in sub-G1 phase and (d) caspase 3 activity. (c) (insert) Western blot of lysates collected 72 h post-transfection showed effective BAD silencing. β-Actin showed equal loading. Result shown in (a and b) are from one representative experiment of at least three. (c and d) are a mean of three experiments performed in duplicates±s.e.

Figure 5
figure5

EGF inactivates BAD and prevents cell apoptosis in LY-treated LNCaP cells. (a) LNCaP cells were serum-starved for 20 h before being incubated with serum-free medium (No serum), EGF in serum-free medium (EGF), LY in serum-free medium (LY), LY and EGF in serum-free medium (LY+EGF) or preincubated with U0126 for 3 h before being incubated with serum-free medium with LY, EGF and U0126 (LY+EGF+UO). Lysates were collected at 1, 3 and 6 h post-treatment for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 antibody. Membrane was stripped and reprobed with BAD antibody as loading control (t-BAD). (b) BAD protein was overexpressed in LNCaP cells by transfection with either BADwt plasmid encoding for murine BAD or control vector pcDNA3.1 as described in Materials and methods. At 48 h post-transfection cells were serum-starved for 20 h before being incubated in 10% serum-containing medium (serum), LY in serum-free medium (LY), LY and EGF in serum-free medium. Cells were harvested 1 h later for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 and antiphospho BAD Ser99 antibodies, which cross-reacts with both the human (23 kDa) and murine (29 kDa) phospho-BAD. β-Actin and total BAD (t-BAD) show equal loading. Results shown in (a and b) are from one representative experiment of at least three.

Serum induces phosphorylation of BAD at Ser75 but serum-mediated inhibition of LY-induced cell death is independent of BAD inactivation

Although addition of serum following 20 h serum starvation induced phosphorylation of BAD at Ser75 to the same level seen with EGF (Figure 5a, compare no serum and EGF and Figure 6, compare: no serum and serum), the presence of LY prevented serum-induced Ser75 phosphorylation of BAD (Figure 6, compare: serum and LY+serum). In contrast, inhibition of MEK with U0126 did not significantly prevent serum-induced BAD phosphorylation. (Figure 6, compare serum and serum+UO). These results suggest that contrary to EGF, serum phosphorylates BAD at Ser75 through a MEK-independent, LY-inhibitable pathway.

Figure 6
figure6

Serum-mediated phosphorylation of BAD is dependant on PI3K but not MEK activity. LNCaP cells were serum-starved for 20 h followed by incubation with serum-free medium (no serum), LY in serum-free medium (LY), medium with 10% serum (serum), LY in medium with 10% serum (LY+serum) or preincubated with U0126 for 3 h before being incubated with medium with 10% serum and U0126 (Serum+UO). Lysates were collected at 1, 3 and 6 h for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 antibody as in Figure 5a. Membrane was stripped and reprobed with BAD antibody as loading control (t-BAD). Results shown are from one representative experiment of at least three.

RSK1 is the EGF activated BAD kinase in LNCaP cells

ERK1/2-dependent phosphorylation of the proapoptotic protein BAD at Ser75 has mainly been attributed to activation of the MAPK-activated protein kinases from the ribosomal S6 kinase (RSK) family. Indeed, incubation of LNCaP cells with EGF following 20 h of serum starvation induces phosphorylation of RSK even in the presence of LY (Figure 7a, compare EGF and LY+EGF). Moreover, EGF-induced RSK phosphorylation is dependent upon MEK activation as shown by the absence of RSK phosphorylation upon incubation with U0126 (Compare Figure 7a and b, EGF and LY+EGF). To the contrary, phosphorylation of RSK was not detected in the presence of serum (Figure 7a, serum).

Figure 7
figure7

EGF activates ribosomal S6 kinase, RSK1 to inhibit apoptosis in LY-treated LNCaP cells. (a and b) LNCaP cells were treated as in Figure 4a and b. Cells were harvested 1 h post-treatment and subjected to SDS–PAGE and immunoblotted with antiphospho RSK Ser380. (c) LNCaP cells were transfected with either control siRNA or siRNA specific for RSK1 or RSK2. At 48 h post-transfection cells were serum starved for 20 h before treatment with 10% serum-containing-medium (serum), LY in 10% serum containing medium (LY+serum), LY in serum-free medium (LY) and LY in serum-free medium and EGF (LY+EGF). Cells were harvested 1 h post-treatment and analysed by SDS–PAGE. Immunoblotting with anti-RSK1 or anti-RSK2 antibodies demonstrated specific silencing of RSK1 and RSK2, respectively. Apoptotic cell death at 24 h post-treatment was analysed by % of cells in sub-G1 phase (d) and caspase 3 activity (e). Results shown in (ac) are from one representative experiment of at least three. (d) and (e) are a mean of three experiments performed in duplicates±s.e.

Both RSK1 and RSK2 have been shown to phosphorylate BAD on Ser75 (Bonni et al., 1999; Shimamura et al., 2000). Unfortunately, the phospho-antibody used to detect phosphorylated RSK crossreacts with phosphorylated form of RSK1 and RSK2. Hence, in order to determine which of these two RSKs (RSK1 or RSK2) were involved in EGF-mediated BAD phosphorylation in LNCaP cells, RSK1 and RSK2 were silenced using siRNA specific for RSK1 or RSK2, respectively. Results show that silencing of RSK1-blocked EGF-induced phosphorylation of BAD, whereas RSK2 knock down only had a minimum effect (Figure 7c, compare: LY and LY+EGF). Similarly, EGF was no longer able to protect LNCaP cells from LY-mediated apoptosis upon silencing of RSK1, whereas silencing of RSK2 had no affect (Figure 7d and e). To the contrary, serum-mediated phosphorylation of BAD at Ser75 was not affected by silencing of either RSK1 or RSK2, whereas presence of LY in serum-containing medium significantly dephosphorylated BAD at Ser75 (Figure 7c, compare: serum and LY+serum).

Serum does not inhibit LY-mediated cell death through an androgen or an EGFR-mediated pathway

Similar to EGF and serum, incubation of LNCaP cells with the synthetic androgen, methyltrienolone, R1881, was previously shown to inhibit LY-mediated apoptosis (Carson et al., 1999). Incubation of LNCaP cells with R1881 induces an increase in the EGFR expression and activity (Torring et al., 2003). Hence, in order to assess if activation of the androgen receptor could be the mechanism involved in serum-mediated inhibition of LY-induced death, serum-starved cells were incubated with LY alone, serum and LY, or 5 nM R1881 and LY. Consistent with earlier findings while the presence of serum significantly blocked LY-induced cell death, R1881 had no effect unless added for 24 h before the death trigger (Torring et al., 2003). Moreover, inhibition of EGFR activity with the EGFR inhibitor, AG1478 (5 μ M), did not have any effect on the ability of serum to rescue cells from LY-induced death (Figure 8). Contrarily, AG1478 completely blocked the effect of EGF on LY-induced death signaling. Moreover, incubation with 10 μ M AG879, an inhibitor of the ErbB2 receptor, was less efficient in inhibiting the effect of EGF on LY-induced death and did not affect serum-induced survival. Taken together, these data are in agreement with previously published reports demonstrating that androgen-mediated inhibition of LY-induced death requires a minimum of 24 h exposure to the hormone in order to be effective (Torring et al., 2003). Moreover, there is evidence to suggest that, neither androgen nor EGFR or ErbB2 activity are involved in serum-mediated inhibition of LY-induced cell death in LNCaP cells.

Figure 8
figure8

Inhibition of EGFR and ErbB2 activity do not prevent serum-mediated inhibition of LY-induced death in LNCaP cells. LNCaP cells serum-starved for 20 h were preincubated for 1 h without (control) or with 5 μ M AG1478 or 5 μ M AG879 before being treated with fresh medium containing 10% serum (serum), medium containing 10% serum and 25 μ M LY (LY+serum), serum-free medium with 25 μ M LY (LY) or serum-free medium containing 25 μ M LY and 100 ng/ml EGF (LY+EGF). (a) 8 h later caspase 3 activity was determined (b) 12 h later % of apoptotic cells was determined by propidium iodide staining as described in Materials and methods. Caspase 3 activity is expressed as time increase over cells incubated in serum alone (X increase versus control cells).

BAX is required for LY-mediated apoptosis in LNCaP cells

In agreement with the requirement for BAX in LY-induced apoptosis in LNCaP cells, silencing of BAX (Figure 9a) prevented LY-mediated apoptosis and caspase 3 activation (Figure 9b and c). It should be pointed out that BAX silencing was more efficient in inhibiting LY-induced apoptosis than BAD silencing. These data strongly argue in favor of an effector function for BAX whereas suggesting a facilitator role for BAD in this system (Letai et al., 2002). Silencing both proteins at the same time gave the same level of inhibition as seen in the absence of BAX alone. Previous reports have shown that, in addition to regulation at the expression level, BAX activity could be affected by its phosphorylation status. BAX could be phosphorylated at Ser184 by AKT (Gardai et al., 2004) or at Ser163 by Gsk3β (Linseman et al., 2004); Ser184 phosphorylation inhibits Bax activity whereas phosphorylation at Ser163 increases it. Using immunoprecipitation of the BAX protein followed by blotting with a general antiphospho serine antibody, no phosphorylation of BAX was detected in the presence of serum or EGF (data not shown).

Figure 9
figure9

BAX is required for induction of apoptosis in LY-treated LNCaP cells. LNCaP were transfected with control siRNA, BAD siRNA, BAX siRNA or both BAD siRNA and BAX siRNA. At 48 h post-transfection, cells were serum starved followed by treatment either 10% serum-containing medium (serum) or LY in serum-free medium (LY). (a) Western blot of lysates collected 72 h post-transfection with anti-BAD and anti-BAX antibodies demonstrated specific silencing of BAD and BAX. β-Actin was used to show equal loading. Apoptosis in LY-treated cells at 6, 12 and 24 h post-treatment were shown by (b) % of cells in sub-G1 phase and (c) caspase 3 activity. Result shown in (a) is 1 representative experiment of at least three. (b and c) are a mean of three experiments performed in duplicates±s.e.

Serum-mediated inhibition of LY-induced apoptosis involves inhibition of BAX and BAD translocation to the mitochondria

Induction of apoptosis by activated BAD and BAX proteins involves the translocation of both proteins from the cytosol to the mitochondria. Figure 10 shows that in LNCaP cells growing in medium containing serum, BAD and BAX proteins are detected in both, the cytosol and the mitochondria, whereas the antiapoptotic protein Bcl-2 is confined to the mitochondrial fraction. However, as expected, phosphorylated BAD at Ser75 is only detected in the cytosol. Although the presence of LY dephosphorylated cytosolic BAD in serum-containing medium, EGF induced a strong Ser75 phosphorylation of the protein (Figure 10a, compare serum, LY+serum and LY+EGF). The quality of cell fractionation was verified by demonstrating the presence of Cu/Zn SOD in only the cytosolic fraction, whereas the voltage-dependent anion selective channel (VDAC), usually expressed in the outer mitochondrial membrane, only appeared in the mitochondrial fraction. Upon serum starvation of cells for 20 h, only slightly more BAD and BAX proteins could be detected in the mitochondria compared to cells grown in serum containing medium (Figure 10a, compare 0 h: serum and no serum). On the other hand, exposure of serum-starved cells to LY resulted in a strong accumulation of BAX and BAD in the mitochondrial fraction, which could be inhibited by the addition of serum (Figure 10a–c, 90 min compare: LY and LY+serum). Interestingly, and in agreement with the inhibition of BAD activation through its phosphorylation at Ser75, the presence of EGF at the time of incubation with LY only prevented BAD but not BAX translocation to the mitochondria (Figure 10a–c, 90 min compare: LY and LY+EGF). These data were confirmed by immunofluorescence staining of activated BAX using an antibody that recognizes the N terminus of the BAX protein (anti-Bax6A7). The amount of activated BAX present in the mitochondria was not significantly different upon incubation of LNCaP cells with either LY or LY and EGF for 2 h. In contrast, in presence of serum no activated BAX could be detected (Figure 10d).

Figure 10
figure10

EGF prevents BAD but not BAX translocation to the mitochondria. (a) LNCaP cells were plated in 100 mm dish for 2 days before either control media (10% serum) or serum-free media (no serum) was added for 20 h. Cells were harvested at this point (0 h) (lane 1–4) for subcellular fractionation. Additionally, serum-starved cells were further treated with 10% serum-containing medium (serum), LY in 10% serum-containing medium (LY+serum), LY in serum-free medium (LY) or LY in serum-free medium and EGF (LY+EGF) for 90 min before harvesting for subcellular fractionation (lane 5–12). Fractionation was performed as in Materials and methods and equal protein amount for cytosol (C) and mitochondria (M) fractions were subjected to SDS–PAGE and immunoblot analysis. Purity of the fractions was determined by blotting for cytosol- or mitochondria-specific protein, Cu/Zn SOD and VDAC, respectively. (b and c) densitometry analysis of the amount of BAD and BAX translocation into the mitochondria. Results are shown as the ratio of mitochondrial BAD or BAX over VDAC converted to % of the ratio obtained in cells incubated with serum (% of relative intensity). (d) Activation of BAX was assessed as described in Materials and methods following cell treatment as described above. Results shown in (a and c) represent one representative experiment of at least three.

Discussion

Data presented here show that EGF and serum employ distinct mechanisms to inactivate BAD and rescue LNCaP cells from death triggered by inhibition of the PI3K/AKT axis; EGF inactivates BAD through its phosphorylation at Ser75, whereas serum prevents its mitochondrial translocation.

EGF activates a MEK/ERK/RSK1-dependent survival pathway in LNCaP cells

In addition to its effect on proliferation through the recruitment of transcription factors and induction of gene expression (Yarden, 2001; Kooijman et al., 2003; Lyons-Darden and Daaka, 2004), here, we report that EGF contributes to survival of LNCaP cells via mechanisms that block activation and/or proapoptotic activity of the Bcl-2 proteins, BAD. In a model of PI3K inhibition-induced cell death, the presence of EGF rescued LNCaP cells by activating the MEK/ERK/RSK1 pathway leading to inactivation of BAD through its phosphorylation at Ser75. In this regard, in an earlier finding, Eisenmann et al. (2003) implicated the same MEK/ERK/RSK1 signaling module as a critical survival pathway for melanoma cells; RSK-mediated BAD inactivation through Ser75 phosphorylation was necessary to maintain survival in melanoma cells but not in melanocytes. Although, the report did not specify if the constitutive activation of the MEK/ERK/RSK1 module and Ser75 phosphorylation of BAD in melanoma cells was due to constitutive activation of the EGF receptor, results from Hutchinson's group (Shahbazi et al., 2002) demonstrated that high EGF production might be important in the development of malignant melanoma. Taken together, these results suggest that EGF-induced activation of the MEK/ERK/RSK1 module and inactivation of BAD may not be specific to prostate cancer cells but, may represent a common EGF driven survival pathway that functions via phosphorylation-dependent loss of apoptotic function of BAD.

Serum phosphorylates BAD at Ser75 through a PI3K-dependent pathway

Contrary to EGF-induced BAD phosphorylation at Ser75, neither the MEK inhibitor nor silencing of RSK1 and RSK2 prevented phosphorylation of BAD at Ser75 upon releasing serum-starved cells in 10% serum. These results implicate a mechanism other than RSK1 or RSK2 in serum-induced phosphorylation of BAD at Ser75. Indeed, results show that serum-induced inactivation of BAD via Ser75 phosphorylation, unlike EGF, is dependent on pathway inhibited by LY whereas EGF is able to activate the MEK/ERK/RSK1 module in the absence of PI3K signaling. The nature of the LY-regulated pathway responsible for serum-mediated BAD phosphorylation at Ser75 remains undetermined.

Potential crosstalk between PI3K and the ERK activation pathway

Interestingly, serum-induced activation of ERK could be inhibited in the presence of the PI3K inhibitor, LY, thereby pointing to possible crosstalk between the PI3K and MAPK pathways in LNCaP cells. In this regard, a recent report from Sato et al. (2004) demonstrated that the PI3K-activated kinase, PDK1, could directly phosphorylate MEK, thus suggesting a possible mechanism leading to PI3K-dependent activation of the MAPK pathway. Notably, the acquisition of androgen-independent phenotype in LNCaP cells has been linked to intact PI3K signaling. To that end, Murillo et al. (2001) showed that androgen ablation alone increased PI3K/AKT activation, and surmised that progression from acute to chronic androgen ablation resulted in survival in case of the former and proliferation in case of the latter. These data suggest that an increase in PI3K activation upon androgen deprivation could induce sufficient PDK1 activation leading to MEK phosphorylation/activation. Activated MEK can then signal phosphorylation of downstream substrates, such as ERK, in a PI3K-dependent manner, thereby inducing an androgen-independent state and bypassing the need for androgen-mediated activation of the MAPK pathway.

Phosphorylation of BAD at Ser75 is a critical regulator of LNCaP cells' survival

Inactivation of the protein BAD has been shown to depend on its phosphorylation at three serine residues, Ser99, Ser75 and Ser119 (human nomenclature). Whereas BAD phosphorylation at Ser75 was easily detected in LNCaP cells maintained in 10% serum or EGF containing medium, weak and no phosphorylation at Ser118 and Ser99, respectively could be detected. Detection of phosphorylated Ser99 was performed with the phospho-BAD antibody (Ser136) from Cell Signaling Technology Inc., USA, which detects BAD only upon overexpression. In agreement with that, we could detect phosphorylation of the transfected wild-type mouse BAD at Ser136 but not at Ser99 in the human homolog. Could it be that the expression of BAD in LNCaP cells is below the limit of detection for phosphorylation at Ser99? A probable answer to this comes from an earlier study which showed efficient BAD phosphorylation at Ser99 and Ser118 in melanocytes while minimally detected in melanoma cell lines, even though the levels of BAD protein was comparable in the two cell types (Eisenmann et al., 2003). Perhaps, regulation of Ser75 phosphorylation and weak phosphorylation on Ser99 and Ser119 is a commonly shared by tumor cells from diverse backgrounds.

Presence of serum induces a non-conducive environment for mitochondrial translocation of BAX and BAD

Using siRNA-mediated silencing of BAD and/or BAX, we show that both proteins are required for LY-induced apoptosis in LNCaP cells in the absence of serum. Serum-mediated phosphorylation of BAD at Ser75 appears to be dependent on an LY sensitive pathway. Therefore, contrary to EGF, survival induced by serum cannot be attributed to the inhibition of BAD inactivation via phosphorylation at Ser75. Supporting this notion, death signal triggered upon dephosphorylation of BAD is tolerated well by LNCaP cells in the presence of serum. This could be a function of the neutralizing effect of Bcl-xL expression in LNCaP cells as previously demonstrated (Yang et al., 2003), or the need for the activation of another proapoptotic protein, such as BAX, together with BAD to induce efficient killing. We provide evidence to refute the former-serum does not induce increased expression of Bcl-xL in LNCaP cells (Chao and Clement, unpublished data),however, the presence of serum not only inhibited mitochondrial localization of BAD but also, that of BAX. The necessity of both proteins to translocate to the mitochondria to induce efficient killing was evidenced by the weak death signal upon translocation of only BAX in the presence of EGF. This might be due to the presence of other antiapoptotic proteins, such as Bcl-2, in LNCaP cells' mitochondria, which counteract the effect of BAX translocation. However, when BAD translocates with BAX, the presence of BAD in the mitochondria could sequester Bcl-2 away from BAX, thus promoting BAX-induced cytochrome c release and apoptosis (Chao and Korsmeyer, 1998; Green and Reed, 1998; Lutz, 2000; Martinou and Green, 2001; Puthalakath and Strasser, 2002). The precise mechanism underlying serum-induced inhibition of BAD and BAX translocation is currently the focus of our ongoing investigations.

In conclusion, our data combined with some data from the literature show the existence of redundant survival pathways in the LNCaP prostate cancer cell line. These may include EGF-mediated inactivation of BAD, decrease in efficient mitochondrial translocation of BAD or BAX or even inhibition of BAD or BAX function by their antiapoptotic counterparts, such as Bcl-xL (Li et al., 2001; Yang et al., 2003) and Bcl-2 (McDonnell et al., 1992; McConkey et al., 1996) or even expression of the inhibitor of apoptotic proteins, IAPs (McEleny et al., 2002; Nomura et al., 2003; Zhang et al., 2005). A better understanding of these pathways could help develop more effective therapies and target-selective therapies against prostate carcinoma in general.

Materials and methods

Cell culture and antibodies

The human prostate cancer cell line, LNCaP-FGC (LNCaP) was purchased from American Type Culture Collection. Cells were maintained in RPMI1640 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate and 1 mM gentamycin. LNCaP cells were plated for 2 days in complete media (70% confluent cells), followed by 20 h of serum deprivation before treatment with LY294002 (25 μ M) (Calbiochem, San Diego, CA, USA) and/or epidermal growth factor (EGF) (100 ng/ml) (Sigma, St Louis, MO, USA) in complete media or serum-free media. For pretreatment with U0126 (Cell Signaling Technology, Beverly, MA, USA), or AG1478 and AG879 (Calbiochem, San Diego, CA, USA) cells were incubated with 10 μ M of U0126, 5 μ M of AG1478, or 5 μ M of AG879 or medium as control for 3 and 1 h, respectively, before adding LY, EGF or serum. Stock solutions of LY294002, U0126, AG1478 and AG879 were made in DMSO. DMSO was added at appropriate concentration in control medium. Antibodies for Phospho-RSK(Ser380), Phospho-AKT(Thr308), Phospho-AKT(Ser473), AKT, Phospho-ERK1/2(Thr202/Tyr204), ERK1/2, Phospho-BAD(Ser136), Phospho-BAD(Ser112), BAD, Phospho-GSK-3α/β(Ser21/9) and GSK-3β were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). β-Actin monoclonal antibody was from Sigma (St Louis, MO, USA), Bcl-2 antibodies were from BD Biosciences (San Jose, CA, USA), RSK1 and RSK2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cu/Zn Superoxide Dismutase (SOD) and BAX-NT antibodies were purchased from Upstate (Lake Placid, NY, USA). Voltage-dependant anion channel (VDAC) antibody was a gift from Dr Catherine Brenner. Peroxidase-conjugated goat anti-rabbit antibody was obtained from DakoCytomation (Glostrup, Denmark), and peroxidase-conjugated goat anti-mouse antibody and peroxide-conjugated rabbit anti-sheep antibody were from Pierce Biotechnology (Rockford, IL, USA).

DNA Fragmentation assay

Cells were harvested by scraping, pelleted and washed once with cold PBS/1% FBS. Cells were fixed with 75% ethanol before they were resuspended in propidium iodide (10 μg/ml) and RNAse A (0.25 mg/ml) (Sigma, St Louis, MO, USA). After 30 min incubation at 37°C in the dark, stained cells were analysed by flow cytometry.

Caspase activity assay

Cells were harvested at different time points after drug treatment and lysed using × 1 Cell Lysis Buffer (BD Biosciences Pharmingen, San Diego, CA, USA). Cell lysate was added to 2 × Reaction Buffer (10 mM HEPES, pH7.4, 2 mM EDTA, 6 mM DTT, 10 mM KCl and 1.5 mM MgCl2) supplemented with protease inhibitors and caspase substrate (50 μ M) (caspase 3 substrate: Ac-DEVD-AFC and caspase 9 substrate: Ac-LEHD-AFC) (AG Scientific, San Diego, CA, USA) in a 96-well plate. Samples were incubated at 37°C for 1 h and AFC fluorescence were measured using SpectrafluorPlus spectrofluorometer. Caspase activity was normalized with protein amount and expressed as caspase activity/μg protein.

Transfection

For transfection of murine BAD, cells were transiently transfected with 4 μg of the control vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) or pcDNA3-BADwt encoding for murine BAD (a kind gift from Dr Stanley J Korsmeyer) using the CalPhos Mammalian Transfection kit (Clontech, Palo Alto, CA, USA) as per the manufacturer's instruction. At 48 h post-transfection, cells were serum starved for 20 h before adding LY, EGF and serum. Cells were harvested after 1 h and subjected to SDS–PAGE and immunoblotting.

In vitro AKT kinase assay

Kinase assay was performed using the nonradioactive AKT Kinase Assay kit from Cell Signaling (Beverly, MA, USA). Briefly, after 1 h of treatment as above, cells were harvested and solubilized using ice-cold 1 × Cell Lysis Buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin) supplemented with 1 mM PMSF for 10 min on ice. After clearing the lysate, 500 μg of lysate were incubated with AKT antibody immobilized to crosslinked hydrazide agarose beads overnight with gentle rocking at 4°C. The beads were washed and incubated with 1 μg of the GSK-3 fusion protein in a kinase buffer (25 mM Tris (pH 7.5), 5 mM glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2) containing 10 mM ATP at 30°C for 30 min. The kinase reaction was stopped by adding 3 × SDS sample buffer and the phosphorylation of GSK-3 was measured by SDS–PAGE and immunoblotting using the antiphospho GSK-3α/β antibody. Blots were reprobed with GSK-3β and AKT to check for equal loading.

SDS–PAGE and immunoblotting

Cells were lysed with RIPA lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, supplemented with 1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin and 1 mM PMSF before use. Equal amounts of protein were resolved by SDS–PAGE and immunoblotted. In experiments involving detection of phosphorylated proteins, blots were stripped with Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL, USA), and reprobed with total protein and β-actin antibody to check for equal protein loading. Densitometry analysis was performed using Multi Gauge Version 3, Fujifilm.

RNA interference

Small interfering RNA (siRNA) inhibition of endogenous BAD, BAX and RSK2 expression was achieved using custom designed siRNA that target the respective DNA sequence: BAD-IndexTermAAGAAGGGACTTCCTCGCCCG (PROLIGO, Boulder, CO, USA), BAX-IndexTermAAGGTGCCGGAACTGATCAGA and RSK2-IndexTermAAGGCCACACTGAAAGTTCGA (Qiagen-Xeragon, Valencia, CA, USA). Validated RSK1 siRNA were purchased from Ambion (Austin, TX, USA). A control siRNA (non-homologous to any known gene sequence) (Qiagen-Xeragon, Valencia, CA, USA) was used as a negative control. Cells were transfected with siRNA using the CalPhos Mammalian Transfection kit and the level of silencing was assessed by Western blot. At 48 h post-transfection, the cells were serum starved and treated as above for indicated times before harvesting for either caspase activity assay or DNA fragmentation assay.

Subcellular fractionation

Cells, 1.25 × 106, were seeded on 100 mm dish and treated as for sub-G1 and caspase determination. After 90 min, cells were harvested and resuspended with extraction buffer (220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT) supplemented with protease inhibitors and incubated on ice for 20 min. The cell extract was homogenized 30 times on ice with a dounce homogenizer and subjected to centrifugation at 700 g, 4°C for 10 min to pellet cell nuclei, cellular debris and intact cells. Supernatant collected was centrifuged at 10 000 g for 30 min, 4°C. The resulting pellet (mitochondria fraction) was washed once with extraction buffer and centrifuged at 10 000 g for 10 min, 4°C; whereas the supernatant (cytosolic fraction) was further centrifuged at 16 000 g for 20 min, 4°C to remove any residual mitochondria. The pellet from mitochondria fraction was dissolved in of extraction buffer containing 0.5% Triton X-100. Equal amounts of protein were subjected to SDS–PAGE and Western blotting. The purity of the fractions was confirmed by assessing localization of the mitochondrial-specific protein (VDAC) and the cytosolic Cu/Zn SOD.

Bax activation assay

Cells grown on coverslips in 12-well-plate and treated as in the subcellular fractionation assay were pretreated with 100 nM of MitoTracker CMXRos (Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed with PBS, and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Then the cells were permeabilized with 0.1% saponin for 10 min at room temperature before incubation with anti-Bax6A7 antibody (BD Biosciences, San Jose, CA, USA) in staining solution (2% BSA+5% FBS+0.05% saponin in PBS) for 1 h, followed by incubation with anti-mouse FITC antibody (Molecular Probes Inc., Eugene, OR, USA) for another 1 h. Bax activation was assessed with confocal laser microscopy. Images were obtained with the Axiophot microscope (Carl Zeiss, Oberkochen, Germany) equipped with epifluorescence optics and the LSM 510 confocal laser optics. The excitation wavelengths for MitoTracker Red CMXRos and FITC were 543 and 488 nm, respectively. A long pass filter (585 nm) was used to measure MitoTracker emission, and a band pass filter (505–530 nm) was used to measure FITC emission. Gains for both fluorophores were set using the control cells. Images in all other conditions were obtained using the identical microscope settings.

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Acknowledgements

We thank Dr C Brenner for the VDAC antibody, Dr Stanley J Korsmeyer laboratory, Dana-Farber Cancer Institute, Boston, MA, for the pcDNA3-BADwt encoding for murine BAD, Ms Wang Ya for useful technical assistance in the immunofluorescence analysis of activated BAX and Dr S Pervaiz for his help in editing the paper. This work was supported by Grant R-183-000-084-213 from The National Medical Research Council of Singapore to MVC.

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Correspondence to M-V Clément.

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Chao, O., Clément, M. Epidermal growth factor and serum activate distinct pathways to inhibit the BH3 only protein BAD in prostate carcinoma LNCaP cells. Oncogene 25, 4458–4469 (2006). https://doi.org/10.1038/sj.onc.1209421

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Keywords

  • LNCaP
  • BAD
  • BAX
  • survival
  • prostate carcinoma
  • phosphatidylinositol 3-kinase

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