a) b) 12000 serum 8000 LY 7000 LY+EGF caspase activity (RFU/µg protein) caspase activity (RFU/µg protein) 14000 10000 8000 6000 * 4000 6000 serum LY LY+EGF 5000 4000 3000 ** 2000 2000 1000 0 pcDNA3.1 plasmid RSK1DN pcDNA3.1 RSK1DN plasmid * : p < 0.01 (vs LY) **: p < 0.02 (vs LY) Figure 26: Transfection of dominant-negative RSK1 attenuates EGF-mediated inhibition of apoptosis LNCaP cells were transfected with either dominant negative mutant of RSK1 or control vector plasmid pcDNA3.1 for fourty-eight hours and treated as in Figure 25 Cells were harvested and analyzed for caspase-3 activity at (a) 10 hours and (b) 20 hours post-treatment Results shown in (a) and (b) are mean of three experiments performed in duplicates ± SE 129 3.1.8 Role of ErbB receptors in EGF- and serum-mediated survival of LNCaP cells There is accumulating evidence supporting the role of ErbB family of receptor tyrosine kinases in the progression of many types of human cancer including prostate cancer (Salomon et al 1995; Nicholson et al 2001; Hynes and Lane 2005) They consist of four members up to date, the epidermal growth factor receptor – EGFR (ErbB-1), ErbB2, ErbB3 and ErbB4, and play a major role in the proliferation, differentiation, migration and survival of mammalian cells (Schlessinger 2000; Jorissen et al 2003) Tumors with alterations in EGFR and ErbB2 receptors most often signal a more aggressive disease and is associated with poor prognosis for the patient (Allred et al 1992; Sjogren et al 1998; Nicholson et al 2001) Increased EGFR expression has been correlated to the progression of prostate cancer to the androgen-independent stage (Di Lorenzo et al 2002; Mimeault et al 2003; Hernes et al 2004) The role of ErbB2 in PCa is more controversial due to conflicting reports of the degree of gene amplification and protein expression in PCa (Ross et al 1997; Signoretti et al 2000; Osman et al 2001; Calvo et al 2003; Hernes et al 2004) However, it has been proposed that ErbB2 role in PCa progression is more significant at the late stages of hormone refractory PCa (Torring et al 2003) It is likely that the importance of ErbB receptors in prostate cancer maintenance is not only attributed by the level of expression but other events as well that contribute to aberrant receptor signaling such as establishment of autocrine signaling (Kambhampati et al 2005), receptor mutation (Olapade-Olaopa et al 2004) and their interaction with the androgen receptor Serum contains a complex mixture of active components that have been shown to protect against apoptosis in various cell types including fibroblasts (Evan et 130 al 1992), Chinese hamster ovary cells (Zanghi et al 1999), neuronal cells (Uto et al 1994; Lucca et al 1997), pancreatic cancer cells (Levitt and Pollak 2002) and human amnion-like WISH (Wistar Institute Susan Hayflick) epithelial cells (Berry et al 2004) Other than growth factors like EGF, IGF and PDGF that promote survival and growth, small peptides in serum like cytokines, endothelin-1, thrombin, bombesin and lysophosphotidic acid have been shown to be able to transactivate the ErbB receptors when they bind to their cognate receptors Cytokine receptors can transactivate the ErbB receptor tyrosine kinase by directly phosphorylating the receptor itself via Janus tyrosine kinases (Jak) (Yamauchi et al 1997; Yamauchi et al 2000), whereas G-protein coupled receptors (GCPR) transactivate the ErbB receptors indirectly by activating metalloproteinases which in turn cleave and release ErbB ligand precursors (e.g: pro-HB-EGF) which then bind and activate ErbB kinase activity (Prenzel et al 1999) As ErbB receptors play such an important role in prostate cancer maintenance, we wanted to investigate if serum-mediated cell survival required activation of the ErbB receptors whether directly or indirectly via transactivation of the receptor In order to assess this, we utilized two widely used ErbB kinase inhibitors – AG1478 and AG879 They both belong to the tryphostin group of tyrosine kinase inhibitors that function by blocking the ATP acceptor site (lysine 721) located within the receptor’s intracellular kinase domain (Levitzki and Gazit 1995) The EGFR kinase inhibitor, AG1478, has a selectivity for EGFR (IC50, 3nM) at least orders of magnitude higher than for ErbB2 (IC50, > 100µM), whereas the ErbB2 kinase inhibitor, AG879, has approximately 500-fold higher selectivity for ErbB2 (IC50, 1µM) than EGFR (IC50, > 500µM) in cell-free systems (Levitzki and Gazit 1995) 131 The dose response of LNCaP cells to both AG1478 and AG879 and a combination of the two inhibitors at various ratios were tested to determine the appropriate concentration that inhibits the kinase activity of the respective ErbB receptors without inducing excessive cell death Serum-starved LNCaP cells were pretreated for hour with various doses of the inhibitors in serum-free media Then a final concentration of 10% serum was added to the media to trigger activation of the receptors After 24 hours incubation, crystal violet assay was performed to determined cell viability Cells were also harvested at 20 hours for determination of caspase-3 activity For AG1478, there was no significant cell death observed with all three doses used (0.5, and 5µM) (Figure 27a) Accordingly, caspase-3 activity measured was no higher than basal level for even the highest dose of AG1478 used (Figure 27d) For the ErbB2 inhibitor, AG879, there was significant decrease in cell viability after 24 hours treatment with 10µM of the inhibitor (Figure 27b), and this was coincides with a noticeable increase in caspase-3 activity at 20 hours compared to basal levels Therefore we proceeded to use a concentration of 5µM for both the AG1478 and AG879, and a combination of both inhibitors at a concentration ratio of 5µM:5µM (AG1478:AG879) (Figure 27c) as there was no significant cell death or caspase-3 activation at these concentrations At the same time, the effects of the ErbB kinase inhibitors on the phosphorylation status of EGFR and ErbB2 receptors were analyzed Serum starved LNCaP cells were pretreated with AG1478, AG879 or a combination of both inhibitors in serum-free media for hour before EGF (100ng/ml) or 10% serum was added Whole cell lysates were analyzed for EGFR and ErbB2 phosphorylation status using antibodies specific for phosphotyrosine site Tyr1173 of EGFR or Tyr1248 of ErbB2 (Figure 28) EGFR Tyr1173 is a major autophosphorylation site activated by 132 b) 100 80 60 40 20 c) 120 % Cell Viability % Cell Viability 120 120 100 100 % Cell Viability a) 80 60 40 80 60 40 20 20 0 AG1478: AG879: 0 0.5 AG1478 conc (µM) 5 AG879 conc (µM) 10 0 1 10 Drug conc (µM) 5 d) 5000 (RFU/µg protein) 4000 caspase activity Figure 27: Dose-response of LNCaP cell viability and caspase-3 activation to ErbB receptor kinase inhibitors LNCaP cells were serum starved for 20 hours before hour preincubation with varying concentration of AG1478, AG879 or a combination of both in fresh serum-free media Then 10% (v/v) serum was added to the media (a), (b) and (c) After 24 hours incubation, crystal violet assay was performed to determined cell viability (d) Cells were also harvested at 20 hours for measurement of caspase-3 activity Results shown are a mean of experiments performed in duplicates ± SE 3000 2000 1000 AG1748 AG879 0.5 0 0 0 0 10 0 5 10 Drug conc (µM) 133 EGF (100ng/mL) AG1478 (5µM) AG879 (5µM) - + - + + + 10%FBS - + - + + + P-EGFR (Tyr1173) EGFR β-actin P-ErbB2 (Tyr1248) ErbB2 β-actin Figure 28: Effects of AG1478 and AG879 on EGFR and ErbB2 phosphorylation by EGF and serum LNCaP cells were serum starved for 20 hours before hour preincubation with AG1478 (5µM), AG879 (5µM) or DMSO control in fresh serum-free media Then EGF (100ng/ml) or 10% (v/v) serum was added to the media Whole cells were harvested after 15 minutes for SDS-PAGE analysis and immunoblotted for phosphorylated EGFR (P-EGFR) and ErbB2 (P-ErbB2) using anti-phospho-EGFR Tyr1173 and anti-phospho-ErbB2 Tyr1248 antibodies respectively Membrane were stripped and reprobed for total EGFR and ErbB2 β-actin was used as loading control Results shown in are from one representative experiment of at least three receptor dimerization (Downward et al 1984) whereas Tyr1248 was previously reported to be an ErbB2 autophosphorylation site (Hazan et al 1990) EGF induced strong phosphorylation of EGFR at Tyr1173 that was inhibited significantly by AG1478 However the EGFR kinase inhibitor seemed to inhibit phosphorylation of the ErbB2 at Tyr1248 as well even to a greater extent than the ErbB2 kinase inhibitor This suggests that this site maybe subjected to transphosphorylation by EGFR kinase region and to a much lesser extent autophosphorylation by ErbB2 kinase in the presence of EGF It is known that EGF binding to EGFR can lead to the formation of both homo- and heterodimers ErbB2 is an orphan receptor without a physiologic ligand and is the preferred partner for heterodimerization for all ErbB proteins (Graus-Porta et al 1997) Therefore the most likely heterodimers formed upon EGF 134 binding would be the EGFR/ErbB2 dimers The use of a combination of EGFR and ErbB2 kinase inhibitors did not have much synergistic effect on both their respective tyrosine phosphorylation sites as AG1478 alone inhibited almost completely EGFinduced phosphorylation on both EGFR and ErbB2 These results support that the phosphorylation of ErbB2 at Tyr1248 is mainly due to transactivation by EGFR kinase domain following EGF-induced heterodimerization In contrast, serum was not able to induce detectable phosphorylation of EGFR at Tyr1173 site while weak phosphorylation of ErbB2 Tyr1248 was detected This was totally inhibited by a combination of AG1478 and AG879 but only partially when either inhibitor was used, suggesting that both EGFR and ErbB2 kinase activity contribute to seruminduced ErbB2 phosphorylation at Tyr1248 These results show that while EGF binding to its receptor induces robust phosphorylation of the EGFR homodimers and EGFR/ErbB2 heterodimers as expected, serum is only able to weakly phosphorylate the ErbB2 receptor via a mechanism that is dependent on both EGFR and ErbB2 kinase activity Coinciding with EGF’s robust phosphorylation of EGFR, we observed a significant decrease in the level of detectable EGFR It has been observed that EGF binding to the EGFR activates the receptor and induces rapid internalization and degradation of the receptor by the ubiquitin system (Ceresa and Schmid 2000) (Waterman and Yarden 2001) Interestingly, incubation with EGFR kinase inhibitor prevented the internalization and degradation of the receptor It is possible that the kinase activity of EGFR of may play a role in the degradation of the receptor as phosphorylation of a single tyrosine residue on EGFR cytoplasmic region (Tyr1045) has been shown to be essential for Cbl-mediated EGFR degradation The Cbl ubiquitin ligase, when recruited to the internalized ligand-receptor complex in early 135 endosomes targets the receptor for lysosomal degradation by promoting receptor ubiquitination (Levkowitz et al 1998) This may explain why in the case of serum, EGFR degradation is not observed, possibly due to its inability to strongly activate the EGFR kinase activity (as seen by the absence of EGFR phosphorylation) We proceeded to examine the relevance of the EGFR and ErbB2 receptors in serum and EGF-mediated survival in LY-treated LNCaP cells Serum-starved LNCaP cells were pretreated with AG1478, AG879 or a combination of both inhibitors as before, followed by addition of LY with either EGF or serum Cells were harvested for PI staining of DNA and caspase-3 activity at various time points Inhibition of EGFR kinase activity with AG1478 did not have any effect on the ability of serum to rescue cells from LY-induced DNA fragmentation (Figure 29a) and caspase-3 activation (Figure 28b) On the contrary, AG1478 completely blocked the effect of EGF on LY-induced cell death The ErbB2 inhibitor, AG879, was less efficient in inhibiting the effect of EGF-mediated survival (Figure 29a and c) Moreover the combination of both inhibitors seems to have the same effect on survival as the presence of EGFR inhibitor alone Taken together with the receptor phosphorylation experiments, these results demonstrate that EGF binding to its receptor, EGFR, induces its activation and ErbB2 transactivation, and subsequent downstream signaling The survival signal of EGF however, seems to be transmitted by the EGFR kinase activity and not ErbB2’s On the other hand, serum-mediated survival signal does not appear to be conveyed via the EGFR or ErbB2 activation as inhibition of their kinase function did block serum’s protection on LY-mediated cell death 136 30 Apoptotic cells (% cells in subG1) 25 caspase activity (RFU/µg protein) a) b) serum LY LY+serum LY+EGF * 20 * * * 15 * * 10 control AG1478 AG879 AG1478:AG879 * : p < 0.01 (vs LY) Figure 29: Inhibition of EGFR and ErbB2 activity not prevent serum-mediated inhibition of LY-induced death in LNCaP cells LNCaP cells serum starved for 20 hours were preincubated for hour without (control) or with AG1478 (5µM) or AG879 (5µM) before being treated with 10% (v/v) serum (serum), LY (25µM) in serum-free media (LY), LY in media with 10% serum (LY+serum) and LY with EGF (100ng/ml) in serum-free media (LY+EGF) (a) 12 hours later % of apoptotic cells was determined by propidium iodide staining (b) and (c) Caspase-3 activity was measured hours post-treatment Results are a mean of three experiments done in duplicates ± SE c) caspase activity (RFU/µg protein) serum 10000 LY 8000 LY+serum LY+EGF 6000 4000 2000 control 30000 AG1478 serum LY 25000 LY+serum LY+EGF 20000 15000 10000 5000 control AG879 AG1478:AG879 137 3.1.9 Bax is required for LY-mediated apoptosis in LNCaP cells While the BH3-only protein, Bad is clearly responsible for EGF-mediated protection in LNCaP cells treated with LY, silencing Bad 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distinct pathways to inhibit the BH3 only protein BAD in prostate carcinoma LNCaP cells Oncogene 25(32): 4458-4469 Poster presentations: Chao, O.S and Clement, M.V Regulation of intracellular pH in the human prostate cancer cell LNCaP: A new survival signaling pathway independent of the activation of Akt th Poster presented at 10 Euro-conference on Apoptosis held at Pasteur Institute, Paris, France in October 2002 Chao, O.S and Clement, M.V Dephosphorylation of the BH3-only protein Bad: Critical for the induction of apoptosis in LNCaP prostate cancer cells Poster presented at the 4th GSS-FOM Meeting held at the Faculty of Medicine, NUS in May 2004 Won the merit award for poster presentation 245 ... SERUM-MEDIATED SURVIVAL IS INDEPENDENT OF MEKERK- AND PI3K-AKT -INDEPENDENT PATHWAY IN LNCAP CELLS Although both EGF and serum inhibited LY-induced apoptosis in LNCaP cells, analysis of survival signaling involved... PI3K-AKT -INDEPENDENT SURVIVAL PATHWAY(S) IN LNCAP CELLS The PI3K-Akt pathway has been proposed to be the major survival pathway in LNCaP cells due to PTEN inactivation in this PCa cell line (Lin et... supporting the essential role of O2·− in regulation of LNCaP cells sensitivity to apoptosis by LY This is in line with other recent findings that have demonstrated that an increase in intracellular