Analysis of p13k independent survival pathways in the prostate cancer cell line LN cap 1

ANALYSIS OF PI3K-INDEPENDENT SURVIVAL PATHWAYS IN THE PROSTATE CANCER CELL LINE LNCaP OLIVIA CHAO SU PING B.App.Sc (Hons.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 i Acknowledgements I wish to express my deepest gratitude to my mentor and supervisor, Associate Professor Marie-Véronique Clément, Department of Biochemistry, for introducing me to the field of apoptosis and generously sharing her vast knowledge on the subject The work in this thesis would never have been completed without her constant guidance, everlasting optimism and unending support in all matters, academic and otherwise Furthermore I want to thank her for her exceptional patience and faith in my abilities to accomplish the work involved I also wish to thank my co-supervisor Associate Professor Ren Ee Chee, Department of Microbiology, for opening the door to the field of scientific research and his support through the years I am also indebted to Professor Shazib Pervaiz and his lab in Department of Physiology for their helpful collaboration in knowledge and expertise My warmest thanks to my lab colleagues and friends for making the sometimes long days in the lab more bearable I want to thank them for being my teachers, my extra pair of hands when two weren’t enough, my ‘cheerleaders’ when the going gets tough and most of all for being my friends Finally, my deepest gratitude to my family for their support and encouragement throughout the years, and a special thanks to Seng, who has stood by me through the good and bad times I would not have been able to this without his unfailing love and faith in me ii Contents Acknowledgements ii Contents iii Summary vi List of Figures viii List of Tables x Abbreviations xi CHAPTER 1: INTRODUCTION 1.1 APOPTOSIS 1.1.1 1.1.2 1.2 1.2.3 1.3 1.4.4 18 Mutations that Confer Apoptosis Resistance 19 ANTI-APOPTOTIC MECHANISMS: GROWTH FACTOR SIGNALING 1.4.1 1.4.2 1.4.3 Role of Mitochondria in Apoptosis Bcl-2 family proteins 10 The Multidomain Pro-survival proteins 12 The Multidomain Pro-apoptotic proteins .13 The BH3-only Pro-apoptotic proteins 15 Interactions Among Bcl-2 family members 17 DEFECTS IN APOPTOSIS AND CANCER 1.3.1 1.4 Overview of Apoptosis Molecular mechanisms of apoptosis: Caspases as the central executioner of Apoptosis Activation of caspases Extrinsic and Intrinsic Apoptotic Pathway Substrates of caspases BCL-2 FAMILY 1.2.1 1.2.2 22 Growth Factor Signaling 22 Aberrant Growth Factors Signaling in Cancer Cells 24 Growth Factors-Regulated Survival Signaling Pathways: PI3KAkt Pathway 27 Aberrations of PI3K-Akt Signaling in Cancer 27 PI3K-Akt Signaling 28 Akt-mediated Survival Signaling 29 Growth Factors-Regulated Survival Signaling Pathways: Ras-RafMEK-ERK Pathway .32 Aberrations of Ras-Raf-MEK-ERK Signaling in Cancer 32 Ras-Raf-MEK-ERK signaling 33 p90 Ribosomal S6 Kinase (RSK) .38 Ras-Raf-MEK-ERK-mediated Survival Signaling 40 iii 1.5 ANTI-APOPTOTIC MECHANISMS: REDOX REGULATION OF CELL SIGNALING 1.5.1 1.5.2 1.5.3 1.5.4 1.6 1.7 Sources of Reactive Oxygen Species (ROS) and Redox Balance 45 ROS in Cell Signaling .48 ROS in Tumorigenesis 54 The Pro-Survival Role of Superoxide anion in Cancer 55 PROSTATE CANCER 1.6.1 1.6.2 1.6.3 AIM OF STUDY MATERIALS 2.1.1 2.1.2 2.1.3 2.1.4 2.2 GROWTH-FACTOR REGULATION OF CELL SURVIVAL 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 68 68 71 Treatment of Cells 71 Cell Viability Assay (Crystal Violet Assay) .72 DNA Fragmentation Assay 73 Caspase Activity Assay 74 Transient Transfection 75 β-galactosidase Survival Assay 76 SDS-PAGE and Western Immunoblotting 77 Intracellular Superoxide Measurement .78 RNA Interference (RNAi) Assay 79 Subcellular Fractionation 79 In vitro Akt Kinase Assay 81 Immunofluorescence Assay for Bax Activation .82 Statistical Analysis 83 CHAPTER 3: RESULTS 3.1 66 Chemicals .68 Antibodies 69 Plasmids used in the study .70 Cell lines and cell culture .71 METHODS 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11 2.2.12 2.2.13 59 Prostate Cancer Development and Progression 59 Survival Signals in Prostate Cancer Development and Progression 63 Growth Factor Signaling and Survival in Prostate Cancer 65 CHAPTER 2: MATERIALS AND METHODS 2.1 45 84 84 PI3K-Akt pathway is the major survival pathway in serumdeprived LNCaP cells 84 Serum and Epidermal Growth Factor activate an alternative survival mechanism that is PI3K-independent .93 EGF but not serum-mediated survival is MEK-dependent .99 EGF inhibits LY-induced apoptosis via inactivation of Bad 106 Serum-mediated inhibition of LY-induced cell death is independent of Bad inactivation 114 LY-mediated apoptosis is Bad-dependent 116 RSK1 is the Bad kinase activated by EGF in LNCaP cells 121 iv 3.1.8 3.1.9 3.1.10 3.2 Role of ErbB receptors in EGF- and serum-mediated survival of LNCaP cells 130 Bax is required for LY-mediated apoptosis in LNCaP cells 138 Serum promotes cell survival in LY-treated LNCaP cells via inhibition of Bad and Bax translocation .141 REACTIVE OXYGEN SPECIES REGULATION OF CELL SURVIVAL 3.2.1 3.2.2 3.2.3 Intracellular superoxide anions modulate serum’s inhibition of LY-induced cell death .147 Intracellular superoxide and activation of MEK-ERK-RSK pathway 153 Bad phosphorylation is regulated by intracellular O2·− level 158 CHAPTER 4: DISCUSSION 4.1 4.2 4.3 4.4 4.5 4.6 4.7 147 164 EGF AND SERUM ACTIVATE PI3K-AKT-INDEPENDENT SURVIVAL PATHWAY(S) IN LNCAP CELLS 165 EGF-MEDIATED SURVIVAL IS DEPENDENT ON MEK-ERK ACTIVATION IN LNCAP CELLS 166 EGF-MEDIATED SURVIVAL REQUIRES EGFR’S TYROSINE KINASE ACTIVITY 167 PHOSPHORYLATION AND INACTIVATION OF BAD IS AN IMPORTANT MECHANISM OF GROWTH FACTOR MEDIATED SURVIVAL IN PROSTATE CANCER CELLS 169 BAD EXPRESSION REGULATES CANCER CELLS SENSITIVITY TO APOPTOTIC TRIGGERS 172 SERUM-MEDIATED SURVIVAL IS INDEPENDENT OF MEKERK- AND PI3K-AKT-INDEPENDENT PATHWAY IN LNCAP CELLS 174 CROSSTALK BETWEEN PI3K-AKT PATHWAY AND MEK-ERK PATHWAY IN LNCAP CELLS 175 4.8 PRESENCE OF SERUM INDUCES A NON-CONDUCIVE ENVIRONMENT FOR TRANSLOCATION OF BAD AND BAX TO THE MITOCHONDRIA 177 4.9 INCREASED LEVEL OF SUPEROXIDE ANION PROMOTES LNCAP CELLS SURVIVAL 179 CONCLUSION 182 4.10 References 184 Publications and Presentations 245 v Summary Understanding the mechanisms behind tumor cells ability to evade cell death when confronted with multiple apoptotic signals during the course of cancer development and progression as well as during treatment with anti-cancer drugs, is of key importance towards development of efficient targeted therapy for different types of cancer In prostate cancer (PCa), one of the most common mutations found is inactivation mutation of PTEN (phosphatase and tensin homologue deleted on chromosome 10), resulting in constitutive activation of PI3K-Akt signaling, recognized as a major survival pathway in PCa cells However, there is increasing evidence supporting the existence of PI3K-Akt-independent survival pathways in PCa Deregulation of growth factor signaling is often observed during the course of PCa, and is proposed to gain importance as the tumor progresses towards androgenindependence In this study, we provide evidence for the role of growth factors- and serum-mediated activation of PI3K-Akt-independent survival signaling in PCa Using LNCaP prostate cancer cell line which harbors a PTEN frameshift mutation, we showed that EGF activated the MEK-ERK signaling pathway to promote LNCaP survival independently of PI3K-signaling Inhibition of apoptosis by EGF was mediated mainly through EGF’s ability to phosphorylate the pro-apoptotic BH3-only Bcl-2 protein, Bad, at Ser75, which has been shown to sequester the protein in the cytosol, preventing Bad from antagonizing pro-survival Bcl-2 functions Moreover we demonstrated that RSK1 as the kinase activated downstream of MEK-ERK signaling responsible for phosphorylating Bad Using siRNA strategy, we demonstrated that silencing Bad inhibited apoptosis similar to the level afforded by EGF, whereas silencing of Bax, a multidomain pro-apoptotic Bcl-2 protein, completely inhibited vi apoptosis, supporting the role of Bad as an “enabler” and Bax as an “effector” of apoptosis in LNCaP cells Moreover, we show that serum-mediated survival, unlike EGF, was independent of MEK-ERK signaling Although serum also phosphorylated Bad on Ser75, it was sensitive to inhibition of PI3K signaling and not MEK signaling, implying that serum phosphorylation of Bad was not the mechanism behind serummediated survival under those conditions We proceeded to demonstrate that serum inhibited translocation of both Bad and Bax to the mitochondria in a PI3Kindependent manner, which likely accounts for serum-mediated survival Additionally we show that while EGF transmits it survival signals through EGFR tyrosine kinase activation (not ErbB2), serum-mediated survival signaling did not require EGFR or ErbB2 tyrosine activity Previous studies in our lab demonstrated the role of increased O2·− in inhibition of apoptosis by diverse apoptotic triggers in tumor cells While others have shown growth factors and serum increase production of O2·− via NADPH oxidase, we found that serum did not induce significant increase in O2·− levels in LNCaP cells However, when LNCaP’s steady-state level of O2·− was decreased using an inhibitor of NADPH oxidase, DPI, serum-mediated survival was abrogated, while increasing O2·− levels using DDC an inhibitor of superoxide dismutase, protected the cells Interestingly, we also show that phosphorylation of Bad and ERK1/2 was sensitive to regulation by O2·− levels However, further studies are required to elucidate the molecular targets of O2·− in promoting survival as well as their regulation of Bad and ERK1/2 vii List of Figures Figure I: Bcl-2 family members 11 Figure II: Schematic diagram of Growth Factor-mediated survival signaling via PI3K-Akt pathway 30 Figure III: Domain structure and regulatory phosphorylations sites of RSK1 .39 Figure IV: Schematic diagram of Growth Factor-mediated survival signaling via Ras-Raf-MEK-ERK pathway .44 Figure V: Production of ROS in cells 48 Figure 1: PI3K-Akt pathway is constitutively activated in LNCaP in the absence of growth factors 86 Figure 2: Restoring a functional PTEN sensitizes LNCaP cells to cell death in the absence of growth factors .88 Figure 3: LY294002, a specific inhibitor of PI3K, sensitizes LNCaP cells to cell death in the absence of growth factor 91 Figure 4: LY-mediated cell death is caspase-3- and caspase-9-dependent but not caspase-8 92 Figure 5: Serum and EGF increased viability of LY-treated LNCaP cells 94 Figure 6: Serum and EGF decrease LY-mediated DNA fragmentation 95 Figure 7: Serum and EGF decrease LY-mediated caspase-3 and caspase-9 activation 96 Figure 8: Serum and EGF does not activate PI3K-Akt pathway in the presence of LY294002 .97 Figure 9: EGF induces robust and sustained ERK phosphorylation in LNCaP cells.100 Figure 10: EGF- and serum induced-ERK phosphorylation in LNCaP cells is MEKdependent 102 Figure 11: EGF but not serum-mediated decrease in DNA fragmentation is inhibited by U0126, a specific inhibitor of MEK1/2 104 Figure 12: EGF but not serum-mediated decrease in caspase-3 activation is inhibited by U0126 105 Figure 13: Serum and EGF does not alter Bcl-2 or Bcl-xL protein expression .108 Figure 14: Serum induces phosphorylation of endogenous Bad at Ser75 109 Figure 15: LY294002 induces total dephosphorylation of Bad .110 Figure 16: EGF activation of the MEK-ERK pathway leads to strong phosphorylation of Bad .112 Figure 17: Ser75 is the major phosphorylation site of Bad by EGF 113 viii Figure 18: Serum-mediated phosphorylation of Bad is dependent on PI3K but not MEK activity 115 Figure 19: Bad is required for LY-mediated cell death execution in LNCaP cells 117 Figure 20: EGF prevents LY-mediated translocation of Bad to the mitochondria 119 Figure 21: RSK is strongly phosphorylated by EGF via MEK-dependent pathway 122 Figure 22: RSK1 not RSK2 is the major Bad kinase activated by EGF in LNCaP cells 124 Figure 23: Silencing RSK1 not RSK attenuates EGF-mediated inhibition of apoptosis .125 Figure 24: Silencing RSK1 or RSK2 does not attenuate serum-mediated inhibition of apoptosis .126 Figure 25: Transfection of dominant-negative RSK1 decreases phosphorylation of Bad by EGF 128 Figure 26: Transfection of dominant-negative RSK1 attenuates EGF-mediated inhibition of apoptosis 129 Figure 27: Dose-response of LNCaP cell viability and caspase-3 activation to ErbB receptor kinase inhibitors 133 Figure 28: Effects of AG1478 and AG879 on EGFR and ErbB2 phosphorylation by EGF and serum .134 Figure 29: Inhibition of EGFR and ErbB2 activity not prevent serum-mediated inhibition of LY-induced death in LNCaP cells 137 Figure 30: Bax is required for induction of apoptosis in LY-treated LNCaP cells 140 Figure 31: LY-induced initial Bax translocation to the mitochondria is inhibited by serum but not EGF 143 Figure 32: LY-induced initial Bax activation is inhibited by serum but EGF 145 Figure 33: DPI decrease O2·− level and abrogates serum’s protection in LY-induced apoptosis .149 Figure 34: DDC increase intracellular O2·− concentration in LNCaP cells 151 Figure 35: DDC-mediated increase intracellular O2·− reverts sensitization to apoptosis induced by DPI .152 Figure 36: Activation of MEK-ERK pathway is regulated by intracellular superoxide 156 Figure 37: DDC increases activation of MEK-ERK-RSK signaling cascade in a dosedependent manner 157 Figure 38: DPI induces Bad dephosphorylation 159 Figure 39: DPI-induced caspase-3 activation in LY-treated LNCaP cells requires Bad 160 Figure 40: DDC induces Bad phosphorylation 160 Figure 41: PMA-induced O2·− production induces Bad phosphorylation and cell survival .163 ix List of Tables Table 1: Properties of the members of the caspase family x 3.1.5 Serum-mediated inhibition of LY-induced cell death is independent of Bad inactivation With our previous observation that serum was able to induce Bad phosphorylation at Ser75 (Figure 14), we wanted to investigate if Bad played a role in serum’s inhibitory effect on LY-induced death While addition of serum following 20 hours serum starvation induced phosphorylation of Bad at Ser75 to the same level seen with EGF (Figure 16: No serum vs EGF; and Figure 18: No serum vs serum), the presence of LY prevented serum induced Ser75 phosphorylation of Bad (Figure 18: Serum vs LY + serum) This suggests that serum-mediated phosphorylation of Bad at Ser75 is via PI3K-dependent mechanism More importantly, as serum was unable to phosphorylate Bad in the presence of LY, serum-mediated inhibition of apoptosis as observed previously is not due to inactivation of Bad This implies that the survival signals triggered by serum allow the cells to survive even when Bad is activated In contrast, inhibition of MEK with U0126 did not significantly prevent serum–induced Bad phosphorylation (Figure 18: serum vs serum + UO) Taken together, these results suggest that contrary to EGF, serum phosphorylates Bad at Ser75 through a MEK-independent, LY-inhibitable pathway 114 No Serum 1hr 3hr LY 6hr 1hr 3hr 6hr Serum LY + Serum 1hr 3hr 6hr 1hr Serum + U0 3hr 6hr 1hr 3hr 6hr P-Bad (S75) t-Bad Figure 18: Serum-mediated phosphorylation of Bad is dependent on PI3K but not MEK activity LNCaP cells were serum-starved for 20 hours followed by incubation with serum-free media (No serum), LY (25µM) in serum-free media (LY), media with 10% serum (Serum), LY in media with 10% serum (LY+Serum) or preincubated with U0126 for hours before being incubated with media with 10% serum (Serum+UO) Lysates were collected at 1, 3, hours post-treatment for SDS-PAGE analysis and immunoblotted with anti-phospho Bad Ser75 antibody Membrane were stripped and reprobed with total Bad antibody (t-Bad) as loading control Results shown are from one representative experiment of at least three 115 3.1.6 LY-mediated apoptosis is Bad-dependent To support further the role of Bad in LY-induced apoptosis in LNCaP cells, expression of the Bad protein was silenced by transfection with siRNA specific for human Bad In our lab, several methods of transfection were tested for LNCaP cell line over a period of 48 and 72 hours to achieve the most efficient level of silencing Cells transfected with 50nM of Bad siRNA utilizing the calcium phosphate transfection method exhibited 98% decrease in the level of Bad protein after 72 hours Therefore this protocol was used for subsequent experiments utilizing the siRNA technique for LNCaP cells Indeed, forty-eight hours post-transfection, cells transfected with the control siRNA or the specific Bad siRNA were serum-starved for 20 hours before LY was added in the presence or absence of EGF and apoptotic cell death and caspase-3 activity were assessed As can be seen in Figure 19a, silencing Bad alone could significantly decrease DNA fragmentation in LY-treated cells as compared to cells transfected with control siRNA The effect was most significant at hours and was sustained up to the 24 hours that was tested Similar results were obtained when caspase-3 activity was assessed (Figure 19b and c) These results show that in agreement with the role of Bad in the induction of apoptosis in LNCaP cells, silencing Bad significantly inhibited LY–induced death and caspase-3 activity To note both control siRNA and Bad siRNA did not induce significant apoptotic cell death or caspase-3 activity in cells grown in control media with 10% serum It demonstrates the control siRNA designed did not have an adverse effect on the cells’ viability and that the cell death induced after treatment with LY was due to the effect of the drug alone Interestingly, the level of inhibition of LY-induced apoptosis and caspase-3 116 activity obtained with Bad siRNA was similar to that observed upon incubation of control-transfected cells (control siRNA) with EGF a) siRNA : Control Bad 60 50 40 β-actin control siRNA Bad siRNA * * * ** 30 ** 20 ** 10 6hr 12hr GF LY +E LY se ru m LY +E GF LY se ru m GF LY +E LY se ru m % Apoptotic cells (% cells in sub-G1) t-Bad 24hr Figure 19: Bad is required for LY-mediated cell death execution in LNCaP cells LNCaP cells were transfected with either Bad siRNA or control siRNA for 48 hours followed by 20 hours of serum withdrawal before treatment with media with 10% serum (serum), serum-free media and LY (25μM) (LY) or serum-free media with LY and EGF (100ng/ml) (LY+EGF) After various timepoints indicated, cells were harvested for assessment of (a) percentage of cells in sub-G1 phase and (b) and (c) caspase-3 activity (a)(insert) Western blot of lysates collected 72 hours posttransfection showed effective Bad silencing β-actin showed equal loading Results are a mean of three experiments performed in duplicates ± SE 117 caspase activity (RFU/μg protein) b) 400 control siRNA 350 * Bad siRNA 300 250 200 150 ** 100 50 serum LY LY+EGF serum 1hr LY LY+EGF 3hr caspase activity (RFU/μg protein) c) 300 control siRNA * BadsiRNA * 250 200 ** 150 ** 100 50 serum LY LY+EGF serum 6hr LY LY+EGF 12hr * : p < 0.02 (control siRNA vs Bad siRNA) ** : p < 0.01 (Bad siRNA- serum vs LY) 118 a) LY+EGF LY serum 90 Bad Mitochondria Fraction Cu/Zn SOD VDAC b) Relative Intensity (%) 250 Bad 200 150 100 50 GF LY LY +E se ru m Figure 20: EGF prevents LY-mediated translocation of Bad to the mitochondria LNCaP cells serum starved for 20 hours before incubation with media with 10% serum (serum), LY (25µM) in serum-free media (LY) and EGF (100ng/ml) and LY in serum-free media (LY+EGF) After 90 minutes, cells were harvested for subcellular fractionation as in Materials and methods (a) Equal amount of mitochondrial protein was subjected to SDS-PAGE and immunoblotted Bad Purity of mitochondrial fraction was determined by blotting for mitochondria- or cytosolspecific protein, VDAC and Cu/Zn SOD, respectively (b) Densitometry analysis of the amount of Bad translocation to the mitochondria Results shown are the ratio of the mitochondrial Bad over VDAC converted to % of ratio obtained in cells incubated with serum (% of relative intensity) Results shown represent one experiment of at least three 119 Once Bad is activated by dephosphorylation of key serine residues, it is free to translocate from the cytosol to the mitochondria where it binds and antagonizes the pro-survival proteins like Bcl-xL and Bcl-2 Translocation to the mitochondria is essential for Bad to exert its pro-apoptotic effects To further confirmed the role of Bad in LY-induced cell death and EGF-mediated inhibition of cell death, translocation of Bad to the mitochondria was assessed Figure 20a and b demonstrate that LY-induced cell death correlates with an increase of Bad translocation to the mitochondria as compared to cells in control media Moreover the addition of EGF into the media in the presence of LY significantly decreased Bad translocation to the mitochondria This data is in line with the previous observation that EGF-mediated inhibition of LY-induced apoptosis could be due to inhibition of Bad activation through Ser75 phosphorylation Taken together, these results strongly implicate that activation of Bad and its translocation to the mitochondria mediates the LY-induced apoptotic cell death via activation of caspase-9 and caspase-3 Additionally, EGF’s ability to phosphorylate Bad at Ser75 and thus decrease its translocation to the mitochondria is likely to be responsible for its inhibitory effect on LY-induced cell death 120 3.1.7 RSK1 is the Bad kinase activated by EGF in LNCaP cells The members of the p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases are MAPK-activated protein kinases that are widely expressed in many human tissues In response to growth factors, polypeptide hormones or neurotransmitters, they are activated via phosphorylation at several key serine/threonine residues (Frodin and Gammeltoft 1999) Phosphorylation of Ser380 of RSK1 has been shown to be critical for the activation of its amino-terminal kinase (NTKD), which is the responsible for the phosphorylation of RSKs’ substrates (Dalby et al 1998; Roux et al 2003) ERK1/2-dependent phosphorylation of Bad at Ser75 has mainly been attributed to the activation of the members of the RSK family (Bonni et al 1999; Shimamura et al 2000) Indeed, incubation of LNCaP cells with EGF following twenty hours of serum starvation induces phosphorylation of RSK at Ser380 even in the presence of LY (Figure 21: EGF vs LY + EGF) Moreover, EGFinduced RSK phosphorylation is dependent upon MEK activation as shown by the absence of RSK phosphorylation upon incubation with U0126 (Figure 21: EGF (control) vs EGF (U0126)) Notably, serum was not able to induce a detectable phosphorylation of RSK supporting previous observation that phosphorylation of Bad by serum is not dependent on MEK The four human isoforms of RSK identified to date (RSK1, RSK2, RSK3 and RSK4) (Frodin and Gammeltoft 1999; Yntema et al 1999) show 73 to 80% amino acid identity Although they are structurally similar and preferentially phosphorylates at serine/threonine residues within the Arg/Lys-Xaa-Arg-Xaa-Xaa-Ser/Thr or ArgArg-Xaa-Ser/Thr peptide motifs (Leighton et al 1995), it is still not clear whether 121 F LY +E G F EG se ru LY m +s er um LY N o se ru m F LY +E G F U0126 (10μM) EG se ru LY m +s er um LY N o se ru m control P-RSK (S380) β-actin Figure 21: RSK is strongly phosphorylated by EGF via MEK-dependent pathway LNCaP cells were serum-starved for 20 hours followed by pretreatment with U0126 (10µM) or DMSO control in fresh serum-free media for hours This is followed by addition of 10% serum, LY (25µM) and/or EGF (100ng/ml) into the media for hour before the cells were harvested and equal amounts of protein were subjected to SDSPAGE (a) Blots were probed with anti-phospho RSK Ser380 antibody and β-actin as loading control Results shown are from one representative experiment of at least three different RSK isoforms have different protein targets and functions depending on cell type or upstream signaling The peptide sequence that surrounds the murine Bad Ser112 corresponds to the sequence that can be phosphorylated by members of the RSK family (Blenis 1993; Alessi et al 1996) Both RSK1 and RSK2 have been shown to phosphorylate Bad at Ser75 in hematopoietic (Shimamura et al 2000) and neuronal (Bonni et al 1999) cell lines, respectively Unfortunately, the phosphoantibody used to detect phosphorylated RSK cross-reacts with phosphorylated form of RSK1 and RSK2 at homologous sites 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 at Ser75 by approximately 80% as compared to cells transfected with control siRNA, whereas RSK2 knock-down had a less significant effect (approximately 35%) (Figure 22a and b) Moreover, LNCaP transfected with 122 increasing amount of RSK1 siRNA followed by stimulation with EGF demonstrated a corresponding reduction in the level of Bad phosphorylation (Figure 22c and d) se ru m LY +s e LY rum LY +E G F se ru m LY +s e LY rum RSK1 RSK2 F se ru m LY +s e LY rum LY +E G F Control siRNA: LY +E G a) RSK1 RSK2 P-Bad (S75) t-Bad 120 100 control 80 RSK1 RSK2 60 40 20 GF LY +E LY LY +s er um se ru m b) Relative Intensity of P-Bad (%) β-actin c) siRNA amount: siRNA: 25nM Ctrl 50nM RSK1 Ctrl 100nM RSK1 Ctrl RSK1 RSK1 P-Bad (S75) β-actin 123 d) Relative Intensity (%) 100 P-Bad (S75) 81.6 80 60.1 60 43.8 40 20 25 50 100 RSK1 siRNA conc (nM) Figure 22: RSK1 not RSK2 is the major Bad kinase activated by EGF in LNCaP cells (a) LNCaP cells were transfected with either control siRNA or siRNA specific for RSK1 or RSK2 Forty-eight hours post-transfection cells were serum starved for 20 hours 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 hour posttreatment and analyzed by SDS-PAGE and immunoblotted with phospho-Bad antibody and total Bad (t-Bad) and β-actin antibody as loading control Immunoblotting with anti-RSK1 or anti-RSK2 antibodies demonstrated specific silencing of RSK1 and RSK2 respectively (b) Densitometry analysis of results from (a) Results are shown as the ratio of phosphorylated Bad over β-actin converted to % of the ratio obtained in control siRNA transfected cells incubated with serum (% of relative intensity) Results shown in are from representative experiment of at least (c) LNCaP cells were transfected with either increasing concentration of control siRNA or RSK1 siRNA Forty-eight hours post-transfection cells were serum starved for 20 hours before adding serum-free media with EGF (100ng/ml) Cells were harvested hour post-treatment and analyzed by SDS-PAGE and immunoblotted with phospho-Bad and RSK1 antibodies β-actin was used as a loading control (b) Densitometry analysis of results from (a) Results are shown as the ratio of phosphorylated Bad over β-actin converted to % of the ratio obtained in control siRNA transfected cells (% of relative intensity) Results shown in are from one representative experiment of at least three To further support the role of RSK1 as the EGF-stimulated Bad kinase, the effects of silencing RSK1 and RSK2 on EGF-mediated survival was determined Similar to the effects of silencing Bad, silencing of RSK1 diminished the ability of 124 EGF to protect LNCaP cells from LY-mediated apoptosis and caspase-3 activation, while silencing of RSK2 had no affect (Figure 23a and b) On the contrary, serummediated phosphorylation of Bad at Ser75 was not affected by silencing of either RSK1 or RSK2, whereas as shown previously presence of LY in serum-containing medium significantly dephosphorylated Bad at Ser75 (Figure 22: serum vs LY + serum) In addition, results shown in Figure 24a and b further confirmed the existence of a serum-dependent survival pathway that does not rely on the phosphorylation of Bad and RSK1 or RSK2 activity Indeed, serum maintained LNCaP viability and inhibited caspase-3 activity upon incubation with LY in absence of RSK1 or RSK2 protein a) b) 50 serum 10000 40 serum LY LY+EGF 30 * * 20 10 caspase activity (RFU/μg protein) % Apoptotic cells (% cells in sub-G1) LY 8000 LY+EGF 6000 * * 4000 2000 0 control RSK1 siRNA RSK2 control RSK1 RSK2 siRNA * : p < 0.01 (vs LY) Figure 23: Silencing RSK1 not RSK attenuates EGF-mediated inhibition of apoptosis LNCaP cells were transfected with either control siRNA or siRNA specific for RSK1 or RSK2 Forty-eight hours post-transfection cells were serum starved for 20 hours before treatment with 10% serum containing-medium (serum), LY in serum-free medium (LY) and LY in serum-free medium and EGF (LY + EGF) Apoptotic cell death was analyzed by (a) % cells in sub-G1 phase 24 hours post-treatment and (b) caspase-3 activity at 12 hours post-treatment (a) and (b) show mean of three experiments performed in duplicates ± SE 125 a) b) 50 10000 serum LY LY+serum ** 30 20 ** ** 10 caspase activity (RFU/μg protein) % Apoptotic cells (% cells in sub-G1) LY 40 serum 8000 LY+serum 6000 4000 ** ** ** 2000 0 control RSK1 siRNA RSK2 control RSK1 RSK2 siRNA ** : p < 0.05 (vs LY) Figure 24: Silencing RSK1 or RSK2 does not attenuate serum-mediated inhibition of apoptosis LNCaP cells were transfected with either control siRNA or siRNA specific for RSK1 or RSK2 Forty-eight hours post-transfection cells were serum starved for 20 hours before treatment with 10% serum containing-medium (serum), LY in serum-free medium (LY) and LY in medium with 10 serum (LY + serum) Apoptotic cell death was analyzed by (a) % cells in sub-G1 phase 24 hours post-treatment and (b) caspase-3 activity at 12 hours post-treatment (a) and (b) show mean of three experiments performed in duplicates ± SE The role of RSK1 in EGF-mediated Bad phosphorylation and survival was further confirmed by overexpressing a dominant negative mutant of RSK1, p90RskD205N, where the ATP-binding site of the amino-terminal kinase domain (NTKD) was disrupted by replacing aspartic acid on position 205 in the DFG consensus sequence with asparagine (Schouten et al 1997) Figure 25a and b show that in LNCaP cells transfected with RSK1 dominant negative mutant, EGF’s ability to phoshorylate Bad at Ser75 was reduced as compared to cells transfected with the control vector pcDNA3.1 Immunoblotting with anti-HA and RSK1 antibody confirmed the transfection of the RSK1 mutant plasmid with carries a HA tag However the repression of EGF-mediated Bad phosphorylation achieved was not as 126 dramatic as the results obtained with RSK1 siRNA most probably because contrary to transfection with siRNA, not all cells were successfully transfected with the plasmid encoding for the dominant negative form of RSK1 Results of caspase-3 activity show that transfection with the mutant RSK1 decreased EGF-mediated inhibition of caspase-3 activation in LY-treated LNCaP cells (Figure 26a and b) Taken together, these results support the critical role of RSK1 activation in EGFmediated survival in LNCaP cells via phosphorylation of Bad Furthermore, these experiments also confirm a serum-dependent survival pathway that does not involve activation of either RSK1 or RSK2 and is independent of Bad phosphorylation at Ser75 127 a) RSK1DN LY F F se ru m LY +E G LY se ru m LY +E G pcDNA3.1 Plasmid: HA RSK1 P-Bad (S75) Bad pcDNA3.1 120 RSK1DN 100 80 60 40 20 GF LY +E LY se ru m b) Relative Intensity of P-Bad (%) β-actin Figure 25: Transfection of dominant-negative RSK1 decreases phosphorylation of Bad by EGF LNCaP cells were transfected with either dominant negative mutant of RSK1 or control vector plasmid pcDNA3.1 as in Materials and methods Fourty-eight hours post-transfection, cells were serum starved for 20 hours before incubation with media containing 10% serum (serum), LY in serum-free media (LY) or LY with EGF in serum-free media (LY + EGF) (a) After hour, cells were harvested for SDS-PAGE and immunoblotted with phospho-Bad, anti-HA and RSK1 antibody β-actin and total Bad (t-Bad) demonstrate equal loading (b) Densitometry analysis of results from (a) Results are shown as the ratio of phosphorylated Bad over β-actin converted to % of the ratio obtained in pcDNA3.1 transfected cells incubated with serum (% of relative intensity) Results shown in are from one representative experiment of at least three 128 ... Role of Superoxide anion in Cancer 55 PROSTATE CANCER 1. 6 .1 1.6.2 1. 6.3 AIM OF STUDY MATERIALS 2 .1. 1 2 .1. 2 2 .1. 3 2 .1. 4 2.2 GROWTH-FACTOR REGULATION OF CELL SURVIVAL 3 .1. 1 3 .1. 2 3 .1. 3 3 .1. 4 3 .1. 5... Bad-dependent 11 6 RSK1 is the Bad kinase activated by EGF in LNCaP cells 12 1 iv 3 .1. 8 3 .1. 9 3 .1. 10 3.2 Role of ErbB receptors in EGF- and serum-mediated survival of LNCaP cells 13 0 Bax... apoptosis in LNCaP cells 13 8 Serum promotes cell survival in LY-treated LNCaP cells via inhibition of Bad and Bax translocation .14 1 REACTIVE OXYGEN SPECIES REGULATION OF CELL SURVIVAL 3.2 .1 3.2.2